CN117377767A - Immunomodulatory molecules and uses thereof - Google Patents

Immunomodulatory molecules and uses thereof Download PDF

Info

Publication number
CN117377767A
CN117377767A CN202280019509.3A CN202280019509A CN117377767A CN 117377767 A CN117377767 A CN 117377767A CN 202280019509 A CN202280019509 A CN 202280019509A CN 117377767 A CN117377767 A CN 117377767A
Authority
CN
China
Prior art keywords
terminus
variant
binding
subunit
domain
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
CN202280019509.3A
Other languages
Chinese (zh)
Inventor
E·吴
X·吴
J·威克菲尔德
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Immune Recovery Co
Original Assignee
Immune Recovery Co
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from PCT/US2021/073107 external-priority patent/WO2022140797A1/en
Application filed by Immune Recovery Co filed Critical Immune Recovery Co
Priority claimed from PCT/US2022/071077 external-priority patent/WO2022192898A2/en
Publication of CN117377767A publication Critical patent/CN117377767A/en
Pending legal-status Critical Current

Links

Abstract

The present application relates to immunomodulatory molecules comprising a first binding domain (e.g., an immunostimulatory cytokine such as IL-2 or IL-12 or a variant thereof) that specifically recognizes a first target molecule (e.g., a receptor for an immunostimulatory cytokine) and a second binding domain (e.g., an inhibitory checkpoint molecule such as PD-1) that specifically recognizes a second target molecule, wherein the first binding domain upregulates an immune response upon binding to the first target molecule, and wherein the second binding domain downregulates an immune response upon binding to the second target molecule. Methods of making and using such immunomodulatory molecules are also provided.

Description

Immunomodulatory molecules and uses thereof
Cross Reference to Related Applications
The present application claims priority from U.S. provisional patent application No. US63/159,441 filed on day 3 and 10 of 2021 and from International patent application No. PCT/US2021/073107 filed on day 23 of 2021, 12, each of which is incorporated herein by reference in its entirety.
Sequence list submitted in ASCII text file
The contents of the following submitted ASCII text files are incorporated herein by reference in their entirety: a Computer Readable Form (CRF) of the sequence listing (file name: 75439000640 seqlist. Txt, date of preservation: 2022, 3, 10 days, size: 789,099 bytes).
Technical Field
The present invention relates to immunoregulatory molecules that can both up-regulate and down-regulate immune responses, methods of their preparation and uses thereof.
Background
Current immunotherapy often induces excessive unwanted immune responses such as immune cell overactivation, cytokine storms, etc.
Cytokines are key regulators of the innate and adaptive immune systems that enable immune cells to communicate with each other. Cytokine therapy that activates the immune system of cancer patients remains an important target area for clinical cancer research. One significant challenge of cytokine monotherapy is achieving an effective anti-tumor response without causing therapeutic limiting toxicity. The low response rate and notorious toxicity of IL-2 and IL-12 therapies well illustrate this dilemma. High doses of IL-2 have been found to induce Vascular Leak Syndrome (VLS), tumor tolerance caused by activation-induced cell death (AICD), and immunosuppression caused by activation of regulatory T cells (tregs). These serious side effects often limit the optimal IL-2 dose, which limits the number of patients who successfully respond to treatment. IL-12 shows moderate anti-tumor responses in clinical trials, but is often accompanied by significant toxicity problems (Lasek et al, cancer Immunol immunother, 2014). IL-12 therapy has been found to be associated with systemic influenza-like symptoms (e.g., fever, chill, fatigue, erythromelalgia and headache), and toxic effects on bone marrow and liver. Dose studies have shown that patients can tolerate only IL-12 below 1. Mu.g/kg, well below a therapeutically effective dose. IL-12 has not demonstrated an effective sustained therapeutic effect in clinical trials, either as monotherapy or in combination with other agents (Lasek et al, 2014).
Several approaches have been taken to overcome the problems of cytokine monotherapy. Recently, NKTR-214, a recombinant human IL-2 conjugated with polyethylene glycol (PEG; "IL-2-PEG"), has shown promising results in animal models. IL-2-PEG provides two benefits. First, the steric hindrance of PEG masks the region of IL-2 that interacts with the IL-2 receptor alpha (IL-2rα) subunit responsible for activating immunosuppressive tregs and biasing the activity of tumor killing cd8+ T cells (Charych et al, clin Cancer res., 2016). Second, conjugation of PEG greatly increases plasma half-life and proteolytic stability, and reduces immunogenicity and liver uptake (Chaffee et al, J Clin invest, 1992;Pyatak et al, res Commun Chem Pathol pharmacol, 1980). Targeted delivery of cytokines (e.g., IL-12) to tumor sites by local injection or use of immune cytokines (cytokines fused to antibodies, antibody fragments, or ligand/receptor-Fc fusion proteins) has also been developed to overcome the side effects of cytokine therapy. The immunocytokines may target the cytokine to a target cell or tissue, such as a tumor cell or immune effector cell (Klein et al, oncominium, 2017; king et al, J Clin Oncol, 2004).
The disclosures of all publications, patents, patent applications, and published patent applications mentioned herein are incorporated by reference in their entirety.
Summary of The Invention
One aspect of the invention provides an immunomodulatory molecule comprising a first binding domain (e.g., an immunostimulatory cytokine such as IL-2 or IL-12 or variant thereof) that specifically recognizes a first target molecule (e.g., an receptor for an immunostimulatory cytokine) and a second binding domain (e.g., an agonist ligand such as PD-L1 or PD-L2 or variant thereof, or an agonist antigen binding fragment such as an anti-PD-1 agonist Fab, scFv, VHH or full-length antibody) that specifically recognizes a second target molecule (e.g., an inhibitory checkpoint molecule such as PD-1), wherein the first binding domain upregulates an immune response upon binding to the first target molecule (e.g., IL-2 or IL-12 receptor), and wherein the second binding domain downregulates an immune response upon binding to the second target molecule (e.g., PD-1).
Another aspect of the present application provides a method of modulating an immune response in an individual comprising administering to the individual an effective amount of any of the immunomodulatory molecules described herein.
Also provided are isolated nucleic acids encoding any of the immunomodulatory molecules described herein, vectors (e.g., lentiviral vectors) comprising such nucleic acids, host cells (e.g., CHO cells) comprising such nucleic acids or vectors, and methods of producing any of the immunomodulatory molecules described herein.
Also provided are compositions (e.g., pharmaceutical compositions), kits, and articles of manufacture comprising any of the immunomodulatory molecules described herein. Also provided are methods of treating a disease or disorder (e.g., cancer, infection, autoimmune disease, allergy, graft rejection, or graft versus host disease (GvHD)) in an individual using an effective amount of any of the immunomodulatory molecules or compositions described herein (e.g., a pharmaceutical composition).
Drawings
FIGS. 1A-1W depict exemplary immunomodulatory molecular structures of the invention. FIG. 1A depicts an exemplary immunomodulatory structure comprising a cytokine or variant thereof fused to the N-terminus of a subunit of the Fc fragment of a parent full length antibody. FIG. 1B depicts a dimeric (homodimeric or heterodimeric) cytokine or variant thereof (e.g., IFN-gamma, IL-10, IL-12, or IL-23) expressed in a single chain and located at the hinge region of one heavy chain of a parent full-length antibody. FIGS. 1A-1B depict exemplary immunostimulatory structures of the invention wherein an immunostimulatory cytokine or variant thereof (e.g., IFN-gamma, IL-2, IL-12, or IL-23) is expressed in a single chain and is located at the hinge region of one heavy chain of a dimeric parent ligand/receptor/Fab-hinge-Fc fusion protein. FIG. 1A shows that the Fab of the dimeric parent ligand/receptor/Fab-hinge-Fc fusion protein can be an agonist. FIG. 1B shows that the Fab of the dimeric parent ligand/receptor/Fab-hinge-Fc fusion protein can be either agonist or non-agonist. FIG. 1C depicts an exemplary immunostimulatory structure of the invention wherein an immunostimulatory cytokine or variant thereof (e.g., IFN-gamma, IL-2, IL-12, or IL-23) is expressed in a single chain and is located at the hinge region of one heavy chain of a parent full-length agonist antibody (e.g., an anti-PD-1 agonist). FIG. 1D depicts an alternative exemplary immunomodulatory structure of the invention, wherein an immunostimulatory cytokine or variant thereof is located between the VH (e.g., within the Fab of an agonist antibody) and a subunit of the Fc fragment. FIG. 1E depicts an exemplary immunomodulatory structure comprising an immunostimulatory cytokine or variant thereof (e.g., IFN-gamma, IL-2, IL-12, or IL-23) located at the hinge region of one polypeptide of a dimeric parent ligand/receptor-hinge-Fc fusion protein. FIG. 1F depicts an exemplary immunomodulatory structure comprising an immunostimulatory cytokine or variant thereof (e.g., IFN-gamma, IL-2, IL-12, or IL-23) fused to the N-terminus of a subunit of an Fc fragment of a parent full-length agonist antibody (e.g., an anti-PD agonist). FIG. 1G depicts an exemplary immunomodulatory structure comprising a cytokine or variant thereof (e.g., IFN-gamma, IL-2, IL-12, or IL-23) located at the hinge region of one polypeptide of a parent ligand/receptor-hinge-Fc fusion protein. FIG. 1H depicts an exemplary immunomodulatory structure comprising an immunostimulatory cytokine or variant thereof (e.g., IFN-gamma, IL-2, IL-12, or IL-23) located at the hinge region of one polypeptide of a dimeric parent ligand/receptor-hinge-Fc fusion protein. FIG. 1I depicts an exemplary immunomodulatory structure of the invention wherein an immunostimulatory cytokine or variant thereof (e.g., IFN-gamma, IL-2, IL-12, or IL-23) is located at the C-terminus of the Fc domain of a parent ligand/receptor-hinge-Fc fusion protein. FIG. 1J depicts an exemplary immunomodulatory structure comprising an immunostimulatory cytokine or variant thereof (e.g., IFN-gamma, IL-2, IL-12, or IL-23) at the C-terminus of the Fc domain of a parent full-length agonist antibody (e.g., an anti-PD-1 agonist). FIG. 1K depicts an exemplary immunomodulatory structure comprising an immunostimulatory cytokine or variant thereof (e.g., IFN-gamma, IL-2, IL-12, or IL-23) located at the C-terminus of the Fc domain of a dimeric parent ligand/receptor/agonist Fab-hinge-Fc fusion protein. FIG. 1L depicts two cytokines or variants thereof, each located in the hinge region of one polypeptide of a parent ligand/receptor-hinge-Fc fusion protein; or dimeric (homodimeric or heterodimeric) cytokines or variants thereof, each subunit being located at the hinge region of one polypeptide of the parent ligand/receptor-hinge-Fc fusion protein. FIG. 1M depicts two cytokines or variants thereof, each located in the hinge region of one polypeptide of a parent ligand/receptor-hinge-Fc fusion protein; or a dimeric (homodimeric or heterodimeric) cytokine or variant thereof, each subunit being located at the hinge region of one polypeptide of the dimeric parent ligand/receptor-hinge-Fc fusion protein. FIGS. 1N-1O depict two cytokines or variants thereof, each located in the hinge region of one polypeptide of a parent full-length agonist antibody; or dimeric (homodimeric or heterodimeric) cytokines or variants thereof, each subunit being located at the hinge region of one polypeptide of the parent full-length agonist antibody. Figure 1N shows Fab identity where they are all from agonist antibodies. FIG. 1O shows that Fab's may be different, one being the Fab of an agonist antibody and the other being a different Fab (which may be a non-agonist or an agonist). FIG. 1P depicts two cytokines or variants thereof, each located at the C-terminus of the Fc domain of a parent ligand/receptor-hinge-Fc fusion protein; or a dimeric (homodimeric or heterodimeric) cytokine or variant thereof, each subunit being located at the C-terminus of one polypeptide of the parent ligand/receptor-hinge-Fc fusion protein and the other being located at the hinge region of one polypeptide of the parent ligand/receptor-hinge-Fc fusion protein. FIGS. 1Q-1R depict two cytokines or variants thereof, each located at the C-terminus of one polypeptide of a parent full length antibody; or a dimeric (homodimeric or heterodimeric) cytokine or variant thereof, each subunit being located at the C-terminus of one polypeptide of the Fc domain of an agonist antibody and the other being located at the C-terminus of one polypeptide of the Fc domain. Figure 1Q shows that Fab may be identical, where both are from agonist antibodies. Fig. 1R shows that Fab may be different, one being Fab of agonist antibodies and the other being different Fab (which may be non-agonist or agonist). FIG. 1S depicts an exemplary immunomodulatory structure comprising an immunostimulatory cytokine or variant thereof fused to the C-terminus of the light chain constant region (CL) of a parent full-length agonist antibody. FIG. 1T depicts an exemplary immunomodulatory structure comprising an immunostimulatory cytokine or variant thereof fused to the N-terminus of a heavy chain variable domain (VH) of a parent full length antibody. FIG. 1U depicts an exemplary immunomodulatory structure comprising an immunostimulatory cytokine or variant thereof fused to the N-terminus of one polypeptide of a dimeric parent ligand/receptor-hinge-Fc fusion protein. FIG. 1V depicts an exemplary immunomodulatory structure comprising an immunostimulatory cytokine or variant thereof fused to the N-terminus of one polypeptide of a parent ligand/receptor/agonist Fab-hinge-Fc fusion protein. FIG. 1W depicts an exemplary immunomodulatory structure comprising an immunostimulatory cytokine or variant thereof fused to the N-terminus of a heavy chain variable domain (VH) of a parent ligand/receptor/agonist Fab-hinge-Fc fusion protein.
FIGS. 2A-2C depict tumor volumes in CT26 syngeneic tumor mice treated with IL-12 (F60A)/PD-L2-Fc, hinge (IW- # 30) immunoregulatory molecules, C-terminal ends of IL-12 (F60A)/PD-L2-Fc, HC (IW- # 34) immunoregulatory molecules, or PBS (negative control). Black arrows indicate days of injection. Individual mouse responses in each group are given in figures 2B-2C.
Figures 3A-3B depict CT26 and EMT6 tumor volume growth over time in cured CT26 mice (previously cured in figures 2A-2C).
FIG. 4A depicts tumor volumes in CT26 syngeneic tumor mice treated with IL-12 (E59A/F60A)/PD-L2-Fc, hinge (IW- # 29) immunoregulatory molecules, IL-12 (F60A)/PD-L2-Fc, hinge (IW- # 30) immunoregulatory molecules, or PBS (negative control). Black arrows indicate days of injection. FIG. 4B depicts a series of photographs taken of a mouse during treatment with an L-12 (F60A)/PD-L2-Fc, hinge (IW- # 30) immunoregulatory molecule.
FIGS. 5A-5D depict tumor volumes in EMT6 syngeneic tumor mice treated with IL-12 (E59A/F60A)/PD-L2-Fc, hinge (IW- # 29) immunoregulatory molecules, IL-12 (F60A)/PD-L2-Fc, hinge (IW- # 30) immunoregulatory molecules, IL-12 (E59A/F60A)/anti-PD-1, hinge (IW- # 48) immunoregulatory molecules, or PBS (negative control). Black arrows indicate days of injection. Individual mouse responses in each group are given in figures 5B-5D.
Figures 6A-6C depict CT26 and EMT6 tumor volume growth over time in cured EMT6 mice (previously cured in figures 5A-5D).
FIGS. 7A-7D depict tumor volumes in 4T1 syngeneic tumor mice treated with increasing concentrations (0, 1, 3, 10 and 50 mg/kg) of IL-12 (F60A)/PD-L2-Fc, hinge (IW- # 30) immunoregulatory molecules, IL-12 (F60A)/PD-L2-Fc, C-terminal ends of HC (IW- # 34) immunoregulatory molecules, IL-12 (F60A)/anti-PD-1, hinge (IW- # 46) immunoregulatory molecules, IL-12 (E59A/F60A)/anti-PD-1, hinge (IW- # 48) immunoregulatory molecules or PBS (negative control). Black arrows indicate days of injection.
FIGS. 8A-8C depict tumor volumes in B16-F10 syngeneic tumor mice treated with IL-12 (F60A)/PD-L2-Fc, hinge (IW- # 30) immunoregulatory molecules, PD-L2-Fc/IL-12 (F60A) (IW- #34, C-terminal fusion) immunoregulatory molecules, or PBS (negative control). Black arrows indicate days of injection. Figure 8A shows the average tumor volume for all mice groups, with the average tumor size (±std) at the time of first treatment administration shown in brackets. FIGS. 8B-8C show tumor volumes of individual mice receiving the indicated IL-12 immunomodulatory molecules.
FIGS. 9A-9C depict tumor volumes in LL2 syngeneic tumor mice treated with IL-12 (F60A)/PD-L2-Fc, hinge (IW- # 30) immunoregulatory molecules, C-terminal ends of IL-12 (F60A)/PD-L2-Fc, HC (IW- # 34) immunoregulatory molecules, or PBS (negative control). Black arrows indicate days of injection. Individual mouse responses in each group are given in figures 9B-9C.
Figure 10 shows two methods of activating the immune system against disease (e.g., cancer). The left panel shows a target independent activation mechanism ("transactivation") in which an immunomodulatory molecule can bind to a target antigen on an immune cell (e.g., a T cell) and can bind to a target antigen on a target cell (e.g., a tumor cell), thereby bringing the immune cell into proximity to the target cell to obtain a therapeutic effect. However, this approach may be associated with systemic toxicity, as the binding domain targeted to immune cells (e.g., wild-type (wt) immunostimulatory cytokines such as IL-12 or IL-2) may stimulate an immune response even in the absence of target cells. The right panel shows a target cell antigen (e.g., tumor antigen) independent activation mechanism ("cis-activation") in which an immunoregulatory molecule can up-and down-regulate an immune response, which more closely mimics the natural regulation and balance of the immune system. In a further aspect, the immunoregulatory molecules in both the left and right panels may also have a "restricted activation mechanism" in which a first binding domain that up-regulates an immune response (e.g., an immunostimulatory cytokine such as IL-12 or IL-2) after binding to an immune cell is modified to reduce activity (binding and/or biological activity), and/or in a "masked" configuration (e.g., at the hinge region) until binding of a second binding domain to a second target antigen (e.g., a tumor antigen or immune cell surface molecule) occurs. Exemplary immunomodulatory molecules of the invention may act via restricted activation, cis-activation, trans-activation, or all mechanisms.
FIGS. 11A-11L depict exemplary multi-specific immunomodulatory molecules of the invention. The immunomodulatory molecule may comprise a combination of binding domain types: i) A first binding domain, labeled "1" in the figure, that upregulates the immune response upon binding to the first target molecule; ii) a second binding domain, labeled "2" in the figure, which down-regulates the immune response upon binding to a second target molecule; and iii) optionally, a third binding domain, labeled "3" in the figures, that facilitates localization of the immunoregulatory molecule to a target site (e.g., tumor microenvironment) by targeting a third target molecule (e.g., T cell depleted marker, T cell surface marker, or tumor antigen). The immunoregulatory molecule may comprise one or more of any of the first, second and/or third binding domains. The plurality of first binding domains may be the same or different from each other. The plurality of second binding domains may be the same or different from each other. The plurality of third binding domains may be the same or different from each other. The various binding domains within the immunoregulatory molecule may be constructed in a variety of configurations, not limited to those shown in FIGS. 11A-11L. As illustrated in FIGS. 11A-11L, the IL-12 moiety located at the C' of one or both Fc subunits (e.g., a mutant (mut) IL-12 moiety having reduced IL-12 activity; constructed as a single chain fusion, or as two separate subunits) can be a first binding domain that upregulates the immune response upon binding to IL-12R on an immune cell. Thus, in FIGS. 11G, 11I and 11J, the IL-12 moiety functions as a "first binding domain". The first binding domain (e.g., an immunostimulatory cytokine moiety or variant thereof) can be located at the hinge region between the Fc subunit and the second binding domain or the third binding domain, as in the exemplary configurations shown in fig. 11A-11F, 11H, 11K, and 11L. Such a "limited proximity" configuration of the first binding domain to its first target molecule may allow for: i) In the absence of binding between the second binding domain and the second target molecule and/or binding between the third binding domain and the third target molecule (whichever is the domain of N' of the first binding domain), reduced, minimized or no binding/activity between the first binding domain and its first target molecule; and ii) a salvaged/restored binding/activity of the first binding domain in the presence of binding between the second binding domain and the second target molecule and/or binding between the third binding domain and the third target molecule (whichever is the binding domain of N' of the first binding domain). The first binding domain (e.g., an immunostimulatory cytokine moiety or variant thereof) can also be located at the C' of one or both Fc subunits of an Fc fusion protein, such as the IL-12 moiety illustrated in FIGS. 11A-11L (constructed as a single chain fusion and fused to one Fc subunit; or as two separate subunits, each fused to one Fc subunit of an Fc domain). Such a configuration does not limit or hardly limit the binding/activity of the first binding domain.
FIGS. 12A-12D depict exemplary immunomodulatory molecules having a first binding domain (e.g., an immunostimulatory cytokine such as IL-12 or variant thereof, e.g., constructed as a single chain fusion) located at the hinge region of one polypeptide chain of a parent (ligand/receptor/antigen binding domain) -hinge-Fc fusion protein, which may be homodimerized or heterodimerized. FIG. 12A depicts an exemplary immunomodulatory molecule wherein the PD-L1 or PD-L2 extracellular domain (wild-type or mutant) is fused via a hinge to the N-terminus of the Fc domain, and an immunostimulatory cytokine moiety (e.g., IL-12 or variant, constructed as a single chain fusion) is located at the hinge region of one of the (PD-L1 or PD-L2) -hinge-Fc polypeptide chains. May be referred to as IL-12/PD-L1-Fc or IL-12/PD-L2-Fc. FIG. 12B depicts an exemplary immunomodulatory molecule wherein a PD-L1 or PD-L2 ectodomain (wild-type or mutant) is fused via a first hinge to the N-terminus of a first Fc subunit, a CD155 ectodomain (wild-type or mutant) is fused via a second hinge to the N-terminus of a second Fc subunit, and an immunostimulatory cytokine moiety (e.g., IL-12 or variant, constructed as a single chain fusion) is located at the hinge region of one of the paired polypeptide chains (e.g., PD-L1/PD-L2-hinge-Fc chain). May be referred to as IL-12/PD-L1-Fc/CD155-Fc or IL-12/PD-L2-Fc/CD155-Fc. FIG. 12C depicts an exemplary immunomodulatory molecule wherein a PD-L1 or PD-L2 extracellular domain (wild-type or mutant) is fused via a first hinge to the N-terminus of a first Fc subunit, an antibody moiety (e.g., sdAb or scFv) that specifically recognizes a target molecule (which may be an agonist, antagonist or neutral antibody, modulates or does not modulate an immune response) is fused via a second hinge to the N-terminus of a second Fc subunit, and an immunostimulatory cytokine moiety (e.g., IL-12 or variant, constructed as a single chain fusion) is located at the hinge region of one of the paired polypeptide chains (e.g., PD-L1/PD-L2-hinge-Fc chain). May be referred to as sdAb/IL-12/PD-L1-Fc or sdAb/IL-12/PD-L2-Fc. FIG. 12D depicts an exemplary immunoregulatory molecule in which a PD-L1 or PD-L2 extracellular domain (wild-type or mutant) is fused via a first hinge to the N-terminus of a first Fc subunit, a Fab which specifically recognizes a target molecule (which may be an agonist, antagonist or neutral antibody, modulates or does not modulate an immune response) is fused via a second hinge to the N-terminus of a second Fc subunit via its CH1, and an immunostimulatory cytokine moiety (e.g., IL-12 or variant, constructed as a single chain fusion) is located at the hinge region of one of the paired polypeptide chains (e.g., PD-L1/PD-L2-hinge-Fc chain). May be referred to as Fab/IL-12/PD-L1-Fc or Fab/IL-12/PD-L2-Fc.
Fig. 13A-13D depict exemplary immunomodulatory molecules having a first binding domain (e.g., an immunostimulatory cytokine such as IL-12 or variant thereof, e.g., constructed as a single chain fusion) located at the C-terminus of the Fc domain (one or both Fc subunits) of a parent (ligand/receptor/antigen binding domain) -hinge-Fc fusion protein, which may be homodimerized or heterodimerized. FIG. 13A depicts an exemplary immunomodulatory molecule wherein the PD-L1 or PD-L2 extracellular domain (wild-type or mutant) is fused to the N-terminus of the Fc domain via an optional hinge and an immunostimulatory cytokine moiety (e.g., IL-12 or variant, constructed as a single chain fusion) is located at the C-terminus of one or both subunits of the Fc domain. May be referred to as PD-L1-Fc/IL-12 or PD-L2-Fc/IL-12. FIG. 13B depicts an exemplary immunomodulatory molecule wherein a PD-L1 or PD-L2 extracellular domain (wild-type or mutant) is fused via a first optional hinge to the N-terminus of a first Fc subunit, a CD155 extracellular domain (wild-type or mutant) is fused via a second optional hinge to the N-terminus of a second Fc subunit, and an immunostimulatory cytokine moiety (e.g., IL-12 or variant, constructed as a single chain fusion) is located at the C-terminus of one or both subunits of the Fc domain (e.g., the C' of the PD-L1/PD-L2-hinge-Fc chain). May be referred to as PD-L1-Fc/CD155-Fc/IL-12 or PD-L2-Fc/CD155-Fc/IL-12. FIG. 13C depicts an exemplary immunomodulatory molecule wherein a PD-L1 or PD-L2 extracellular domain (wild-type or mutant) is fused to the N-terminus of a first Fc subunit via a first optional hinge, an antibody moiety (e.g., an sdAb or scFv) that specifically recognizes a target molecule (which may be an agonist, antagonist or neutral antibody, modulates or does not modulate an immune response) is fused to the N-terminus of a second Fc subunit via a second optional hinge, and an immunostimulatory cytokine moiety (e.g., IL-12 or variant, constructed as a single chain fusion) is located at the C-terminus of one or both subunits of the Fc domain (e.g., the C' of the PD-L1/PD-L2-hinge-Fc chain). May be referred to as sdAb/PD-L1-Fc/IL-12 or sdAb/PD-L2-Fc/IL-12. FIG. 13D depicts an exemplary multi-target immunomodulatory molecule, wherein a PD-L1 or PD-L2 extracellular domain (wild-type or mutant) is fused via a first optional hinge to the N-terminus of a first Fc subunit, a Fab which specifically recognizes the target molecule (which may be an agonist, antagonist or neutral antibody, modulates or does not modulate an immune response) is fused via a second optional hinge to the N-terminus of a second Fc subunit via its CH1, and an immunostimulatory cytokine moiety (e.g., IL-12 or variant, constructed as a single chain fusion) is located at the C-terminus of one or both subunits of the Fc domain (e.g., C' of the PD-L1/PD-L2-hinge-Fc chain). May be referred to as Fab/PD-L1-Fc/IL-12 or Fab/PD-L2-Fc/IL-12.
FIGS. 14A-14D depict exemplary immunomodulatory molecules (e.g., immunostimulatory cytokines such as IL-12, IL-2, or variants thereof, e.g., constructed as single chain fusions) having two first binding domains, each located at the hinge region of one polypeptide chain of a parent (ligand/receptor/antigen binding domain) -hinge-Fc fusion protein, which may be homodimerized or heterodimerized. FIG. 14A depicts an exemplary immunomodulatory molecule wherein a PD-L1 or PD-L2 extracellular domain (wild-type or mutant) is fused via a hinge to the N-terminus of an Fc domain, a first immunostimulatory cytokine moiety (e.g., IL-12 or variant, constructed as a single chain fusion) is located at the hinge region of one of the (PD-L1 or PD-L2) -hinge-Fc polypeptide chains, and a second immunostimulatory cytokine moiety (e.g., IL-2 or variant thereof) is located at the hinge region of the other of the (PD-L1 or PD-L2) -hinge-Fc polypeptide chains. May be referred to as IL-12/IL-2/PD-L1-Fc or IL-12/IL-2/PD-L2-Fc. FIG. 14B depicts an exemplary immunomodulatory molecule wherein a PD-L1 or PD-L2 extracellular domain (wild-type or mutant) is fused via a first hinge to the N-terminus of a first Fc subunit, a CD155 extracellular domain (wild-type or mutant) is fused via a second hinge to the N-terminus of a second Fc subunit, a first immunostimulatory cytokine moiety (e.g., IL-12 or variant, constructed as a single chain fusion) is located at the hinge region of one of the paired polypeptide chains (e.g., PD-L1/PD-L2-hinge-Fc chain), and a second immunostimulatory cytokine moiety (e.g., IL-2 or variant thereof) is located at the hinge region of the other of the paired polypeptide chains (e.g., CD 155-hinge-Fc chain). May be referred to as IL-12/IL-2/PD-L1-Fc/CD155-Fc or IL-12/IL-2/PD-L2-Fc/CD155-Fc. FIG. 14C depicts an exemplary immunoregulatory molecule in which a PD-L1 or PD-L2 extracellular domain (wild-type or mutant) is fused via a first hinge to the N-terminus of a first Fc subunit, an antibody moiety (e.g., sdAb or scFv) that specifically recognizes a target molecule (which may be an agonist, antagonist or neutral antibody, modulates or does not modulate an immune response) is fused via a second hinge to the N-terminus of a second Fc subunit, a first immunostimulatory cytokine moiety (e.g., IL-12 or variant, constructed as a single chain fusion) is located at the hinge region of one of the paired polypeptide chains (e.g., PD-L1/PD-L2-hinge-Fc chain), and a second immunostimulatory cytokine moiety (e.g., IL-2 or variant thereof) is located at the hinge region of the other of the paired polypeptide chains (e.g., CD 155-hinge-Fc chain). May be referred to as sdAb/IL-12/IL-2/PD-L1-Fc or sdAb/IL-12/IL-2/PD-L2-Fc. FIG. 14D depicts an exemplary immunoregulatory molecule in which a PD-L1 or PD-L2 extracellular domain (wild-type or mutant) is fused via a first hinge to the N-terminus of a first Fc subunit, a Fab which specifically recognizes a target molecule (which may be an agonist, antagonist or neutral antibody, modulates or does not modulate an immune response) is fused via a second hinge to the N-terminus of a second Fc subunit via its CH1, a first immunostimulatory cytokine moiety (e.g., IL-12 or variant, constructed as a single chain fusion) is located at the hinge region of one of the paired polypeptide chains (e.g., PD-L1/PD-L2-hinge-Fc chain), and a second immunostimulatory cytokine moiety (e.g., IL-2 or variant thereof) is located at the hinge region of the other of the paired polypeptide chains (e.g., CD 155-hinge-Fc chain). May be referred to as Fab/IL-12/IL-2/PD-L1-Fc or Fab/IL-12/IL-2/PD-L2-Fc.
FIGS. 15A-15D depict exemplary immunomodulatory molecules (e.g., immunostimulatory cytokines such as IL-12, IL-2, or variants thereof, e.g., constructed as single chain fusions) having two first binding domains, one located at the hinge region of one polypeptide chain of a parent (ligand/receptor/antigen binding domain) -hinge-Fc fusion protein, and the other located at the C-terminus of one or both Fc subunits of the parent (ligand/receptor/antigen binding domain) -hinge-Fc fusion protein. FIG. 15A depicts an exemplary immunomodulatory molecule wherein a PD-L1 or PD-L2 extracellular domain (wild-type or mutant) is fused via a hinge to the N-terminus of an Fc domain, a first immunostimulatory cytokine moiety (e.g., IL-2 or variant) is located at the hinge region of one of the (PD-L1 or PD-L2) -hinge-Fc polypeptide chains, and a second immunostimulatory cytokine moiety (e.g., IL-12 or variant, constructed as a single chain fusion) is located at the C' of the Fc subunit of the other of the (PD-L1 or PD-L2) -hinge-Fc polypeptide chains. May be referred to as IL-2/PD-L1-Fc/IL-12 or IL-2/PD-L2-Fc/IL-12. FIG. 15B depicts an exemplary immunomodulatory molecule wherein a PD-L1 or PD-L2 extracellular domain (wild-type or mutant) is fused via a first hinge to the N-terminus of a first Fc subunit, a CD155 extracellular domain (wild-type or mutant) is fused via a second hinge to the N-terminus of a second Fc subunit, a first immunostimulatory cytokine moiety (e.g., IL-12 or variant, constructed as a single chain fusion) is located on the C' chain of the Fc subunit of one of the paired polypeptide chains (e.g., PD-L1/PD-L2-hinge-Fc chain), and a second immunostimulatory cytokine moiety (e.g., IL-2 or variant thereof) is located on the hinge region of the other chain of the paired polypeptide chains (e.g., CD 155-hinge-Fc chain). May be referred to as IL-2/PD-L1-Fc/CD155-Fc/IL-12 or IL-2/PD-L2-Fc/CD155-Fc/IL-12. FIG. 15C depicts an exemplary immunomodulatory molecule wherein a PD-L1 or PD-L2 extracellular domain (wild-type or mutant) is fused via a first hinge to the N-terminus of a first Fc subunit, an antibody moiety (e.g., sdAb or scFv) that specifically recognizes a target molecule (which may be an agonist, antagonist or neutral antibody, modulates or does not modulate an immune response) is fused via a second hinge to the N-terminus of a second Fc subunit, a first immunostimulatory cytokine moiety (e.g., IL-12 or variant, constructed as a single chain fusion) is located at the C' of the Fc subunit of one of the paired polypeptide chains (e.g., PD-L1/PD-L2-hinge-Fc chain), and a second immunostimulatory cytokine moiety (e.g., IL-2 or variant thereof) is located at the hinge region of the other of the paired polypeptide chains (e.g., CD 155-hinge-Fc chain). May be referred to as sdAb/IL-2/PD-L1-Fc/IL-12 or sdAb/IL-2/PD-L2-Fc/IL-12. FIG. 15D depicts an exemplary immunomodulatory molecule wherein PD-L1 or PD-L2 (wild-type or mutant) is fused via a first hinge to the N-terminus of a first Fc subunit, a Fab which specifically recognizes a target molecule (which may be an agonist, antagonist or neutral antibody, modulates or does not modulate an immune response) is fused via a second hinge to the N-terminus of a second Fc subunit via its CH1, a first immunostimulatory cytokine moiety (e.g., IL-12 or variant, constructed as a single chain fusion) is located at the C' of the Fc subunit of one of the paired polypeptide chains (e.g., PD-L1/PD-L2-hinge-Fc chain), and a second immunostimulatory cytokine moiety (e.g., IL-2 or variant thereof) is located at the hinge region of the other of the paired polypeptide chains (e.g., CD 155-hinge-Fc chain). May be referred to as Fab/IL-2/PD-L1-Fc/IL-12 or Fab/IL-2/PD-L2-Fc/IL-12.
FIG. 16 shows 4T1 murine breast cancer tumors extracted from mammary fat pads of mice treated with IL-12 (E59A/F60A)/PD-L2-Fc (IW- # 29), IL-12 (F60A)/PD-L2-Fc (IW- # 30), a combination of anti-PD-1 and anti-CTLA-4 antibodies or PBS (negative control).
FIG. 17 depicts 4T1 murine breast cancer cells that were injected with 4T1 cells in mammary fat pads and transferred to the lung in mice treated with IL-12 (E59A/F60A)/PD-L2-Fc (IW- # 29), IL-12 (F60A)/PD-L2-Fc (IW- # 30), a combination of anti-PD-1 and anti-CTLA-4 antibodies, or PBS (negative control).
FIG. 18 depicts tumor volumes in 4T1 syngeneic tumor mice treated with IL-12 (E59A/F60A)/anti-PD-1 (IW- # 48), IL-12 (E59A/F60A)/PD-L2-Fc (IW- # 29), IL-12 (E59A/F60A)/IL-2 (R38D/K43E/E61R)/anti-PD-1 (IW- # 54) immunoregulatory molecules or PBS (negative control). Black arrows indicate days of injection.
FIG. 19 depicts tumor volumes in EMT6 syngeneic tumor mice treated with IL-12 (E59A/F60A)/anti-PD-1 (IW- # 48), IL-12 (E59A/F60A)/PD-L2-Fc (IW- # 29), IL-2 (R38D/K43E/E61R)/PD-L2-Fc (IW- # 11) immunoregulatory molecules or PBS (negative control). Black arrows indicate days of injection.
Detailed Description
Current immunotherapy often causes excessive adverse immune responses such as immune cell overactivation, cytokine storms, etc. For example, cytokine therapies (e.g., for treating cancer) have shown limited success due to severe toxicity, which limits the dose to levels well below therapeutically effective doses. Immunocytokines, which are constructs of cytokines fused to antibodies, antigen binding fragments, ligand-Fc fusion proteins or receptor-Fc fusion proteins (hereinafter collectively referred to as "ligand/receptor-Fc fusion proteins" or "ligand/receptor-hinge-Fc fusion proteins"), can be delivered to target cells (e.g., tumor cells or immune effector cells) or tissues and recognize target antigens in immune modulatory molecules through antibodies or antigen binding fragments (e.g., antibody fragments, ligands, or receptors), which can both reduce non-specific (off-target) cytokine activity and/or associated toxicity (e.g., toxicity to healthy cells or tissues) and concentrate cytokine therapeutic effects at target sites (e.g., disease sites). Activation of the immunomodulatory molecule may occur via transactivation, which requires specific binding of the antibody or antigen binding fragment to a target antigen on a tumor cell; or via cis-activation, which requires specific binding of the antibody or antigen binding fragment to a target antigen on an immune cell (see figure 10). Most immunocytokines now developed fuse cytokine moieties to the N-or C-terminus of the heavy or light chain of a full length antibody (e.g., hu14.8-IL2, NHS-IL2LT, NHS-IL12, BC1-IL12; see, e.g., FIGS. 1C-1E), or to the N-or C-terminus of an antigen binding fragment (e.g., diabodies, scFvs such as L19-IL2 or F16-IL 2), so that cytokine-receptor binding/activation can still occur even in the absence of antibody-antigen recognition, resulting in off-target toxicity. Immune checkpoint inhibitors (e.g., anti-PD-1, anti-CTLA-4 antibodies) developed in recent years, while exhibiting some great clinical success in cancer patients, have focused on up-regulating immune responses, which can exacerbate systemic toxicity if further used with pro-inflammatory cytokines.
The present invention provides immunomodulatory molecules with adverse effects in modulating immune responses, demonstrating significantly better toxicity profiles and therapeutic efficacy. The immunomodulatory molecule comprises a first binding domain (e.g., an immunostimulatory cytokine or variant thereof, such as IL-12, IL-2, IFN- γ) that specifically recognizes a first target molecule (e.g., a receptor for an immunostimulatory cytokine or variant thereof) and a second binding domain (e.g., a ligand such as PD-L1, PD-L2, CD155 extracellular domain or variant thereof) that specifically recognizes a second target molecule (e.g., PD-1 or TIGIT on an immune effector cell), wherein the first binding domain upregulates an immune response upon binding to the first target molecule, and wherein the second binding domain downregulates an immune response upon binding to the second target molecule. For example, when an IL-12 cytokine (pro-inflammatory) is located in the hinge region of a PD-L2 ectodomain-hinge-Fc fusion protein, the resulting IL-12/PD-L2-Fc immunoregulatory molecule not only specifically targets IL-12 activity (e.g., activity that binds to the IL-12 receptor, and/or IL-12 pro-inflammatory activity) to PD-1+ target cells, but also stimulates PD-1 inhibitory immune checkpoint signaling via PD-L2-PD-1 binding, thereby generating an immunosuppressive signal that "balances" or "counteracts" the immunostimulatory activity of IL-12. Any agonist antibody or ligand (e.g., PD-L2, PD-L1, CD80, or CD 86) that can activate or stimulate an immunosuppressive signaling pathway (e.g., by binding to an inhibitory immune checkpoint molecule such as PD-1 or CTLA-4), or any antagonist antibody, ligand, or receptor that can reduce or block an immune stimulatory signaling pathway (e.g., by binding to a stimulatory immune checkpoint molecule such as CD27 or CD28, or an immunostimulatory receptor such as IL-2R), can be used in combination with an immunostimulatory cytokine or variant thereof (e.g., IL-2, IL-12, IFN- γ, or IL-23) to construct an immunomodulatory molecule having any of the immunomodulatory molecular configurations described herein. Any antagonist antibody, ligand or receptor that can reduce or block an immunosuppressive signaling pathway (e.g., by binding to an inhibitory immune checkpoint molecule such as PD-1 or CTLA-4), or any agonist antibody or ligand (e.g., CD70, CD80, CD86 or IL-2) that can activate or stimulate an immune stimulatory signaling pathway (e.g., by binding to a stimulatory immune checkpoint molecule such as CD27 or CD28 or an immune stimulatory receptor such as IL-2R) can be combined with an immunosuppressive cytokine or variant thereof (e.g., IL-10, IL-27, IL-35, TGF- β) to construct an immunomodulatory molecule having any of the immunomodulatory molecular configurations described herein. The immunomodulatory molecules described herein may comprise one or more first binding domains, and/or one or more second binding domains, to achieve multiple immune response modulation. The plurality of first binding domains may be the same or different. The plurality of second binding domains may be the same or different. See FIGS. 1A-1W and 11A-15D for examples.
The first binding domain can include a molecule such as an immunostimulatory cytokine, ligand, or agonist antibody (e.g., a ligand or agonist antibody that stimulates a stimulatory checkpoint molecule such as OX 40) that targets immune cells such as T cells, NK cells, DC cells, macrophages, and B cells. The invention provides in some embodiments a first binding domain having reduced activity (e.g., reduced binding to its target or reduced stimulatory activity), as compared to an unmodified parent first binding domain. For example, see cytokine variants described herein that exhibit substantially reduced activity compared to wild-type cytokines. Decreasing the binding affinity of the first binding domain may bias the mechanism of action towards target-dependent activation (cis-activation) and away from target-independent activation (trans-activation).
The second binding domain may comprise a molecule such as an immunosuppressive cytokine, ligand or agonist antibody (e.g., a ligand (e.g., PD-L1, PD-L2, CD 155) or agonist Ab that stimulates an inhibitory checkpoint molecule such as PD-1 or TIGIT for down-regulation of an immune response; in some embodiments, the invention provides an anti-PD-1 antibody (antagonist antibody) with reduced binding affinity for PD-1, thus reducing the immune response that may be induced by a wild-type anti-PD-1 antibody (antagonist antibody, e.g., nalmezor Wu Liyou) (see example 22). In some embodiments, the invention also provides a ligand with increased binding affinity for an inhibitory checkpoint molecule such as PD-1 as compared to the wild-type ligand, which may further down-regulate the immune response; e.g., see mutant PD-L1 and PD-L2 molecules generated in example 23. As compared to those immune regulatory molecules with wild-type PD-L1 or PD-L2 extracellular domain, an immune response comprising PD-L1 or PD-L2 extracellular domain as a non-regulatory domain is greatly reduced in affinity for PD-L1 or a mutant (PD-L2) compared to wild-type PD-L10) -8 M K d ) Or with wild-type anti-PD-1 antibodies (less than 10 -9 M K d ) Low binding affinity compared to mutant anti-PD-1 antibodies (antagonist antibodies; greater than 10 -8 M K d ) Allowing their immunomodulatory molecules to target cancer cells expressing higher levels of PD-1, such as T cell depletion and tumor microenvironment that attempts to bypass anti-tumor activity, rather than any PD-1 positive cells.
For example, IL-12 (E59A/F60A)/PD-L2 (S58V) -Fc immunoregulatory molecules described herein provide a positive signal (IL-12/IL-12R signaling) and a negative signal (PD-1/PD-L2 signaling). The counter acting immunomodulatory molecules described herein allow for mimicking the natural T cell activation process, modulating the T cell activation process, and overcoming the overactivation of the immune system.
The immunomodulatory molecules comprising the first and second binding domains described herein may further comprise a third binding domain that specifically recognizes a third target molecule. The third binding domain can aid in the localization of the immunoregulatory molecule to a target site (e.g., tumor microenvironment) by binding to a third target molecule (e.g., a T cell depleted marker, a T cell surface marker, or a tumor antigen). The third binding domain, upon binding to the third target molecule, can: i) Up-regulating the above or other immune response, or ii) down-regulating the above or other immune response; or iii) does not modulate any immune response by binding itself. For example, the third binding domain may serve as a tumor antigen targeting domain alone to bring the immunoregulatory molecule to the tumor site, or as an immunoeffector targeting domain to bring the immunoregulatory molecule to the immunoeffector cell or to enhance its binding to the immunoeffector cell. Intratumoral microenvironments contain relatively high levels of depleted T cells that express several markers, such as TIGIT, TIM3, LAG3, and PD-1. Because of the large differences in expression patterns and levels of the depletion marker in Tumor Microenvironments (TMEs), the third binding domain can be used to target other depletion markers to broadly target TMEs. Alternatively, the third binding domain may be used to target a particular cancer against a particular tumor antigen, including but not limited to: her2, CEACAM, her3, EGFR, trop2, CLDN18.2, prostate specific antigen, MUC1, epCAM, GPC3, mesothelin (MSLN), conjugated to 4, folate receptor alpha, tissue factor, and the like. The third binding domain may target T cell markers including, but not limited to: CD4, CD8, CD3, CD2, CD5, CD7, CD40L, CD, CD137, CD69, CTLA4, CD127, ICOS, etc. The third binding domain may also target dendritic cell markers including, but not limited to: CD1c, CD11c, CD141, CD123, BDCA-2, BDCA-4, CLEC9A, XCR1, CD80, CD86, PD-L1, PD-L2, etc. The third binding domain may also target monocyte/macrophage markers including, but not limited to: CSF1R, CD, cd86, cd11, cd14, cd68, cd163, cd16, cd32, cd64, etc. The third binding domain may also target neutrophil markers, including but not limited to: CD11, CD16, CD32, etc. The immunomodulatory molecules described herein may comprise one or more third binding domains to achieve multiple immune response modulation or for enhanced targeting. The plurality of third binding domains may be the same or different.
In addition, the present invention provides immunomodulatory molecules having certain unique configurations that address the problems faced by current cytokine/immunocytokine therapies. Specifically, some immunomodulatory molecules of the invention reduce the nonspecific activity (i.e., antibody or antigen binding fragment-independent binding) of a first binding domain (immunostimulatory cytokine) and increase the specific activity (i.e., antibody or antigen binding fragment-dependent binding) of the first binding domain (immunostimulatory cytokine), i.e., by locating the first binding domain (e.g., cytokine or variant thereof) at the hinge region between the second binding domain (e.g., ligand, receptor, VHH, scFv, or Fab) and an Fc domain subunit or portion thereof (e.g., CH2-CH3 fragment, or CH2 alone, or CH3 alone), e.g., between the scFv and an Fc domain subunit (e.g., antigen binding polypeptide comprising VH-VL-cytokine-Fc subunit, or VL-VH-cytokine-Fc subunit), or between the Fab and Fc domain of a full-length antibody (e.g., antigen binding polypeptide comprising VH-CH 1-cytokine-Fc subunit), or between the ligand (or receptor) and an Fc domain subunit (e.g., antigen binding polypeptide comprising ligand-cytokine-Fc subunit, or antigen binding subunit). Without being bound by theory, it is believed that steric hindrance of the second binding domain (e.g., ligand, receptor, VHH, scFv, fab) and Fc domain or portion thereof reduces proximity of the first binding domain (e.g., immunomodulatory cytokine or variant thereof) to its target molecule (e.g., receptor for immunomodulatory cytokine), or "masks" the binding of the first binding domain to its first target molecule in the absence of binding of the second binding domain to the second target molecule. On the other hand, upon binding of the second binding domain to the second target molecule, the first binding domain becomes activated. Surprisingly, unlike other immunocytokine designs that "expose" a cytokine moiety at their N-terminus or C-terminus, the unique immunoregulatory molecule configurations of the present invention require that the second binding domain (e.g., ligand, receptor, VHH, scFv or Fab) first bind to its second target molecule (e.g., receptor) before the first binding domain (e.g., immunoregulatory cytokine moiety) binds to its first target molecule (e.g., receptor), thereby ensuring that the upregulation of the immune response (e.g., cytokine signaling) is entirely second binding domain binding-dependent (at the target). With this enhanced targeting-specific design, and optionally further in combination with the reduced activity of the first binding domain discussed above (e.g., cytokine variants described herein), a desired immune response (e.g., cytokine signaling activation) can be safely delivered to a target site (e.g., tumor cells or immune cells) to achieve a therapeutic effect. Such unique target-specific designs add an additional regulatory layer to the "balance" or "offset" of current immune response designs, further fine tuning the biological activity and toxicity of the immunomodulatory molecules described herein.
Accordingly, in one aspect the present application provides an immunomodulatory molecule comprising: a first binding domain (e.g., a ligand, VHH, scFv or VH, e.g., a receptor for an immunostimulatory cytokine) that specifically recognizes a first target molecule (e.g., a cell surface antigen or receptor, e.g., an immunostimulatory cytokine such as IL-2 or IL-12), and a second binding domain (e.g., a ligand, VHH, scFv or VH, e.g., an inhibitory checkpoint molecule such as PD-1), that specifically recognizes a second target molecule (e.g., a ligand, VHH, scFv or VH, e.g., an agonist ligand such as PD-L1 or PD-L2, or an agonist antigen-binding fragment such as an anti-PD-1 agonist Fab, scFv, VH, VHH or full-length antibody), wherein the first binding domain upregulates an immune response upon binding to the first target molecule, and wherein the second binding domain downregulates an immune response upon binding to the second target molecule.
Also provided are isolated nucleic acids encoding such immunomodulatory molecules, vectors comprising such nucleic acids, host cells comprising such nucleic acids or vectors, methods of producing such immunomodulatory molecules, pharmaceutical compositions and articles of manufacture comprising such immunomodulatory molecules, methods of modulating an immune response with such immunomodulatory molecules or pharmaceutical compositions thereof, and methods of treating diseases (e.g., cancer, viral infection, autoimmune diseases) with such immunomodulatory molecules or pharmaceutical compositions thereof.
I. Definition of the definition
Unless specifically stated to the contrary, practice of the present invention will employ conventional methods of virology, immunology, microbiology, molecular biology and recombinant DNA techniques by those skilled in the art, many of which are described below for purposes of illustration. Such techniques are well explained in the literature. See, e.g., current Protocols in Molecular Biology or Current Protocols in Immunology, john Wiley & Sons, new York, n.y. (2009); ausubel et al Short Protocols in Molecular Biology,3rd ed., john Wiley & Sons,1995; sambrook and Russell, molecular Cloning: A Laboratory Manual (3 rd edition, 2001); maniatis et al Molecular Cloning: A Laboratory Manual (1982); DNA Cloning: A Practical Approach, vol.I & II (D.Glover, ed.); oligonucleotide Synthesis (n.gait, ed., 1984); nucleic Acid Hybridization (b.hames & s.higgins, eds., 1985); transcription and Translation (b.hames & s.higgins, eds., 1984); animal Cell Culture (r.freshney, ed., 1986); perbal, A Practical Guide to Molecular Cloning (1984) and other similar references.
As used herein, the term "immunocytokine" refers to a form of an antigen binding protein (e.g., an antibody or antigen binding fragment (e.g., a ligand, receptor, or antibody fragment)) that is fused to a cytokine molecule. The antigen binding protein (e.g., antibody or antigen binding fragment (e.g., ligand, receptor, or antibody fragment)) form can be any of the forms described herein, and the cytokines can be fused directly, or through a linker or chemically coupled to the antigen binding protein form.
The term "cytokine storm", also known as "cytokine cascade" or "hypercytokinemia", is a potentially fatal immune response, usually consisting of a positive feedback loop between the cytokine and the immune cells, with greatly elevated levels of various cytokines (e.g., INF-gamma, IL-10, IL-6, CCL2, etc.).
As used herein, when a binding domain (e.g., an antibody, antigen binding fragment, or ligand) is referred to as an "antagonist" of a target molecule (e.g., a receptor or immune checkpoint molecule), it means that the binding domain (e.g., antibody, antigen binding fragment, or ligand) blocks, inhibits, or reduces (e.g., reduces by at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%) the biological activity (e.g., blocks receptor signaling) of the target molecule following binding of the target antigen. For example, an anti-PD-1 antagonist antibody is an antibody that reduces or blocks PD-1 signaling; antagonist ligands for IL-12 receptors reduce or block IL-12 receptor signaling. When a binding domain (e.g., an antibody, antigen binding fragment, or ligand) is referred to as an "agonist" of a target molecule (e.g., a receptor or immune checkpoint molecule), it means that the binding domain (e.g., antibody, antigen binding fragment, or ligand) stimulates, activates, or enhances (e.g., enhances at least about any one of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more) the biological activity (e.g., activates receptor signaling) of the target molecule upon binding of the target antigen. For example, wild-type PD-L2 ligand (e.g., extracellular domain) is an agonist that activates PD-1 signaling. For example, an anti-PD-1 agonist antibody is an antibody that induces or enhances PD-1 signaling.
For purposes of the present invention, "treating" also includes alleviating the pathological consequences of the disease, including, but not limited to, alleviating one or more symptoms caused by the disease, reducing the extent of the disease, stabilizing the disease (e.g., preventing or delaying exacerbation of the disease), preventing or delaying the spread of the disease (e.g., metastasis), preventing or delaying the recurrence of the disease, delaying or slowing the progression of the disease, improving the disease state, providing remission (part or all) of the disease, reducing the dosage of one or more other drugs needed to treat the disease, delaying the progression of the disease, increasing the quality of life, and/or prolonging the life span.
The terms "prevent" and similar words such as "prevention" refer to a method for preventing, inhibiting or reducing the likelihood of recurrence of a disease or condition (e.g., cancer). It also refers to delaying the recurrence of a disease or condition or delaying the recurrence of symptoms of a disease or condition. As used herein, "prevention" and similar terms also include reducing the intensity, impact, symptoms and/or burden of a disease or condition prior to recurrence of the disease or condition.
As used herein, "delaying" the progression of a disease means delaying, impeding, slowing, retarding, stabilizing, and/or delaying the progression of the disease. This delay may be of varying lengths of time, depending on the history of the disease and/or the individual receiving the treatment. A method of "delaying" disease progression is a method of reducing the probability of disease progression over a given time frame and/or reducing the extent of disease over a given time frame, as compared to not using the method. Such comparisons are typically based on clinical studies, using a statistically significant number of individuals. The development of cancer can be detected using standard methods including, but not limited to: computed axial tomography (CAT scan), magnetic Resonance Imaging (MRI), abdominal ultrasound, coagulation tests, angiography or biopsy. Progression may also refer to the progression of a disease (e.g., cancer) that may not be detected initially, including occurrence, recurrence, and onset.
As used herein, the term "effective amount" refers to an amount of an agent or combination of agents sufficient to treat a particular disorder, condition, or disease, such as to ameliorate, reduce, and/or delay one or more symptoms thereof. With respect to cancer, an effective amount includes an amount sufficient to cause tumor shrinkage and/or reduce the rate of tumor growth (e.g., inhibit tumor growth) or prevent or delay other unwanted cell proliferation. In some embodiments, the effective amount is an amount sufficient to delay progression. In some embodiments, the effective amount is an amount sufficient to prevent or delay recurrence. The effective amount may be administered in one or more administrations. An effective amount of the drug or composition may be: (i) reducing the number of cancer cells; (ii) reducing tumor size; (iii) Inhibit, retard, slow and preferably prevent infiltration of cancer cells to peripheral organs to some extent; (iv) Inhibit (i.e., slow down to some extent and preferably prevent) tumor metastasis; (v) inhibiting tumor growth; (vi) preventing or delaying the onset and/or recurrence of a tumor; (vii) To some extent, alleviate one or more symptoms associated with cancer; (viii) Stimulating or activating immune cells (e.g., immune effector cells), e.g., for immune responses, such as production of cytokines, or for immune cell proliferation and/or differentiation; and/or (ix) prevent, reduce or eliminate inflammatory or autoimmune responses, such as inhibiting pro-inflammatory cytokine secretion. In the case of viral infection, an effective amount of the agent may inhibit (i.e., reduce to some extent and preferably eliminate) viral activity; controlling and/or attenuating and/or inhibiting inflammation or cytokine storm caused by said viral pathogen; preventing exacerbation, preventing and/or ameliorating at least one symptom of or damage to the subject or an organ or tissue of the subject, the symptom resulting from or associated with the viral infection; controlling, reducing and/or inhibiting cell necrosis in infected and/or uninfected tissues and/or organs; controlling, ameliorating and/or preventing infiltration of inflammatory cells (e.g., NK cells, cytotoxic T cells, neutrophils) in infected or uninfected tissues and/or organs; and/or stimulating or activating immune cells (e.g., immune effector cells), e.g., for immune responses, such as production of cytokines, or for immune cell proliferation and/or differentiation.
As used herein, "individual" or "subject" refers to a mammal, including but not limited to: humans, cattle, horses, cats, dogs, rodents or primates. In some embodiments, the individual is a human.
The term "antibody" is used in its broadest sense and covers a variety of antibody structures, including but not limited to: monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), full length antibodies, and antigen-binding fragments thereof, so long as they exhibit the desired antigen-binding activity. The term "antibody" includes conventional 4-chain antibodies, single domain antibodies, and antigen-binding fragments thereof.
The basic 4-chain antibody unit is a hetero-tetrameric glycoprotein consisting of two identical light (L) chains and two identical heavy (H) chains. IgM antibodies consist of 5 basic hetero-tetrameric units and one additional polypeptide called the J chain and contain 10 antigen binding sites, whereas IgA antibodies contain 2-5 basic 4-chain units that can polymerize to form multivalent aggregates that bind to the J chain. In the case of IgG, the 4-chain unit is typically about 150,000 daltons. Each L chain is linked to the H chain by one covalent disulfide bond, while the two H chains are linked to each other by one or more disulfide bonds depending on the H chain isotype. Each H and L chain also has regularly spaced intrachain disulfide bonds. Each H chain has a variable domain at the N-terminus (v H ) Followed by three constant domains (C H ) Four C of the mu and epsilon isoforms H A domain. Each L chain has a variable domain at the N-terminus (v L ) Followed by a constant domain at the other end. V (V) L And V is equal to H Alignment, C L With the first constant domain of the heavy chain (C H 1) Alignment. Specific amino acid residues are believed to form an interface between the light chain variable domain and the heavy chain variable domain. V (V) H And V L Paired together to form a single antigen binding site. For the structure and properties of different classes of antibodies, see, e.g., basic and Clinical Immunology,8th Edition,Daniel P.Sties,Abba I.Terr and Tristram G.Parsolw (eds), appleton&Lange, norwalk, conn, 1994,page 71and Chapter 6. Based on the amino acid sequence of its constant domain, the L chain of any vertebrate species can be divided into one of two distinct types, called kappa and lambda. According to its heavy chain (C) H ) Amino acid sequences of constant domains, immunoglobulins can be assigned to different classes or isotypes. There are five classes of immunoglobulins: igA, igD, igE, igG and IgM, the heavy chains of which are designated α, δ, ε, γ and μ, respectively. Based on C H The relatively small differences in sequence and function, the gamma and alpha classes are further divided into subclasses, e.g., humans express the following subclasses: igG1, igG2A, igG2B, igG3, igG 4. IgA1 and IgA2.
An "isolated" antibody (or construct) is an antibody that has been identified, isolated, and/or recovered from a component of its production environment (e.g., natural or recombinant). Preferably, the isolated polypeptide does not bind to all other components in its production environment. Contaminant components of the production environment, such as those produced by recombinant transfected cells, are materials that generally interfere with the research, diagnostic or therapeutic uses of the antibody, and may include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes. In a preferred embodiment, the polypeptide will be purified: (1) Up to greater than 95% by weight of antibody, as determined by, for example, the Lowry method, and in some embodiments, up to greater than 99% by weight; (2) To a sufficient extent to obtain an N-terminal at least 15 residues or internal amino acid sequence using a rotating cup sequencer; or (3) homogeneity is detected by SDS-PAGE under non-reducing or reducing conditions using Coomassie blue or preferably silver staining. Isolated antibodies (or constructs) include in situ antibodies within recombinant cells because at least one component of the natural environment of the antibody is not present. However, typically an isolated polypeptide, antibody or construct will be prepared by at least one purification step.
"variable region" or "variable domain" of an antibody refers to the amino-terminal domain of the heavy or light chain of the antibody. The variable domains of the heavy and light chains, respectively, may be referred to as "V H "and" V L ". These domains are typically the most variable parts of an antibody (relative to other antibodies of the same class) and contain antigen binding sites. Heavy chain-only antibodies from camelid species have a single heavy chain variable region, termed "V H H). Thus V H H is a special type of V H 。
The term "variable" refers to the fact that certain segments of the variable domain differ greatly in sequence between antibodies. The V domain mediates antigen binding and defines the specificity of a particular antibody for its particular antigen. However, the variability is not evenly distributed throughout the variable domains. Instead, it is concentrated in three segments in the heavy and light chain variable domains called Complementarity Determining Regions (CDRs) or hypervariable regions (HVRs). The more highly conserved parts of the variable domains are called Framework Regions (FR). The variable domains of the natural heavy and light chains each comprise four FR regions, principally employing a β -sheet structure, joined by three CDRs, which form a loop junction, in some cases forming part of the β -sheet structure. The CDRs in each chain are held tightly together by the FR regions and, together with CDRs from the other chain, contribute to the formation of the antigen binding site of the antibody (see Kabat et al Sequences of Immunological Interest, fifth Edition National Institute of Health, bethesda, md. (1991)). The constant domains are not directly involved in binding of antibodies to antigens, but exhibit a variety of effector functions, such as antibodies involved in antibody-dependent cytotoxicity.
As used herein, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations and/or post-translational modifications that may be present in minor amounts (e.g., isomerization, amidation). Monoclonal antibodies are highly specific, being directed against a single antigenic site. In contrast to polyclonal antibody preparations, which typically comprise different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, monoclonal antibodies have the advantage that they are synthesized by hybridoma cultures and are not contaminated with other immunoglobulins. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, monoclonal Antibodies to be used in accordance with the present invention can be made by a variety of techniques, including, for example, the Hybridoma method (Kohler and Milstein., nature,256:495-97 (1975); hongo et al, hybrid, 14 (3): 253-260 (1995), harlow et al, antibodies: ALaboratory Manual, (Cold Spring Harbor Laboratory Press, 2) nd ed.1988); hammerling et al, in Monoclonal Antibodies and T-Cell hybrid 563-681 (Elsevier, N.Y., 1981)), recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567), phage display techniques (see, e.g., clackson et al, nature,352:624-628 (1991); marks et al, J.mol. Biol.222:581-597 (1992); sidhu et al, J.mol. Biol.338 (2): 299-310 (2004); lee et al, J.mol.biol.340 (5): 1073-1093 (2004); felloose, proc. Natl. Acad. Sci. USA 101 (34): 12467-12472 (2004); and Lee et al, J.Immunol. Methods 284 (1-2): 119-132 (2004), and techniques for producing human or human-like antibodies in animals having a partial or complete human immunoglobulin locus or gene encoding a human immunoglobulin sequence (see, e.g., WO 1998/24893; WO 1996/34096; WO 1996/33735; WO 1991/10741;Jakobovits et al., proc. Natl. Acad. Sci. USA 90:2551 (1993), jakobovits et al, nature 362:255-258 (1993), bruggemann et al, year in Immunol.7:33 (1993), U.S. Pat. Nos.5,545,807;5,545,806;5,569,825;5,625,126;5,633,425;and 5,661,016;Marks et al, bio/Technology 10:779-783 (1992), lonberg et al 368:856-859 (1994), morrison, nature 362:255-813, 1996:93, and Biotechnology (1996:14-93; nature 84:356.1995, 1995).
The terms "full-length antibody", "whole antibody" or "complete antibody" are used interchangeably and refer to an antibody in substantially complete form, as opposed to an antibody fragment. In particular, full length 4 chain antibodies include antibodies having a heavy chain and a light chain comprising an Fc region. Full length heavy chain-only antibodies include heavy chain variable domains (e.g., V H H) And an Fc region. The constant domain may be a natural sequence constant domain (e.g., a human natural sequence constant domain) or an amino acid sequence variant thereof. In some cases, an intact antibody may have one or more effector functions. It is to be understood that for the purposes of the present invention, reference to "full length antibody" also includes a full length antibody backbone or parent full length antibody (e.g., a full length 4 chain antibody, or a full length heavy chain only antibody) having a hinge region with a first binding domain (e.g., cytokine moiety) located therein (see, e.g., fig. 1C, 1D, 1N, 1O).
An "antibody fragment", "antigen binding domain" or "antigen binding fragment" comprises a portion of an intact antibody, preferably the antigen binding and/or variable regions of an intact antibody. Anti-cancer agentExamples of body segments include, but are not limited to: fab, fab ', F (ab') 2 And Fv fragments; a double body; linear antibodies (see U.S. Pat. No. 5,641,870, example 2; zapata et al, protein Eng.8 (10): 1057-1062 (1995)); a single chain antibody (scFv) molecule; single domain antibodies (e.g.V H H) And multispecific antibodies formed from antibody fragments. Papain digestion of antibodies produces two identical antigen binding fragments, a "Fab" fragment and a residual "Fc" fragment, the names reflecting the ability to crystallize readily. Fab fragments consist of the variable domains of the whole L and H chain (v H ) And a first constant domain of a heavy chain (C H 1) Composition is prepared. Each Fab fragment is monovalent in terms of antigen binding, i.e., it has a single antigen binding site. Pepsin treatment of antibodies produced single large F (ab') 2 A fragment which corresponds approximately to two disulfide-linked Fab fragments with different antigen binding activities and which is still capable of cross-linking the antigen. Fab' fragments differ from Fab fragments in that: at C H The carboxy terminus of the 1 domain has some additional residues, including one or more cysteines from the antibody hinge region. Fab '-SH is the designation herein for Fab' wherein the cysteine residue of the constant domain bears a free thiol group. F (ab') 2 Antibody fragments were initially produced as pairs of Fab' fragments with hinge cysteines between them. Other chemical couplings of antibody fragments are also known. It is to be understood that for the purposes of the present invention, reference to an "antigen binding domain" or "antigen binding fragment" also includes ligands that can specifically recognize a target receptor, or receptors that can specifically recognize a target ligand.
The term "constant domain" refers to a portion of an immunoglobulin molecule that has a more conserved amino acid sequence relative to other portions of the immunoglobulin (the variable domain, which comprises the antigen binding site). Constant domain comprising C of heavy chain H 1、C H 2 and C H 3 domain (collectively referred to as C H ) And CHL (or C) of light chain L ) A domain.
The "heavy chain" of an antibody (immunoglobulin) can be divided into three functional regions: fd region, hinge region and FRegion c (crystallizable fragment). Fd region contains V H And a CH1 domain, and combines with the light chain to form a Fab-antigen binding fragment. The Fc fragment is responsible for immunoglobulin effector functions including, for example, complement fixation and homologous Fc receptor binding to effector cells. The hinge region found in the IgG, igA and IgD immunoglobulin classes acts as a flexible spacer region that allows the Fab portion to move freely in space relative to the Fc region. In contrast to the constant region, the hinge domain is structurally diverse, differing in sequence and length between immunoglobulin class and subclass. For heavy chain-only antibodies, "heavy chain" includes heavy chain variable domains (e.g., V H H) A hinge region and an Fc region. It is to be understood that for the purposes of the present invention, reference to a "heavy chain" also includes heavy chains comprising a VH domain, a hinge region, and an Fc domain or portion thereof (e.g., a VL-VH-hinge-Fc domain or a VH-VL-hinge-Fc domain subunit), as well as heavy chains comprising a first binding domain (e.g., a cytokine portion) located at the hinge region (e.g., a heavy chain of a full length 4 chain antibody, an antibody comprising a VH-hinge-Fc, or a heavy chain-only antibody) (see, e.g., fig. 1C, 1D, 1N, 1O).
Based on the amino acid sequences of their constant domains, the "light chains" of antibodies (immunoglobulins) from any mammalian species can be divided into one of two distinct types, termed "kappa" and "lambda".
"Fv" is the smallest antibody fragment that contains the complete antigen recognition and binding site. The fragment consists of a dimer of one heavy and one light chain variable region domain, which is tightly, non-covalently bound. Six hypervariable loops (3 loops for each of the H and L chains) are created from the folding of these two domains, which provide amino acid residues for antigen binding and confer antigen binding specificity to the antibody. However, even though a single variable domain (or half Fv comprising only three CDRs specific for an antigen) has the ability to recognize and bind antigen, its affinity is lower than for the entire binding site.
"Single chain Fv" also abbreviated "sFv" or "scFv" is a polypeptide comprising V linked into a single polypeptide chain H And V L Antibody fragments of antibody domains. Preferably, the scFv polypeptide further comprises V H And V L Polypeptide linkers between the domains that enable the scFv to form the desired structure for antigen binding. For reviews of scFv, see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol.113, rosenburg and Moore eds., springer-Verlag, new York, pp.269-315 (1994).
The term "diabody" refers to a polypeptide produced by a polypeptide producing a polypeptide of interest H And V L Construction of small antibody fragments with short linkers (about 5-10 residues) between domains (see paragraph above) allows the interchain rather than intrachain pairing of the V domains to be achieved to produce bivalent fragments, i.e., fragments with two antigen binding sites. Bispecific diabodies are heterodimers of two "cross" sFv fragments, wherein the V of both antibodies H And V L The domains are present on different polypeptide chains. Diabodies are described for more detail, see, for example, EP404,097; WO 93/11161; hollinger et al, proc.Natl. Acad.Sci.USA 90:6444-6448 (1993).
Monoclonal antibodies herein include in particular "chimeric" antibodies (immunoglobulins) in which a portion of the heavy and/or light chain is identical or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) are identical or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (U.S. Pat. No. 4,816,567; morrison et al, proc.Natl. Acad. Sci. USA,81:6851-6855 (1984)). "humanized antibodies" are used as a subset of "chimeric antibodies".
A "humanized" form of a non-human (e.g., llama or camel) antibody is a chimeric antibody that contains minimal sequences derived from a non-human immunoglobulin. In some embodiments, the humanized antibody is a human immunoglobulin (recipient antibody) in which residues from CDRs (defined below) of the recipient are replaced with residues from CDRs of a non-human species (donor antibody) such as mouse, rat, rabbit, camel, llama, alpaca, or a non-human primate having the desired specificity, affinity, and/or capacity. In some cases, the framework ("FR") residues of the human immunoglobulin are replaced with corresponding non-human residues. In addition, humanized antibodies may comprise residues not found in the recipient antibody or the donor antibody. These modifications may be made to further improve antibody properties, such as binding affinity. In general, a humanized antibody will comprise substantially all (at least one, and typically two) variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin sequence, and all or substantially all of the FR regions are those of a human immunoglobulin sequence, although the FR regions may comprise one or more substitutions of individual FR residues, to enhance antibody performance, such as binding affinity, isomerization, immunogenicity, or the like. The number of these amino acid substitutions in the FR is typically no more than 6 in the H chain and no more than 3 in the L chain. The humanized antibody optionally will also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see, e.g., jones et al, nature321:522-525 (1986); riechmann et al, nature 332:323-329 (1988); and Presta, curr.Op.struct.biol.2:593-596 (1992). See also, e.g., vaswani and Hamilton, ann. Allergy, asthma & Immunol.1:105-115 (1998); harris, biochem. Soc. Transactions 23:1035-1038 (1995); hurle and Gross, curr.op.Biotech.5:428-433 (1994); and U.S. Pat. nos. 6,982,321 and 7,087,409.
A "human antibody" is an antibody having an amino acid sequence that corresponds to the amino acid sequence of a human-produced antibody and/or has been prepared using any of the techniques disclosed herein for preparing human antibodies. This definition of human antibodies specifically excludes humanized antibodies that comprise non-human antigen binding residues. Human antibodies can be produced using a variety of techniques known in the art, including phage display libraries. Hoogenboom and Winter, J.mol.biol.,227:381 (1991); marks et al, J.mol.biol.,222:581 (1991). Methods that can also be used to prepare human monoclonal antibodies are described in Cole et al Monoclonal Antibodies and Cancer Therapy, alan r.list, p.77 (1985); boerner et al, J.Immunol.,147 (1): 86-95 (1991). See also van Dijk and van de Winkel, curr. Opin. Pharmacol.5:368-74 (2001). Human anti-human antibodies can be prepared by administering an antigen to a transgenic animalThe animal has been modified to produce such antibodies in response to antigen challenge, but its endogenous locus has been disabled, e.g., immunized xenograft mice (see, e.g., for xenomouise TM Technical U.S. Pat. nos. 6,075,181 and 6,150,584). See also, e.g., li et al, proc.Natl.Acad.Sci.USA,103:3557-3562 (2006), which pertains to human antibodies produced via human B cell hybridoma technology.
The term "hypervariable region", "HVR" or "HV", as used herein, refers to a region of an antibody variable domain that is hypervariable in sequence and/or forms a structurally defined loop. In general, a single domain antibody comprises three HVRs (or CDRs): HVR1 (or CDR 1), HVR2 (or CDR 2), and HVR3 (or CDR 3). HVR3 (or CDR 3) shows the greatest diversity among the three HVRs and is believed to play a unique role in conferring good specificity to antibodies. See, e.g., hamers-Casterman et al, nature 363:446-448 (1993); sheiff et al, nature Structure. Biol.3:733-736 (1996).
The term "complementarity determining region" or "CDR" is used to refer to a hypervariable region defined by the Kabat system. See Kabat et al Sequences of Proteins of Immunological Interest,5th Ed.Public Health Service,National Institutes of Health,Bethesda,Md (1991).
Many HVR descriptions are in use and are encompassed herein. Kabat Complementarity Determining Regions (CDRs) are based on sequence variability and are most commonly used (Kabat et al Sequences of Proteins of Immunological Interest,5th Ed.Public Health Service,National Institutes of Health,Bethesda,Md (1991)). Chothia refers to the position of the structural ring (Chothia and Lesk, J. Mol. Biol.196:901-917 (1987)). AbM HVR represents a compromise between Kabat HVR and Chothia structural loops and was used by Oxford Molecular AbM antibody modeling software. The "Contact" HVR is based on an analysis of existing complex crystal structures. Residues from each of these HVRs are noted in table a below.
Table a.hvr description
The HVR may include the following "extended HVR": v (V) L 24-36 or 24-34 (LI), 46-56 or 50-56 (L2) and 89-97 or 89-96 (L3), and V H In (C) is selected from the group consisting of (1) 26-35 (H), 50-65 or 49-65 (H2) and 93-102, 94-102 or 95-102 (H3). For each of these definitions, variable domain residues are numbered according to Kabat et al, supra.
The expression "variable domain residue number in Kabat" or "amino acid position number in Kabat" and variants thereof refers to the numbering system for heavy chain variable domains or light chain variable domains in the antibody assembly of Kabat et al, supra. Using this numbering system, the actual linear amino acid sequence may comprise fewer or more amino acids, which corresponds to a shortening or insertion of the FR or HVR of the variable domain. For example, the heavy chain variable domain may include a single amino acid inserted after residue 52 of H2 (residue 52a according to Kabat) and residues inserted after heavy chain FR residue 82 (e.g., residues 82a, 82b, and 82c according to Kabat, etc.). The Kabat residue number of a given antibody can be determined by alignment between the homologous region of the antibody sequence and a "standard" Kabat numbering sequence.
Unless otherwise indicated herein, the numbering of residues in the immunoglobulin heavy chain is that of the EU index, as in Kabat et al, supra. The "EU index in Kabat" refers to the residue numbering of the human IgG1 EU antibody.
"framework" or "FR" residues are those variable domain residues that differ from the HVR residues defined herein.
"human consensus framework" or "recipient human framework" is representative of human immunoglobulin V of choice L Or V H The most frequently occurring amino acid residues in the framework sequence. In general, human immunoglobulin V L Or V H The selection of sequences is from a subset of variable domain sequences. In general, a subset of sequences is Kabat et al, sequences of Proteins of Immunological Interest,5 th Subgroup of Ed.public Health Service, national Institutes of Health, bethesda, md. (1991). Examples include, forV L The subgroup may be subgroup κi, κii, κiii or κiv, as in Kabat et al above. Furthermore, for VH, the subgroup may be subgroup I, subgroup II or subgroup III, as in Kabat et al. Alternatively, a human consensus framework can be obtained from above, wherein a particular residue, such as when a human framework residue is selected based on its homology to the donor framework by aligning the donor framework sequence with a collection of multiple human framework sequences. The recipient human framework "derived from" a human immunoglobulin framework or human consensus framework may comprise the same amino acid sequence thereof, or it may contain pre-existing amino acid sequence changes. In some embodiments, the number of pre-existing amino acid changes is 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less.
An "affinity matured" antibody is an antibody having one or more alterations in one or more of its CDRs, which results in an improved affinity of the antibody for the antigen as compared to the parent antibody without such alterations. In some embodiments, the affinity matured antibody has nanomolar or even picomolar affinity for the target antigen. Affinity matured antibodies are produced by procedures known in the art. For example, marks et al, bio/Technology 10:779-783 (1992) describe the passage of V H -and V L Affinity maturation of domain rearrangements. Random mutagenesis of CDR and/or framework residues is described, for example, in Barbas et al Proc Nat. Acad. Sci. USA 91:3809-3813 (1994); schier et al Gene169:147-155 (1995); yelton et al J.Immunol.155:1994-2004 (1995); jackson et al, J.Immunol.154 (7): 3310-9 (1995); and Hawkins et al, J.mol.biol.226:889-896 (1992).
The term "epitope" means a protein determinant capable of specifically binding to an antibody or antigen binding fragment (e.g., ligand, receptor, VHH, scFv, fab, etc.). Epitopes are typically composed of a chemically active surface group of molecules, such as amino acids or sugar side chains, and typically have specific three-dimensional structural features, as well as specific charge features. Conformational and non-conformational epitopes differ in that binding to the former but not to the latter is lost in the presence of denaturing solvents.
As used herein, the terms "specific binding," "specific recognition," or "specific for …" refer to a measurable and reproducible interaction, such as binding between a target molecule and a binding domain (or cytokine and cytokine receptor), which is the determination of the presence of a target molecule (or cytokine) in the presence of a heterogeneous population of molecules, including biomolecules. For example, an antigen binding protein (e.g., fab) that specifically binds to a target molecule (which may be an epitope) is one that binds to the target with greater affinity, avidity, ease, and/or longer duration than it binds to other target molecules. Cytokines that specifically bind to a cytokine receptor are cytokines that bind to the cytokine receptor with greater affinity, avidity, ease, and/or longer duration than they bind to other cytokine receptors. In some embodiments, the extent of binding of the binding domain (or cytokine) to an unrelated target molecule (or unrelated cytokine receptor) is less than about 10% of binding of the binding domain (or cytokine) to the target molecule (or cytokine receptor), e.g., as measured by a Radioimmunoassay (RIA). In some embodiments, the antigen binding protein that specifically binds to the target (or cytokine that specifically binds to cytokine receptor) has a value of 10 or less -5 M、≤10 -6 M、≤10 -7 M、≤10 -8 M、≤10 -9 M、≤10 -10 M、≤10 -11 M or less than or equal to 10 -12 Dissociation constant of M (K D ). In some embodiments, the antigen binding protein (or cytokine receptor) specifically binds to an epitope on a protein (or cytokine) that is conserved among proteins from different species. In some embodiments, specific binding may include, but is not required to be, unique binding. The binding specificity of an antigen binding protein or binding domain (or cytokine and cytokine receptor) can be determined experimentally by any protein binding method known in the art. Such methods include, but are not limited to: western blot, ELISA-, RIA-, ECL-, IRMA-, EIA-, BIACORE TM Test and peptide scan.
The term "specific" refers to the selective recognition of a particular epitope of a target molecule by a binding domain. For example, natural antibodies are monospecific. As used herein, the term "multispecific" means that an antigen-binding protein has multiple epitope specificities (i.e., is capable of specifically binding to two, three, or more different epitopes on one biomolecule, or is capable of specifically binding to an epitope on two, three, or more different biomolecules). As used herein, "bispecific" means that an antigen binding protein has two different antigen binding specificities. The order of antigens to which bispecific antibodies bind is arbitrary unless otherwise indicated. That is, for example, the terms "anti-CD 3/HER2", "anti-HER 2/CD3", "CD3 x HER2" and "HER2 x CD3" are used interchangeably to refer to bispecific antibodies that specifically bind CD3 and HER 2. As used herein, the term "monospecific" refers to an antigen binding protein having one or more binding sites, each binding site binding to the same epitope of the same antigen.
As used herein, the term "valency" means the presence of a specific number of binding sites in an antigen binding protein. For example, a natural antibody or a full length antibody has two binding sites and is bivalent. Thus, the terms "trivalent", "tetravalent", "pentavalent" and "hexavalent" mean that there are two binding sites, three binding sites, four binding sites, five binding sites and six binding sites, respectively, in the antigen binding protein.
"antibody effector functions" refer to those biological activities that are attributed to the Fc region of an antibody (either the native sequence Fc region or the amino acid sequence variant Fc region) and vary with the antibody isotype. Examples of antibody effector functions include: c1q binding and complement dependent cytotoxicity; fc receptor binding; antibody dependent cell-mediated cytotoxicity (aDCC); phagocytosis; down-regulation of cell surface receptors (e.g., B cell receptors); b cell activation. By "reduced or minimized" antibody effector function is meant a reduction of at least 50% (or 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) compared to a wild-type or unmodified antibody. Determination of antibody effector function is readily determined by the art The ordinarily skilled artisan determines and measures. In preferred embodiments, complement binding, complement dependent cytotoxicity, and antibody dependent cytotoxicity are affected by antibody effector function. In some embodiments, effector functions, e.g., "null effector mutations," are eliminated by mutations in the constant region that eliminate glycosylation. In some embodiments, the null point mutation is C H The N297A or DANA mutation in region 2 (d265 a+n297A). Shields et al, J.biol. Chem.276 (9): 6591-6604 (2001). Alternatively, other mutations that result in reducing or eliminating effector function include: K322A and L234A/L235A (LALA). Alternatively, effector functions may be reduced or eliminated by production techniques, such as expression in host cells that are not glycosylated (e.g., E.coli) or where altered glycosylation patterns result such that they are ineffective or less effective in promoting effector function (e.g., shinkawa et al, J.biol. Chem.278 (5): 3466-3473 (2003).
"antibody-dependent cell-mediated cytotoxicity" or ADCC refers to a form of cytotoxicity in which secreted Ig bound onto Fc receptors (fcrs) present on certain cytotoxic cells (e.g., natural Killer (NK) cells, neutrophils, and macrophages) enables these cytotoxic effector cells to specifically bind to antigen-bearing target cells and subsequently kill the target cells with cytotoxins. Antibodies "arm" cytotoxic cells and are necessary to kill target cells by this mechanism. Primary cells, NK cells, that mediate ADCC express fcyriii only, whereas monocytes express fcyri, fcyrii and fcyriii. Fc expression on hematopoietic cells is summarized in Table 2 at page 464 of Ravetch and Kinet, annu. Rev. Immunol.9:457-92 (1991). In order to assess ADCC activity of a target molecule, an in vitro ADCC assay may be performed, as described in U.S. Pat. No. 5,500,362 or U.S. Pat. No. 5,821,337. Effector cells useful in such assays include Peripheral Blood Mononuclear Cells (PBMCs) and Natural Killer (NK) cells. Alternatively, or in addition, ADCC activity of the target molecule may be assessed in vivo, for example in an animal model as disclosed in Clynes et al, PNAS USA 95:652-656 (1998).
"complement-dependent cytotoxicity" or "CDC" refers to the lysis of target cells in the presence of complement. Activation of the classical complement pathway is initiated by the binding of the first component of the complement system (C1 q) to antibodies (appropriate subclasses) that bind to their cognate antigens. To assess complement activation, CDC assays can be performed, for example, as described in Gazzano-Santoro et al, J.Immunol. Methods 202:163 (1996). Antibody variants with altered amino acid sequences of the Fc region and increased or decreased C1q binding capacity are described in U.S. Pat. No. US6,194,551B1 and WO99/51642. The disclosures of those patents are specifically incorporated herein by reference. See also Idusogene et al J.Immunol.164:4178-4184 (2000).
The terms "Fc region", "fragment crystallizable region", "Fc fragment" or "Fc domain" are used herein to define the C-terminal region of an immunoglobulin heavy chain, including native sequence Fc regions and variant Fc regions. Although the boundaries of the immunoglobulin heavy chain Fc region may vary, the human IgG heavy chain Fc region is generally defined as extending from amino acid residue 226 of Cys or from Pro230 to its carboxy terminus. For example, the C-terminal lysine (residue 447 according to the EU numbering system) of the Fc region may be removed during production or purification of the antibody or Fc fusion protein, or by recombinant engineering of nucleic acid encoding the heavy chain of the antibody or Fc fusion protein. Thus, a composition of intact antibodies may comprise a population of antibodies that have all K447 residues removed, a population of antibodies that have no K447 residues removed, and a population of antibodies that have a mixture of antibodies with and without K447 residues. Native sequence Fc regions suitable for use in the immunomodulatory molecules described herein include human IgG1, igG2 (IgG 2A, igG 2B), igG3, and IgG4.
As used herein, the term IgG "isotype" or "subclass" is intended to mean any subclass of immunoglobulin defined by the chemistry and antigenic characteristics of its constant region. Immunoglobulins fall into five main categories: igA, igD, igE, igG and IgM, several of which can be further divided into subclasses (isotypes), for example, igG1, igG2, igG3, igG4, igA1 and IgA2. The heavy chain constant domains corresponding to the different classes of immunoglobulins are called α, γ, ε, γ and μ, respectively. The subunit structure and three-dimensional configuration of different classes of immunoglobulins are well known and are generally described, for example, in Abbas et al cellular and mol.immunology,4th ed. (w.b. samners, co., 2000).
"Fc receptor" or "FcR" describes a receptor that binds to the Fc region of an antibody or Fc fusion protein. The preferred FcR is a native sequence human FcR. Furthermore, preferred fcrs are the following: it binds IgG antibodies (gamma receptors) and includes receptors of the fcyri, fcyrii and fcyriii subclasses, including allelic and alternatively spliced forms of these receptors, fcyrii receptors including fcyriia ("activating receptor") and fcyriib ("inhibiting receptor"), which have similar amino acid sequences, differing primarily in their cytoplasmic domains. The activation receptor fcyriia comprises an immunoreceptor tyrosine-based activation motif (iTAM) in its cytoplasmic domain. The inhibitory receptor fcyriib comprises an immunoreceptor tyrosine-based inhibitory motif (ikim) in its cytoplasmic domain. (see M. Annu.Rev.Immunol.15:203-234 (1997). FcR is reviewed in Ravetch and Kinet, annu. Rev. Immunol.9:457-92 (1991); capel et al, immunomethods 4:25-34 (1994); and de Haas et al, J.Lab.Clin.Med.126:330-41 (1995). Other fcrs, including those identified in the future, are encompassed by the term "FcR" herein.
The term "Fc receptor" or "FcR" also includes the neonatal receptor FcRn, which is responsible for transferring maternal IgG to the fetus. Guyer et al, J.Immunol.117:587 (1976) and Kim et al, J.Immunol.24:249 (1994). Methods for measuring binding to FcRn are known (see, e.g., ghetie and Ward, immunol. Today 18 (12): 592-8 (1997); ghetie et al, nature Biotechnology (7): 637-40 (1997); hinton et al, J. Biol. Chem.279 (8): 6213-6 (2004); WO 2004/92219 (Hinton et al); binding to FcRn in vivo and serum half-life of human FcRn high affinity binding polypeptides can be determined, e.g., in transgenic mice or transfected human cell lines expressing human FcRn, or in primates administered with polypeptides having variant Fc regions WO 2004/42072 (Presta) describes antibody variants that improve or reduce binding to FcRn see, e.g., shields et al, J. Biol. Chem.9 (2): 91-6604 (2001).
"binding affinity" generally refers to the strength of the sum of non-covalent interactions between a molecule (e.g., an antibody, antigen binding fragment (e.g., ligand, receptor, VHH, scFv, etc.) or cytokine) and its binding partner (e.g., an antigen (e.g., cell surface molecule, receptor, ligand, etc.) or cytokine receptor) at a single binding site. As used herein, unless otherwise indicated, "binding affinity" refers to an intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair. Binding affinity can be achieved by K d 、K off 、K on Or K a And (3) representing. The term "K", as used herein Off "is intended to mean the dissociation rate constant of an antibody (or antigen binding fragment) from an antibody (or antigen binding fragment)/antigen complex (e.g., ligand-receptor complex), or the dissociation rate constant of a cytokine from a cytokine/cytokine receptor complex, as determined by a kinetic selection set-up, in s -1 Expressed in units. The term "K", as used herein on "is intended to mean the binding rate constant of an antibody (or antigen binding fragment) to an antigen to form an antibody (or antigen binding fragment)/antigen complex, or the binding rate constant of a cytokine to a cytokine receptor to form a cytokine/cytokine receptor complex, at M -1 s -1 Expressed in units. As used herein, the term equilibrium dissociation constant "K D "or" K d "refers to the dissociation constant of a particular antibody (or antigen binding fragment) -antigen interaction (or cytokine-cytokine receptor interaction) and describes the concentration of antigen (or cytokine) required to occupy half of all antibody binding domains (or antigen binding fragments) present in an antibody (or antigen binding fragment) molecule (or cytokine receptor) solution at equilibrium and is equal to K off /K on Expressed in units of M. K (K) d It is assumed that all binders are in solution. In the case where the antibody (or antigen binding fragment) is tethered to the cell wall, for example, in a yeast expression system, the corresponding equilibrium rate constant is expressed as the EC50, which gives K d Is a good approximation of (a). Affinity constant K a Is the dissociation constant K d To the reciprocal of (1)M -1 Expressed in units. Dissociation constant (K) D Or K d ) Used as an indicator of the affinity of an antibody (or antigen binding fragment) for an antigen (or cytokine for a cytokine receptor). For example, by using the Scatchard (Scatchard) method of antibodies (or antigen binding fragments) labeled with various labeling agents, and by using BIACORE TM X (manufactured by AmershamBiosciences), which is an over-the-counter, measurement kit, or the like, simple analysis is possible according to the user manual and experimental procedure attached to the kit. K which can be derived using these methods D The values are expressed in units of M (Mol). Antibodies or antigen binding fragments (or cytokines) thereof that specifically bind to a target (or cytokine receptor) may have a dissociation constant (K d ) For example, 10 +. -5 M、≤10 -6 M、≤10 -7 M、≤10 -8 M、≤10 -9 M、≤10 -10 M、≤10 -11 M or less than or equal to 10 -12 M。
Half maximal Inhibitory Concentration (IC) 50 ) Is a measure of the effectiveness of a substance (e.g., an antibody or antigen binding fragment) in inhibiting a particular biological or biochemical function. It indicates how much particular drug or other substance (inhibitor, such as an antibody or antigen binding fragment) is required to inhibit a given biological process by half. These values are generally expressed as molar concentrations. IC (integrated circuit) 50 "EC" corresponding to agonist drugs or other substances (e.g., antibodies, antigen binding fragments, or cytokines) 50 ”。EC 50 And also represents the plasma concentration required to obtain 50% of the maximum effect in vivo. As used herein, "IC 50 "is used to indicate the effective concentration of antibody or antigen binding fragment required to neutralize 50% of the antigen biological activity in vitro. IC (integrated circuit) 50 Or EC (EC) 50 Inhibition of ligand binding may be measured by bioassays, such as by FACS analysis (competitive binding assay), cell-based cytokine release assay, or amplified luminescent proximity homogeneous assay (alphaLISA).
As used herein, "covalent bond" refers to a stable bond between two atoms sharing one or more electrons. Examples of covalent bonds include, but are not limited to, peptide bonds and disulfide bonds. As used herein, "peptide bond" refers to a covalent bond formed between a carboxyl group of an amino acid and an amine group of an adjacent amino acid. As used herein, "disulfide" refers to a covalent bond formed between two sulfur atoms, such as two Fc fragments (or cytokine subunits) through a combination of one or more disulfide bonds. One or more disulfide bonds may be formed between the two fragments by linking thiol groups in the two fragments. In some embodiments, one or more disulfide bonds may be formed between one or more cysteines of the two Fc fragments. Disulfide bonds may be formed by oxidation of two thiol groups. In some embodiments, the covalent linkage is directly linked by a covalent bond. In some embodiments, the covalent bond is directly linked through a peptide bond or disulfide bond.
"percent (%) amino acid sequence identity" and "homology" are defined with respect to peptide, polypeptide, or antibody sequences as the percentage of amino acid residues in a candidate sequence that are identical to amino acid residues in a particular peptide or polypeptide sequence, with a space (if desired) introduced after the alignment to obtain the maximum percent sequence identity, and without regard to any conservative substitutions as part of the sequence identity. Alignment aimed at determining percent identity of amino acid sequences can be accomplished in a variety of ways within the skill of the art, e.g., using publicly available computer software such as BLAST, BLAST-2, ALIGN or MEGALIGN TM (DNASTAR) software. One skilled in the art can determine appropriate parameters for measuring the alignment, including any algorithms needed to achieve maximum alignment over the full length of the sequences being compared.
As used herein, the "C-terminus" of a polypeptide refers to the last amino acid residue of the polypeptide that provides for the formation of a peptide bond with the carboxyl group of its adjacent amino acid residue. As used herein, the "N-terminus" of a polypeptide refers to the first amino acid of the polypeptide, which provides that its carboxyl group forms a peptide bond with the amine group of its adjacent amino acid residue.
An "isolated" nucleic acid molecule encoding a construct, antibody or antigen binding fragment thereof described herein is a nucleic acid molecule identified and isolated from at least one contaminant nucleic acid molecule with which it is ordinarily associated in the environment in which it is produced. Preferably, the isolated nucleic acid does not bind to all components associated with the production environment. Isolated nucleic acid molecules encoding the constructs, polypeptides, and antibodies described herein are in a form that is different from the form or environment in which they are found in nature. Thus, an isolated nucleic acid molecule differs from nucleic acids encoding constructs, polypeptides, and antibodies described herein that naturally occur in a cell. An isolated nucleic acid includes a nucleic acid molecule contained in a cell that normally contains the nucleic acid molecule, but the nucleic acid molecule is present extrachromosomally or at a chromosomal location different from its natural chromosomal location.
The term "control sequences" refers to DNA sequences necessary for expression of an operably linked coding sequence in a particular host organism. Control sequences suitable for use in prokaryotes, for example, include a promoter, an optional operator sequence, and a ribosome binding site. Eukaryotic cells are known to utilize promoters, polyadenylation signals and enhancers.
A nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, if a polypeptide is expressed as a preprotein that participates in the secretion of the polypeptide, then the DNA of the prepro sequence or secretory leader is operably linked to the DNA of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or operably linked to the coding sequence if the ribosome binding site is positioned so as to facilitate translation. In general, "operably linked" means that the DNA sequences being linked are contiguous and, in the case of secretory leader, contiguous and in the read phase. However, the enhancers do not have to be contiguous. Ligation is accomplished by ligation at convenient restriction sites. If such a site is not present, the synthetic oligonucleotide adaptors or linkers are used in accordance with conventional practice.
As used herein, the term "vector" refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes vectors that are self-replicating nucleic acid structures and vectors that incorporate the genome of a host cell into which the vector has been introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operably linked. Such vectors are referred to herein as "expression vectors".
As used herein, the term "transfected" or "transformed" or "transduced" refers to the process of transferring or introducing an exogenous nucleic acid into a host cell. A "transfected" or "transformed" or "transduced" cell is a cell that has been transfected, transformed or transduced with an exogenous nucleic acid. The cells include primary test cells and their progeny.
The terms "host cell", "host cell line" and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells," which include primary transformed cells and progeny derived therefrom, independent of passage number. The nucleic acid content of the progeny cells may not be exactly the same as the parent cell, but may contain mutations. Mutant progeny having the same function or biological activity as selected or selected in the originally transformed cell are included herein.
The term "pharmaceutical formulation" of a "pharmaceutical composition" refers to a formulation in a form that allows the biological activity of the active ingredient to be effective and that is free of additional ingredients that have unacceptable toxicity to the subject to whom the formulation is to be administered. Such formulations are sterile. "sterile" formulations are sterile or free of all living microorganisms and spores thereof.
It should be understood that the embodiments of the invention described herein include embodiments that "consist of … …" and/or "consist essentially of … …".
Reference herein to "about" a value or parameter includes (and describes) a variation for that value or parameter itself. For example, a description referring to "about X" includes a description of "X".
As used herein, reference to "not" a value or parameter generally means and describes a "different" value or parameter. For example, the method not being used to treat type X cancer means that the method is used to treat types of cancer other than type X.
As used herein, the term "about X-Y" has the same meaning as "about X to about Y".
As used herein and in the appended claims, the singular forms "a," "an," "or," and "the" include plural referents unless the context clearly dictates otherwise.
Immune modulating molecules
In one aspect, the invention provides an immunomodulatory molecule comprising a first binding domain that specifically recognizes a first target molecule (e.g., a receptor for an immunostimulatory cytokine) (e.g., an immunostimulatory cytokine such as IL-2 or IL-12 or variant thereof) and a second binding domain that specifically recognizes a second target molecule (e.g., an inhibitory checkpoint molecule such as PD-1) (e.g., an agonist ligand such as PD-L1 or PD-L2 or variant thereof, or an agonist antigen binding fragment such as an anti-PD-1 agonist Fab, scFv, VHH, or full-length antibody), wherein the first binding domain upregulates an immune response upon binding to the first target molecule, and wherein the second binding domain downregulates an immune response upon binding to the second target molecule. In some embodiments, the immunoregulatory molecule further comprises a third binding domain (e.g., an antigen binding fragment) that specifically recognizes a third target molecule, such as a cell surface antigen on an immune effector cell (e.g., CD3, PD-1, CTLA-4) or a cancer cell (e.g., a tumor antigen). In some embodiments, the third binding domain upregulates or downregulates the immune response upon binding to the third target molecule. In some embodiments, the third binding domain does not modulate an immune response upon binding to the third target molecule.
In some embodiments, the first binding domain and/or the second binding domain and/or the third binding domain is a VHH. In some embodiments, the first binding domain and/or the second binding domain and/or the third binding domain is an scFv. In some embodiments, the first binding domain and/or the second binding domain and/or the third binding domain is a Fab. In some embodiments, the first binding domain and/or the second binding domain and/or the third binding domain is a single-chain ligand (e.g., a PD-L2 extracellular domain or cytokine) or receptor. For example, the first domain may be a dimeric cytokine moiety formed from a first cytokine subunit recombinantly linked to a second cytokine subunit via an optional linker. In some embodiments, the first binding domain and/or the second binding domain and/or the third binding domain is a ligand or receptor formed from two polypeptide chains. For example, the first domain may be a dimeric cytokine moiety formed from a first cytokine subunit in one polypeptide chain and a second cytokine subunit in another polypeptide chain. In some embodiments, the first binding domain or portion thereof is fused to the N-terminus of the second binding domain or portion thereof. In some embodiments, the first binding domain or portion thereof is fused to the C-terminus of the second binding domain or portion thereof. In some embodiments, the first binding domain or portion thereof is fused to the N-terminus of the third binding domain or portion thereof. In some embodiments, the first binding domain or portion thereof is fused to the C-terminus of the third binding domain or portion thereof. In some embodiments, the third binding domain or portion thereof is fused to the N-terminus of the second binding domain or portion thereof. In some embodiments, the third binding domain or portion thereof is fused to the C-terminus of the second binding domain or portion thereof. The immunoregulatory molecule may have any of the configurations/components illustrated in figures 1A-1W and 11A-15D and described in any of the examples and sequence listings herein.
In some embodiments, the first binding domain is a VHH. In some embodiments, the first binding domain is an scFv. In some embodiments, the first binding domain is a single-chain ligand (e.g., PD-L2 or cytokine) or receptor. In some embodiments, the second binding domain is a Fab. In some embodiments, the first binding domain is fused to the N-terminus of the VH of the Fab. In some embodiments, the first binding domain is fused to the N-terminus of the VL of the Fab. In some embodiments, the first binding domain is fused to the C-terminus of the CH of the Fab. In some embodiments, the first binding domain is fused to the C-terminus of the CL of the Fab. In some embodiments, the first binding domain is a Fab.
In some embodiments, the second binding domain is a VHH. In some embodiments, the second binding domain is an scFv. In some embodiments, the second binding domain is a single chain ligand (e.g., PD-L2 or cytokine) or receptor. In some embodiments, the first binding domain is a Fab. In some embodiments, the second binding domain is fused to the N-terminus of the VH of the Fab. In some embodiments, the second binding domain is fused to the N-terminus of the VL of the Fab. In some embodiments, the second binding domain is fused to the C-terminus of the CH of the Fab. In some embodiments, the second binding domain is fused to the C-terminus of the CL of the Fab. In some embodiments, the second binding domain is a Fab.
In some embodiments, the third binding domain is a VHH. In some embodiments, the third binding domain is an scFv. In some embodiments, the third binding domain is a Fab. In some embodiments, the third binding domain is a ligand or receptor (e.g., an extracellular domain of a ligand or receptor).
In some embodiments, the first binding domain is located in the hinge region of the immunomodulatory molecule, such as the hinge region between the second binding domain and the Fc domain subunit or portion thereof. In some embodiments, the first binding domain is not located at the hinge region of the immunomodulatory molecule, such as at the C' of one or both Fc subunits of the parent Fc fusion protein or Fc-containing parent antibody.
In some embodiments, the immunoregulatory molecule comprises: i) An antigen binding protein comprising an antigen binding polypeptide; and ii) a first binding domain (e.g., an immunostimulatory cytokine such as IL-2 or IL-12 or variant thereof), wherein the antigen binding polypeptide comprises, from N-terminus to C-terminus: a second binding domain or portion thereof (e.g., an agonist ligand such as PD-L1 or PD-L2 or variant thereof, or an agonist antigen binding fragment such as anti-PD-1 agonist Fab, scFv, VHH), a hinge region, and an Fc domain subunit or portion thereof, and wherein the first binding domain is located at the hinge region. Thus, in some embodiments, an immunomodulatory molecule is provided comprising: i) An antigen binding protein comprising an antigen binding polypeptide; and ii) a first binding domain (e.g., an immunostimulatory cytokine such as IL-2 or IL-12 or variant thereof) that specifically recognizes a first target molecule (e.g., a receptor for an immunostimulatory cytokine), wherein the antigen binding polypeptide comprises from N-terminus to C-terminus: a second binding domain or portion thereof (e.g., an agonist ligand such as PD-L1 or PD-L2 or variant thereof, or an agonist antigen binding fragment such as anti-PD-1 agonist Fab, scFv, VHH), a hinge region, and an Fc domain subunit or portion thereof that specifically recognizes a second target molecule (e.g., an inhibitory checkpoint molecule such as PD-1), wherein the first binding domain is located at the hinge region, wherein the first binding domain upregulates an immune response upon binding to the first target molecule, and wherein the second binding domain downregulates an immune response upon binding to the second target molecule. In some embodiments, the activity of the first binding domain is increased by at least about 20% (e.g., any of at least about 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500% or more) in the presence of binding of the second binding domain to the second target molecule, as compared to the absence of binding of the second binding domain to the second target molecule. In some embodiments, in the absence of binding of the second binding domain to the second target molecule, the activity of the first binding domain at the hinge region is no more than about 70% (e.g., no more than about any of 60%, 50%, 40%, 30%, 20%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.9%,0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1% or 0%) of the activity of the corresponding first binding domain in the free state. In some embodiments, the antigen binding protein comprises two antigen binding polypeptides, each comprising a hinge region, and wherein only one antigen binding polypeptide comprises a first binding domain located at the hinge region. In some embodiments, the antigen binding protein comprises two antigen binding polypeptides, each antigen binding polypeptide comprising a hinge region, and wherein each antigen binding polypeptide comprises a first binding domain located at the hinge region. In some embodiments, the immunomodulatory molecule comprises two or more first binding domains, wherein the two or more first binding domains are positioned in tandem at the hinge region of the antigen binding polypeptide. In some embodiments, the first binding domain is an immunostimulatory cytokine or variant thereof. In some embodiments, the immunostimulatory cytokine is selected from the group consisting of: IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-12, IL-15, IL-17, IL-18, IL-21, IL-22, IL-23, IL-27, IFN-alpha, IFN-beta, IFN-gamma, TNF-alpha, erythropoietin, thrombopoietin, G-CSF, M-CSF, SCF, and GM-CSF. In some embodiments, the first binding domain is an immunostimulatory cytokine variant, and wherein the immunostimulatory cytokine variant in the free state has an activity of no more than about 80% (e.g., no more than any of about 70%, 60%, 50%, 40%, 30%, 20%, 10%, or 5%) of the activity of a corresponding wild-type immunostimulatory cytokine in the free state. In some embodiments, the immunostimulatory cytokine or variant thereof is a monomeric immunostimulatory cytokine or variant thereof. In some embodiments, the immunostimulatory cytokine or variant thereof is a dimeric immunostimulatory cytokine or variant thereof. In some embodiments, two subunits of the dimeric immunostimulatory cytokine or variant thereof are positioned in tandem in the hinge region of the antigen binding polypeptide. In some embodiments, the antigen binding protein comprises two antigen binding polypeptides, each comprising a hinge region, wherein one subunit of the dimeric immunostimulatory cytokine or variant thereof is located in the hinge region of one antigen binding polypeptide, and wherein the other subunit of the dimeric immunostimulatory cytokine or variant thereof is located in the hinge region of the other antigen binding polypeptide. In some embodiments, the immunostimulatory cytokine or variant thereof is IL-2 or variant thereof. In some embodiments, the IL-2 variant comprises one or more mutations at a position selected from the group consisting of: f24, K35, R38, F42, K43, E61 and P65. In some embodiments, the IL-2 variant comprises one or more mutations selected from the group consisting of: F24A, R D, K3543, E, E R and P65L. In some embodiments, the IL-2 variant comprises an R38D/K43E/E61R mutation relative to wild-type IL-2. In some embodiments, the immunostimulatory cytokine or variant thereof is IL-12 or a variant thereof. In some embodiments, the IL-12 variant comprises one or more mutations within the p40 subunit at a position selected from the group consisting of: e45, Q56, V57, K58, E59, F60, G61, D62, A63, G64, Q65 and C177. In some embodiments, the IL-12 variant comprises one or more mutations within the p40 subunit, relative to the wild-type p40 subunit, selected from the group consisting of: Q56A, V57A, K A, E3559A, F60A, G61A, D A, A63S, G a and Q65A. In some embodiments, the IL-12 variant comprises an E59A/F60A mutation within the p40 subunit relative to the wild-type p40 subunit. In some embodiments, the IL-12 variant comprises an F60A mutation within the p40 subunit relative to the wild-type p40 subunit. In some embodiments, IL-12 or its variants of the p40 subunit and p35 subunit connected by a linker. In some embodiments, two or more first binding domains are identical. In some embodiments, the two or more first binding domains are different. In some embodiments, the second binding domain is an agonist ligand of an inhibitory checkpoint molecule or a variant thereof. In some embodiments, the inhibitory checkpoint molecule is selected from the group consisting of: PD-1, PD-L2, CTLA-4, LAG-3, TIM-3, HHA 2, CD47, CXCR4, CD160, CD73, BLTA, B7-H4, TIGIT, siglec7, siglec9 and VISTA. In some embodiments, the second binding domain is PD-L1 or a variant thereof. In some embodiments, the PD-L1 variant has increased binding affinity for PD-1 as compared to wild-type PD-L1. In some embodiments, the PD-L1 variant comprises one or more mutations at a position selected from the group consisting of: i54, Y56, E58, R113, M115, S117, and G119. In some embodiments, the PD-L1 variant comprises one or more mutations relative to wild-type PD-L1 selected from the group consisting of: I54Q, Y56F, E58M, R113T, M115L, S a and G119K. In some embodiments, the PD-L1 variant comprises an I54Q/Y56F/E58M/R113T/M115L/S117A/G119K mutation relative to wild-type PD-L1. In some embodiments, the second binding domain is PD-L2 or a variant thereof. In some embodiments, the PD-L2 variant has increased binding affinity for PD-1 as compared to wild-type PD-L2. In some embodiments, the second binding domain is an agonist antibody or antigen binding fragment thereof of an inhibitory checkpoint molecule. In some embodiments, the inhibitory checkpoint molecule is selected from the group consisting of: PD-1, PD-L2, CTLA-4, LAG-3, TIM-3, HHA 2, CD47, CXCR4, CD160, CD73, BLTA, B7-H4, TIGIT, siglec7, siglec9 and VISTA. In some embodiments, the agonist antibody or antigen-binding fragment thereof specifically recognizes PD-1 ("anti-PD-1 agonist antibody or antigen-binding fragment thereof"). In some embodiments, the agonist antibody or antigen binding fragment thereof is a Fab. In some embodiments, the agonist antibody or antigen binding fragment thereof is a scFv. In some embodiments, the antigen binding protein comprises two or more second binding domains. In some embodiments, two or more second binding domains, or portions thereof, are positioned in tandem at the N-terminus of the antigen binding polypeptide. In some embodiments, the antigen binding protein comprises two antigen binding polypeptides, each comprising a hinge region, and wherein only one antigen binding polypeptide comprises two or more second binding domains, or portions thereof, in tandem at the N-terminus of the antigen binding polypeptide. In some embodiments, the antigen binding protein comprises two antigen binding polypeptides, each antigen binding polypeptide comprising a hinge region, and wherein each antigen binding polypeptide comprises one or more second binding domains, or portions thereof, at the N-terminus of each antigen binding polypeptide. In some embodiments, the antigen binding protein comprises two antigen binding polypeptides, each antigen binding polypeptide comprising a hinge region, wherein a first antigen binding polypeptide comprises one or more second binding domains or portions thereof at the N-terminus of the first antigen binding polypeptide, wherein a second antigen binding polypeptide comprises a third binding domain or portion thereof at the N-terminus of the second antigen binding polypeptide, and wherein the third binding domain specifically recognizes a third target molecule. In some embodiments, the third binding domain and the second binding domain are the same. In some embodiments, the third binding domain and the second binding domain are different. In some embodiments, the third target molecule and the second target molecule are the same. In some embodiments, the third target molecule and the second target molecule are different.
In some embodiments, there is provided an immunomodulatory molecule comprising: i) A first antigen binding polypeptide comprising, from N-terminus to C-terminus: a first second binding domain (e.g., PD-L2 or PD-L1 or variant thereof), a first binding domain located at a first hinge region (e.g., the p35 subunit and the p40 subunit of IL-12 or variant thereof (e.g., E59A/F60A or F60A in p 40) connected in series), and a first subunit of an Fc domain or portion thereof; ii) a second antigen binding polypeptide comprising, from N-terminus to C-terminus: VH, optionally CH1, a second hinge region, and a second subunit of an Fc domain, or portion thereof; and iii) a third antigen binding polypeptide comprising, from N-terminus to C-terminus: VL and optionally CL; wherein VH and VL, and optionally CH1 and CL, form a third binding domain that specifically recognizes a third target molecule, wherein the first binding domain specifically recognizes a first target molecule, wherein the second binding domain specifically recognizes a second target molecule (e.g., PD-1), wherein the first binding domain upregulates an immune response upon binding to the first target molecule (e.g., IL-12 receptor), and wherein the first and/or second binding domain (e.g., PD-L2 or PD-L1, or variant thereof) downregulates an immune response upon binding to the second target molecule. See, for example, fig. 1B. In some embodiments, the third binding domain is an agonist antigen binding fragment that specifically recognizes PD-1. See, for example, fig. 1A. In some embodiments, the first and second binding domains are identical. In some embodiments, the first and second binding domains are different. In some embodiments, the first and second binding domains specifically recognize the same epitope. In some embodiments, the first and second binding domains specifically recognize different epitopes.
In some embodiments, there is provided an immunomodulatory molecule comprising: i) A first antigen binding polypeptide comprising, from N-terminus to C-terminus: a first VH, an optional first CH1, a first binding domain located at a first hinge region (e.g., a p35 subunit and a p40 subunit in tandem connection with IL-12 or a variant thereof), and a first subunit of an Fc domain or portion thereof; ii) a second antigen binding polypeptide comprising, from N-terminus to C-terminus: a second VH, optionally a second CH1, a second hinge region, and a second subunit of an Fc domain, or portion thereof; iii) A third antigen binding polypeptide comprising, from N-terminus to C-terminus: a first VL and optionally a first CL; and iv) a fourth antigen binding polypeptide comprising, from N-terminus to C-terminus: a second VL and optionally a second CL, wherein the first VH and the first VL, and optionally the first CH1 and the first CL, form a second binding domain that specifically recognizes a second target molecule (e.g., an agonist antigen binding fragment that specifically recognizes PD-1), wherein the first binding domain specifically recognizes a first target molecule (e.g., an IL-12 receptor), wherein the second VH and the second VL, and optionally the second CH1 and the second CL, form a third binding domain that specifically recognizes a third target molecule, wherein the first binding domain upregulates an immune response upon binding to the first target molecule (e.g., an IL-12 receptor), and wherein the second binding domain downregulates an immune response upon binding to the second target molecule (e.g., PD-1). See, for example, fig. 1D. In some embodiments, the third binding domain is an agonist antigen binding fragment that specifically recognizes PD-1. Thus, in some embodiments, there is provided an immunomodulatory molecule comprising: i) A first antigen binding polypeptide comprising, from N-terminus to C-terminus: a first VH, optionally a first CH1, a p35 subunit and a p40 subunit in tandem with IL-12 or variant thereof at a first hinge region, and a first subunit of an Fc domain or portion thereof; ii) a second antigen binding polypeptide comprising, from N-terminus to C-terminus: a second VH, optionally a second CH1, a second hinge region, and a second subunit of an Fc domain, or portion thereof; iii) A third antigen binding polypeptide comprising, from N-terminus to C-terminus: a first VL and optionally a first CL; and iv) a fourth antigen binding polypeptide comprising, from N-terminus to C-terminus: a second VL and optionally a second CL, wherein the first VH and the first VL, and optionally the first CH1 and the first CL, form a second binding domain that is an agonist antigen-binding fragment that specifically recognizes PD-1, and wherein the second VH and the second VL, and optionally the second CH1 and the second CL, form a third binding domain that is an agonist antigen-binding fragment 1 that specifically recognizes PD-1, wherein IL-12 or the variant upregulates an immune response upon binding to the IL-12 receptor, and wherein the second binding domain and/or the third binding domain downregulates an immune response upon binding to PD-1. See, for example, fig. 1C. In some embodiments, the third binding domain and the second binding domain are the same. In some embodiments, the third binding domain and the second binding domain are different. In some embodiments, the third binding domain and the second binding domain specifically recognize the same epitope. In some embodiments, the third binding domain and the second binding domain specifically recognize different epitopes.
In some embodiments, there is provided an immunomodulatory molecule comprising: i) A first antigen binding polypeptide comprising, from N-terminus to C-terminus: a first second binding domain (e.g., PD-L2 or PD-L1 or variant thereof), a first binding domain located at a first hinge region (e.g., p35 subunit and p40 subunit of IL-12 or variant thereof connected in series), and a first subunit of an Fc domain or portion thereof; and ii) a second antigen binding polypeptide comprising, from N-terminus to C-terminus: a second binding domain (e.g., PD-L2 or PD-L1 or variant thereof), a second hinge region, and a second subunit of an Fc domain or portion thereof, wherein the first binding domain specifically recognizes a first target molecule (e.g., an IL-12 receptor), wherein the first binding domain upregulates an immune response upon binding to the first target molecule (e.g., an IL-12 receptor), and wherein the second binding domain downregulates an immune response upon binding to the second target molecule (e.g., PD-1). See, for example, fig. 1G. In some embodiments, the first and second binding domains are identical. In some embodiments, the first and second binding domains are different. In some embodiments, the first and second binding domains specifically recognize the same epitope. In some embodiments, the first and second binding domains specifically recognize different epitopes.
In some embodiments, there is provided an immunomodulatory molecule comprising: i) A first antigen binding polypeptide comprising, from N-terminus to C-terminus: a first second binding domain (e.g., PD-L2 or PD-L1 or variant thereof), a first binding domain located at a first hinge region (e.g., in tandem with the p35 subunit and the p40 subunit of IL-12 or variant thereof), and a first subunit of an Fc domain or portion thereof; and ii) a second antigen binding polypeptide comprising, from N-terminus to C-terminus: a third second binding domain (e.g., PD-L2 or PD-L1 or variant thereof), a fourth second binding domain (e.g., PD-L2 or PD-L1 or variant thereof), a second hinge region, and a second subunit of an Fc domain or portion thereof, wherein the first binding domain specifically recognizes a first target molecule (e.g., an IL-12 receptor), wherein the first, second, third, and/or fourth second binding domain specifically recognizes a second target molecule (e.g., PD-1), wherein the first binding domain upregulates an immune response upon binding to the first target molecule (e.g., an IL-12 receptor), and wherein the first, second, third, and/or fourth second binding domain downregulates an immune response upon binding to the second target molecule (e.g., PD-1). See, for example, fig. 1H. In some embodiments, the first, second, third, and/or fourth second binding domains are identical. In some embodiments, the first, second, third, and/or fourth second binding domains are different. In some embodiments, the first, second, third, and/or fourth second binding domains specifically recognize the same epitope. In some embodiments, the first, second, third, and/or fourth second binding domains specifically recognize different epitopes.
In some embodiments, there is provided an immunomodulatory molecule comprising: i) A first antigen binding polypeptide comprising, from N-terminus to C-terminus: a first second binding domain (e.g., PD-L2 or PD-L1 or variant thereof), a portion of the first binding domain located at the first hinge region (e.g., the p35 subunit of IL-12 or variant thereof), and a first subunit of an Fc domain or portion thereof; and ii) a second antigen binding polypeptide comprising, from N-terminus to C-terminus: a second binding domain (e.g., PD-L2 or PD-L1 or variant thereof), another portion of the first binding domain (e.g., the p40 subunit of IL-12 or variant thereof) at the second hinge region, and a second subunit of the Fc domain or portion thereof, wherein the first binding domain specifically recognizes a first target molecule (e.g., an IL-12 receptor), wherein the first and second binding domains specifically recognize a second target molecule (e.g., PD-1), wherein the first binding domain upregulates an immune response upon binding to the first target molecule (e.g., an IL-12 receptor), and wherein the first and/or second binding domains downregulate an immune response upon binding to the second target molecule (e.g., PD-1). See, for example, fig. 1L. In some embodiments, the first and second binding domains are identical. In some embodiments, the first and second binding domains are different. In some embodiments, the first and second binding domains specifically recognize the same epitope. In some embodiments, the first and second binding domains specifically recognize different epitopes.
In some embodiments, there is provided an immunomodulatory molecule comprising: i) A first antigen binding polypeptide comprising, from N-terminus to C-terminus: a portion of a first binding domain (e.g., the p35 subunit or the p40 subunit of IL-12 or a variant thereof) and a first subunit of an Fc domain, or a portion thereof, at a first hinge region; and ii) a second antigen binding polypeptide comprising, from N-terminus to C-terminus: a first second binding domain (e.g., PD-L2 or PD-L1 or variant thereof), a second binding domain (e.g., PD-L2 or PD-L1 or variant thereof), another portion of the first binding domain (e.g., the p40 subunit or the p35 subunit of IL-12 or variant thereof) at the second hinge region, and a second subunit of the Fc domain or portion thereof, wherein the first binding domain specifically recognizes a first target molecule (e.g., an IL-12 receptor), wherein the first and second binding domains specifically recognize a second target molecule (e.g., PD-1), wherein the first binding domain upregulates an immune response upon binding to the first target molecule (e.g., an IL-12 receptor), and wherein the first and/or second binding domain downregulates an immune response upon binding to the second target molecule (e.g., PD-1). See, for example, fig. 1M. In some embodiments, the first and second binding domains are identical. In some embodiments, the first and second binding domains are different. In some embodiments, the first and second binding domains specifically recognize the same epitope. In some embodiments, the first and second binding domains specifically recognize different epitopes.
In some embodiments, there is provided an immunomodulatory molecule comprising: i) A first antigen binding polypeptide comprising, from N-terminus to C-terminus: a first VH, optionally a first CH1, a portion of a first binding domain located at a first hinge region (e.g., the p35 subunit or the p40 subunit of IL-12 or a variant thereof), and a first subunit of an Fc domain, or portion thereof; ii) a second antigen binding polypeptide comprising, from N-terminus to C-terminus: a second VH, an optional second CH1, another portion of the first binding domain located at a second hinge region (e.g., the p40 subunit or the p35 subunit of IL-12 or a variant thereof), and a second subunit of an Fc domain or portion thereof; iii) A third antigen binding polypeptide comprising, from N-terminus to C-terminus: a first VL and optionally a first CL; and iv) a fourth antigen binding polypeptide comprising, from N-terminus to C-terminus: a second VL and optionally a second CL, wherein the first VH and the first VL, and optionally the first CH1 and the first CL, form a second binding domain that specifically recognizes a second target molecule (e.g., an agonist antigen binding fragment that specifically recognizes PD-1), wherein the second VH and the second VL, and optionally the second CH1 and the second CL, form a third binding domain that specifically recognizes a third target molecule, wherein the first binding domain specifically recognizes the first target molecule (e.g., an IL-12 receptor), wherein the first binding domain upregulates an immune response upon binding to the first target molecule (e.g., an IL-12 receptor), and wherein the second binding domain downregulates an immune response upon binding to the second target molecule (e.g., PD-1). See, for example, fig. 1O. In some embodiments, the third binding domain is an agonist antigen binding fragment that specifically recognizes PD-1. See, for example, fig. 1N. In some embodiments, the third binding domain and the second binding domain are the same. In some embodiments, the third binding domain and the second binding domain are different. In some embodiments, the third binding domain and the second binding domain specifically recognize the same epitope. In some embodiments, the third binding domain and the second binding domain specifically recognize different epitopes.
In some embodiments, the immunomodulatory molecule comprises an antigen binding protein comprising an antigen binding polypeptide, wherein the antigen binding polypeptide comprises, from N-terminus to C-terminus: a first binding domain or portion thereof, a second binding domain or portion thereof, an optional hinge region, and an Fc domain subunit or portion thereof. Thus, in some embodiments, there is provided an immunomodulatory molecule comprising an antigen-binding protein comprising an antigen-binding polypeptide, wherein the antigen-binding polypeptide comprises, from N 'to C': a first binding domain or portion thereof (e.g., an immunostimulatory cytokine such as IL-2 or IL-12 or variant thereof), a second binding domain or portion thereof (e.g., an agonist ligand such as PD-L1 or PD-L2 or variant thereof, or an agonist antigen binding fragment such as an anti-PD-1 agonist Fab, scFv, VHH), an optional hinge region, and an Fc domain subunit or portion thereof, wherein the first binding domain specifically recognizes a first target molecule (e.g., an IL-12 receptor), wherein the second binding domain specifically recognizes a second target molecule (e.g., PD-1), wherein the first binding domain upregulates an immune response upon binding to the first target molecule (e.g., an IL-12 receptor), and wherein the second binding domain downregulates an immune response upon binding to the second target molecule (e.g., PD-1). In some embodiments, the second binding domain is an agonist Fab or agonist scFv that specifically recognizes an inhibitory checkpoint molecule. In some embodiments, the second binding domain is an agonist ligand of an inhibitory checkpoint molecule or a variant thereof. In some embodiments, the second binding domain is PD-L1 or PD-L2 or a variant thereof. In some embodiments, the first binding domain is an immunostimulatory cytokine or variant thereof. In some embodiments, the immunostimulatory cytokine or variant thereof is IL-2 or IL-12 or variant thereof. In some embodiments, wherein the antigen binding protein comprises two antigen binding polypeptides, each comprising a hinge region, wherein the first antigen binding polypeptide comprises, from N-terminus to C-terminus: a first binding domain or portion thereof, a second binding domain or portion thereof, a first hinge region, and a first subunit of an Fc domain or portion thereof; wherein the second antigen binding polypeptide comprises, from N-terminus to C-terminus: a third binding domain or portion thereof, a second hinge region, and a second subunit of an Fc domain or portion thereof; and wherein the third binding domain specifically recognizes a third target molecule. In some embodiments, the third binding domain and the second binding domain are the same. In some embodiments, the third binding domain and the second binding domain are different. In some embodiments, the third target molecule and the second target molecule are the same. In some embodiments, the third target molecule and the second target molecule are different.
In some embodiments, there is provided an immunomodulatory molecule comprising: i) A first antigen binding polypeptide comprising, from N-terminus to C-terminus: a first binding domain that specifically recognizes a first target molecule (e.g., the p35 subunit and the p40 subunit of IL-12 or a variant thereof fused in tandem), a first VH, optionally a first CH1, a first hinge region, and a first subunit of an Fc domain, or portion thereof; ii) a second antigen binding polypeptide comprising, from N-terminus to C-terminus: a second VH, optionally a second CH1, a second hinge region, and a second subunit of an Fc domain, or portion thereof; iii) A third antigen binding polypeptide comprising, from N-terminus to C-terminus: a first VL and optionally a first CL; and iv) a fourth antigen binding polypeptide comprising, from N-terminus to C-terminus: a second VL and optionally a second CL, wherein the first VH and the first VL, and optionally the first CH1 and the first CL, form a second binding domain that specifically recognizes a second target molecule (e.g., an agonist antigen-binding fragment that specifically recognizes PD-1), wherein the second VH and the second VL, and optionally the second CH1 and the second CL, form a third binding domain that specifically recognizes a third target molecule, wherein the first binding domain upregulates an immune response upon binding to the first target molecule (e.g., an IL-12 receptor), and wherein the second binding domain downregulates an immune response upon binding to the second target molecule (e.g., PD-1). In some embodiments, the third binding domain is an agonist antigen binding fragment that specifically recognizes PD-1. See, for example, fig. 1T. In some embodiments, the third binding domain and the second binding domain are the same. In some embodiments, the third binding domain and the second binding domain are different. In some embodiments, the third target molecule and the second target molecule are the same. In some embodiments, the third target molecule and the second target molecule are different.
In some embodiments, there is provided an immunomodulatory molecule comprising: i) A first antigen binding polypeptide comprising, from N-terminus to C-terminus: a first binding domain that specifically recognizes a first target molecule (e.g., p35 subunit and p40 subunit of IL-12 or a variant thereof fused in tandem), a first second binding domain (e.g., PD-L2 or PD-L1 or a variant thereof), a first hinge region, and a first subunit of an Fc domain or portion thereof; and ii) a second antigen binding polypeptide comprising, from N-terminus to C-terminus: a third second binding domain (e.g., PD-L2 or PD-L1 or variant thereof), a fourth second binding domain (e.g., PD-L2 or PD-L1 or variant thereof), a second hinge region, and a second subunit of an Fc domain or portion thereof, wherein the first, second, third, and/or fourth second binding domain specifically recognizes a second target molecule (e.g., PD-1), wherein the first binding domain upregulates an immune response upon binding to the first target molecule (e.g., IL-12 receptor), and wherein the first, second, third, and/or fourth second binding domain downregulates an immune response upon binding to the second target molecule (e.g., PD-1). See, e.g., fig. 1U. In some embodiments, the first, second, third, and/or fourth second binding domains are identical. In some embodiments, the first, second, third, and/or fourth second binding domains are different. In some embodiments, the first, second, third, and/or fourth second binding domains specifically recognize the same epitope. In some embodiments, the first, second, third, and/or fourth second binding domains specifically recognize different epitopes.
In some embodiments, there is provided an immunomodulatory molecule comprising: i) A first antigen binding polypeptide comprising, from N-terminus to C-terminus: a first binding domain that specifically recognizes a first target molecule (e.g., p35 subunit and p40 subunit of IL-12 or a variant thereof fused in tandem), a first second binding domain (e.g., PD-L2 or PD-L1 or a variant thereof), a first hinge region, and a first subunit of an Fc domain or portion thereof; ii) a second antigen binding polypeptide comprising, from N-terminus to C-terminus: VH, optionally CH1, a second hinge region, and a second subunit of an Fc domain, or portion thereof; and iii) a third antigen binding polypeptide comprising, from N-terminus to C-terminus: VL and optionally CL, wherein VH and VL, and optionally CH1 and CL form a third binding domain that specifically recognizes a third target molecule, wherein the first and/or second binding domain specifically recognizes a second target molecule (e.g., PD-1), wherein the first binding domain upregulates an immune response upon binding to the first target molecule (e.g., IL-12 receptor), and wherein the first and/or second binding domain downregulates an immune response upon binding to the second target molecule (e.g., PD-1). In some embodiments, the third binding domain is an agonist antigen binding fragment that specifically recognizes PD-1. See, for example, fig. 1V. In some embodiments, the first and second binding domains are identical. In some embodiments, the first and second binding domains are different. In some embodiments, the first and second binding domains specifically recognize the same epitope. In some embodiments, the first and second binding domains specifically recognize different epitopes.
In some embodiments, there is provided an immunomodulatory molecule comprising: i) A first antigen binding polypeptide comprising, from N-terminus to C-terminus: a first binding domain that specifically recognizes a first target molecule (e.g., the p35 subunit and the p40 subunit of IL-12 or variant thereof fused in tandem), VH, optionally CH1, a first hinge region, and a first subunit of an Fc domain, or portion thereof; ii) a second antigen binding polypeptide comprising, from N-terminus to C-terminus: a first third binding domain (e.g., PD-L2 or PD-L1 or variant thereof), a second hinge region, and a second subunit of an Fc domain or portion thereof; and iii) a third antigen binding polypeptide comprising, from N-terminus to C-terminus: VL and optionally CL, wherein VH and VL and optionally CH1 and CL form a second binding domain that specifically recognizes a second target molecule (e.g., an agonist antigen binding fragment that specifically recognizes PD-1), wherein the first and/or second third binding domain specifically recognizes a third target molecule (e.g., PD-1), wherein the first binding domain upregulates an immune response upon binding to the first target molecule (e.g., IL-12 receptor), and wherein the second binding domain downregulates an immune response upon binding to the second target molecule (e.g., PD-1). See, for example, fig. 1W. In some embodiments, the first and second third binding domains are identical. In some embodiments, the first and second third binding domains are different. In some embodiments, the first and second third binding domains specifically recognize the same epitope. In some embodiments, the first and second third binding domains specifically recognize different epitopes.
In some embodiments, there is provided an immunomodulatory molecule comprising: i) A first antigen binding polypeptide comprising, from N-terminus to C-terminus: a first VH, optionally a first CH1, a first hinge region, and a first subunit of an Fc domain, or portion thereof; ii) a second antigen binding polypeptide comprising, from N-terminus to C-terminus: a second VH, optionally a second CH1, a second hinge region, and a second subunit of an Fc domain, or portion thereof; iii) A third antigen binding polypeptide comprising, from N-terminus to C-terminus: a first binding domain that specifically recognizes a first target molecule (e.g., the p35 subunit and the p40 subunit of IL-12 or a variant thereof fused in tandem), a first VL, and optionally a first CL; and iv) a fourth antigen binding polypeptide comprising, from N-terminus to C-terminus: a second VL and optionally a second CL, wherein the first VH and the first VL, and optionally the first CH1 and the first CL, form a second binding domain that specifically recognizes a second target molecule (e.g., an agonist antigen-binding fragment that specifically recognizes PD-1), and wherein the second VH and the second VL, and optionally the second CH1 and the second CL, form a third binding domain that specifically recognizes a third target molecule, wherein the first binding domain upregulates an immune response upon binding to the first target molecule (e.g., an IL-12 receptor), and wherein the second binding domain downregulates an immune response upon binding to the second target molecule (e.g., PD-1). In some embodiments, the third binding domain is an agonist antigen binding fragment that specifically recognizes PD-1. In some embodiments, the third binding domain and the second binding domain are the same. In some embodiments, the third binding domain and the second binding domain are different. In some embodiments, the third target molecule and the second target molecule are the same. In some embodiments, the third target molecule and the second target molecule are different.
In some embodiments, there is provided an immunomodulatory molecule comprising: i) A first antigen binding polypeptide comprising, from N-terminus to C-terminus: VH, optionally CH1, a first hinge region, and a first subunit of an Fc domain, or portion thereof; ii) a second antigen binding polypeptide comprising, from N-terminus to C-terminus: a first third binding domain (e.g., PD-L2 or PD-L1 or variant thereof), a second hinge region, and a second subunit of an Fc domain or portion thereof; and iii) a third antigen binding polypeptide comprising, from N-terminus to C-terminus: a first binding domain that specifically recognizes a first target molecule (e.g., the p35 subunit and the p40 subunit of IL-12 or a variant thereof fused in tandem), a VL, and optionally a CL, wherein VH and VL, and optionally CH1 and CL form a second binding domain that specifically recognizes a second target molecule (e.g., an agonist antigen binding fragment that specifically recognizes PD-1), wherein the first and/or second third binding domain specifically recognizes a third target molecule (e.g., PD-1), wherein the first binding domain upregulates an immune response upon binding to the first target molecule (e.g., IL-12 receptor), and wherein the second binding domain downregulates an immune response upon binding to the second target molecule (e.g., PD-1). In some embodiments, the first and second third binding domains are identical. In some embodiments, the first and second third binding domains are different. In some embodiments, the first and second third binding domains specifically recognize the same epitope. In some embodiments, the first and second third binding domains specifically recognize different epitopes.
In some embodiments, the immunomodulatory molecule comprises an antigen-binding protein comprising a first antigen-binding polypeptide and a second antigen-binding polypeptide, wherein the first antigen-binding polypeptide comprises from N-terminus to C-terminus: a second antigen binding domain or portion thereof, a first hinge domain, and a first subunit of an Fc domain or portion thereof; wherein the second antigen binding polypeptide comprises, from N-terminus to C-terminus: a first antigen binding domain or portion thereof, a second hinge domain, and a second subunit of an Fc domain or portion thereof. Thus, in some embodiments, an immunomodulatory molecule is provided comprising an antigen-binding protein comprising a first antigen-binding polypeptide and a second antigen-binding polypeptide, wherein the first antigen-binding polypeptide comprises from N-terminus to C-terminus: a second antigen binding domain or portion thereof, a first hinge domain, and a first subunit of an Fc domain or portion thereof; wherein the second antigen binding polypeptide comprises, from N-terminus to C-terminus: a first antigen binding domain or portion thereof, a second hinge domain, and a second subunit of an Fc domain or portion thereof, wherein the first binding domain specifically recognizes a first target molecule, wherein the second binding domain specifically recognizes a second target molecule, wherein the first binding domain upregulates an immune response upon binding to the first target molecule (e.g., an IL-12 receptor), and wherein the second binding domain downregulates an immune response upon binding to the second target molecule (e.g., PD-1). In some embodiments, the second binding domain is an agonist Fab or agonist scFv that specifically recognizes an inhibitory checkpoint molecule. In some embodiments, the second binding domain is an agonist ligand of an inhibitory checkpoint molecule or a variant thereof. In some embodiments, the second binding domain is PD-L1 or PD-L2 or a variant thereof. In some embodiments, the first binding domain is an immunostimulatory cytokine or variant thereof. In some embodiments, the immunostimulatory cytokine or variant thereof is IL-2 or IL-12 or variant thereof.
In some embodiments, there is provided an immunomodulatory molecule comprising: i) A first antigen binding polypeptide comprising, from N-terminus to C-terminus: VH, optionally CH1, a first hinge region, and a first subunit of an Fc domain, or portion thereof; ii) a second antigen binding polypeptide comprising, from N-terminus to C-terminus: a first binding domain that specifically recognizes a first target molecule (e.g., the p35 subunit and the p40 subunit of IL-12 or a variant thereof fused in tandem), a second hinge region, and a second subunit of an Fc domain or portion thereof; and iii) a third antigen binding polypeptide comprising, from N-terminus to C-terminus: VL and optionally CL, wherein VH and VL and optionally CH1 and CL form a second binding domain that specifically recognizes a second target molecule (e.g., an agonist antigen binding fragment that specifically recognizes PD-1), wherein the first binding domain upregulates an immune response upon binding to a first target molecule (e.g., IL-12 receptor), and wherein the second binding domain downregulates an immune response upon binding to a second target molecule (e.g., PD-1). See, for example, fig. 1F.
In some embodiments, there is provided an immunomodulatory molecule comprising: i) A first antigen binding polypeptide comprising, from N-terminus to C-terminus: a first second binding domain (e.g., PD-L2 or PD-L1 or variant), a second binding domain (e.g., PD-L2 or PD-L1 or variant thereof), a first hinge region, and a first subunit of an Fc domain or portion thereof; and ii) a second antigen binding polypeptide comprising, from N-terminus to C-terminus: a first binding domain that specifically recognizes a first target molecule (e.g., the p35 subunit and the p40 subunit of IL-12 or a variant thereof fused in tandem), a second hinge region, and a second subunit of an Fc domain or portion thereof, wherein the first and/or second binding domain specifically recognizes a second target molecule (e.g., PD-1), wherein the first binding domain upregulates an immune response upon binding to the first target molecule (e.g., IL-12 receptor), and wherein the first and/or second binding domain downregulates an immune response upon binding to the second target molecule (e.g., PD-1). See, for example, fig. 1E. In some embodiments, the first and second binding domains are identical. In some embodiments, the first and second binding domains are different. In some embodiments, the first and second binding domains specifically recognize the same epitope. In some embodiments, the first and second binding domains specifically recognize different epitopes.
In some embodiments, the immunomodulatory molecule comprises an antigen binding protein comprising an antigen binding polypeptide, wherein the antigen binding polypeptide comprises, from N-terminus to C-terminus: a second binding domain or portion thereof, an optional hinge region, an Fc domain subunit or portion thereof, and a first binding domain or portion thereof. Thus, in some embodiments, an immunomodulatory molecule is provided comprising an antigen-binding protein comprising an antigen-binding polypeptide, wherein the antigen-binding polypeptide comprises from N-terminus to C-terminus: a second binding domain or portion thereof, optionally a hinge region, an Fc domain subunit or portion thereof, and a first binding domain or portion thereof, wherein the first binding domain specifically recognizes a first target molecule, wherein the second binding domain specifically recognizes a second target molecule (e.g., PD-1), wherein the first binding domain upregulates an immune response upon binding to the first target molecule (e.g., IL-12 receptor), and wherein the second binding domain downregulates an immune response upon binding to the second target molecule (e.g., PD-1). In some embodiments, the second binding domain is an agonist Fab or agonist scFv that specifically recognizes an inhibitory checkpoint molecule. In some embodiments, the second binding domain is an agonist ligand of an inhibitory checkpoint molecule or a variant thereof. In some embodiments, the second binding domain is PD-L1 or PD-L2 or a variant thereof. In some embodiments, the first binding domain is an immunostimulatory cytokine or variant thereof. In some embodiments, the immunostimulatory cytokine or variant thereof is IL-2 or IL-12 or variant thereof. In some embodiments, the immunostimulatory cytokine or variant thereof is a monomeric immunostimulatory cytokine or variant thereof. In some embodiments, the immunostimulatory cytokine or variant thereof is a dimeric immunostimulatory cytokine or variant thereof. In some embodiments, the two subunits of the dimeric immunostimulatory cytokine or variant thereof are positioned in tandem at the C-terminus of the antigen binding polypeptide. In some embodiments, the antigen binding protein comprises two antigen binding polypeptides, each antigen binding polypeptide comprising a hinge region and an Fc domain subunit or portion thereof, wherein one subunit of a dimeric immunostimulatory cytokine or variant thereof is fused to the C-terminus of the Fc domain subunit of one antigen binding polypeptide or portion thereof, and wherein the other subunit of a dimeric immunostimulatory cytokine or variant thereof is fused to the C-terminus of the Fc domain subunit of the other antigen binding polypeptide or portion thereof. In some embodiments, the antigen binding polypeptide comprising no second binding domain or portion thereof comprises, from N-terminus to C-terminus: a third binding domain or portion thereof that specifically recognizes a third target molecule, a hinge region, an Fc domain subunit or portion thereof, and a subunit of a dimeric immunostimulatory cytokine or variant thereof. In some embodiments, the antigen binding protein comprises a first antigen binding polypeptide and a second antigen binding polypeptide, wherein the first antigen binding polypeptide comprises from N-terminus to C-terminus: a second binding domain or portion thereof, a first hinge region, a first subunit of an Fc domain or portion thereof, and a first binding domain or portion thereof; wherein the second antigen binding polypeptide comprises, from N-terminus to C-terminus: specifically recognizes a third binding domain of a third target molecule or portion thereof, a second hinge region, and a second subunit of an Fc domain or portion thereof. In some embodiments, the third binding domain and the second binding domain are the same. In some embodiments, the third binding domain and the second binding domain are different. In some embodiments, the third target molecule and the second target molecule are the same. In some embodiments, the third target molecule and the second target molecule are different.
In some embodiments, there is provided an immunomodulatory molecule comprising: i) A first antigen binding polypeptide comprising, from N-terminus to C-terminus: a first second binding domain (e.g., PD-L2 or PD-L1 or variant thereof), a first hinge region, a first subunit of an Fc domain or portion thereof, and a first binding domain that specifically recognizes a first target molecule (e.g., p35 subunit and p40 subunit of IL-12 or variant thereof fused in tandem); and ii) a second antigen binding polypeptide comprising, from N-terminus to C-terminus: a second binding domain (e.g., PD-L2 or PD-L1 or variant thereof), a second hinge region, and a second subunit of an Fc domain or portion thereof, wherein the first and/or second binding domain specifically recognizes a second target molecule (e.g., PD-1), wherein the first binding domain upregulates an immune response upon binding to the first target molecule (e.g., IL-12 receptor), and wherein the first and/or second binding domain downregulates an immune response upon binding to the second target molecule (e.g., PD-1). See, for example, fig. 1I.
In some embodiments, there is provided an immunomodulatory molecule comprising: i) A first antigen binding polypeptide comprising, from N-terminus to C-terminus: a first VH, optionally a first CH1, a first hinge region, a first subunit of an Fc domain or portion thereof, and a first binding domain that specifically recognizes a first target molecule (e.g., a p35 subunit and a p40 subunit of IL-12 or variant thereof fused in tandem); ii) a second antigen binding polypeptide comprising, from N-terminus to C-terminus: a second VH, optionally a second CH1, a second hinge region, and a second subunit of an Fc domain, or portion thereof; iii) A third antigen binding polypeptide comprising, from N-terminus to C-terminus: a first VL and optionally a first CL; and iv) a fourth antigen binding polypeptide comprising, from N-terminus to C-terminus: a second VL and optionally a second CL, wherein the first VH and the first VL, and optionally the first CH1 and the first CL, form a second binding domain that specifically recognizes a second target molecule (e.g., an agonist antigen-binding fragment that specifically recognizes PD-1), wherein the second VH and the second VL, and optionally the second CH1 and the second CL, form a third binding domain that specifically recognizes a third target molecule, wherein the first binding domain upregulates an immune response upon binding to the first target molecule (e.g., an IL-12 receptor), and wherein the second binding domain downregulates an immune response upon binding to the second target molecule (e.g., PD-1). In some embodiments, the third binding domain is an agonist antigen binding fragment that specifically recognizes PD-1. See, for example, fig. 1J. In some embodiments, the third binding domain and the second binding domain are the same. In some embodiments, the third binding domain and the second binding domain are different. In some embodiments, the third target molecule and the second target molecule are the same. In some embodiments, the third target molecule and the second target molecule are different.
In some embodiments, there is provided an immunomodulatory molecule comprising: i) A first antigen binding polypeptide comprising, from N-terminus to C-terminus: VH, optionally CH1, a first hinge region, a first subunit of an Fc domain, or portion thereof, and a first binding domain that specifically recognizes a first target molecule (e.g., p35 subunit and p40 subunit of IL-12 or variant thereof fused in tandem); ii) a second antigen binding polypeptide comprising, from N-terminus to C-terminus: a first third binding domain (e.g., PD-L2 or PD-L1 or variant thereof), a second hinge region, and a second subunit of an Fc domain or portion thereof; and iii) a third antigen binding polypeptide comprising, from N-terminus to C-terminus: VL and optionally CL, wherein VH and VL, and optionally CH1 and CL, form a second binding domain that specifically recognizes a second target molecule (e.g., an agonist antigen binding fragment that specifically recognizes PD-1), wherein the first binding domain upregulates an immune response upon binding to a first target molecule (e.g., IL-12 receptor), and wherein the second binding domain downregulates an immune response upon binding to a second target molecule (e.g., PD-1). See, for example, fig. 1K. In some embodiments, the first and second third binding domains are identical. In some embodiments, the first and second third binding domains are different. In some embodiments, the first and second third binding domains specifically recognize the same epitope. In some embodiments, the first and second third binding domains specifically recognize different epitopes.
In some embodiments, there is provided an immunomodulatory molecule comprising: i) A first antigen binding polypeptide comprising, from N-terminus to C-terminus: a first second binding domain (e.g., PD-L2 or PD-L1 or variant thereof), a first hinge region, a first subunit of an Fc domain or portion thereof, and a portion of the first binding domain (e.g., p35 subunit or p40 subunit of IL-12 or variant thereof); and ii) a second antigen binding polypeptide comprising, from N-terminus to C-terminus: a second binding domain (e.g., PD-L2 or PD-L1 or variant thereof), a second hinge region, a second subunit of an Fc domain or portion thereof, and another portion of the first binding domain (e.g., the p40 subunit or the p35 subunit of IL-12 or variant thereof), wherein the first binding domain specifically recognizes a first target molecule (e.g., an IL-12 receptor), wherein the first and/or second binding domain specifically recognizes a second target molecule, wherein the first binding domain upregulates an immune response upon binding to the first target molecule (e.g., an IL-12 receptor), and wherein the first and/or second binding domain downregulates an immune response upon binding to the second target molecule (e.g., PD-1). See, for example, fig. 1P. In some embodiments, the first and second binding domains are identical. In some embodiments, the first and second binding domains are different. In some embodiments, the first and second binding domains specifically recognize the same epitope. In some embodiments, the first and second binding domains specifically recognize different epitopes.
In some embodiments, there is provided an immunomodulatory molecule comprising: i) A first antigen binding polypeptide comprising, from N-terminus to C-terminus: a first VH, optionally a first CH1, a first hinge region, a first subunit of an Fc domain or portion thereof, and a portion of a first binding domain (e.g., the p35 subunit or p40 subunit of IL-12 or variant thereof); ii) a second antigen binding polypeptide comprising, from N-terminus to C-terminus: a second VH, optionally a second CH1, a second hinge region, a second subunit of an Fc domain or portion thereof, and another portion of the first binding domain (e.g., the p40 subunit or the p35 subunit of IL-12 or variant thereof); iii) A third antigen binding polypeptide comprising, from N-terminus to C-terminus: a first VL and optionally a first CL; and iv) a fourth antigen binding polypeptide comprising, from N-terminus to C-terminus: a second VL and optionally a second CL, wherein the first VH and the first VL, and optionally the first CH1 and the first CL, form a second binding domain that specifically recognizes a second target molecule (e.g., an agonist antigen-binding fragment that specifically recognizes PD-1), and wherein the second VH and the second VL, and optionally the second CH1 and the second CL, form a third binding domain that specifically recognizes a third target molecule, wherein the first binding domain specifically recognizes the first target molecule (e.g., an IL-12 receptor), wherein the first binding domain upregulates an immune response upon binding to the first target molecule (e.g., an IL-12 receptor), and wherein the first and/or second binding domains downregulate an immune response upon binding to the second target molecule (e.g., PD-1). See, for example, fig. 1R. In some embodiments, the third binding domain is an agonist antigen binding fragment that specifically recognizes PD-1. See, for example, fig. 1Q. In some embodiments, the third binding domain and the second binding domain are the same. In some embodiments, the third binding domain and the second binding domain are different. In some embodiments, the third target molecule and the second target molecule are the same. In some embodiments, the third target molecule and the second target molecule are different.
In some embodiments, the immunomodulatory molecule comprises an antigen-binding protein comprising a first antigen-binding polypeptide and a second antigen-binding polypeptide, wherein the first antigen-binding polypeptide comprises from N-terminus to C-terminus: VH, CH1, optional hinge region, fc domain subunit or portion thereof; wherein the second antigen binding polypeptide comprises, from N-terminus to C-terminus: VL, CL, and a first binding domain or portion thereof; and wherein VH and VL, and optionally CH1 and CL, form the second binding domain. Thus, in some embodiments, there is provided an immunomodulatory molecule comprising: i) A first antigen binding polypeptide and a second antigen binding polypeptide, wherein the first antigen binding polypeptide comprises from N-terminus to C-terminus: VH, CH1, optional hinge region, fc domain subunit or portion thereof; wherein the second antigen binding polypeptide comprises, from N-terminus to C-terminus: VL, CL, and a first binding domain or portion thereof; and wherein VH and VL, and optionally CH1 and CL, form a second binding domain that specifically recognizes a second target molecule, wherein the first binding domain specifically recognizes a first target molecule (e.g., an IL-12 receptor), wherein the first binding domain upregulates an immune response upon binding to the first target molecule (e.g., an IL-12 receptor), and wherein the second binding domain downregulates an immune response upon binding to the second target molecule (e.g., PD-1). In some embodiments, the first antigen binding polypeptide comprises, from N-terminus to C-terminus: VH, CH1, first hinge region, first subunit of Fc domain, or portion thereof; wherein the antigen binding protein further comprises a third antigen binding polypeptide comprising, from N-terminus to C-terminus: specifically recognizes a third binding domain of a third target molecule or portion thereof, a second hinge region, and a second subunit of an Fc domain or portion thereof. In some embodiments, the third binding domain and the second binding domain are the same. In some embodiments, the third binding domain and the second binding domain are different. In some embodiments, the third target molecule and the second target molecule are the same. In some embodiments, the third target molecule and the second target molecule are different. In some embodiments, the immunomodulatory molecule comprises an antigen binding protein comprising four antigen binding polypeptides, wherein the first antigen binding polypeptide comprises, from N-terminus to C-terminus: a first VH, a first CH1, a first hinge region, a first subunit of an Fc domain, or a portion thereof; wherein the second antigen binding polypeptide comprises, from N-terminus to C-terminus: a first VL, a first CL, and a first binding domain or portion thereof; wherein the third antigen binding polypeptide comprises, from N-terminus to C-terminus: a second VH, a second CH1, a second hinge region, and a second subunit of an Fc domain, or portion thereof; wherein the fourth antigen binding polypeptide comprises, from N-terminus to C-terminus: a second VL and a second CL; wherein the first VH and first VL and the first CH1 and first CL form a second binding domain; and wherein the second VH and the second VL and the second CH1 and the second CL form a third binding domain that specifically recognizes a third target molecule. In some embodiments, the first binding domain is an immunostimulatory cytokine or variant thereof. In some embodiments, the immunostimulatory cytokine or variant thereof is IL-2 or IL-12 or variant thereof. In some embodiments, the immunostimulatory cytokine or variant thereof is a monomeric immunostimulatory cytokine or variant thereof. In some embodiments, the immunostimulatory cytokine or variant thereof is a dimeric immunostimulatory cytokine or variant thereof. In some embodiments, the dimeric immunostimulatory cytokine or variant thereof is located in tandem at the C-terminus of the second antigen binding polypeptide and/or the fourth antigen binding polypeptide. In some embodiments, one subunit of the dimeric immunostimulatory cytokine or variant thereof is fused to the C-terminus of the first CL of the second antigen binding polypeptide, and wherein the other subunit of the dimeric immunostimulatory cytokine or variant thereof is fused to the second CL of the fourth antigen binding polypeptide.
In some embodiments, there is provided an immunomodulatory molecule comprising: i) A first antigen binding polypeptide comprising, from N-terminus to C-terminus: a first VH, a first CH1, a first hinge region, and a first subunit of an Fc domain, or portion thereof; ii) a second antigen binding polypeptide comprising, from N-terminus to C-terminus: a first VL, a first CL, and a first binding domain (e.g., p35 subunit and p40 subunit of IL-12 or a variant thereof fused in tandem); iii) A third antigen binding polypeptide comprising, from N-terminus to C-terminus: a second VH, a second CH1, a second hinge region, and a second subunit of an Fc domain, or portion thereof; and iv) a fourth antigen binding polypeptide comprising, from N-terminus to C-terminus: a second VL, a second CL, and a second first binding domain (e.g., p35 subunit and p40 subunit of IL-12 or variant thereof fused in tandem); wherein the first VH and the first VL and the first CH1 and the first CL form a second binding domain that specifically recognizes a second target molecule (e.g., an agonist antigen-binding fragment that specifically recognizes PD-1), and wherein the second VH and the second VL and the second CH1 and the second CL form a third binding domain that specifically recognizes a third target molecule, wherein the first and/or second first binding domain specifically recognizes the first target molecule (e.g., an IL-12 receptor), wherein the first and/or second first binding domain upregulates an immune response upon binding to the first target molecule (e.g., an IL-12 receptor), and wherein the second binding domain downregulates an immune response upon binding to the second target molecule (e.g., PD-1). In some embodiments, the third binding domain is an agonist antigen binding fragment that specifically recognizes PD-1. See, for example, fig. 1S. In some embodiments, the third binding domain and the second binding domain are the same. In some embodiments, the third binding domain and the second binding domain are different. In some embodiments, the third target molecule and the second target molecule are the same. In some embodiments, the third target molecule and the second target molecule are different. In some embodiments, the first and second first binding domains are identical. In some embodiments, the first and second first binding domains are different. In some embodiments, the first and second first binding domains specifically recognize the same epitope. In some embodiments, the first and second first binding domains specifically recognize different epitopes.
In some embodiments, there is provided an immunomodulatory molecule comprising: i) A first antigen binding polypeptide comprising, from N-terminus to C-terminus: a second binding domain, a first binding domain, a first hinge region, a first subunit of an Fc domain or portion thereof, and a second first binding domain (e.g., in tandem with the p35 subunit and the p40 subunit of IL-12 or variant thereof); and ii) a second antigen binding polypeptide comprising, from N-terminus to C-terminus: a third binding domain, optionally a third first binding domain, a second hinge region, and a second subunit of an Fc domain, or a portion thereof, wherein the first binding domain specifically recognizes a first target molecule, wherein the second first binding domain specifically recognizes a second target molecule (e.g., an IL-12 receptor), wherein the second binding domain specifically recognizes a third target molecule, wherein the third binding domain specifically recognizes a fourth target molecule, optionally wherein the optional third first binding domain recognizes a fifth target molecule, wherein the first binding domain upregulates an immune response upon binding to the first target molecule, wherein the second first binding domain upregulates an immune response upon binding to the second target molecule, wherein the second binding domain downregulates an immune response upon binding to the third target molecule, wherein the third binding domain localizes the immune modulating molecule to the tumor microenvironment upon binding to the fourth target molecule, and optionally wherein the third first binding domain upregulates an immune response upon binding to the fifth target molecule. See, for example, FIGS. 11A-11B. In some embodiments, the first, second, and/or third first binding domains are different. In some embodiments, the first, second and/or third first binding domains specifically recognize the same epitope. In some embodiments, the first, second and/or third first binding domains specifically recognize different epitopes.
In some embodiments, there is provided an immunomodulatory molecule comprising: i) A first antigen binding polypeptide comprising, from N-terminus to C-terminus: a second binding domain, a first binding domain, a first hinge region, a first subunit of an Fc domain or portion thereof, and a second first binding domain subunit (e.g., a p35 subunit or a p40 subunit of IL-12 or variant thereof); and ii) a second antigen binding polypeptide comprising, from N-terminus to C-terminus: a third binding domain, optionally a third first binding domain, a second hinge region, a second subunit of an Fc domain or a portion thereof, and a second first binding domain subunit (e.g., the p35 subunit or the p40 subunit of IL-12 or a variant thereof), wherein the first binding domain specifically recognizes a first target molecule, wherein the second first binding domain specifically recognizes a second target molecule (e.g., an IL-12 receptor), wherein the second binding domain specifically recognizes a third target molecule, wherein the third binding domain specifically recognizes a fourth target molecule, optionally wherein the optional third first binding domain recognizes a fifth target molecule, wherein the first binding domain upregulates an immune response upon binding to the first target molecule, wherein the second first binding domain upregulates an immune response upon binding to the second target molecule, wherein the second binding domain downregulates an immune response upon binding to the third target molecule, wherein the third binding domain localizes an immune response to the tumor microenvironment upon binding to the fourth target molecule, and optionally wherein the third first binding domain upregulates an immune response upon binding to the fifth target molecule. See, for example, FIGS. 11C-11F. In some embodiments, the first, second, and/or third first binding domains are different. In some embodiments, the first, second and/or third first binding domains specifically recognize the same epitope. In some embodiments, the first, second and/or third first binding domains specifically recognize different epitopes.
In some embodiments, there is provided an immunomodulatory molecule comprising: i) A first antigen binding polypeptide comprising, from N-terminus to C-terminus: a second binding domain, a first hinge region, a first subunit of an Fc domain or portion thereof, and a first binding domain subunit (e.g., the p35 subunit or the p40 subunit of IL-12 or variant thereof); and ii) a second antigen binding polypeptide comprising, from N-terminus to C-terminus: a third binding domain, optionally a second first binding domain, a second hinge region, a second subunit of an Fc domain, or a portion thereof, and a first binding domain subunit (e.g., the p35 subunit or the p40 subunit of IL-12 or a variant thereof), wherein the first binding domain specifically recognizes a first target molecule (e.g., an IL-12 receptor), wherein the second binding domain specifically recognizes a second target molecule, wherein the third binding domain specifically recognizes a third target molecule, optionally wherein the optional second first binding domain recognizes a fourth target molecule, wherein the first binding domain upregulates an immune response upon binding to the first target molecule, wherein the second binding domain downregulates an immune response upon binding to the second target molecule, wherein the third binding domain localizes the immune modulating molecule to the tumor microenvironment upon binding to the third target molecule, and optionally wherein the second first binding domain upregulates an immune response upon binding to the fourth target molecule. See, for example, FIGS. 11I-11L. In some embodiments, the first and/or second first binding domains are different. In some embodiments, the first and/or second first binding domain specifically recognizes the same epitope. In some embodiments, the first and/or second first binding domain specifically recognizes a different epitope.
In some embodiments, there is provided an immunomodulatory molecule comprising: i) A first antigen binding polypeptide comprising, from N-terminus to C-terminus: a first second binding domain (e.g., PD-L2 or PD-L1 or variant thereof) located at the first hinge region and a first subunit of an Fc domain or portion thereof; and ii) a second antigen binding polypeptide comprising, from N-terminus to C-terminus: a second binding domain (e.g., PD-L2 or PD-L1 or variant thereof), a first binding domain (e.g., p35 subunit and p40 subunit of IL-12 or variant thereof connected in series), a second hinge region, and a second subunit of an Fc domain or portion thereof, wherein the first binding domain specifically recognizes a first target molecule (e.g., an IL-12 receptor), wherein the first and/or second binding domain specifically recognizes a second target molecule (e.g., PD-1), wherein the first binding domain upregulates an immune response upon binding to the first target molecule (e.g., an IL-12 receptor), and wherein the first and/or second binding domain downregulates an immune response upon binding to the second target molecule (e.g., PD-1). See, for example, fig. 12A. In some embodiments, the first and/or second binding domains are identical. In some embodiments, the first and/or second binding domains are different. In some embodiments, the first and/or second binding domains specifically recognize the same epitope. In some embodiments, the first and/or second binding domains specifically recognize different epitopes.
In some embodiments, there is provided an immunomodulatory molecule comprising: i) A first antigen binding polypeptide comprising, from N-terminus to C-terminus: a first second binding domain (e.g., CD155 or variant thereof) located at the first hinge region and a first subunit of an Fc domain or portion thereof; and ii) a second antigen binding polypeptide comprising, from N-terminus to C-terminus: a second binding domain (e.g., PD-L2 or PD-L1 or variant thereof), a first binding domain (e.g., p35 subunit and p40 subunit of IL-12 or variant thereof connected in series), a second hinge region, and a second subunit of an Fc domain or portion thereof, wherein the first binding domain specifically recognizes a first target molecule (e.g., IL-12 receptor), wherein the first second binding domain specifically recognizes a second target molecule (e.g., TIGIT), wherein the second binding domain specifically recognizes a third target molecule (e.g., PD-1), wherein the first binding domain upregulates an immune response upon binding to the first target molecule (e.g., IL-12 receptor), and wherein the first and/or second binding domain downregulates an immune response upon binding to the second target molecule (e.g., TIGIT and/or PD-1). See, for example, fig. 12B.
In some embodiments, there is provided an immunomodulatory molecule comprising: i) A first antigen binding polypeptide comprising, from N-terminus to C-terminus: a third binding domain (e.g., sdAb), a first hinge region, and a first subunit of an Fc domain, or portion thereof; and ii) a second antigen binding polypeptide comprising, from N-terminus to C-terminus: a second binding domain (e.g., PD-L2 or PD-L1 or variant thereof), a first binding domain (e.g., p35 subunit and p40 subunit in tandem connection with IL-12 or variant thereof), a second hinge region, and a second subunit of an Fc domain or portion thereof, wherein the first binding domain upregulates an immune response upon binding to a first target molecule (e.g., IL-12 receptor), wherein the second binding domain specifically recognizes a second target molecule (e.g., PD-1), wherein the third binding domain specifically recognizes a third target molecule (e.g., TIGIT, TIM3, LAG3, CTLA4, CD16A, HER2, conjugated to-4, trop2, or CLDN18.2 or variant thereof), wherein the first binding domain upregulates an immune response upon binding to a first target molecule (e.g., IL-12 receptor), and wherein the second binding domain downregulates an immune response upon binding to a third target molecule (e.g., LAG3, CTLA4, CD16A, HER, or CLDN18.2 or variant thereof), and wherein the third binding domain specifically recognizes a third target molecule (e.g., TIGIT, LAG3, CTLA4, CTLA 2, or a variant thereof). See, for example, fig. 12C.
In some embodiments, there is provided an immunomodulatory molecule comprising: i) A first antigen binding polypeptide comprising, from N-terminus to C-terminus: a third binding domain (e.g., a Fab comprising a VH and optionally a CH 1), a first hinge region, and a first subunit of an Fc domain, or portion thereof, and ii) a second antigen-binding polypeptide comprising, from N-terminus to C-terminus: a second binding domain (e.g., PD-L2 or PD-L1 or variant thereof), a first binding domain (e.g., p35 subunit and p40 subunit in tandem connection with IL-12 or variant thereof), a second hinge region, and a second subunit of an Fc domain or portion thereof, wherein the first binding domain upregulates an immune response upon binding to a first target molecule (e.g., IL-12 receptor), wherein the second binding domain specifically recognizes a second target molecule (e.g., PD-1), wherein the third binding domain specifically recognizes a third target molecule (e.g., TIGIT, TIM3, LAG3, CTLA4, CD16A, HER2, conjugated to-4, trop2, or CLDN18.2 or variant thereof), wherein the first binding domain upregulates an immune response upon binding to the first target molecule (e.g., IL-12 receptor), and wherein the second binding domain downregulates an immune response upon binding to the second target molecule (e.g., PD-1), and wherein the third binding domain specifically recognizes a third target molecule (e.g., TIGIT, LAG3, CTLA4, CTLA 3, CD16A, HER2, trop 4, trop2, or CLDN 18.2) and a variant thereof. See, for example, fig. 12D.
In some embodiments, there is provided an immunomodulatory molecule comprising: i) A first antigen binding polypeptide comprising, from N-terminus to C-terminus: a first second binding domain (e.g., PD-L2 or PD-L1 or variant thereof), a first subunit of an Fc domain or portion thereof, and a first binding domain (e.g., p35 subunit and p40 subunit of IL-12 or variant thereof connected in series) at a first hinge region; and ii) a second antigen binding polypeptide comprising, from N-terminus to C-terminus: a second binding domain (e.g., PD-L2 or PD-L1 or variant thereof), a second hinge region, and a second subunit of an Fc domain or portion thereof, wherein the first binding domain specifically recognizes a first target molecule (e.g., an IL-12 receptor), wherein the first and second binding domains specifically recognize a second target molecule (e.g., PD-1), wherein the first binding domain upregulates an immune response upon binding to the first target molecule (e.g., an IL-12 receptor), and wherein the first and/or second binding domain downregulates an immune response upon binding to the second target molecule (e.g., PD-1). See, for example, fig. 13A. In some embodiments, the first and/or second binding domains are identical. In some embodiments, the first and/or second binding domains are different. In some embodiments, the first and/or second binding domains specifically recognize the same epitope. In some embodiments, the first and/or second binding domains specifically recognize different epitopes.
In some embodiments, there is provided an immunomodulatory molecule comprising: i) A first antigen binding polypeptide comprising, from N-terminus to C-terminus: a first second binding domain (e.g., CD155 or variant thereof) located at the first hinge region and a first subunit of an Fc domain or portion thereof; and ii) a second antigen binding polypeptide comprising, from N-terminus to C-terminus: a second binding domain (e.g., PD-L2 or PD-L1 or variant thereof), a second hinge region, a second subunit of an Fc domain or portion thereof, and a first binding domain (e.g., p35 subunit and p40 subunit of IL-12 or variant thereof connected in series), wherein the first binding domain specifically recognizes a first target molecule (e.g., IL-12 receptor), wherein the first second binding domain specifically recognizes a second target molecule (e.g., TIGIT), wherein the second binding domain specifically recognizes a third target molecule (e.g., PD-1), wherein the first binding domain upregulates an immune response upon binding to the first target molecule (e.g., IL-12 receptor), and wherein the first and/or second binding domain downregulates an immune response upon binding to the second target molecule (e.g., TIGIT and/or PD-1). See, for example, fig. 13B.
In some embodiments, there is provided an immunomodulatory molecule comprising: i) A first antigen binding polypeptide comprising, from N-terminus to C-terminus: a second binding domain (e.g., PD-L2 or PD-L1 or variant thereof) located at the first hinge region, a first subunit of an Fc domain or portion thereof, and a first binding domain (e.g., p35 subunit and p40 subunit of IL-12 or variant thereof connected in series); and ii) a second antigen binding polypeptide comprising, from N-terminus to C-terminus: a third binding domain (e.g., sdAb), a second hinge region, and a second subunit of an Fc domain, or a portion thereof, wherein the first binding domain upregulates an immune response upon binding to a first target molecule (e.g., an IL-12 receptor), wherein the second binding domain specifically recognizes a second target molecule (e.g., PD-1), wherein the third binding domain specifically recognizes a third target molecule (e.g., TIGIT, TIM3, LAG3, CTLA4, CD16A, HER2, binder-4, trop2, or CLDN18.2, or a variant thereof), wherein the first binding domain upregulates an immune response upon binding to the first target molecule (e.g., an IL-12 receptor), and wherein the second binding domain downregulates an immune response upon binding to the second target molecule (e.g., PD-1), and wherein the third binding domain modulates an immune response upon binding to the third target molecule (e.g., TIGIT, LAG3, CTLA4, CD16A, HER, nep-4, trop2, or CLDN 18.2). See, for example, fig. 13C.
In some embodiments, there is provided an immunomodulatory molecule comprising: i) A first antigen binding polypeptide comprising, from N-terminus to C-terminus: a second binding domain (e.g., PD-L2 or PD-L1 or variant thereof) located at the first hinge region, a first subunit of an Fc domain or portion thereof, and a first binding domain (e.g., p35 subunit and p40 subunit of IL-12 or variant thereof connected in series); and ii) a second antigen binding polypeptide comprising, from N-terminus to C-terminus: a third binding domain (e.g., a Fab comprising VH and optionally CH 1), a second hinge region, and a second subunit of an Fc domain, or a portion thereof, wherein the first binding domain upregulates an immune response upon binding to the first target molecule (e.g., an IL-12 receptor), wherein the second binding domain specifically recognizes the second target molecule (e.g., PD-1), wherein the third binding domain specifically recognizes the third target molecule (e.g., TIGIT, TIM3, LAG3, CTLA4, CD16A, HER2, conjugated-4, trop2, or CLDN18.2, or a variant thereof), wherein the first binding domain upregulates an immune response upon binding to the first target molecule (e.g., an IL-12 receptor), wherein the second binding domain downregulates an immune response upon binding to the second target molecule (e.g., a PD-1), and wherein the third binding domain specifically recognizes the third target molecule (e.g., TIGIT, LAG3, CTLA4, CD16A, HER2, conjugated-4, op2, or CLDN 18.2) modulates an immune response to a micro-tumor environment upon binding to the third target molecule (e.g., TIGIT, LAG3, LAG 4, CTLA4, op2, or CLDN 18.2). See, for example, fig. 13D.
In some embodiments, there is provided an immunomodulatory molecule comprising: i) A first antigen binding polypeptide comprising, from N-terminus to C-terminus: a first second binding domain (e.g., PD-L2 or PD-L1 or variant thereof), a first binding domain (e.g., IL-2 or variant thereof), a first hinge region, a first subunit of an Fc domain, or a portion thereof; and ii) a second antigen binding polypeptide comprising, from N-terminus to C-terminus: a second binding domain (e.g., PD-L2 or PD-L1 or variant thereof), a second first binding domain (e.g., p35 subunit and p40 subunit of IL-12 or variant thereof connected in series), a second hinge region, and a second subunit of an Fc domain or portion thereof, wherein the first binding domain specifically recognizes a first target molecule (e.g., IL-2 receptor), wherein the second first binding domain specifically recognizes a second target molecule (e.g., IL-2 receptor), wherein the first and second binding domains specifically recognize a third target molecule (e.g., PD-1), wherein the first binding domain upregulates an immune response upon binding to the first target molecule (e.g., IL-2 receptor), wherein the second first binding domain upregulates an immune response upon binding to the second target molecule (e.g., IL-12 receptor), and wherein the first and/or second binding domain downregulates an immune response upon binding to the third target molecule (e.g., PD-1). See, for example, fig. 14A. In some embodiments, the first and/or second first binding domains are identical. In some embodiments, the first and/or second first binding domains are different. In some embodiments, the first and/or second first binding domain specifically recognizes the same epitope. In some embodiments, the first and/or second first binding domain specifically recognizes a different epitope. In some embodiments, the first and/or second binding domains are identical. In some embodiments, the first and/or second binding domains are different. In some embodiments, the first and/or second binding domains specifically recognize the same epitope. In some embodiments, the first and/or second binding domains specifically recognize different epitopes.
In some embodiments, there is provided an immunomodulatory molecule comprising: i) A first antigen binding polypeptide comprising, from N-terminus to C-terminus: a first second binding domain (e.g., CD155 or variant thereof), a first binding domain (e.g., IL-2 or variant thereof), a first hinge region, a first subunit of an Fc domain, or a portion thereof; and ii) a second antigen binding polypeptide comprising, from N-terminus to C-terminus: a second binding domain (e.g., PD-L2 or PD-L1 or variant thereof), a second first binding domain (e.g., in tandem with the p35 subunit and the p40 subunit of IL-12 or variant thereof), a second hinge region, and a second subunit of an Fc domain or portion thereof, wherein the first binding domain specifically recognizes a first target molecule (e.g., IL-2 receptor), wherein the second first binding domain specifically recognizes a second target molecule (e.g., IL-12 receptor), wherein the first second binding domain specifically recognizes a third target molecule (e.g., TIGIT), wherein the second binding domain specifically recognizes a fourth target molecule (e.g., PD-1), wherein the first binding domain upregulates an immune response upon binding to the first target molecule (e.g., IL-2 receptor), wherein the first second binding domain upregulates an immune response upon binding to the second target molecule (e.g., IL-12 receptor), wherein the first second binding domain downregulates an immune response upon binding to the third target molecule (e.g., TIGIT), and wherein the second binding to the fourth target molecule (e.g., TIGIT) downregulates an immune response upon binding to the second target molecule (e.g., PD-1). See, for example, fig. 14B.
In some embodiments, there is provided an immunomodulatory molecule comprising: i) A first antigen binding polypeptide comprising, from N-terminus to C-terminus: a third binding domain (e.g., sdAb), a first binding domain (e.g., IL-2 or variant thereof), a first hinge region, a first subunit of an Fc domain, or a portion thereof; and ii) a second antigen binding polypeptide comprising, from N-terminus to C-terminus: a second binding domain (e.g., PD-L2 or PD-L1 or variant thereof), a second first binding domain (e.g., p35 subunit and p40 subunit of IL-12 or variant thereof in tandem), a second hinge region, and a second subunit of an Fc domain, or a portion thereof, wherein the first binding domain specifically recognizes a first target molecule (e.g., IL-2 receptor), wherein the second first binding domain specifically recognizes a second target molecule (e.g., IL-12 receptor), wherein the second binding domain specifically recognizes a third target molecule (e.g., PD-1), wherein the third binding domain specifically recognizes a fourth target molecule (e.g., TIGIT, TIM3, LAG3, CTLA4, CD16A, HER2, binding agent-4, trop2 or CLDN 18.2), wherein the first binding domain upregulates an immune response upon binding to the first target molecule (e.g., IL-2 receptor), wherein the second binding domain specifically recognizes a second target molecule (e.g., IL-12 receptor), wherein the second binding domain specifically recognizes a third target molecule (e.g., PD-1), wherein the third binding domain specifically recognizes a fourth target molecule (e.g., TIGIT, tig 3, LAG 4, CTLA 2, or clp 4) and wherein the third binding domain binds to the fourth target molecule (tig 3, tig 4, trop2, or clp 2.2) at a binding to the environment. See, for example, fig. 14C.
In some embodiments, there is provided an immunomodulatory molecule comprising: i) A first antigen binding polypeptide comprising, from N-terminus to C-terminus: a third binding domain (e.g., a Fab comprising VH and optionally CH 1), a first binding domain (e.g., IL-2 or variant thereof), a first hinge region, a first subunit of an Fc domain, or a portion thereof; and ii) a second antigen binding polypeptide comprising, from N-terminus to C-terminus: a second binding domain (e.g., PD-L2 or PD-L1 or variant thereof), a second first binding domain (e.g., p35 subunit and p40 subunit of IL-12 or variant thereof in tandem), a second hinge region, and a second subunit of an Fc domain, or a portion thereof, wherein the first binding domain specifically recognizes a first target molecule (e.g., IL-2 receptor), wherein the second first binding domain specifically recognizes a second target molecule (e.g., IL-12 receptor), wherein the second binding domain specifically recognizes a third target molecule (e.g., PD-1), wherein the third binding domain specifically recognizes a fourth target molecule (e.g., TIGIT, TIM3, LAG3, CTLA4, CD16A, HER2, binding element-4, trop2, or CLDN 18.2), wherein the first binding domain upregulates an immune response upon binding to the first target molecule (e.g., IL-2 receptor), wherein the second first binding domain upregulates an immune response upon binding to the second target molecule (e.g., IL-12 receptor), wherein the second binding domain specifically binds to a third target molecule (e.g., PD-1), wherein the third binding domain specifically binds to tig 3, LAG3, CTLA4, or clp 4, trop2, or CLDN18.2 is located upon binding to the third target molecule (e.g., tig 3, tig 4, tig 3, or binding to the fourth target molecule). See, for example, fig. 14D.
In some embodiments, there is provided an immunomodulatory molecule comprising: i) A first antigen binding polypeptide comprising, from N-terminus to C-terminus: a first second binding domain (e.g., PD-L2 or PD-L1 or variant thereof), a first subunit of an Fc domain or portion thereof, and a first binding domain (e.g., p35 subunit and p40 subunit in tandem connection of IL-12 or variant thereof) at a first hinge region; and ii) a second antigen binding polypeptide comprising, from N-terminus to C-terminus: a second binding domain (e.g., PD-L2 or PD-L1 or variant thereof), a second first binding domain (e.g., IL-2 or variant thereof), a second hinge region, and a second subunit of an Fc domain or portion thereof, wherein the first binding domain specifically recognizes a first target molecule (e.g., IL-12 receptor), wherein the second first and second binding domains specifically recognize a second target molecule (e.g., IL-2 receptor), wherein the first binding domain specifically recognizes a third target molecule (e.g., PD-1), wherein the first binding domain upregulates an immune response upon binding to the first target molecule (e.g., IL-12 receptor), wherein the second first binding domain upregulates an immune response upon binding to the second target molecule (e.g., IL-2 receptor), and wherein the first and/or second binding domain downregulates an immune response upon binding to the third target molecule (e.g., PD-1). See, e.g., fig. 15A. In some embodiments, the first and/or second first binding domains are identical. In some embodiments, the first and/or second first binding domains are different. In some embodiments, the first and/or second first binding domain specifically recognizes the same epitope. In some embodiments, the first and/or second first binding domain specifically recognizes a different epitope. In some embodiments, the first and/or second binding domains are identical. In some embodiments, the first and/or second binding domains are different. In some embodiments, the first and/or second binding domains specifically recognize the same epitope. In some embodiments, the first and/or second binding domains specifically recognize different epitopes.
In some embodiments, there is provided an immunomodulatory molecule comprising: i) A first antigen binding polypeptide comprising, from N-terminus to C-terminus: a first second binding domain (e.g., PD-L2 or PD-L1 or variant thereof) located at the first hinge region, a first subunit of an Fc domain or portion thereof, and a first binding domain (e.g., p35 subunit and p40 subunit in tandem connection with IL-12 or variant thereof); and ii) a second antigen binding polypeptide comprising, from N-terminus to C-terminus: a second binding domain (e.g., CD155 or variant thereof), a second first binding domain (e.g., IL-2 or variant thereof), a second hinge region, and a second subunit of an Fc domain or portion thereof, wherein the first binding domain specifically recognizes a first target molecule (e.g., IL-12 receptor), wherein the second first binding domain specifically recognizes a second target molecule (e.g., IL-2 receptor), wherein the first second binding domain specifically recognizes a third target molecule (e.g., PD-1), wherein the second binding domain recognizes a fourth target molecule (e.g., TIGIT), wherein the first binding domain upregulates an immune response upon binding to the first target molecule (e.g., IL-12 receptor), wherein the second first binding domain upregulates an immune response upon binding to the second target molecule (e.g., IL-2 receptor), wherein the first second binding domain downregulates an immune response upon binding to the third target molecule (e.g., PD-1), and wherein the second binding domain downregulates an immune response upon binding to the third target molecule (e.g., TIGIT). See, for example, fig. 15B.
In some embodiments, there is provided an immunomodulatory molecule comprising: i) A first antigen binding polypeptide comprising, from N-terminus to C-terminus: a first second binding domain (e.g., PD-L2 or PD-L1 or variant thereof) located at the first hinge region, a first subunit of an Fc domain or portion thereof, and a first binding domain (e.g., p35 subunit and p40 subunit in tandem connection with IL-12 or variant thereof); and ii) a second antigen binding polypeptide comprising, from N-terminus to C-terminus: a third binding domain (e.g., sdAb), a second first binding domain (e.g., IL-2 or variant thereof), a second hinge region, and a second subunit of an Fc domain or portion thereof, wherein the first binding domain specifically recognizes a first target molecule (e.g., IL-12 receptor), wherein the second first binding domain specifically recognizes a second target molecule (e.g., IL-2 receptor), wherein the second binding domain specifically recognizes a third target molecule (e.g., PD-1), wherein the third binding domain specifically recognizes a fourth target molecule (e.g., TIGIT, TIM3, LAG3, CTLA4, CD16A, HER2, conjugated to 4, trop2, or CLDN 18.2), wherein the first binding domain upregulates an immune response upon binding to the first target molecule (e.g., IL-12 receptor), wherein the second first binding domain upregulates an immune response upon binding to the second target molecule (e.g., IL-2 receptor), wherein the second binding domain specifically recognizes a third target molecule (e.g., PD-1), wherein the third binding domain specifically recognizes a fourth target molecule (e.g., TIGIT, TIM3, LAG3, CTLA4, CD16A, HER2, conjugated to a four target molecule (e.g., tig 3, trop2, trop 3, trop2, or CLDN 18.2), and wherein the third binding domain binds to the tig 3, trop2, 3.374. See, for example, fig. 15C.
In some embodiments, there is provided an immunomodulatory molecule comprising: i) A first antigen binding polypeptide comprising, from N-terminus to C-terminus: a first second binding domain (e.g., PD-L2 or PD-L1 or variant thereof), a first subunit of an Fc domain or portion thereof, and a first binding domain (e.g., p35 subunit and p40 subunit in tandem connection of IL-12 or variant thereof) at a first hinge region; and ii) a second antigen binding polypeptide comprising, from N-terminus to C-terminus: a third binding domain (e.g., a Fab comprising VH and optionally CH 1), a second first binding domain (e.g., IL-2 or variant thereof), a second hinge region, and a second subunit of an Fc domain or portion thereof, wherein the first binding domain specifically recognizes a first target molecule (e.g., IL-12 receptor), wherein the second first binding domain specifically recognizes a second target molecule (e.g., IL-2 receptor), wherein the second binding domain specifically recognizes a third target molecule (e.g., PD-1), wherein the third binding domain specifically recognizes a fourth target molecule (e.g., TIGIT, CTLA 3, LAG3, CTLA4, CD16A, HER2, binder-4, trop2, or CLDN 18.2), wherein the first binding domain upregulates an immune response upon binding to the first target molecule (e.g., IL-12 receptor), wherein the second first binding domain upregulates an immune response upon binding to the second target molecule (e.g., IL-2 receptor), wherein the second binding domain specifically recognizes a third target molecule (e.g., PD-1), wherein the third binding domain specifically recognizes a fourth target molecule (e.g., TIGIT, CTLA 3, LAG 4, CD16A, HER2, binder 4, trop2, or CLDN 18.2), wherein the first binding domain binds to the fourth target molecule and binds to the tumor 3, trop 2. See, for example, fig. 15D.
In some embodiments, an immunomodulatory molecule as described in any of figures 1A-1W and 11A-15D, examples, and sequence listing herein is provided.
Binding domains that specifically recognize target molecules
The immunomodulatory molecules described herein comprise a first binding domain that specifically recognizes a first target molecule and a second binding domain that specifically recognizes a second target molecule, wherein the first binding domain upregulates an immune response upon binding to the first target molecule, and wherein the second binding domain downregulates an immune response upon binding to the second target molecule.
In some embodiments, the first binding domain upregulates the immune response upon binding to the first target molecule by one or more activities ("upregulation activities") selected from the group consisting of: up-regulating the release of immunostimulatory cytokines, down-regulating the release of immunosuppressive cytokines, up-regulating immune cell proliferation, up-regulating immune cell differentiation, up-regulating immune cell activation, up-regulating cytotoxicity against tumor cells, and up-regulating elimination of infectious pathogens.
In some embodiments, the second binding domain down-regulates the immune response upon binding to the second target molecule by one or more activities ("down-regulating activities") selected from the group consisting of: down-regulating the release of immunostimulatory cytokines, up-regulating the release of immunosuppressive cytokines, down-regulating immune cell proliferation, down-regulating immune cell differentiation, down-regulating immune cell activation, down-regulating cytotoxicity against tumor cells, and down-regulating elimination of infectious pathogens.
In some embodiments, the first binding domain modulates (e.g., modulates at least about any one of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more) the immune response by one or more activities independently selected from the group consisting of: cytokine release, immune cell proliferation, immune cell differentiation, immune cell activation, cytotoxicity to tumor cells, and up-regulation elimination of infectious pathogens. For example, in some embodiments, the first binding domain, upon binding to the first target molecule, upregulates (e.g., upregulates (or downregulates in the case of release of immunosuppressive cytokines) at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more) the immune response by an activity selected from one or more of: up-regulating the release of immunostimulatory cytokines, down-regulating the release of immunosuppressive cytokines, up-regulating immune cell proliferation, up-regulating immune cell differentiation, up-regulating immune cell activation, up-regulating cytotoxicity to tumor cells, and up-regulating elimination of infectious pathogens. In some embodiments, the second binding domain down-regulates (e.g., down-regulates (or up-regulates in the case of release of immunosuppressive cytokines) at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% of the immune response) upon binding to the second target molecule by one or more activities selected from the group consisting of: down-regulating the release of immunostimulatory cytokines, up-regulating the release of immunosuppressive cytokines, down-regulating immune cell proliferation, down-regulating immune cell differentiation, down-regulating immune cell activation, down-regulating cytotoxicity against tumor cells, and down-regulating elimination of infectious pathogens. In some embodiments, the immunostimulatory cytokine is selected from the group consisting of: IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-12, IL-15, IL-17, IL-18, IL-21, IL-22, IL-23, IL-27, IFN-alpha, IFN-beta, IFN-gamma, TNF-alpha, erythropoietin, thrombopoietin, G-CSF, M-CSF, SCF, and GM-CSF. In some embodiments, the immunosuppressive cytokine is selected from the group consisting of: IL-1Ra, IL-4, IL-5, IL-6, IL-10, IL-11, IL-13, IL-27, IL-33, IL-35, IL-37, IL-39, IFN-alpha, LIF and TGF-beta.
In some embodiments, the first target molecule and/or the second target molecule is a stimulatory checkpoint molecule. In some embodiments, the stimulatory checkpoint molecule is selected from the group consisting of: CD27, CD28, CD40, CD122, CD137, OX40, GITR and ICOS. In some embodiments, the first binding domain is an agonist antibody or antigen binding fragment thereof. In some embodiments, the agonist ligand is selected from the group consisting of: CD27L (TNFSF 7, CD 70), CD40L (CD 154), CD80, CD86, CD137L, OX L (CD 252), GITRL and ICOSLG (CD 275). In some embodiments, the first binding domain is a variant of an agonist ligand, and wherein the variant of an agonist ligand has increased (e.g., by at least about any one of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more) activity (e.g., binding affinity and/or biological activity) on the first target molecule as compared to the agonist ligand. In some embodiments, the first binding domain is a variant of an agonist ligand, and wherein the variant of an agonist ligand has reduced (e.g., by at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%) activity (e.g., binding affinity and/or biological activity) on the first target molecule as compared to the agonist ligand. In some embodiments, the second binding domain is an antagonist antibody or antigen-binding fragment thereof (e.g., VH, VHH, scFv, fab, full-length antibody). In some embodiments, the second binding domain is an antagonist ligand or variant thereof. In some embodiments, the second binding domain is a variant of an antagonist ligand, and wherein the variant of the antagonist ligand has increased (e.g., by at least about any one of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more) activity (e.g., binding affinity and/or biological activity) on the second target molecule as compared to the antagonist ligand. In some embodiments, the second binding domain is a variant of an antagonist ligand, and wherein the variant of an antagonist ligand has reduced (e.g., by at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%) activity (e.g., binding affinity and/or biological activity) on the second target molecule as compared to the antagonist ligand.
In some embodiments, the first target molecule and/or the second target molecule is a receptor for an immunostimulatory cytokine. In some embodiments, the immunostimulatory cytokine is selected from the group consisting of: IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-12, IL-15, IL-17, IL-18, IL-21, IL-22, IL-23, IL-27, IFN-alpha, IFN-beta, IFN-gamma, TNF-alpha, erythropoietin, thrombopoietin, G-CSF, M-CSF, SCF, and GM-CSF. In some embodiments, the first binding domain is an immunostimulatory cytokine or variant thereof. In some embodiments, the first binding domain is a variant of an immunostimulatory cytokine, and wherein the variant of an immunostimulatory cytokine has an increased (e.g., an increase of any of at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more) or decreased (e.g., a decrease of any of at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100%) activity (e.g., binding affinity and/or biological activity) to the first target molecule as compared to the immunostimulatory cytokine. In some embodiments, the first binding domain is IL-2 or a variant thereof. In some embodiments, the first binding domain is an IL-2 variant that has reduced (e.g., by at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%) activity (e.g., binding affinity and/or biological activity) for an IL-2 receptor as compared to wild-type IL-2. In some embodiments, the first binding domain is IL-12 or a variant thereof. In some embodiments, the first binding domain is an IL-12 variant that has reduced (e.g., by at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%) activity (e.g., binding affinity and/or biological activity) to an IL-12 receptor as compared to wild-type IL-12. In some embodiments, the first binding domain is an agonist antibody or antigen-binding fragment thereof (e.g., VH, VHH, scFv, fab, full-length antibody, such as an agonist of IL-12 receptor signaling). In some embodiments, the second binding domain is an antagonist antibody or antigen-binding fragment thereof (e.g., VH, VHH, scFv, fab, full-length antibody). In some embodiments, the second binding domain is an antagonist ligand or variant thereof (e.g., blocks or reduces IL-12 receptor signaling). In some embodiments, the second binding domain is a variant of an antagonist ligand, and wherein the variant of an antagonist ligand has an increased (e.g., by at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more) or decreased (e.g., by at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100%) activity (e.g., binding affinity and/or biological activity) relative to the second target molecule.
IL-2 receptor, interleukin-2 receptor (IL-2R), is a type of receptorHeterotrimeric proteins are expressed on the surface of certain immune cells, such as lymphocytes. IL-2R has a chain consisting of an alpha chain (IL-2Ralpha, CD25, tac antigen), a beta chain (IL-2Rbeta, CD 122) and a gamma chain (IL-2Rgamma, gamma) c Normal gamma chain or CD 132). IL-2Rα binds IL-2 with low affinity, and the complex of IL-2Rβ and IL-2Rγ binds IL-2 with moderate affinity, acting primarily on memory T cells and NK cells. All complexes of alpha, beta and gamma chains bind IL-2 with high affinity to activated T cells and regulatory T cells (tregs). CD25 (IL-2 ra) plays a key role in the development and maintenance of tregs and can play a role in the expression of CD62L of tregs, CD62L being necessary for tregs to enter the lymph nodes (Malek and Bayer, 2004). CD25 is a marker of activated T cells and tregs. Experimental data indicate that the immunosuppressive ability of the antagonist against CD25 significantly delays rejection of heart allografts in mice (Kirkman et al, 1985) and kidney allografts in non-human primates (Reed et al, 1989). Exemplary antagonist anti-CD 25 antibodies include, but are not limited to, basiliximab (e.g., ) Daclizumab (e.g.)>)。
In some embodiments, the first target molecule and/or the second target molecule is an activated immune cell surface receptor. In some embodiments, the activated immune cell surface receptor is selected from the group consisting of: CD2, CD3, CD4, CD8, CD16, CD56, CD96, CD161, CD226, NKG2C, NKG2D, NKG2E, NKG2F, NKG2H, NKp, NKp44, NKp46, CD11c, CD11b, CD13, CD45RO, CD33, CD123, CD62L, CD45RA, CD36, CD163 and CD206. In some embodiments, the first binding domain is an agonist antibody or antigen-binding fragment thereof (e.g., VH, VHH, scFv, fab, full-length antibody). In some embodiments, the first binding domain is an agonist ligand or variant thereof. In some embodiments, the first binding domain is a variant of an agonist ligand, and wherein the variant of an agonist ligand has an increased (e.g., by any of at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more) or decreased (e.g., by any of at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100%) activity (e.g., binding affinity and/or biological activity) to the first target molecule as compared to the agonist ligand. In some embodiments, the second binding domain is an antagonist antibody or antigen-binding fragment thereof (e.g., VH, VHH, scFv, fab, full-length antibody). In some embodiments, the second binding domain is an antagonist ligand or variant thereof. In some embodiments, the second binding domain is a variant of an antagonist ligand, and wherein the variant of an antagonist ligand has an increased (e.g., by at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more) or decreased (e.g., by at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100%) activity (e.g., binding affinity and/or biological activity) relative to the second target molecule.
In some embodiments, the first target molecule and/or the second target molecule is an inhibitory checkpoint molecule. In some embodiments, the inhibitory checkpoint molecule is selected from the group consisting of: PD-1, PD-L2, CTLA-4, LAG-3, TIM-3, HHA 2, CD47, CXCR4, CD160, CD73, BLTA, B7-H4, TIGIT, siglec7, siglec9 and VISTA. In some embodiments, the first binding domain is an antagonist ligand or variant thereof (e.g., blocks or reduces PD-1 signaling). In some embodiments, the first binding domain is an antagonist ligand of PD-1 or a variant thereof. In some embodiments, the first binding domain is a variant of an antagonist ligand, and wherein the variant of an antagonist ligand has an increased (e.g., by at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more) or decreased (e.g., by at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100%) activity (e.g., binding affinity and/or biological activity) relative to the first target molecule. In some embodiments, the first binding domain is an antagonist antibody or antigen-binding fragment thereof (e.g., VH, VHH, scFv, fab, full-length antibody). In some embodiments, the first binding domain is an antagonist anti-PD-1 antibody or antigen-binding fragment thereof. In some embodiments, the second binding domain is an agonist antibody or antigen-binding fragment thereof (e.g., VH, VHH, scFv, fab, full-length antibody). In some embodiments, the agonist antibody or antigen binding fragment thereof specifically recognizes PD-1, TIGIT, LAG-3, TIM-3 or CTLA-4. In some embodiments, the second binding domain is an agonist ligand or variant thereof. In some embodiments, the second target molecule is PD-1 and the second binding domain is PD-L1, PD-L2, or a variant thereof. In some embodiments, the second target molecule is TIGIT and the second binding domain is CD112 (PVRL 2, integrin-2), CD155 (PVR), or a variant thereof. In some embodiments, the second target molecule is LAG-3, and wherein the second binding domain is MHC II, lseclin, or a variant thereof. In some embodiments, the second target molecule is TIM-3, and wherein the second binding domain is galectin-9, caecam-1, HMGB-1, phosphatidylserine or a variant thereof. In some embodiments, the second target molecule is CTLA-4, and wherein the second binding domain is CD80, CD86, or variant thereof. In some embodiments, the second binding domain is a variant of an agonist ligand, and wherein the variant of an agonist ligand has an increased (e.g., by any of at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more) or decreased (e.g., by any of at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100%) activity (e.g., binding affinity and/or biological activity) relative to the second target molecule as compared to the agonist ligand. In some embodiments, the second binding domain is a variant of PD-L1 (or PD-L2) that has an increased (e.g., an increase of at least about any one of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more) or a decreased (e.g., a decrease of at least about any one of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100%) activity (e.g., binding affinity and/or biological activity) to PD-1 as compared to wild-type PD-L1 (or PD-L2). In some embodiments, the second binding domain comprises an extracellular domain of an agonist ligand or variant thereof.
PD-1 (programmed cell death protein 1) is part of the B7/CD28 family of costimulatory molecules, which can modulate T cell activation and tolerance, and thus antagonistic anti-PD-1 antibodies or PD-1 ligand-Fc fusion proteins can be used to overcome tolerance. PD-1 has been defined as the receptor for B7-4. B7-4 can inhibit immune cell activation after binding to an inhibitory receptor on an immune cell. The involvement of the PD-1/PD-L1 pathway results in inhibition of T cell effector function, cytokine secretion and proliferation (Turnis et al, oncomelanology 1 (7): 1172-1174, 2012). High levels of PD-1 are associated with depleted or chronically stimulated T cells. Furthermore, increased PD-1 expression is associated with decreased survival in cancer patients. Agents for down regulating the interaction of PD-1, B7-4, and B7-4 and PD-1 inhibitory signals in immune cells may enhance immune responses. Exemplary antagonist anti-PD-1 antibodies include, but are not limited to: palbociclizumab (e.g.,) Semipril Li Shan anti (cemiplimab)/(cemiplimab)>And Na Wu Liyou mab (Na Wu Liyou mab) (e.g., european Diwa,/->)。
In some embodiments, the second binding domain comprises an anti-PD-1 antibody fragment derived from nal Wu Liyou mab (antagonist). In some embodiments, the anti-PD-1 antibody fragment comprises: VH-CDR1, VH-CDR2 and VH-CDR3 of VH containing the sequence of SEQ ID No. 48, VL-CDR1, VL-CDR2 and VL-CDR3 of VL containing the sequence of SEQ ID No. 49. In some embodiments, VH-CDR3 further comprises any one of the following mutations relative to SEQ ID No. 48: d100N, D100G, D100R, N99G, N99A or N99M. In some embodiments, an anti-PD-1 antibody fragment comprising such a VH-CDR3 mutation has reduced binding affinity for PD-1 (e.g., reduced by at least about any one of 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, 100, 1000, or more fold) compared to nal Wu Liyou mab.
In some embodiments, the second binding domain is an agonist antibody or antigen-binding fragment thereof that specifically recognizes PD-1 ("anti-PD-1 agonist antibody or antigen-binding fragment thereof").
PD-L1 (programmed cell death ligand 1) is also known as cluster of differentiation 274 (CD 274) or B7 homolog 1 (B7-H1). PD-L1 acts as a ligand for PD-1 and plays an important role in suppressing the immune system during specific events (such as pregnancy, tissue allography, autoimmune diseases and other disease states such as hepatitis and cancer). Formation of the PD-1 receptor/PD-L1 ligand complex transmits an inhibitory signal, thereby reducing CD8 at lymph nodes + Proliferation of T cells. Exemplary antagonist anti-PD-L1 antibodies include, but are not limited to: atilizumab (atezolizumab) (e.g.,) Avermeab (e.g., +)>) And divaline You Shan anti (durvalumab) (e.g., IMFINZI TM )。
In some embodiments, the second binding domain is PD-L1 or a variant thereof. In some embodiments, the wild-type PD-L1 extracellular domain comprises the sequence of SEQ ID NO: 121. In some embodiments, the second binding domain is a PD-L1 variant, and the PD-L1 variant has an increased (e.g., an increase of at least about any one of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more) activity (e.g., binding affinity and/or biological activity) on PD-1 as compared to wild-type PD-L1. In some embodiments, the PD-L1 variant comprises one or more mutations at a position selected from the group consisting of: i54, Y56, E58, R113, M115, S117, and G119. In some embodiments, the PD-L1 variant comprises one or more mutations relative to wild-type PD-L1 (SEQ ID NO: 120) selected from the group consisting of: I54Q, Y56F, E58M, R113T, M115L, S a and G119K. In some embodiments, the PD-L1 variant comprises an I54Q/Y56F/E58M/R113T/M115L/S117A/G119K mutation relative to wild type PD-L1 (SEQ ID NO: 120). In some embodiments, the mutated PD-L1 extracellular domain comprises the sequence of any one of SEQ ID NOs 122-129.
PD-L2 (programmed cell death 1 ligand 2, B7-DC, CD 273) is another immune checkpoint receptor ligand for PD-1. PD-L2 plays a role in the down regulation of the adaptive immune response. Engagement of PD-L2 with PD-1 significantly inhibited T Cell Receptor (TCR) -mediated proliferation and cytokine production by T cells. At low antigen concentrations, the PD-L2-PD-1 interaction inhibits the strong B7-CD28 signal. In contrast, at high antigen concentrations, PD-L2-PD-1 interactions reduce cytokine production, but do not inhibit T cell proliferation.
In some embodiments, the second binding domain is PD-L2 or a variant thereof. In some embodiments, the second binding domain is a PD-L2 variant, and the PD-L2 variant has an increased (e.g., an increase of at least about any one of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more) activity (e.g., binding affinity and/or biological activity) on PD-1 as compared to wild-type PD-L2.
In some embodiments, the PD-L2 extracellular domain comprises the sequence of SEQ ID NO. 106. In some embodiments, the PD-L2 ectodomain or portion thereof is derived from wild-type (e.g., wild-type human) PD-L2. In some embodiments, the PD-L2 ectodomain or portion thereof comprises one or more mutations (e.g., deletions, insertions, or substitutions). In some embodiments, the PD-L2 variant comprises one or more mutations at a position selected from the group consisting of: t56, S58, and Q60 (e.g., T56V, S58V, Q L/T56V, S58V/Q60L). In some embodiments, the mutant PD-L2 ectodomain or portion thereof has an increased (e.g., an increase of about any one of 2, 3, 4, 5, 10, 50, 100-fold, greater than 100-fold) binding affinity for PD-1 as compared to the wild-type PD-L2 ectodomain or portion thereof. In some embodiments, the mutated PD-L2 extracellular domain comprises the sequence of any one of SEQ ID NOS: 107-110. In some embodiments, the mutant PD-L2 ectodomain or portion thereof has a reduced (e.g., reduced by about any one of 2, 3, 4, 5, 10, 50, 100, less than 100-fold) binding affinity to PD-1 as compared to the wild-type PD-L2 ectodomain or portion thereof.
Cytotoxic T lymphocyte-associated protein 4 (CTLA-4 or CD 152) is a homolog of CD28 and is known as an inhibitory immune checkpoint molecule that is upregulated on activated T cells. CTLA-4 also binds to B7-1 and B7-2, but with higher affinity than CD 28. The interaction between B7 and CTLA-4 inhibits T cell activation, which constitutes an important mechanism for tumor immune escape. Antagonist anti-CTLA-4 antibody therapy has shown promise in a variety of cancers such as melanoma. Exemplary antagonist anti-CTLA-4 antibodies include, but are not limited to, ipilimumab (ipilimumab) (e.g.,)。
in some embodiments, the second binding domain is CD155 (e.g., extracellular domain) or a variant thereof. In some embodiments, the extracellular domain of wild-type human CD155 comprises the sequence of SEQ ID NO: 137. CD155 can bind TIGIT and down regulate immune responses.
In some embodiments, the first target molecule and/or the second target molecule is a receptor for an immunosuppressive cytokine. In some embodiments, the immunosuppressive cytokine is selected from the group consisting of: IL-1Ra, IL-4, IL-5, IL-6, IL-10, IL-11, IL-13, IL-27, IL-33, IL-35, IFN-alpha, LIF and TGF-beta. In some embodiments, the second binding domain is an immunosuppressive cytokine or variant thereof. In some embodiments, the second binding domain is a variant of an immunosuppressive cytokine, and the variant of an immunosuppressive cytokine has an increased (e.g., an increase of any of at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more) or decreased (e.g., a decrease of any of at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100%) activity (e.g., binding affinity and/or biological activity) on the second target molecule as compared to the immunosuppressive cytokine. In some embodiments, the second binding domain is IL-10 or a variant thereof. In some embodiments, the second binding domain is TGF- β or a variant thereof. In some embodiments, the second binding domain is an agonist antibody or antigen-binding fragment thereof (e.g., VH, VHH, scFv, fab, full-length antibody). In some embodiments, the first binding domain is an antagonist antibody or antigen-binding fragment thereof (e.g., VH, VHH, scFv, fab, full-length antibody). In some embodiments, the first binding domain is an antagonist ligand or variant thereof. In some embodiments, the first binding domain is a variant of an antagonist ligand, and wherein the variant of an antagonist ligand has an increased (e.g., by at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more) or decreased (e.g., by at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100%) activity (e.g., binding affinity and/or biological activity) relative to the first target molecule.
In some embodiments, the first target molecule and/or the second target molecule is an inhibitory immune cell surface receptor. In some embodiments, the inhibitory immune cell surface receptor is selected from the group consisting of: CD5, NKG2A, NKG2B, KLRG, FCRL4, siglec2, CD72, CD244, GP49B, lair-1, pirB, PECAM-1, CD200R, ILT2 and KIR2DL. In some embodiments, the second binding domain is an agonist antibody or antigen-binding fragment thereof (e.g., VH, VHH, scFv, fab, full-length antibody). In some embodiments, the second binding domain is an agonist ligand or variant thereof. In some embodiments, the second binding domain is a variant of an agonist ligand, wherein the variant of an agonist ligand has an increased (e.g., an increase of any of at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more) or decreased (e.g., a decrease of any of at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100%) activity (e.g., binding affinity and/or biological activity) relative to the second target molecule. In some embodiments, the first binding domain is an antagonist antibody or antigen-binding fragment thereof (e.g., VH, VHH, scFv, fab, full-length antibody, such as blocking or reducing NKG2B signaling). In some embodiments, the first binding domain is an antagonist ligand or variant thereof. In some embodiments, the first binding domain is a variant of an antagonist ligand, and wherein the variant of an antagonist ligand has an increased (e.g., by at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more) or decreased (e.g., by at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100%) activity (e.g., binding affinity and/or biological activity) relative to the first target molecule.
In some embodiments, the first binding domain is IL-12 or a variant thereof, and the second binding domain is an agonist antibody or antigen-binding fragment thereof that specifically recognizes PD-1 (e.g., VH, VHH, scFv, fab, full-length antibody). Such an immunomodulatory molecule is also referred to hereinafter as an "IL-12/anti-PD-1 agonist antibody". In some embodiments, the first binding domain is IL-12 or a variant thereof, and wherein the second binding domain is PD-L1 (or an extracellular domain thereof) or a variant thereof. Such an immunoregulatory molecule is also referred to hereinafter as "IL-12/PD-L1 immunoregulatory molecule" or "IL-12/PD-L1 immunocytokine". In some embodiments, the second binding domain is a variant of PD-L1, and wherein the variant of PD-L1 has an increased (e.g., an increase of any of at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more) or decreased (e.g., a decrease of any of at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100%) activity (e.g., binding affinity and/or biological activity) on PD-1 as compared to wild-type PD-L1. In some embodiments, the first binding domain is IL-12 or a variant thereof, and wherein the second binding domain is PD-L2 (or an extracellular domain thereof) or a variant thereof. Such an immunoregulatory molecule is also referred to hereinafter as "IL-12/PD-L2 immunoregulatory molecule" or "IL-12/PD-L2 immunocytokine". In some embodiments, the second binding domain is a variant of PD-L2, and wherein the variant of PD-L2 has an increased (e.g., an increase of any of at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more) or decreased (e.g., a decrease of any of at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100%) activity (e.g., binding affinity and/or biological activity) on PD-1 as compared to wild-type PD-L2. In some embodiments, the first binding domain is an IL-12 variant, wherein the IL-12 variant has an increased (e.g., an increase of at least about any one of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more) or decreased (e.g., a decrease of at least about any one of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100%) activity (e.g., binding affinity and/or biological activity) at the IL-12 receptor as compared to wild-type IL-12.
In some embodiments, the first binding domain is IL-2 or a variant thereof, and the second binding domain is an agonist antibody or antigen-binding fragment thereof that specifically recognizes PD-1 (e.g., VH, VHH, scFv, fab, full-length antibody). Such immunomodulatory molecules are also referred to below as "IL-2/anti-PD-1 agonist antibodies". In some embodiments, the first binding domain is IL-2 or a variant thereof, and wherein the second binding domain is PD-L1 (or an extracellular domain thereof) or a variant thereof. Such an immunoregulatory molecule is also referred to hereinafter as "IL-2/PD-L1 immunoregulatory molecule" or "IL-2/PD-L1 immunocytokine". In some embodiments, the second binding domain is a variant of PD-L1, and wherein the variant of PD-L1 has an increased (e.g., an increase of any of at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more) or decreased (e.g., a decrease of any of at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100%) activity (e.g., binding affinity and/or biological activity) on PD-1 as compared to wild-type PD-L1. In some embodiments, the first binding domain is IL-2 or a variant thereof, and wherein the second binding domain is PD-L2 (or an extracellular domain thereof) or a variant thereof. Such an immunoregulatory molecule is also referred to hereinafter as "IL-2/PD-L2 immunoregulatory molecule" or "IL-2/PD-L2 immunocytokine". In some embodiments, the second binding domain is a variant of PD-L2, and wherein the variant of PD-L2 has an increased (e.g., an increase of any of at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more) or decreased (e.g., a decrease of any of at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100%) activity (e.g., binding affinity and/or biological activity) on PD-1 as compared to wild-type PD-L2. In some embodiments, the first binding domain is an IL-2 variant, wherein the IL-2 variant has an increased (e.g., an increase of at least about any one of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more) or decreased (e.g., a decrease of at least about any one of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100%) activity (e.g., binding affinity and/or biological activity) at the IL-2 receptor as compared to wild-type IL-2.
In some embodiments, the immunomodulatory molecule further comprises a third binding domain that specifically recognizes a third target molecule. In some embodiments, the third binding domain and the second binding domain are the same. In some embodiments, the third binding domain and the second binding domain are different. In some embodiments, the third target molecule and the second target molecule are the same. In some embodiments, the third target molecule and the second target molecule are different.
In some embodiments, the target molecule is a cell surface molecule (e.g., an extracellular domain of a receptor/ligand). In some embodiments, the target molecule serves as a cell surface marker on a target cell (e.g., immune cell) associated with a particular disease state. Target molecules specifically recognized by the binding domain may be involved directly or indirectly in the disease.
The binding domains described herein may be in any form known in the art, or derived from any suitable antibody or molecule. In some embodiments, the first binding domain is located at the hinge region between the second binding domain and the Fc domain subunit of the immunomodulatory molecule, or a portion thereof, and the second binding domain is located to ensure that in the absence of binding of the second target molecule to the second binding domain A reduced binding form of a binding domain (e.g., an immunostimulatory cytokine moiety) to its first target molecule (e.g., a cytokine receptor), e.g., without binding to a second target molecule, reduces the activity of the first binding domain at the hinge region (e.g., binding affinity and/or biological activity for a cytokine receptor) to no more than about 70% of the activity of the corresponding first binding domain (e.g., a cytokine or variant thereof) in the free state. For example, the binding domain may be an antigen binding fragment selected from the group consisting of: scFv, VH, VL, scFv-scFv, fv, fab, fab ', (Fab') 2, minibody, diabody, domain antibody variant (dAb), single domain antibody (sdAb) such as camelid antibody (VHH) or V NAR Fibronectin 3 domain variants, ankyrin repeat variants, and other target molecule specific binding domains derived from other protein scaffolds. In some embodiments, the antigen binding fragment is an scFv. In some embodiments, the antigen binding fragment is a Fab. In some embodiments, the antigen-binding fragment is formed from a VH from a first polypeptide chain and a VL from a second polypeptide chain. In some embodiments, the antigen binding fragment is human. In some embodiments, the antigen binding fragment is humanized. In some embodiments, the antigen binding fragment is chimeric. In some embodiments, the antigen binding fragment is derived from a monoclonal antibody of mouse, rat, monkey, or rabbit, or the like.
In some embodiments, the immunomodulatory molecule comprises two or more first binding domains (e.g., immunostimulatory cytokine moieties). In some embodiments, the immunomodulatory molecule comprises two or more second binding domains (e.g., PD-L1 or PD-L2 extracellular domain, or anti-PD-1 agonist Fab, scFv, sdAb, etc.). In some embodiments, the immunoregulatory molecule further comprises one or more third binding domains. In some embodiments, two or more first binding domains (e.g., antigen binding fragments or cytokine moieties) are connected in series via an optional linker. In some embodiments, the two or more first binding domains are on different antigen binding polypeptides. In some embodiments, two or more second binding domains (e.g., antigen binding fragments or cytokine moieties) are connected in series via an optional linker. In some embodiments, the two or more second binding domains are on different antigen binding polypeptides. In some embodiments, two or more third binding domains (e.g., antigen binding fragments or cytokine moieties) are connected in tandem via an optional linker. In some embodiments, the two or more third binding domains are on different antigen binding polypeptides. In some embodiments, two or more first binding domains are identical. In some embodiments, the two or more first binding domains are different. In some embodiments, the target molecule epitopes specifically recognized by two or more first binding domains are identical. In some embodiments, the target molecule epitopes specifically recognized by the two or more first binding domains are different. In some embodiments, two or more second binding domains are identical. In some embodiments, the two or more second binding domains are different. In some embodiments, the target molecule epitopes specifically recognized by two or more second binding domains are identical. In some embodiments, the target molecule epitopes specifically recognized by the two or more second binding domains are different. In some embodiments, two or more third binding domains are identical. In some embodiments, two or more third binding domains are different. In some embodiments, the target molecule epitopes specifically recognized by two or more third binding domains are identical. In some embodiments, the target molecule epitopes specifically recognized by the two or more third binding domains are different. For example, in some embodiments, the immunomodulatory molecule comprises, from N-terminus to C-terminus: fab 1-optional linker 1-Fab 2-optional linker 2- (optional hinge or portion thereof-first binding domain (e.g., immunostimulatory cytokine portion) -optional hinge or portion thereof) -Fc subunit. For example, CH1 or CL of Fab1 is linked to VH or VL of Fab2 via optional linker 1. In some embodiments, the immunomodulatory molecule comprises, from N-terminus to C-terminus: scFvl (or sdAb 1) -optional linker 1-scFv2 (or sdAb 2) -optional linker 2- (optional hinge or portion thereof-first binding domain (e.g., immunostimulatory cytokine portion) -optional hinge or portion thereof) -Fc subunit. In some embodiments, the immunomodulatory molecule comprises, from N-terminus to C-terminus: ligand 1 (e.g., PD-L2) -optional linker 1-ligand 2 (e.g., PD-L2) -optional linker 2- (optional hinge or portion thereof-first binding domain (e.g., immunostimulatory cytokine portion) -optional hinge or portion thereof) -Fc subunit. For other paired immunomodulatory molecule chains, the first binding domain in brackets (e.g., immunostimulatory cytokine moiety) may not be present. For example, an immunomodulatory molecule may comprise a first polypeptide chain comprising, from N-terminus to C-terminus: scFv1 (or sdAb 1) -optionally linker 1-scFv2 (or sdAb 2) -optionally linker 2-a first binding domain (e.g., an immunostimulatory cytokine moiety) -hinge or portion thereof-Fc subunit 1; and a second polypeptide chain comprising, from N-terminus to C-terminus: scFv3 (or sdAb 3) -optionally linker 3-scFv4 (or sdAb 4) -optionally linker 4-hinge or part thereof-Fc subunit 2.
The binding affinity of a binding domain (e.g., scFv, fab, VHH, ligand or receptor) and its target molecule can be determined experimentally by any suitable antibody/antigen binding assay or other protein binding assay known in the art (e.g., ligand-receptor binding), such as western blotting, ELISA, MSD electrochemiluminescence, bead-based MIA, RIA, SPR, ECL, IRMA, EIA, biacore assay, octet assay, peptide scanning, FACS, and the like. For exemplary methods, see also the "binding affinity" section below. In some embodiments, the Kd of the binding between the antibody or antigen binding fragment and its target molecule is about.ltoreq.10 -5 M、≤10 -6 M、≤10 -7 M、≤10 -8 M、≤10 -9 M、≤10 -10 M、≤10 -11 M or less than or equal to 10 -12 M.
Amino acid sequence variants of an antigen binding protein or binding domain (e.g., antigen binding fragment) can be prepared by introducing appropriate modifications into a nucleic acid sequence encoding the antigen binding protein or binding domain or by peptide synthesis. Such modifications include, for example, deletions and/or insertions and/or substitutions of residues within the amino acid sequence of the antigen binding protein or binding domain. Any combination of deletions, insertions, and substitutions may be made to arrive at the final construct, provided that the final construct has the desired characteristics, such as target molecule binding.
In some embodiments, the antigen binding protein (e.g., antibody or ligand/receptor-hinge-Fc fusion protein) or binding domain (e.g., scFv, fab, VHH, ligand or receptor) has one or more amino acid substitutions. Target sites for substitution mutagenesis include HVRs (or CDRs) and FRs of antibodies or antigen binding fragments. Conservative substitutions are shown under the heading of "preferred substitutions" in table B. More substantial variations are provided under the heading of "exemplary substitutions" in table B, and are further described below with reference to the amino acid side chain class. Amino acid substitutions can be introduced into the target binding domain and the product screened for the desired activity, e.g., retaining/improving target molecule binding, reducing immunogenicity.
TABLE B amino acid substitutions
Amino acids can be grouped according to common side chain characteristics: (1) hydrophobic: norleucine, met, ala, val, leu, ile; (2) neutral hydrophilic: cys, ser, thr, asn, gln; (3) acidic: asp, glu; (4) alkaline: his, lys, arg; (5) residues that affect chain orientation: gly, pro; (6) aromatic: trp, tyr, phe. Non-conservative substitutions may require that members of one class be replaced by another class.
One class of substitution variants involves substitution of one or more HVR residues of a parent antibody or antigen-binding fragment thereof. In general, the resulting variants selected for further investigation will have modifications (e.g., improvements) in certain biological properties (e.g., increased affinity, reduced immunogenicity) relative to the parent antibody or antigen-binding fragment thereof, and/or will have certain biological properties of the parent antibody or antigen-binding fragment thereof that are substantially preserved. Exemplary substitution variants are affinity matured antibodies that can be conveniently generated, for example, using phage display-based affinity maturation techniques, such as those described herein. Briefly, one or more HVR residues are mutated and variant antibodies are displayed on phage and screened for a particular biological activity (e.g., binding affinity).
In some embodiments, substitutions, insertions, or deletions may occur within one or more HVRs, provided that such changes do not substantially reduce the ability of the antibody or antigen binding fragment thereof to bind to an antigen. For example, conservative changes (e.g., conservative substitutions as provided herein) may be made in the HVR that do not substantially reduce binding affinity. Such alterations may be outside of the HVR "hot spot" or CDR.
Alterations (e.g., substitutions) may be made in the HVR, for example, to increase antibody affinity. Such changes may be made in HVR "hot spots", i.e. residues encoded by codons that are mutated at high frequencies during somatic maturation (see, e.g., chordhury, methods mol. Biol.207:179-196 (2008)) and/or SDR (a-CDRs), wherein the resulting variant VH or VL is tested for binding affinity. Affinity maturation by construction and reselection from secondary libraries has been described, for example, in Hoogenboom et al Methods in Molecular Biology 178:1-37 (O' Brien et al ed., human Press, totowa, N.J. (2001)). In some embodiments of affinity maturation, diversity is introduced into the variable gene selected for maturation by any of a variety of methods (e.g., error-prone PCR, strand shuffling, or oligonucleotide-directed mutagenesis). A secondary library is then created. The library is then screened to identify any antibody variants with the desired affinity. Another approach to introducing diversity involves HVR-directed approaches, in which several HVR residues (e.g., 4-6 residues at a time) are randomly assigned. HVR residues involved in antigen binding can be specifically identified, for example, using alanine scanning mutagenesis or modeling. CDR-H3 and CDR-L3 are particularly often targeted.
One method that may be used to identify residues or regions of antibodies that can be targeted for mutagenesis is referred to as "alanine scanning mutagenesis" as described in Cunningham and Wells (1989) Science, 244:1081-1085. In this method, a residue or set of target residues (e.g., charged residues such as Arg, asp, his, lys and Glu) are identified and replaced with neutral or negatively charged amino acids (e.g., alanine or polyalanine) to determine whether the interaction of the antibody with the antigen is affected. Further substitutions may be introduced at amino acid positions that exhibit functional sensitivity to the initial substitution. Alternatively, or in addition, the crystal structure of the antigen-antibody complex to recognize the point of contact between the antibody and the antigen. Such contact residues and adjacent residues may be targeted or deleted as candidates for substitution. Variants may be screened to determine whether they contain the desired characteristics.
In some embodiments, the first binding domain is an immunostimulatory cytokine moiety or variant thereof, such as any of the cytokine moieties described herein (e.g., any of SEQ ID NOS: 26-30, 41, 63-65, and 140). In some embodiments, the immunostimulatory cytokine moiety or variant thereof is selected from the group consisting of: IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-12, IL-15, IL-17, IL-18, IL-21, IL-22, IL-23, IL-27, IFN-alpha, IFN-beta, IFN-gamma, TNF-alpha G-CSF, M-CSF, SCF and GM-CSF. In some embodiments, the first binding domain is an agonist antibody directed against a T cell surface antigen, including, but not limited to: CD3 epsilon, CD3 delta or CD3 gamma; or CD2, CD4, CD8, CD27, CD28, CD40, CD134, CD137 and CD278. In some embodiments, the first binding domain is an agonist antibody directed against an NK cell surface antigen, including, but not limited to: CD16a, CD56 (NCAM), NKp46, NKp44, CD244, CD226, TIGIT, CD96, LAG3, TIM3, PD-1, KLRG1, CD161, CD94/NKG2, KIR, NKG2D and NKG 30. In some embodiments, the first binding domain is an agonist antibody directed against any one of CD27, CD28, CD137, OX40, GITR, and HVEM. In some embodiments, the first binding domain is an agonist ligand, such as CD80, CD86, or 4-1BB.
In some embodiments, the second binding domain is an agonist antibody directed against an inhibitory checkpoint molecule (e.g., PD-1, TIGIT, or CTLT-4). In some embodiments, the second binding domain is a ligand of an inhibitory checkpoint molecule, such as PD-L1, PD-L2, CD155, or a variant thereof. In some embodiments, the second binding domain comprises the sequence of any one of SEQ ID NOS: 106-110, 121-128, and 137.
In some embodiments, the third binding domain is an antibody (agonist, antagonist, or neutral) to a T cell surface antigen, including, but not limited to: CD3 epsilon, CD3 delta or CD3 gamma; or CD2, CD4, CD8, CD27, CD28, CD40, CD134, CD137 and CD278. In some embodiments, the third binding domain is an antibody (agonist, antagonist or neutral) to an NK cell surface antigen, including, but not limited to: CD16a, CD56 (NCAM), NKp46, NKp44, CD244, CD226, TIGIT, CD96, LAG3, TIM3, PD-1, KLRG1, CD161, CD94/NKG2, KIR, NKG2D and NKG 30. In some embodiments, the third binding domain is an antibody (agonist, antagonist or neutral) to a T cell depletion marker, including, but not limited to: PD-1, TIGIT, CTLA-4, LAG3 and TIM3. In some embodiments, the third binding domain is an antibody (agonist, antagonist, or neutral) to a tumor antigen, including, but not limited to: her2, her3, CEA, trop2, CLDN18.2. In some embodiments, the third binding domain is a ligand for an immune cell surface antigen (e.g., PD-1 or TIGIT as an antigen), such as PD-L1, PD-L2, CD155, or a variant thereof. In some embodiments, the third binding domain comprises the sequence of any one of SEQ ID NOS: 106-110, 121-128, and 137.
Cytokines or variants thereof
Cytokines (also interchangeably referred to as "cytokine molecules" or "cytokine proteins") are secreted proteins that regulate the cellular activity of the immune system. Examples of cytokines include interleukins, interferons, chemokines, lymphokines, tumor necrosis factors, colony stimulating factors of immune cell precursors, and the like. In some embodiments, the cytokine is a wild-type cytokine. In some embodiments, the cytokine is a naturally occurring cytokine variant. In some embodiments, the cytokine is a naturally occurring cytokine subtype. "cytokine variant" as used herein refers to any cytokine molecule that does not occur in nature, such as a cytokine-active fragment (e.g., a cytokine fragment that retains at least about 10% of the biological activity or cytokine receptor binding activity of a full-length cytokine), a mutant, or a derivative thereof. "cytokine or variant thereof" is also referred to herein interchangeably as "cytokine moiety," which may be a cytokine molecule, or a variant, subtype, active fragment, mutant or derivative thereof.
As used herein, "heterodimeric cytokine" or "cytokine heterodimer" refers to a cytokine that consists of two distinct protein subunits. Currently, the IL-12 family (including IL-12, IL-23, IL-27 and IL-35) is the only naturally occurring heterodimeric cytokine family known. However, artificial heterodimeric cytokines can be constructed. For example, soluble fragments of IL-6 and IL-6R may combine to form heterodimeric cytokines, such as CNTF and CNTF-Rα may also be (Trincheri (1994) Blood 84:4008). "homodimerized cytokine" or "cytokine homodimer" refers herein to a cytokine consisting of two identical protein subunits, such as IFN-gamma or IL-10. "monomeric cytokine" or "cytokine monomer" refers to a cytokine consisting of one unit of cytokine molecule. In some embodiments, the cytokine or variant thereof is a monomeric cytokine or variant thereof. In some embodiments, the cytokine or variant thereof is a homodimerized cytokine or variant thereof. In some embodiments, the cytokine or variant thereof is a heterodimeric cytokine or variant thereof.
In some embodiments, the cytokine moiety is a full-length cytokine molecule. In some embodiments, the cytokine moiety is a functional fragment of a cytokine molecule that is capable of producing some (e.g., at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%,90%, or 95%) or all of the biological activity and/or cytokine receptor binding activity of the full-length cytokine molecule. In some embodiments, the cytokine moiety is a precursor cytokine molecule. In some embodiments, the cytokine moiety is a mature cytokine molecule (e.g., no signal peptide). In some embodiments, the cytokine moiety is a wild-type cytokine. In some embodiments, the cytokine moiety is a naturally occurring cytokine variant. In some embodiments, the cytokine moiety is a naturally occurring cytokine subtype. In some embodiments, the cytokine moiety is a cytokine variant, such as a mutant cytokine that is capable of producing some (e.g., at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%,90%, or 95%) or all of the biological activity and/or cytokine receptor binding activity of the wild-type cytokine. In some embodiments, the cytokine variant is a modified cytokine, such as a glycosylated cytokine. The cytokines or variants thereof described herein may be cytokines isolated from a variety of sources, such as from human tissue types or other sources, or prepared by recombinant or synthetic methods. In some embodiments, the cytokine moiety is a recombinant cytokine. In some embodiments, the cytokine moiety described herein can be a cytokine derived from any organism, such as a mammal, including but not limited to: livestock animals (e.g., cattle, sheep, goats, cats, dogs, donkeys, and horses), primates (e.g., humans and non-human primates such as monkeys or chimpanzees), rabbits, and rodents (e.g., mice, rats, gerbils, and hamsters). In some embodiments, the cytokine moiety is a human cytokine, such as a recombinant human cytokine. In some embodiments, the cytokine moiety is a murine cytokine, such as a recombinant murine cytokine. In some embodiments, the cytokine moiety is a mature human cytokine. In some embodiments, the cytokine moiety comprises a signal peptide at the N-terminus of the cytokine molecule, the signal peptide being from a different molecule or from the same cytokine molecule.
The cytokine variant may be a truncated version, a post-translationally modified version, a hybrid variant, a peptidomimetic, a biologically active fragment, a deletion variant, a substitution variant, or an additional variant that retains at least some degree (e.g., at least about 10%) of the parent cytokine activity (cytokine receptor binding activity and/or biological activity). "parental cytokine" or "parental cytokine" as described herein refers to a cytokine reference sequence from which a cytokine variant is engineered, modified or derived.
When an immunomodulatory molecule of the invention is described as comprising two or more different cytokines (and optionally including additional protein moieties), this means that the immunomodulatory molecule comprises two or more different cytokine molecules (rather than two or more different cytokine subunits). For example, homodimerized cytokines (e.g., IFN- α, IFN- β, IFN- γ, IL-5, IL-8, etc.) refer herein to a single cytokine molecule. For example, an immunomodulatory molecule comprising two IL-5 monomers/subunits (either on the same polypeptide chain as a single chain fusion or on different polypeptide chains) is considered to comprise only one cytokine molecule, IL-5. Similarly, a heterodimeric cytokine such as IL-12, while it comprises different subunits, is a single cytokine. For example, an immunomodulatory molecule comprising a p35 subunit and a p40 subunit (either on the same polypeptide chain as a single chain fusion or on different polypeptide chains) is considered to contain only one cytokine molecule, i.e., IL-12. Furthermore, the heterodimeric form of a normal homodimeric cytokine, such as MCP-1/MCP-2 heterodimer, or both alleles of a normal homodimeric cytokine (e.g., zhang, J. Biol. Chem. [1994] 269:15948-24) is a single cytokine. In some embodiments, a cytokine subunit on one polypeptide chain of an immunomodulatory molecule (e.g., p35 of IL-12) can dimerize with a counterpart cytokine subunit on the same polypeptide chain within the same immunomodulatory molecule or on a different polypeptide chain (e.g., p 40). In some embodiments, the cytokine subunit of the immunoregulatory molecule (e.g., p35 of IL-12) can dimerize with a counterpart cytokine subunit of a nearby immunoregulatory molecule (e.g., p 40).
In some embodiments, the cytokine variants comprise mutations or modifications (e.g., post-translational modifications) that result in the selectivity of a first type of receptor (e.g., a trimeric receptor or a higher affinity receptor) versus a second type of receptor (e.g., a dimeric receptor or a weaker affinity receptor) for the corresponding cytokine molecule, as measured as the ratio of activation of cells expressing the first type of receptor versus activation of cells expressing the second type of receptor. For example, in some embodiments, the cytokine variants are mutant IL-2 (or post-translationally modified IL-2) that bind IL-2rβγ with greater affinity (e.g., at least about any of 2, 3, 4, 5, 6, 7, 8, 9, or 10-fold greater affinity) than IL-2rβγ, or activate more (e.g., at least about any of 2, 3, 4, 5, 6, 7, 8, 9, or 10-fold greater activation) cells that express IL-2rβγ; or vice versa. In some embodiments, the preferred mutation or alteration increases activation of the cytokine portion of immune effector cells (e.g., cd8+ T cells for the treatment of cancer) depending on the type of disease to be treated. For example, in some embodiments, the IL-2 variant has a mutation (or post-translational modification) that reduces activation of the IL-2 variant by cells expressing the IL-2Rβγ receptor relative to activation of the IL-2 variant by cells expressing the IL-2Rβγ receptor.
In some embodiments, the mutation or modification of the cytokine variant results in a differential effect (e.g., such as reduced binding or cell activation) compared to an immunomodulatory molecule without the mutation or modification of such cytokine moiety. In one aspect, differential effects are measured by proliferation response of a cell or cell line that is dependent on the growth of a cytokine (e.g., IL-2). This response to the immunoregulatory molecule is expressed as an EC50 value, which is obtained by plotting a dose response curve and determining the concentration of protein that results in half maximal response. In some embodiments, for an immunomodulatory molecule of the invention (e.g., an IL-2 variant immunomodulatory molecule), the ratio of EC50 values obtained for cells expressing a first receptor type (e.g., an IL-2rβγ receptor) to cells expressing a second receptor type (e.g., an IL-2rββγ receptor) relative to the ratio of EC50 values for a reference immunomodulatory molecule (e.g., an IL-2 wild-type immunomodulatory molecule of the same configuration) gives a measure of the differential effect of the immunomodulatory molecule. In some embodiments, the EC50 values obtained for an immunomodulatory molecule of the invention (e.g., an IL-2 variant immunomodulatory molecule) relative to EC50 values for a reference immunomodulatory molecule (e.g., an IL-2 wild-type immunomodulatory molecule of the same configuration) give a measure of the differential effects of measuring the immunomodulatory molecule.
In some embodiments, the cytokine variants include mutations of one or more amino acids of a parent cytokine molecule (e.g., mature wild-type cytokine). In one embodiment, the cytokine variant comprises amino acid substitutions at one or more amino acid positions of the cytokine. In another embodiment, the cytokine variant comprises an amino acid deletion or insertion at one or more amino acid positions of the cytokine. In some embodiments, the cytokine variant comprises a modification of one or more amino acids of the cytokine.
In some embodiments, the cytokine or variant thereof is selected from the group consisting of: IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12, IL-15, IL-17, IL-18, IL-21, IL-22, IL-23, IL-27, IL-35, IFN- α, IFN- β, IFN- γ, TNF- α, TGF- β, VEGF, erythropoietin, thrombopoietin, G-CSF, M-CSF, SCF, and GM-CSF, or natural variants or subtypes thereof. In some embodiments, the cytokine or variant thereof is an anti-inflammatory or immunosuppressive cytokine or variant thereof, such as IL-1Ra, IL-4, IL-5, IL-6, IL-10, IL-11, IL-13, IL-27, IL-33, IL-35, IL-37, IL-39, IFN- α, LIF, or TGF- β. In some embodiments, the cytokine or variant thereof is a pro-inflammatory or immunostimulatory cytokine or variant thereof, such as IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-12, IL-15, IL-17, IL-18, IL-21, IL-22, IL-23, IL-27, IFN-alpha, IFN-beta, IFN-gamma, TNF-alpha, erythropoietin, thrombopoietin, G-CSF, M-CSF, SCF, or GM-CSF, or a variant or subclass thereof. In some embodiments, the cytokine or variant thereof is selected from the group consisting of: IL-2, IL-10, IL-12, IL-23, IFN-alpha (e.g., IFN-alpha 2 or IFN-alpha 2 b), IFN-beta and IFN-gamma. In some embodiments, the immunostimulatory cytokine is IL-12 and the cytokine subunits are p35 and p40. In some embodiments, the immunostimulatory cytokine is IL-23 and the cytokine subunits are p40 and p19. In some embodiments, the cytokine is IL-27 and the cytokine subunits are Epstein-Barr (Epstein-Barr) virus-induced gene 3 (EBI 3) and IL-27p28. In some embodiments, the immunosuppressive cytokine is IL-35, and the cytokine subunits are IL-12α (p 35) and IL-27β. In some embodiments, the cytokine variant is a single chain fusion of two or more subunits from different cytokines.
IL-2
In some embodiments, the immunostimulatory cytokine or variant thereof is IL-2 or variant thereof. Interleukin 2 (IL-2), also known as T Cell Growth Factor (TCGF), is a 15.5kDa monomeric protein that plays a key role in lymphopoiesis, survival and homeostasis. It participates in the body's natural response to microbial infection and distinguishes between "self" and "non-self. IL-2 is an interleukin that belongs to a family of cytokines that includes IL-4, IL-7, IL-9, IL-15, and IL-21.IL-2 mediates its actions by binding to IL-2 receptors (IL-2R) expressed on lymphocytes. Activated CD4 + T cells and activated CD8 + T cells are the primary source of IL-2. It can expand lymphocyte populations and increase the effector functions of these cells, which makes IL-2 an attractive anticancer therapy. IL-2 has been suggested for the treatment of acute myelogenous leukemia (aML), non-Hodgkin's lymphoma (NHL), cutaneous T-cell lymphoma (CTCL), breast cancer, and bladder cancer.
The IL-2 receptor (IL-2R) is a complex consisting of three chains, α (CD 25, p 55), β (CD 122, p 75) and γ (CD 132, p 65). The gamma chain is shared by all IL-2 cytokine family members. Dimeric CD122/CD132 IL-2R (IL-2 Rβγ, kd-10) with moderate affinity for IL-2 -9 M) or high affinity trimeric CD25/CD122/CD132 IL-2R (IL-2 Rαβγ, kd.about.10) -11 M) binding may result in signal transduction, whereas binding to CD25 alone may not. The beta strand is complexed with Janus kinase 1 (JAK 1). The gamma chain is complexed with JAK 3. After IL-2 binds to IL-2R, JAK1 and JAK3 are activated and are able to add phosphate groups to the molecule, thereby initiating three intracellular signaling pathways: MAP kinase pathway, phosphoinositide 3-kinase (PI 3K) pathway and JAK-STAT pathway. Dimeric IL-2Rβγ is derived from memory CD8 + T cells, NK cells and B cells, whereas high levels of trimeric IL-2rαβγ are expressed by regulatory T cells (tregs) and activated T cells.
Aldi-interleukinsIs recombinant human IL-2, one of the first cancer immunotherapy and the first recombinant protein, approved by the FDA in 1992. Currently, aldesleukins are used to treat metastatic renal cell carcinoma (mRCC) and metastatic melanoma (mM) by intravenous infusion. The administration of aldesleukin occurs in a clinical setting due to the need for frequent intravenous infusion at multiple doses. The aldesleukins have been demonstrated to treat complete Cancer regression in about 10% of patients for metastatic melanoma and renal Cancer (klapp er et al, cancer,2008;Rosenberg,Sci Transl Med, 2012; smith et al, clin Cancer res, 2008). Approximately 70% of fully-responsive patients have cured, remaining fully resolved for more than 25 years after initial treatment (Atkins et al, J Clin oncol, 1999;Klapper et al, cancer,2008;Rosenberg,Sci Transl Med, 2012;Rosenberg et al, ann surg, 1998; smith et al, clin Cancer res, 2008). However, high doses of IL-2 can induce Vascular Leak Syndrome (VLS), tumor tolerance by activation-induced cell death (aod), and immunosuppression by activation of Tregs. Another problem with systemic IL-2 therapy is associated with serious side effects following intravenous administration, including severe cardiovascular, pulmonary edema, hepatic, gastrointestinal (GI), neurological and hematological events (Proleukin) product profile [ SmPC) ]: http:// www.medicines.org.uk/emc/media/19322/SPC). Serious side effects often limit optimal IL-2 doses, which limit the number of patients responding to successful treatment. For more general use in the future, it is desirable to address the problem of the toxicity and short half-life of IL-2.
The natural human IL-2 precursor polypeptide consists of 153 amino acid residues (amino acids 1-20 are signal peptides), while the mature polypeptide consists of 133 amino acid residues (SEQ ID NO: 25). In some embodiments, the IL-2 moiety is human mature IL-2. In some embodiments, the IL-2 moiety is a polypeptide that is substantially homologous to the amino acid sequence of wild-type human IL-2 (SEQ ID NO: 25), e.g., has at least about 85% (e.g., at least about any of 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) amino acid sequence identity to wild-type human IL-2 (SEQ ID NO: 25). In some embodiments, the IL-2 moiety is not glycosylated. In some embodiments, the IL-2 moiety is glycosylated.
In some embodiments, the IL-2 moiety is (or consists essentially of) an aldesleukin (e.g.,see, for example,https://www.drugbank.ca/drugs/DB00041). Alterleukin (des-alanyl-1, serine-125 human interleukin-2) is an antitumor (anticancer) biological response modifier approved by the FDA. Its molecular weight is about 15.3kDa, synonymous being recombinant interleukin-2 human, interleukin-2 aldesleukin, 125-L-serine-2-133-interleukin-2 (reduced human) or interleukin-2 (2-133), 125-serine. Aldi interleukin is a recombinant IL-2 that differs from natural IL-2 in the following ways: a) The aldesleukin is not glycosylated because it is produced by escherichia coli (e.coli); b) Albumin lacks the N-terminal alanine (A); c) Aldesleukin has a cysteine to serine substitution at position 125 (C125S); d) The aggregation state of aldesleukin may be different from that of native IL-2. Thus, in some embodiments, the IL-2 variant comprises a cysteine to serine substitution (C125S) at position 125 from the mature form of human IL-2.
Sauv et al (Proc Natl Acad Sci U S A1991; 88 (11): 4636-4640) found that amino acid residues K35, R38, F42 and K43 of wild-type IL-2 were found to be critical for IL-2 receptor binding (IL-2Rα, low affinity form), and that the R38A and F24A mutations retained a large number of IL-2 biological activities. R. Vazquez-Lombardi et al (Nat Commun.2017; 8:15373) found that the R38D, K E and E61R mutations in IL-2 promoted CD25 compared to wild-type IL-2 - The cytotoxic subpopulations are strongly amplified, while Treg amplification is minimal. The P65L mutation in IL-2 was found to have reduced systemic toxicity and higher antitumor efficacy compared to wild-type IL-2 (Chen et al, cell Death Dis.2018;9 (10): 989).
In some embodiments, the IL-2 variant comprises one or more mutations at a position selected from the group consisting of: l18, Q22, F24, K35, R38, F42, K43, E61, P65, Q126, and S130. In some embodiments, the IL-2 variant comprises one or more mutations relative to wild-type IL-2 (SEQ ID NO: 25) selected from the group consisting of: L18R, Q22E, F A, R38D, K E, E61R, P65L, Q T and S130R. In some embodiments, the IL-2 variant comprises an R38D/K43E/E61R mutation relative to wild-type IL-2 (SEQ ID NO: 25). In some embodiments, the IL-2 variant comprises the sequence of SEQ ID NO. 26. In some embodiments, the IL-2 variant comprises a L18R/Q22E/R38D/K43E/E61R mutation relative to SEQ ID NO. 25. In some embodiments, the IL-2 variant comprises the sequence of SEQ ID NO. 27. In some embodiments, the IL-2 variant comprises an R38D/K43E/E61R/Q126T mutation relative to SEQ ID NO. 25. In some embodiments, the IL-2 variant comprises the sequence of SEQ ID NO. 28. In some embodiments, the IL-2 variant comprises a L18R/Q22E/R38D/K43E/E61R/Q126T mutation relative to SEQ ID NO. 25. In some embodiments, the IL-2 variant comprises the sequence of SEQ ID NO. 29. In some embodiments, the IL-2 variant comprises a L18R/Q22E/R38D/K43E/E61R/Q126T/S130R mutation relative to SEQ ID NO. 25. In some embodiments, the IL-2 variant comprises the sequence of SEQ ID NO. 30.
IFN-α
In some embodiments, the immunostimulatory cytokine or variant thereof is IFN- α or a variant thereof, such as IFN- α2 or a variant thereof, or IFN- α2b or a variant thereof. Human type I Interferon (IFN) is a broad class of IFN that helps regulate the activity of the immune system. They bind to a specific cell surface receptor complex called IFN-alpha receptor (iFNAR) consisting of IFNAR1 and IFNAR2 chains. The type I IFN of mammals contains IFN-alpha, IFN-beta, IFN-kappa, IFN-delta, IFN-epsilon, IFN-tau, IFN-omega and IFN-zeta (also known as limiting hormone (limitin)).
IFN- α proteins are produced primarily by plasmacytoid dendritic cells (pDC) and are primarily involved in innate immunity against viral infections. IFN-alpha protein is a monomeric protein of 19-26kDa, which has been widely used for the treatment of cancer and viral diseases such as hepatitis B and hepatitis C. There are 13 genes responsible for the synthesis of 13 IFN- α subtypes: IFNA1, IFNA2, IFNA4, IFNA5, IFNA6, IFNA7, IFNA8, IFNA10, IFNA13, IFNA14, IFNA16, IFNA17, IFNA21.
Human IFN- α2a, IFN- α2b and IFN- α2c represent allelic variants of the same gene. IFN-alpha 2a and IFN-alpha 2b have lysine and arginine, respectively, at position 23 of the mature protein. Human IFN-alpha 2a and IFN-alpha 2b are the only IFN-alpha subtypes with an O-glycosylation site (on Thr 106). Interferon alpha-2 a (IFN-. Alpha.2a; produced by Hoffmann-La Roche) Sold) and interferon alpha-2 b (IFN-alpha 2b, recombinant forms of IFN-alpha 2; by Schering-Plough +.>Sold) have been approved for the treatment of hairy cell leukemia, melanoma, follicular lymphoma, renal cell carcinoma, aids-related Kaposi's sarcoma (Kaposi's sarcoma) and chronic granulocytic leukemia (m.ferroni et al, biochimie. Jun-Jul 2007;89 (6-7):884-893). Recent studies have emphasized new immunomodulatory effects of IFN- α, including activity on T cells and dendritic cells, which may lead to the generation of a durable anti-tumor response. However, the use of IFN- α in clinical oncology is still generally based on the use of the antiproliferative and antiangiogenic activity of these cytokines. Fully exploiting the role of IFN- α as a regulator of immune response and tumor immunity would require new methods of using these cytokines.
hIFN- α2b is a glycoprotein consisting of 166 amino acids with an O-glycosylated threonine at position 106. Each rhIFN-2b consists of five alpha helices (called helices A through E), joined by loops AB, BC, CD and DE. Residues important in receptor binding are the AB loop (Arg 22, leu26, phe27, leu30, lys31, arg33 and His 34), helix B (Ser 68), helix C (Thr 79, lys83, tyr85 and Tyr 89), helix D (Arg 120, lys121, gln124, lys131 and Glu 132) and helix E (Arg 144 and Glu 146). Important amino acid residues in biological activity are Leu30, lys31, arg33, his34, phe36, arg120, lys121, gln124, tyr122, tyr129, lys131, glu132, arg144 and Glu146 (Ratih Asmana Ningrum, scientifica (Cairo).2014; 2014:970315).
In some embodiments, IFN-alpha moiety is IFN-alpha 2. In some embodiments, IFN-alpha moiety is IFN-alpha 2a. In some embodiments, IFN-alpha moiety is IFN-alpha 2b. In some embodiments, IFN-alpha moiety is IFN-alpha 2c. In some embodiments, IFN- α moieties are mature IFN- α. The natural human IFN-alpha 2b precursor polypeptide consists of 188 amino acid residues (amino acids 1-23 are signal peptides), while the mature polypeptide consists of 165 amino acid residues (SEQ ID NO: 31). In some embodiments, the IFN- α moiety is a polypeptide that is substantially homologous to the amino acid sequence of wild-type IFN- α (SEQ ID NO: 31), e.g., has at least about 85% (e.g., at least about any of 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) amino acid sequence identity to wild-type IFN- α (SEQ ID NO: 31). In some embodiments, the IFN- α moiety is not glycosylated. In some embodiments, IFN- α moieties are glycosylated.
In some embodiments, the IFN- α variant (e.g., IFN- α2b variant) comprises one or more mutations at a position selected from the group consisting of: r22, L26, F27, L30, K31, D32, R33, H34, D35, F36, S68, T79, K83, Y85, Y89, R120, K121, Y122, Q124, Y129, K131, E132, R144, and E146. In some embodiments, the IFN- α variant (e.g., IFN- α2b variant) comprises one or more mutations selected from the group consisting of: L30A, K31A, D A, R33A, H a and D35A. In some embodiments, the IFN- α variant (e.g., IFN- α2b variant) comprises an L30A mutation relative to IFN- α (e.g., IFN- α2b; SEQ ID NO: 31). In some embodiments, IFN- α variants comprise the amino acid sequence of SEQ ID NO. 32. In some embodiments, IFN-alpha variants (e.g., IFN-alpha 2b variants) containing SEQ ID NO 32-37 any sequence.
IFN-β
Two types of IFN- βs have been described, IFN- β1 and IFN- β3. In some embodiments, the immunostimulatory cytokine or variant thereof is IFN- β or variant thereof, such as IFN- β1, IFN- β3 or variant thereof. In some embodiments, the immunostimulatory cytokine or variant thereof is IFN- β1a or variant thereof. In some embodiments, the IFN- β moiety is mature IFN- β. In some embodiments, the IFN- β moiety is a wild-type (e.g., wild-type human) IFN- β. In some embodiments, the IFN beta moiety is a mutant (e.g., mutant human) IFN-beta. In some embodiments, the IFN- β moiety is not glycosylated. In some embodiments, the IFN- β moiety is glycosylated.
IFN-γ
In some embodiments, the immunostimulatory cytokine or variant thereof is IFN- γ or a variant thereof. Interferon gamma (ifnγ) is a disulfide-linked dimeric soluble cytokine that is the only member of class II interferons. IFN-gamma is an approximately 25kDa homodimer with tertiary folding built around unusual interdigital alpha helical patterns. It is produced primarily by T cells and NK cells in response to a variety of inflammatory or immune stimuli. IFN-gamma is useful as an immune system activator and inhibitor. Studies have shown that cancer immunotherapy (checkpoint inhibitors) acts in part by increasing IFN- γ expression, resulting in the elimination of cancer cells. Resistance to immunotherapy is due to a defect in IFN- γ signaling. However, IFN-gamma may also promote cancer escape by promoting tumorigenesis and angiogenesis, triggering expression of tolerance molecules (e.g., PD-L1), and inducing homeostasis. IFN-gamma has not been approved by the FDA for the treatment of Cancer patients (L.Ni and J.Lu, cancer Med.2018;7 (9): 4509-4516) other than malignant osteoporosis due to its opposite and competing effects on the immune system.
The monomeric natural human IFN-gamma (hIFN-gamma) prepropolypeptide consists of 166 amino acid residues (amino acids 1-23 are signal peptides); the monomeric mature polypeptide consists of 138 amino acid residues (SEQ ID NO: 38), which corresponds to amino acids 24-161 of the prepropolypeptide; amino acids 162-166 are the propeptide sequence of the prepro polypeptide. In some embodiments, the monomeric IFN-gamma moiety is monomeric mature IFN-gamma. In some embodiments, the monomeric IFN-gamma moiety is a polypeptide that is substantially homologous to the amino acid sequence of wild-type IFN-gamma (SEQ ID NO: 38), e.g., has at least about 85% (e.g., at least about any of 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) amino acid sequence identity to wild-type IFN-gamma (SEQ ID NO: 38). In some embodiments, the IFN-gamma moiety (or subunit) is not glycosylated. In some embodiments, the IFN-gamma moiety (or subunit) is glycosylated. In some embodiments, the IFN-gamma moiety comprises two identical IFN-gamma monomers/subunits. In some embodiments, the IFN-gamma moiety comprises two different IFN-gamma monomers/subunits. For example, in some embodiments, the IFN-gamma moiety comprises a wild-type IFN-gamma monomer and an IFN-gamma variant monomer. In some embodiments, IFN-gamma moieties comprise two IFN-gamma monomers (e.g., two IFN-gamma variants or wild type monomers) linked together, such as by a peptide linker (e.g., any of SEQ ID NOS: 227-229, 245 and 246) or a chemical linker.
IFN-gamma amino acid residues S20, A23, H111 and Q115 are important for receptor binding; amino acid residues V5, S20, A23, G26 and H111 are important for IFN- γ bioactivity (M.Randal and A.A.Kossiakoff, structure.2001;9 (2): 155-63). Lander et al (J Mol biol.2000;299 (1): 169-79) developed a biologically active single chain variant of hIFN-gamma (IFN-. Gamma.SC1) by ligating two monomers IFN-. Gamma.with one 7-amino acid residue linker and changing His111 in the first IFN-. Gamma.monomer to an aspartic acid residue. Because of the H111D mutation, IFN-. Gamma.SC1 binds only one IFN-. Gamma.but can fully retain its biological activity in cell proliferation, MHC class I induction and antiviral assays.
In some embodiments, the monomer IFN-gamma comprises the sequence of SEQ ID NO: 38. In some embodiments, the IFN-gamma variant comprises one or more mutations within one or both IFN-gamma subunits relative to the wild-type IFN-gamma subunit (SEQ ID NO: 38) at a position selected from the group consisting of: v5, S20, D21, V22, a23, D24, N25, G26, H111, and Q115. In some embodiments, the IFN-gamma variant comprises one or more mutations within one or both IFN-gamma subunits, relative to the wild-type IFN-gamma subunit (SEQ ID NO: 38), selected from the group consisting of: S20A, D21A, D K, V22A, A S, A23E, A23Q, A23V, D A, D24E, N25A, N K and H111D. In some embodiments, the IFN-gamma variant comprises one or more mutations within one or both IFN-gamma subunits, relative to the wild-type IFN-gamma subunit (SEQ ID NO: 38), selected from the group consisting of: S20A/D21A, D21K, V22A/A23S, D24A/N25A, A E/D24E/N25K, A Q and A23V. In some embodiments, one or both subunits of IFN-gamma variants comprise the sequence of any one of SEQ ID NOs 39-45. In some embodiments, the IFN-gamma variant comprises an A23V mutation within one or both IFN-gamma subunits relative to the wild-type IFN-gamma subunit (SEQ ID NO: 38). In some embodiments, one or both subunits of the IFN-gamma variant comprise the sequence of SEQ ID NO. 41. In some embodiments, two subunits of IFN-gamma or a variant thereof are linked by a linker (e.g., any of SEQ ID NOS: 227-229, 245 and 246). In some embodiments, IFN-gamma variants comprise the sequence of SEQ ID NO. 47 or 252. In some embodiments, both subunits of IFN-gamma comprise the sequence of SEQ ID NO: 38. In some embodiments, the IFN-gamma moiety is a recombinant "wild-type" IFN-gamma comprising two wild-type IFN-gamma subunits linked by a linker (e.g., any of SEQ ID NOS: 227-229, 245 and 246), such as a sequence comprising SEQ ID NO:46 or 251.
IL-10
In some embodiments, the immunosuppressive cytokine or variant thereof is IL-10 or variant thereof. Interleukin 10 (IL-10) is an alpha helical cytokine expressed as a non-covalently linked homodimer of about 37kDa, also known as human Cytokine Synthesis Inhibitory Factor (CSIF). It plays a key role in inducing and maintaining tolerance. IL-10 signals through the JAK-STAT complex. The IL-10 receptor (IL-10R) has two subunits, one of which is predominantly expressed on immune cells, particularly the highest on monocytes and macrophages, and the other is the ubiquitously expressed beta subunit. IL-10 is produced primarily by monocytes and in small amounts by lymphocytes, including type II T helper cells (TH 2), mast cells, CD4 + CD25 + Foxp3 + Regulatory T cells, and subpopulations of activated T cells and B cells. Dendritic cells and NK cells can also beIL-10 production. IL-10 inhibits the secretion of pro-inflammatory cytokines such as TNFα, IL-1, IL-6, IL-12 and Th1 cytokines such as IL-2 and IFN- γ, and controls the differentiation and proliferation of macrophages, B cells and T cells (Glocker, E.O.et al., ann.N.Y. Acad.Sci.1246,102-107 (2011); moore, K.W.et al., annu.Rev.Immunol.19,683-765 (2001); R.de Waal Malefyt et al., J.Exp.Med.174,915-924 (1991); williams, L.M.et al., immunology 113,281-292 (2004)). In addition, it is a potent antigen presenting inhibitor that inhibits MHC II expression and the upregulation of the co-stimulatory molecules CD80 and CD86 (Mosser, D.M. &Zhang, X.immunological Reviews 226,205-218 (2008)). If IL-10 is absent or nonfunctional, inflammation may not be controlled. This makes IL-10 an attractive therapeutic candidate for autoimmune diseases. However, clinical trials using IL-10 and the development of recombinant IL-10 (ilotecakin,Schering-Plough Research Institue, kenilworth, N.J.), has been discontinued due to lack of efficacy. Recent studies have demonstrated the potential role of IL-10 in tumor therapy (Fujii et al, (October 2001), "Intereukin-10promotes the maintenance of antitumor CD8 (+) T-cell effector function in situ". Blood.98 (7): 2143-51).
The monomeric native human IL-10 precursor polypeptide consists of 178 amino acid residues (amino acids 1-18 are signal peptides) whereas the monomeric mature IL-10 polypeptide consists of 160 amino acid residues (SEQ ID NO: 52). In some embodiments, the monomeric IL-10 moiety is monomeric mature IL-10. In some embodiments, the monomeric IL-10 moiety is a polypeptide that is substantially homologous to the amino acid sequence of wild-type IL-10 (SEQ ID NO: 52), e.g., has at least about 85% (e.g., at least about any of 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) amino acid sequence identity to wild-type IL-10 (SEQ ID NO: 52). In some embodiments, the IL-10 moiety (or subunit) is not glycosylated. In some embodiments, the IL-10 moiety (or subunit) is glycosylated. In some embodiments, the IL-10 moiety comprises two identical IL-10 monomers/subunits. In some embodiments, the IL-10 moiety comprises two different IL-10 monomers/subunits. For example, in some embodiments, the IL-10 moiety comprises a wild-type IL-10 monomer and an IL-10 variant monomer. In some embodiments, the IL-10 moiety comprises two IL-10 monomers (e.g., two IL-10 variants or wild-type monomers) linked together, such as via a peptide linker (e.g., any of SEQ ID NOS: 227-229, 245 and 246) or a chemical linker, see, e.g., biologically active single chain IL-10 in US20130316404, the contents of which are incorporated herein by reference in their entirety.
IL-10 amino acid residues N21, M22, R24, R32, H90, S31 and S93 are important in IL-10 receptor binding; r24 residues are critical for the biological activity of IL-10 (Yoon et al, J Biol chem.2006;281 (46): 35088-35096; E.S. Acuner-Ozbabacan et al, BMC genomics.2014;15Suppl 4 (Suppl 4): S2).
In some embodiments, the monomer IL-10 comprises the sequence of SEQ ID NO: 52. In some embodiments, the IL-10 variant comprises one or more mutations within one or both IL-10 subunits at a position selected from the group consisting of: n21, M22, R24, D25, L26, R27, D28, a29, F30, S31, R32, H90, and S93. In some embodiments, the IL-10 variant comprises one or more mutations within one or both IL-10 subunits, relative to the wild-type IL-10 subunit (SEQ ID NO: 52), selected from the group consisting of: R24A, D25A, L26A, R27A, D A, A29S, F30A, S a and R32A. In some embodiments, the IL-10 variant comprises one or more mutations within one or both IL-10 subunits, relative to the wild-type IL-10 subunit (SEQ ID NO: 52), selected from the group consisting of: R24A, D A/L26A, R27A, D A/A29S, F A/S31A and R32A. In some embodiments, one or both subunits of the IL-10 variant comprise the sequence of any one of SEQ ID NOs 53-58. In some embodiments, the IL-10 variant comprises an R27A mutation within one or both IL-10 subunits relative to the wild-type IL-10 subunit (SEQ ID NO: 52). In some embodiments, one or both subunits of the IL-10 variant comprise the sequence of SEQ ID NO. 55. In some embodiments, the IL-10 variant comprises the sequence of SEQ ID NO. 60. In some embodiments, both subunits of IL-10 comprise the sequence of SEQ ID NO. 52. In some embodiments, the IL-10 variant or variant thereof is linked by a linker. In some embodiments, the IL-10 moiety is a recombinant "wild-type" IL-10 comprising two wild-type IL-10 monomers, such as a sequence comprising SEQ ID NO:59, linked by a linker (e.g., any of SEQ ID NO:227-229, 245 and 246).
IL-12
In some embodiments, the immunostimulatory cytokine or variant thereof is IL-12 or a variant thereof. IL-12 is a 70kDa heterodimeric protein consisting of two covalently (disulfide) linked p35 (IL-12A) and p40 (IL-12B) subunits. The P40 subunit is shared between IL-12 and IL-23. Active heterodimers (referred to as "p 70") and homodimers of p40 are formed after protein synthesis. IL-12 is an interleukin belonging to the IL-12 family, the only family comprising heterodimeric cytokines, including IL-12, IL-23, IL-27, and IL-35.IL-12 is produced by dendritic cells, macrophages, neutrophils and human B lymphoblastic cells (NC-37) in response to antigen stimulation. IL-12 functions by binding to the IL-12 receptor (IL-12R), a heterodimeric receptor formed by IL-12Rβ1 and IL-12Rβ2, and in turn results in activation of the JAK-STAT pathway. IL-12 promotes the development of Th1 responses and greatly induces IFNγ production by T and NK cells. The ability of IL-12 to activate innate (NK cells) and adaptive (cytotoxic T lymphocytes) immunity makes it a promising candidate for cancer immunotherapy. Despite the positive results achieved in animal trials, IL-12 shows only modest anti-tumor responses in clinical trials and is often accompanied by significant toxicity problems (Lasek et al, cancer Immunol immunother, 2014). Treatment with IL-12 is associated with systemic influenza-like symptoms (fever, chill, fatigue, erythromelalgia or headache) and toxic effects on bone marrow and liver. Dose studies have shown that patients can only tolerate doses below 1pg/kg, well below therapeutic doses. As a result, clinical trials using IL-12, either as monotherapy or in combination with other drugs, failed to demonstrate an effective sustained therapeutic effect ((Lasek et al, cancer Immunol immunother, 2014).
The natural human p35 (IL-12A) precursor polypeptide consists of 219 amino acid residues (amino acids 1-22 are signal peptides), while the mature polypeptide consists of 197 amino acid residues (SEQ ID NO: 61). The natural human p40 (IL-12B) precursor polypeptide consists of 328 amino acid residues (amino acids 1-22 are signal peptides), while the mature polypeptide consists of 306 amino acid residues (SEQ ID NO: 62). In some embodiments, IL-12 part (or IL-12 subunit) is mature IL-12 (or mature subunit). In some embodiments, IL-12A (p 35) subunit or variant thereof is a polypeptide that is substantially homologous to the amino acid sequence of wild-type IL-12A (p 35) (SEQ ID NO: 61), e.g., has at least about 85% (e.g., at least about any of 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) amino acid sequence identity to wild-type IL-12A (p 35) (SEQ ID NO: 61). In some embodiments, IL-12B (p 40) subunit or variant thereof is a polypeptide that is substantially homologous to the amino acid sequence of a wild-type IL-12B (p 40) subunit (SEQ ID NO: 62), e.g., has at least about 85% (e.g., at least about any of 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) amino acid sequence identity to a wild-type IL-12B (p 40) subunit (SEQ ID NO: 62). In some embodiments, IL-12 (or subunit) or variants thereof are not glycosylated. In some embodiments, IL-12 (or subunit) or variants thereof are glycosylated. In some embodiments, IL-12 variants include a wild-type subunit (e.g., wt p 35) and a mutant subunit (e.g., variant p 40). In some embodiments, IL-12 variants include two variant subunits (p 35 variants and p40 variants). In some embodiments, IL-12 variants contain two wild-type subunits (e.g., wt p35 and wt p 40), which are linked together via a synthetic peptide linker (e.g., any of SEQ ID NOS: 227-229, 245 and 246) or a chemical linker.
Within the p40 subunit, the amino acid residues important for IL-12 receptor binding are C177, E45, E59 and D62 (Luo et al J Mol biol.2010;402 (5): 797-812). Studies have shown that the accessible N-terminus of the p40 subunit is important for the biological activity of IL-12. Lieschke et al constructed single chain IL-12 (scIL-12) and noted that the order of subunits influences the bioactivity of IL-12: IL-12 activity is greatly reduced when the p35 subunit is N-terminal to the p40 subunit; when the p40 subunit is located at the N-terminus of the p35 subunit, the scIL-12 has a biological activity comparable to that of rIL-12 (Lieschke et al Nat Biotechnol.1997;15 (1): 35-40).
In some embodiments, the IL-12 moiety comprises a wild-type p35 subunit (SEQ ID NO: 61). In some embodiments, IL-12 part contains variant p35 subunit. In some embodiments, the IL-12 moiety comprises a wild-type p40 subunit (SEQ ID NO: 62). In some embodiments, IL-12 part contains variant p40 subunit. In some embodiments, IL-12 part contains through peptide linker (e.g., SEQ ID NO:227-229, 245 and 246 in any one of the wild type or variant p35 subunit and wild type or variant p40 subunit. In some embodiments, the IL-12 moiety from N-terminal to C-terminal contains: wild-type or variant p40 subunit-linker (e.g., any of SEQ ID NOS: 227-229, 246 and 246) -wild-type or variant p35 subunit. In some embodiments, the IL-12 moiety from N-terminal to C-terminal contains: wild-type or variant p35 subunit-linker (e.g., any of SEQ ID NOS: 227-229, 245 and 246) -wild-type or variant p40 subunit. In some embodiments, the IL-12 variant comprises one or more mutations within the p40 subunit at a position selected from the group consisting of: e45, Q56, V57, K58, E59, F60, G61, D62, A63, G64, Q65 and C177. In some embodiments, the IL-12 variant comprises one or more mutations within the p40 subunit, relative to the wild-type p40 subunit (SEQ ID NO: 62), selected from the group consisting of: Q56A, V57A, K3558A, E59A, F60A, F60D, G A, D62A, A63S, G a and Q65A. In some embodiments, IL-12 variant p40 subunit contains SEQ ID NO 63-66 and 140 any sequence. In some embodiments, the IL-12 variant comprises an E59A/F60A mutation within the p40 subunit relative to the wild-type p40 subunit (SEQ ID NO: 62). In some embodiments, IL-12 variant p40 subunit contains SEQ ID NO:63 sequence. In some embodiments, the IL-12 variant comprises an F60A mutation within the p40 subunit relative to the wild-type p40 subunit (SEQ ID NO: 62). In some embodiments, the p40 subunit of the IL-12 variant comprises the sequence of SEQ ID NO: 65. In some embodiments, the IL-12 variant comprises an F60D mutation within the p40 subunit relative to the wild-type p40 subunit (SEQ ID NO: 62). In some embodiments, IL-12 variant p40 subunit contains SEQ ID NO:140 sequence. In some embodiments, IL-12 or variants thereof p40 subunit and p35 subunit connected by a linker (e.g., SEQ ID NO:227-229, 246 and 246 any one). In some embodiments, IL-12 variants include the sequence of any one of SEQ ID NOS 68-71 and 254. In some embodiments, IL-12 part is recombinant "wild-type" IL-12, which comprises a wild-type p35 subunit and a wild-type p40 subunit, such as comprising the sequence of SEQ ID NO:67 or 253, linked by a linker (e.g., any of SEQ ID NO:227-229, 245 and 246).
In some embodiments, IL-12 part derived from mouse IL-12. The mouse p35 subunit and/or p40 subunit may be wild-type or variant. In some embodiments, the mouse IL-12 variant comprises one or two mutations at one or both positions E59 and F60 within the p40 subunit relative to the mouse wild-type p40 subunit. In some embodiments, the p40 subunit and the p35 subunit of mouse IL-12 or a variant thereof are linked by a linker (e.g., any one of SEQ ID NOS: 227-229, 245 and 246). In some embodiments, the mouse IL-12 variant comprises the sequence of SEQ ID NO: 72.
IL-23
In some embodiments, the immunostimulatory cytokine or variant thereof is IL-23 or variant thereof. Interleukin 23 (IL-23) belongs to the IL-12 cytokine family, a heterodimeric cytokine consisting of the IL12B (p 40) subunit (shared with IL-12) and the IL23A (p 19) subunit. IL-23 functions by binding to the IL-23 receptor consisting of IL-12Rβ1 and IL-23R (p 19 subunit binds IL-23R and p40 subunit binds IL-12Rβ1), resulting in Janus kinase 2 and tyrosine kinase 2 kinase recruitment and phosphorylation of STAT3 and STAT4, resulting in gene activation. STAT3 is responsible for key Th17 developmental features such as roryt expression, or transcription of Th17 cytokines such as IL-17, IL-21, IL-22 and GM-CSF, which mediate protection against fungi and bacteria and are involved in barrier immunity. IL-23 is secreted by activated dendritic cells, macrophages or monocytes stimulated primarily by antigen stimulators. IL-23 receptor Expressed on Th17 and NK cells. Autoimmune and cancer diseases were found to be associated with IL-23 imbalance and increase. The most important function of IL-23 is its role in the development and differentiation of effector Th17 cells. In the context of chronic inflammation, activated DCs and macrophages produce IL-23, which promotes development of Th17 cells. Recent findings of autoimmune diseases such as psoriasis, crohn's disease, rheumatoid arthritis or multiple sclerosis and of the diseases caused by the expression of IL-23 receptor T H -17 and other lymphocyte subpopulations promote IL-23 mediated signal transduction.
The natural human p19 (IL-23A) precursor polypeptide consists of 189 amino acid residues (amino acids 1-19 are signal peptides), while the mature polypeptide consists of 170 amino acid residues (SEQ ID NO: 73). The natural human p40 (IL-12B) precursor polypeptide consists of 328 amino acid residues (amino acids 1-22 are signal peptides), while the mature polypeptide consists of 306 amino acid residues (SEQ ID NO: 62). In some embodiments, the IL-23 moiety (or IL-23 subunit) is mature IL-23 (or IL-23 mature subunit). In some embodiments, IL-23A (p 19) or a variant thereof is a polypeptide that is substantially homologous to an amino acid sequence of wild-type IL-23A (p 19) (SEQ ID NO: 73), e.g., has at least about 85% (e.g., at least about any of 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) amino acid sequence identity to wild-type IL-23A (p 19) (SEQ ID NO: 73). In some embodiments, IL-12B (p 40) subunit or variant thereof is a polypeptide that is substantially homologous to the amino acid sequence of wild-type IL-12B (p 40) (SEQ ID NO: 62), e.g., has at least about 85% (e.g., at least about any of 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) amino acid sequence identity to wild-type IL-12B (p 40) (SEQ ID NO: 62). In some embodiments, IL-23 (or subunit) or variant thereof is not glycosylated. In some embodiments, IL-23 (or subunit) or variant thereof is glycosylated. In some embodiments, the IL-23 variant comprises one wild-type subunit (e.g., wt p 19) and one mutant subunit (e.g., variant p 40). In some embodiments, the IL-23 variant comprises two variant subunits (a p19 variant and a p40 variant). In some embodiments, IL-23 variants comprise two wild-type subunits (e.g., wt p19 and p 40) that are linked together via a synthetic peptide linker (e.g., any of SEQ ID NOS: 227-229, 245 and 246) or a chemical linker. Within the p40 subunit, the amino acid residues important for IL-23 receptor binding are C177, E45, E59 and D62 (Luo et al J Mol biol.2010;402 (5): 797-812).
In some embodiments, the IL-23 portion comprises a wild-type p19 subunit (SEQ ID NO: 73). In some embodiments, the IL-23 moiety comprises a variant p19 subunit. In some embodiments, the IL-23 portion comprises a wild-type p40 subunit (SEQ ID NO: 62). In some embodiments, the IL-23 moiety comprises a variant p40 subunit. In some embodiments, the IL-23 moiety comprises a wild-type or variant p19 subunit and a wild-type or variant p40 subunit linked by a peptide linker (e.g., any of SEQ ID NOS: 227-229, 245 and 246). In some embodiments, the IL-23 moiety comprises from N-terminus to C-terminus: wild-type or variant p40 subunit-linker (e.g., any of SEQ ID NOS: 227-229, 245 and 246) -wild-type or variant p19 subunit. In some embodiments, the IL-23 moiety comprises from N-terminus to C-terminus: wild-type or variant p19 subunit-linker (e.g., any of SEQ ID NOS: 227-229, 245 and 246) -wild-type or variant p40 subunit. In some embodiments, the IL-23 variant comprises one or more mutations within the p40 subunit at a position selected from the group consisting of: e45, Q56, V57, K58, E59, E60, G61, D62, A63, G64, Q65 and C177. In some embodiments, the IL-23 variant comprises one or more mutations within the p40 subunit, relative to the wild-type p40 subunit (SEQ ID NO: 62), selected from the group consisting of: Q56A, V57A, K3558A, E59A, F60A, F60D, G A, D62A, A63S, G a and Q65A. In some embodiments, the p40 subunit of the IL-23 variant comprises the sequence of any one of SEQ ID NOS 63-66 and 140. In some embodiments, the IL-23 variant comprises an E59A/F60A mutation within the p40 subunit relative to the wild-type p40 subunit (SEQ ID NO: 62). In some embodiments, the p40 subunit of the IL-23 variant comprises the sequence of SEQ ID NO. 63. In some embodiments, the IL-23 variant comprises an F60A mutation within the p40 subunit relative to the wild-type p40 subunit. In some embodiments, the p40 subunit of the IL-23 variant comprises the sequence of SEQ ID NO: 65. In some embodiments, the IL-23 variant comprises an F60D mutation within the p40 subunit relative to the wild-type p40 subunit (SEQ ID NO: 62). In some embodiments, the p40 subunit of the IL-23 variant comprises the sequence of SEQ ID NO: 140. In some embodiments, the p40 subunit and the p19 subunit of IL-23 or a variant thereof are linked by a linker (e.g., any of SEQ ID NOS: 227-229, 245, and 246). In some embodiments, the IL-23 variant comprises the sequence of SEQ ID NO. 75. In some embodiments, the IL-23 moiety is a recombinant "wild-type" IL-23 comprising a wild-type p35 subunit and a wild-type p40 subunit, such as a sequence comprising SEQ ID NO:74, linked by a linker (e.g., any of SEQ ID NO:227-229, 245 and 246).
IL-17
In some embodiments, the immunostimulatory cytokine or variant thereof is IL-17 or variant thereof. The IL-17 family includes IL17A, IL-17B, IL-17C, IL-17D, IL-17E (also known as IL-25) and IL-17F. Interleukin 17A (IL-17A or IL-17) is a disulfide-linked, homodimeric, secreted glycoprotein with a molecular weight of about 35kDa. Each subunit of the homodimer is about 15-20kDa. IL-17A is a pro-inflammatory cytokine produced by T helper 17 (Th 17) cells in response to stimulation by IL-23. IL-17 interacts with IL-17R and activates some of the signaling cascade, which in turn leads to chemokine induction. These chemokines act as chemoattractants, recruiting immune cells such as monocytes and neutrophils to the site of inflammation.
Target molecules or target antigens
As used herein, a "target antigen" or "target epitope" may refer to any protein or polypeptide that can be specifically recognized by an antigen binding protein, antigen binding polypeptide, or antigen binding fragment/domain (used interchangeably) described herein, such as a tumor antigen or epitope, a pathogen antigen or epitope, an antigen or epitope involved in autoimmune disease, allergy, and/or graft rejection, a ligand or receptor or portion thereof (e.g., the extracellular domain of a ligand/receptor), an immune cell surface antigen or epitope, and the like. In some embodiments, the antigen binding protein is monovalent and monospecific. In some embodiments, the antigen binding protein is multivalent (e.g., bivalent) and monospecific. In some embodiments, the antigen binding proteins are multivalent (e.g., bivalent) and multispecific (e.g., bispecific). The titers and specificity of antigen binding proteins herein refer to the titers and specificity of antigen binding fragments of immune cytokines (e.g., ligands, receptors, VHHs, scFv, or Fab), excluding the titers or specificity of cytokines or variants thereof.
In some embodiments, the target antigen is a cell surface molecule (e.g., an extracellular domain of a receptor/ligand). In some embodiments, the target antigen serves as a cell surface marker on target cells (e.g., tumor cells, immune cells) associated with a particular disease state. The target antigen specifically recognized by the antigen binding domain (e.g., tumor antigen, extracellular domain of receptor/ligand) may be an antigen on a single diseased cell or an antigen expressed on different cells each contributing to the disease. The target antigen specifically recognized by the antigen binding domain may be directly or indirectly involved in the disease.
Tumor antigens
In some embodiments, the target antigen or epitope (e.g., a third target molecule) is a tumor antigen or epitope.
Tumor antigens are proteins produced by tumor cells that elicit an immune response, particularly a T cell-mediated immune response. The choice of the targeting antigen of the invention will depend on the particular type of cancer to be treated. Exemplary tumor antigens include, for example, glioma-associated antigen, BCMA (B cell maturation antigen), carcinoembryonic antigen (CEA), beta-human chorionic gonadotrophin, alpha Fetoprotein (AFP), lectin-reactive AFP, thyroglobulin, RAGE-1, MN-CAIX, human telomerase reverse transcriptase, RU1, RU2 (AS), enterocarboxylesterase, mut hsp70-2, M-CSF, prostase, prostate Specific Antigen (PSA), PAP, NY-ESO-1, LAGE-la, p53, prostein, PSMA, HER/neu, survivin and telomerase, prostate cancer tumor antigen-1 (PCTA-1), MAGE, ELF2M, neutrophil elastase, ephrinB2, CD22, insulin Growth Factor (IGF) -I, IGF-II, IGF-I receptor, and mesothelin. In some embodiments, the tumor antigen comprises one or more antigenic cancer epitopes associated with a malignancy. Malignant tumors express a variety of proteins and can be used as target antigens for immune attack. These molecules include, but are not limited to, tissue specific antigens such as MART-1, tyrosinase and gp100 in melanoma, and Prostatic Acid Phosphatase (PAP) and Prostate Specific Antigen (PSA) in prostate cancer. Other target molecules belong to the group of transformation related molecules, such as the oncogene HER2/Neu/ErbB-2. Another group of target antigens are tumor embryonal antigens, such as carcinoembryonic antigen (CEA). In B-cell lymphomas, tumor-specific idiotype immunoglobulins constitute the actual tumor-specific immunoglobulin antigen specific to the individual tumor. B cell differentiation antigens (such as CD19, CD20 and CD 37) are other candidates for target antigens in B cell lymphomas.
In some embodiments, the tumor antigen is a Tumor Specific Antigen (TSA) or a Tumor Associated Antigen (TAA). TSA is specific to tumor cells and does not occur on other cells in the body. TAAs are not specific to tumor cells, but are also expressed on normal cells under conditions that are incapable of inducing an immune tolerance state to an antigen. Expression of an antigen on a tumor may occur under conditions that enable the immune system to respond to the antigen. TAAs may be antigens expressed on normal cells during fetal development that fail to respond when the immune system is immature, or they may be antigens that are normally present at very low levels on normal cells but at higher levels on tumor cells. Non-limiting examples of TSA or TAA antigens include the following: differentiation antigens such as MART-1/melanA (MART-1), gp 100 (Pmel 17), tyrosinase, TRP-1, TRP-2, and tumor-specific multilineage antigens such as MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, pl5; overexpressed embryonic antigens such as CEA; overexpressed oncogenes and mutated tumor suppressor genes such as p53, ras, HER2/neu; unique tumor antigens resulting from chromosomal translocation such as BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR; and viral antigens such as Epstein Barr virus antigen EBVA and Human Papilloma Virus (HPV) antigens E6 and E7. Other large protein-based antigens include: TSP-180, MAGE-4, MAGE-5, MAGE-6, RAGE, NY-ESO, pl85erbB2, pl80erbB-3, C-met, nm-23HI, PSA, TAG-72, CA 19-9, CA 72-4, CAM 17.1, nuMa, K-ras, β -catenin, CDK4, mum-1, P15, P16, 43-9F, 5T4 (TPBG), 791Tgp72, alpha fetoprotein, β -HCG, BCA225, BTA, CA 125, CA 15-3\CA 27.29\BCA, CA 195, CA 242, CA-50, CAM43, CD68\P1, CO-029, FGF-5, G250, ga\EpCAM, HTgp-175, M344, MA-50, MG7-Ag, MOV18, NB/70-CO-1, RC35\1, RC16\16\TAAG 2, TAAG-related loop binding proteins, TAAG 2, TAAG proteins.
In some embodiments, the tumor antigen is selected from the group consisting of: FIXa, FX, DLL3, DLL4, ang-2, conjugated-4, folra, GPNMB, CD56 (NCAM), TACSTD2 (TROP-2), tissue factor, ENPP3, P-cadherin, STEAP1, CEACAM5, mucin 1 (sialoglycoCA 6), guanylate Cyclase C (GCC), SLC44A4, LIV1 (ZIP 6), naPi2B, SLITRK6, SC-16, fibronectin, off-domain B (EDB), endothelial receptor ETB, ROBO4, collagen IV, periostin, tenascin C, CD74, CD98, mesothelin, TSHR, CD19, CD123, CD22, CD30, CD171, CS-1, CLL-1, CD33, EGFRvIII, GD2, GD3, BCMA, tn Ag, prostate Specific Membrane Antigen (PSMA), ROR1, FLT3, FAP 72, CD38, CD 796, IL 7, ra 3, IL-45H 3, IL-11, and the like PSCA, PRSS21, VEGFR2 (CD 309), lewis Y, CD24, platelet derived growth factor receptor-beta (PDGFR-beta), SSEA-4, CD20, folate receptor alpha, ERBB2 (HER 2/neu), MUC1, epidermal Growth Factor Receptor (EGFR), NCAM, prostase, PAP, ELF2M, ephrin B2, IGF-I receptor, CAIX, LMP2, gp100, bcr-abl, tyrosinase, ephA2, fucosyl GM1, sLe, GM3, TGS5, HMWMAA, o-acetyl-GD 2, folate receptor beta, TEM1/CD248, TEM 7.6, CLDN18.2, GPRC5D, CXORF, CD97, CD179a, ALK, polysialic acid, PLAC1, globoH, NY-BR-1, UPK2, HAVCR1, RB3, PANX3, PAAD20, SLE 6, GLE 2, TALY 1, ESLY 1, ESY-51, GPR-1, GPR-YY 1, ESGE 1 MAGE-A1, legumain, HPV E6, E7, MAGE A1, ETV6-AML, sperm protein 17, XAGE1, tie 2, MAD-CT-1, MAD-CT-2, fos-associated antigen-1, p53 mutant, Prostein, survivin and telomerase, PCTA-1/galectin-8, melanA/MART1, ras mutant, hTERT, sarcoma translocation breakpoint, ML-IAP, ERG (TMPRSS 2 ETS fusion gene), NA17, PAX3, androgen receptor, cyclin B1, MYCN, rhoC, TRP-2, CYP1B1, BORIS, SART3, PAX5, OY-TES1, LCK, AKAP-4, SSX2, RAGE-1, human telomerase reverse transcriptase, RU1, RU2, intestinal carboxylesterase, mut hsp70-2, CD79a, CD79B, CD72, LAIR1, FCAR, LRLIA 2, CD300LF, CLEC12A, BST2, EMR2, LY75, GPC3, FCRL5 and IGLL1. In some embodiments, the tumor antigen is selected from the group consisting of: BCMA, ephA2, HER2, GD2, glypican-3, 5T4, 8H9, alpha v β 6 Integrins, B7-H3, B7-H6, CAIX, CA9, CD19, CD20, CD22, CD30, CD33, CD38, CD44v6, CD44v7/8, CD70 (TNFSF 7), CD123, CD138, CD171, CEA, CSPG4, EGFR, EGFRvIII, EGP2, EGP40, epCAM, ERBB3, ERBB4, erbB3/4, FAP, FAR, FBP, fetal achR, folate receptor a, GD2, GD3, HLA-AI MAGE A1, HLA-A2, IL11Ra, IL13Ra2, KDR, lewis-Y, MCSP, mesothelin, muc1, muc16, NCAM, NKG2D ligand, NY-ESO-1, PRAME, PSCA, PSC1, PSMA, ROR1, SURVIVIN, TAG72, TEM1, HMW 8, VEGFR2, carcinoembryonic antigen and TEM-MAA. See also Shim H. (biomalecules.2020Mar; 10 (3): 360), and Diamantis N.and Banerji U.Br J cancer.2016;114 (4) exemplary tumor antigens described in 362-367 (the contents of which are incorporated herein by reference in their entirety).
In some embodiments, the tumor antigen is HER2. In some embodiments, the third binding domain that specifically recognizes HER2 is derived from trastuzumab (e.g.,) Pertuzumab (e.g.)>) Either macrituximab or 7C2. In some embodiments, the third binding domain that specifically recognizes HER2 comprises a heavy chain CDR, a light chain CDR, or all of either trastuzumab, pertuzumab, mactuximab, or 7C26 CDRs. In some embodiments, the third binding domain that specifically recognizes HER2 comprises a VH and/or VL of trastuzumab, pertuzumab, mactuximab, or 7C2. In some embodiments, the immune cytokine comprises a parent anti-HER 2 antibody (e.g., a full length antibody).
Pathogen antigen
In some embodiments, the target antigen or epitope (e.g., a third target molecule) is a pathogen antigen or epitope, such as a fungal, viral, bacterial, protozoan, or other parasite antigen or epitope.
In some embodiments, the fungal antigen is from aspergillus or candida. Fungal antigens for use with the compositions and methods of the invention include, but are not limited to, for example, candida fungal antigen components; an aspergillus fungal antigen; histoplasma antigens such as heat shock protein 60 (HSP 60) and other histoplasma antigen components; cryptococcus fungal antigens such as capsular polysaccharides and other cryptococcus fungal antigen components; coccidioidomycosis antigens such as glomeruli antigen and other components of coccidioidomycosis antigens; tinea fungus antigens such as trichostatin and other crude coccidioidomycosis fungus antigen components.
Bacterial antigens for use with the immunocytokines disclosed herein include, but are not limited to, for example, bacterial antigens such as pertussis toxin, filiform hemagglutinin, pertactin, FIM2, FIM3, adenylate cyclase, and other pertussis bacterial antigen components; diphtheria antigens such as diphtheria toxin or toxoid and other diphtheria antigen components; tetanus bacterial antigens such as tetanus toxins or toxoids and other tetanus bacterial antigen components; streptococcal bacterial antigens such as M protein and other streptococcal bacterial antigen components; gram-negative bacterial antigens such as lipopolysaccharide and other gram-negative bacterial antigen components, mycobacterium tuberculosis (Mycobacterium tuberculosis) bacterial antigens such as mycolic acid, heat shock protein 65 (HSP 65), 30kDa major secreted protein, antigen 85A and other mycobacterial antigen components; helicobacter pylori (Helicobacter pylori) bacterial antigen component; pneumococcal bacterial antigens such as pneumolysin, pneumococcal capsular polysaccharide and other pneumococcal bacterial antigen components; haemophilus influenzae (haemophilus influenza) bacterial antigens such as capsular polysaccharides and other haemophilus influenzae bacterial antigen components; anthrax bacterial antigens such as anthrax protective antigens and other anthrax bacterial antigen components; rickettsia bacterial antigens such as rompA and other rickettsia bacterial antigen components. Bacterial antigens described herein also include any other bacterial, mycobacterial, mycoplasma, rickettsial, or chlamydia antigen. Some or all pathogens may also be: haemophilus influenzae; plasmodium falciparum (Plasmodium falciparum); neisseria meningitidis (neisseria meningitidis); streptococcus pneumoniae (streptococcus pneumoniae); neisseria gonorrhoeae (neisseria gonorrhoeae); salmonella serotype typhoid; shigella spp; vibrio cholerae (vibrio cholerae); dengue fever; encephalitis; japanese encephalitis; lyme disease; yersinia pestis (Yersinia pestis); west nile virus; yellow fever; rabbit febrile disease; hepatitis (viral; bacterial); RSV (respiratory syncytial virus); HPIV 1 and HPIV 3; adenoviruses; ceiling; allergies and cancers.
Examples of protozoan and other parasite antigens include, but are not limited to, for example, plasmodium falciparum antigens such as merozoite surface antigens, sporozoite surface antigens, circumsporozoite antigens, gametophyte/gamete surface antigens, blood stage antigens pf 155/RESA, and other plasmodium antigen components; toxoplasma antigens such as SAG-1, p30 and other toxoplasma antigen components; schistosome antigens such as glutathione-S-transferase, paramyoglobulin and other schistosome antigen components; major leishmania (leishmania major) and other leishmania antigens such as gp63, lipoglycans and related proteins and other leishmania antigen components; and trypanosoma cruzi (trypanosoma cruzi) antigens such as 75-77kDa antigen, 56kDa antigen and other trypanosoma antigen components.
In some embodiments, the viral antigen is from Herpes Simplex Virus (HSV), respiratory Syncytial Virus (RSV), metapneumovirus (hMPV), rhinovirus, parainfluenza virus (PIV), epstein Barr Virus (EBV), cytomegalovirus (CMV), JC virus (john cunningham virus), BK virus, HIV, zika virus, human coronavirus, norovirus, encephalitis virus, or ebola virus. In some embodiments, the virus is an orthomyxoviridae virus selected from the group consisting of: influenza a virus, influenza b virus, influenza c virus and any subtype or reassortant strain thereof. In some embodiments, the virus is an influenza a virus or any subtype or reassortant thereof, such as influenza a virus subtype H1N1 (H1N 1) or influenza a virus subtype H5N1 (H5N 1). In some embodiments, the virus is a coronaviridae virus selected from the group consisting of: alpha coronavirus 229E (HCoV-229E), neem coronavirus NL63 (HCoV-NL 63), beta coronavirus OC43 (HCoV-OC 43), coronavirus HKU1 (HCoV-HKU 1), severe acute respiratory syndrome coronavirus (SARS-CoV), middle east respiratory syndrome coronavirus (MERS-CoV) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In some embodiments, the virus is SARS-CoV, MERS-CoV or SARS-CoV-2. In some embodiments, the virus is a filoviridae virus selected from ebola virus (EBOV) and marburg virus (MARV). In some embodiments, the virus is a flaviviridae virus selected from the group consisting of: ziKV, west Nile Virus (WNV), dengue Virus (DENV) and Yellow Fever Virus (YFV).
Antigens involved in autoimmune diseases, allergies and graft rejection
In some embodiments, the target antigen or epitope (e.g., a third target molecule) is an antigen or epitope involved in autoimmune disease, allergy, and/or graft rejection. For example, antigens involved in any one or more of the following autoimmune diseases or disorders may be used in the present invention: the present invention relates to a method for treating diabetes mellitus, diabetes, arthritis (including rheumatoid arthritis, juvenile rheumatoid arthritis, osteoarthritis, psoriatic arthritis), multiple sclerosis, myasthenia gravis, systemic lupus erythematosus, autoimmune thyroiditis, dermatitis (including atopic dermatitis and eczematous dermatitis), psoriasis, sjogren's syndrome (including keratoconjunctivitis sicca secondary to sjogren's syndrome), alopecia areata, allergic reactions caused by arthropod bite reactions, crohn's disease, aphthous ulcers, iritis, conjunctivitis, keratoconjunctivitis, ulcerative colitis, asthma, allergic asthma, cutaneous lupus erythematosus, scleroderma, vaginitis, proctitis, drug eruptions, leprosy reversal reactions, leprosy erythema, autoimmune uveitis, allergic encephalomyelitis, acute necrotizing hemorrhagic encephalopathy, idiopathic bilateral hearing loss, aplastic anemia, pure erythrocytic anemia, idiopathic thrombocytopenia, polychondritis, wegener's granulomatosis, chronic granulomatosis, stens's disease, post-pranditis, crohn's disease, sarcoidosis, che, ocular disease, transient inflammatory disease, inflammatory bowel disease, crohn's disease, inflammatory bowel disease, sarcoidosis, and inflammatory disease. Examples of antigens involved in autoimmune diseases include: glutamate decarboxylase 65 (GAD 65), natural DNA, myelin basic protein, myelin proteolipid protein, acetylcholine receptor components, thyroglobulin, and Thyroid Stimulating Hormone (TSH) receptors. Examples of antigens associated with allergy include: pollen antigens (e.g., japanese cedar pollen antigens, ragweed pollen antigens, ryegrass pollen antigens), animal-derived antigens (e.g., dust mite antigens and cat antigens), histocompatibility antigens, penicillins and other therapeutic agents. Examples of antigens involved in graft rejection include the antigen component of the graft to be transplanted into a graft recipient (e.g., heart, lung, liver, pancreas, kidney), as well as the nerve graft component. The antigen may be an altered peptide ligand for use in the treatment of autoimmune disease. In some embodiments, the target antigen is CD3, CD4, CD123, or CD8.
Immune checkpoint molecules
In some embodiments, the target antigen or epitope (e.g., first, second, and/or third target molecule) is an immune checkpoint molecule. Immune checkpoints are regulators of the immune system.
In some embodiments, the immune checkpoint molecule is a stimulatory immune checkpoint molecule. In some embodiments, the stimulatory immune checkpoint molecule is selected from the group consisting of: CD27, CD28, OX40, ICOS, GITR, 4-1BB, CD27, CD40, CD3 and HVEM. Thus, in some embodiments, the first binding domain described herein is an activator of a stimulatory immune checkpoint molecule, which can stimulate, activate, or increase the intensity of an immune response mediated by the stimulatory immune checkpoint molecule. The antibodies or antigen binding fragments described herein may be derived from any antibody known in the art that is capable of activating a stimulatory immune checkpoint molecule. In some embodiments, the first binding domain is a ligand or receptor for a stimulatory immune checkpoint molecule, e.g., may activate stimulatory immune checkpoint signaling. In some embodiments, the second binding domain (e.g., an antibody, antigen binding domain, or ligand/receptor-Fc fusion protein) described herein is an antagonist of a stimulatory immune checkpoint molecule, which can reduce or block the intensity of an immune response mediated by the stimulatory immune checkpoint molecule.
In some embodiments, the immune checkpoint molecule is an inhibitory immune checkpoint molecule. In some embodiments, the inhibitory immune checkpoint molecule is selected from the group consisting of: PD-1, PD-L2, CTLA-4, LAG-3, TIM-3, HHA 2, CD47, CXCR4, CD160, CD73, BLTA, B7-H4, TIGIT and VISTA. In some embodiments, the inhibitory immune checkpoint molecule is PD-1, PD-L2, or PD-L1. In some embodiments, the inhibitory immune checkpoint molecule is CTLA-4. In some embodiments, the inhibitory immune checkpoint molecule is TIGIT. Thus, in some embodiments, an antigen binding protein (e.g., an antibody, antigen binding domain, or ligand/receptor-Fc fusion protein) described herein is an immune checkpoint inhibitor that reduces, inhibits, or interferes with, in whole or in part, one or more inhibitory immune checkpoint molecules. The antibodies or antigen binding domains described herein may be derived from any antibody known in the art that is capable of acting as an immune checkpoint inhibitor. In some embodiments, the antigen binding fragment is a ligand (e.g., CD155, PD-L2, or PD-L1) or receptor of an inhibitory immune checkpoint molecule (e.g., TIGIT or PD-1), e.g., can activate or stimulate inhibitory immune checkpoint signaling (e.g., TIGIT or PD-1 signaling). In some embodiments, the antigen binding proteins (e.g., antibodies, antigen binding domains, or ligand/receptor-Fc fusion proteins) described herein are agonists of an inhibitory immune checkpoint molecule, which may stimulate, activate, or increase the intensity of an immune response mediated by the inhibitory immune checkpoint molecule.
Cell surface ligands or receptors
In some embodiments, the target antigen or epitope (e.g., a third target molecule) is a ligand or receptor or portion thereof, such as the extracellular domain of a ligand/receptor. In some embodiments, the ligand or receptor is derived from a molecule selected from the group consisting of: IL-2, IL-2Rα (CD 25), IL-3Rα (CD 123), PD-1, PD-L2, CD155, NKG2A, NKG2C, NKG2F, NKG2D, BCMA, APRIL, BAFF, IL-3, IL-13, LLT1, AICL, DNAM-1 and NKp80. In some embodiments, the ligand is derived from APRIL and/or BAFF that can bind BCMA. In some embodiments, the receptor is an FcR and the ligand is an Fc-containing molecule. In some embodiments, the FcR is an fcγ receptor (fcγr). In some embodiments, fcγr is selected from the group consisting of: fcyria (CD 64A), fcyrib (CD 64B), fcyric (CD 64C), fcyriia (CD 32A), fcyriib (CD 32B), fcyriiia (CD 16 a) and fcyriiib (CD 16B).
IL-2 receptor, interleukin-2 receptor (IL-2R), is a heterotrimeric protein expressed on the surface of certain immune cells, such as lymphocytes. IL-2R has an alpha chain (IL-2 Ralpha, CD25, tac antigen), a beta chain (IL-2 Rbeta, CD 122) and a gamma chain (IL-2 Rgamma, gamma) c Normal gamma chain or CD 132). IL-2Rα binds IL-2 with low affinity, and the complex of IL-2Rβ and IL-2Rγ binds IL-2 with moderate affinity, acting primarily on memory T cells and NK cells. All complexes of alpha, beta and gamma chains bind IL-2 with high affinity to activated T cells and regulatory T cells (tregs). CD25 (IL-2 ra) plays a key role in Treg development and maintenance, and can play a role in Treg expression of CD62L, CD62L being necessary for Treg entry into lymph nodes (Malek and Bayer, 2004). CD25 is a marker of activated T cells and tregs.
Immune cell surface antigen
In some embodiments, the target antigen or epitope (e.g., a third target molecule) is an immune cell surface antigen or epitope. Immune cells have different cell surface molecules. For example, CD3 is a cell surface molecule on T cells, whileCD16, NKG2D or NKp30 are cell surface molecules on NK cells, while CD3 or the constant T Cell Receptor (TCR) are cell surface molecules on NKT cells. In some embodiments, wherein the immune cells are T cells, the activating molecules are one or more of CD3, such as CD3 epsilon, CD3 delta, or CD3 gamma; or CD2, CD4, CD8, CD27, CD28, CD40, CD134, CD137, CD278, inhibitory immune checkpoint molecules (e.g., CTLA-4, PD-1, TIM3, BTLA, VISTA, LARG-3, or TIGIT), and stimulatory immune checkpoint molecules (CD 27, CD28, CD137, OX40, GITR, or HVEM). In some embodiments, wherein the immune cell is a B cell, the cell surface molecule is CD19, CD20, or CD138. In other embodiments, wherein the immune cell is an NK cell, the cell surface molecule is CD16, CD56 (NCAM), NKp46, NKp44, CD244, CD226, TIGIT, CD96, LAG3, TIM3, PD-1, KLRG1, CD161, CD94/NKG2, KIR, NKG2D, or NKp30. In some embodiments, wherein the immune cell is a NKT cell and the cell surface molecule is CD3 or a constant TCR. In some embodiments, wherein the immune cell is a myeloid dendritic cell (mDC), the cell surface molecule is CD11c, CD11b, CD13, CD45RO, or CD33. In some embodiments, wherein the immune cells are plasma dendritic cells (pdcs), the cell surface molecule is CD123, CD62L, CD45RA, or CD36. In some embodiments, wherein the immune cell is a macrophage and the cell surface molecule is CD163 or CD206. In some embodiments, the immune cell is selected from the group consisting of: monocytes, dendritic cells, macrophages, B cells, killer T cells (T c Cytotoxic T lymphocytes or CTLs), helper T cells (T h ) Regulatory T cells (tregs), γδ T cells, natural Killer T (NKT) cells and Natural Killer (NK) cells.
In some embodiments, the immune cell surface antigen is selected from the group consisting of: CD3 (e.g., CD3 epsilon, CD3 delta, CD3 gamma), CD4, CD5, CD8, CD16, CD27, CD28, CD40, CD64, CD89, CD134, CD137, CD278, NKp46, NKp30, NKG2D, TCR alpha, TCR beta, TCR gamma, and TCR delta. In some embodiments, the immune cell surface antigen is CD3, CD4, or CD8.
Exemplary anti-CD 4 antibodies include, but are not limited to: ibalizumab (e.g.,) Max.16h5, and IT1208. Exemplary anti-CD 3 antibodies include, but are not limited to OKT3. Exemplary anti-CD 8 antibodies include, but are not limited to: g10-1, OKT8, YTC182.20, 4B11 and DK25.
Activity of binding domains or cytokines or variants thereof
"Activity" of a binding domain (e.g., to its target molecule) as described herein includes the binding affinity of the binding domain to the corresponding target molecule; and/or the biological activity (biological activity/bioactivity) of a binding domain (e.g., a cytokine or variant thereof)), such as, upon binding of the binding domain/target molecule thereof, inducing or inhibiting signal transduction, inducing or inhibiting cell proliferation, differentiation and/or activation, inducing or inhibiting secretion of a influencing cytokine (e.g., a pro-inflammatory cytokine), inducing or inhibiting cytotoxicity to tumor cells, inducing or inhibiting elimination of an infectious agent, and the like. These biological activities are also referred to herein as direct biological activities. In some embodiments, the biological activity of the binding domain (e.g., to its target molecule) also includes indirect biological activity, such as any biological activity resulting from direct biological activity.
The "activity" of a cytokine or variant thereof as described herein includes: binding affinity of the cytokine or variant thereof to the corresponding cytokine receptor; and/or biological activity (biological activity/bioactivity) of the cytokine or variant thereof), such as inducing or inhibiting signal transduction, inducing or inhibiting cell proliferation, differentiation and/or activation, inducing or inhibiting secretion of influencing cytokines (e.g., pro-inflammatory cytokines), etc., after cytokine/cytokine receptor binding. These biological activities are also referred to herein as direct biological activities. In some embodiments, the biological activity of a cytokine or variant thereof also includes indirect biological activity, such as any biological activity resulting from direct biological activity. For example, in some embodiments, biological activity also includes cancer cell killing by immune cells that are attracted to the tumor site due to secreted effector cytokines such as inflammatory markers IL-6, MIP-2 (GRO-P)/CXCL 2, G-CSF/CSF3, TIMP-1, KC (GRO-a)/CXCLl, and the like.
In some embodiments, the first binding domain or portion thereof is located at the hinge region (within the N ', C' of the hinge or hinge) between the second binding domain or portion thereof of the immunomodulatory molecule and the Fc domain subunit or portion thereof. In some embodiments, the activity (binding affinity to a first target molecule, such as a cytokine receptor, and/or biological activity) of a first binding domain (e.g., an immunostimulatory cytokine or variant thereof) is increased by at least about 20% (e.g., at least about any of 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500% or more) in the presence of binding of a second binding domain (e.g., ligand, receptor, VHH, scFv, or Fab) of an immunomodulatory molecule described herein to a second target antigen, as compared to the absence of binding of the second binding domain to the second target molecule. In some embodiments, in the presence of binding of a second binding domain (e.g., ligand, receptor, VHH, scFv, or Fab) of an immunomodulatory molecule described herein to a second target molecule, the activity (binding affinity to a first target molecule, such as a cytokine receptor, and/or biological activity) of a first binding domain (e.g., an immunostimulatory cytokine or variant thereof) is increased by at least about 2-fold (e.g., at least about any of 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100-fold) of the activity in the absence of binding of the second binding domain to the second target molecule.
In some embodiments, in the absence of binding of a second binding domain (e.g., ligand, receptor, VHH, scFv, or Fab) of an immunomodulatory molecule described herein to a second target antigen, the activity (binding affinity to a first target molecule such as a cytokine receptor, and/or biological activity) of a first binding domain (e.g., immunostimulatory cytokine or variant thereof) of fig. 1A-1D, 1G, 1H, 1L-1O, is no more than about 70% (e.g., no more than about 60%, 50%, 40%, 30%, 20%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1.9%, 0.7%, 0.0%, 0.0.0%, or any of the activities of a corresponding first binding domain (e.g., immunostimulatory cytokine or variant thereof) in a free state, in the hinge region of the antigen binding polypeptide (e.g., the hinge region of a heavy chain of an antibody (e.g., full length antibody), or the hinge region between the second binding domain (e.g., ligand, receptor, VHH, scFv, or Fab) and an Fc domain subunit (or portion thereof).
In some embodiments, the "corresponding first binding domain" (e.g., "corresponding cytokine or variant thereof") is the same as the first binding domain (e.g., cytokine or variant thereof) located at the hinge region, but is expressed in a different state or at a different location. By a first binding domain (e.g., a cytokine or variant thereof) in a "free state" is meant herein a first binding domain (e.g., a cytokine or variant thereof) in a soluble form, without attachment of any portion such as the N-terminus or the C-terminus of a cell membrane or another molecule (e.g., an Fc fragment, or a full-length antibody or antigen-binding fragment (e.g., ligand, receptor, VHH, scFv, or Fab).
In some embodiments, the activity (binding affinity to a first target molecule, such as a cytokine receptor, or subunit thereof, and/or biological activity) of a first binding domain (e.g., a cytokine or variant thereof) located in the heavy chain hinge region of a full-length antibody in the absence of binding of a second binding domain of the full-length antibody to a second target antigen is no more than about 50% (e.g., no more than about any of 40%, 30%, 20%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, or 0%) of the activity of the corresponding first binding domain (e.g., cytokine or variant thereof) expressed at any of: i) N-terminal of VH of full length antibody, ii) N-terminal of VL of full length antibody, iii) C-terminal of heavy chain of full length antibody, iv) C-terminal of CL of full length antibody, and v) N-terminal of Fc domain subunit of full length antibody. In some embodiments, the activity (binding affinity to a first target molecule, such as a cytokine receptor, and/or biological activity) of a first binding domain (e.g., cytokine or variant thereof) of a hinge region located between the second binding domain (e.g., scFv or Fab) and an Fc domain subunit (or portion thereof) in the absence of binding of the second binding domain (e.g., scFv or Fab) to the second target molecule is no more than about 50% (e.g., no more than about any of 40%, 30%, 20%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1% or 0%) of the activity of the corresponding first binding domain (e.g., cytokine or variant thereof) expressed at any of: i) N-terminal of VH of the second binding domain (e.g., scFv or Fab), ii) N-terminal of VL of the second binding domain (e.g., scFv or Fab), iii) C-terminal of Fc domain subunit (or portion thereof), iv) C-terminal of CL of the second binding domain (Fab), and v) N-terminal of Fc domain subunit. In some embodiments, the activity (binding affinity to a first target molecule, such as a cytokine receptor or subunit thereof, and/or biological activity) of a first binding domain (e.g., a cytokine or variant thereof) located in the hinge region between the second binding domain (e.g., VHH, ligand or receptor) and the Fc domain subunit (or portion thereof) in the absence of binding of the second binding domain (e.g., VHH, ligand or receptor) to the second target antigen is no more than about 50% (e.g., no more than about any of 40%, 30%, 20%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1% or 0%) of the activity of the corresponding first binding domain (e.g., cytokine or variant thereof) expressed at any of: i) The N-terminus of a second binding domain (e.g., VHH, ligand, or receptor), ii) the C-terminus of an Fc domain subunit (or portion thereof), and iii) the N-terminus of an Fc domain subunit.
In some embodiments, the activity (binding affinity to a first target molecule, such as a cytokine receptor, or subunit thereof, and/or biological activity) of a first binding domain (e.g., a cytokine or variant thereof) located in the heavy chain hinge region of a full-length antibody in the presence of binding of a second binding domain of the full-length antibody to a second target molecule is at least about 70% (e.g., at least about any of 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 300%, 400%, 500% or more) of the activity of the corresponding first binding domain (e.g., cytokine or variant thereof) expressed at any of: i) N-terminal of VH of full length antibody, ii) N-terminal of VL of full length antibody, iii) C-terminal of heavy chain of full length antibody, iv) C-terminal of CL of full length antibody, and v) N-terminal of Fc subunit of full length antibody. In some embodiments, the activity (binding affinity to a first target molecule, such as a cytokine receptor or subunit thereof, and/or biological activity) of a first binding domain (e.g., cytokine or variant thereof) located in the hinge region between the second binding domain (e.g., scFv or Fab) and the Fc domain subunit (or portion thereof) is at least about 70% (e.g., at least about any of 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 300%, 400%, 500% or more) of the activity of a corresponding first binding domain (e.g., cytokine or variant thereof) expressed at any of: i) N-terminal of VH of the second binding domain (e.g., scFv or Fab), ii) N-terminal of VL of the second binding domain (e.g., scFv or Fab), iii) C-terminal of Fc domain subunit (or portion thereof), iv) C-terminal of CL of the second binding domain (Fab), and v) N-terminal of Fc domain subunit. In some embodiments, the activity (binding affinity to a first target molecule, such as a cytokine receptor or subunit thereof, and/or biological activity) of a first binding domain (e.g., a cytokine or variant thereof) of a hinge region between the second binding domain (e.g., VHH, ligand, or receptor) and an Fc domain subunit (or portion thereof) in the presence of binding of the second binding domain (e.g., VHH, ligand, or receptor) to a second target molecule is at least about 70% (e.g., at least about any one of 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 300%, 400%, 500% or more) of the activity of the corresponding first binding domain (e.g., cytokine or variant thereof) expressed at any one of: i) The N-terminus of a second binding domain (e.g., VHH, ligand, or receptor), ii) the C-terminus of an Fc domain subunit (or portion thereof), and iii) the N-terminus of an Fc domain subunit.
In some embodiments, the first binding domain is a ligand or variant thereof. In some embodiments, the first binding domain is a cytokine (e.g., an immunostimulatory cytokine) or variant thereof. In some embodiments, the immunostimulatory cytokine is selected from the group consisting of: IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-12, IL-15, IL-17, IL-18, IL-21, IL-22, IL-23, IL-27, IFN-alpha, IFN-beta, IFN-gamma, TNF-alpha, erythropoietin, thrombopoietin, G-CSF, M-CSF, SCF, and GM-CSF. In some embodiments, the activity (binding affinity to the corresponding cytokine receptor or subunit thereof, and/or biological activity) of the cytokine variant in the free state is no more than about 80% (e.g., no more than any of about 70%, 60%, 50%, 40%, 30%, 20%, 10%, or 5%) of the activity of the corresponding wild-type cytokine in the free state. In some embodiments, the activity (binding affinity to the corresponding cytokine receptor or subunit thereof, and/or biological activity) of the cytokine variant in the free state is the same as or similar to (e.g., within about ±20% difference from) the activity of the corresponding wild-type cytokine in the free state. In some embodiments, the cytokine or variant thereof is a cytokine variant. In some embodiments, the first binding domain is an immunostimulatory cytokine variant, and wherein the activity of the immunostimulatory cytokine variant in the free state (binding affinity to a first target molecule, such as a corresponding cytokine receptor or subunit thereof, and/or biological activity) is no more than about 80% (e.g., no more than any of about 70%, 60%, 50%, 40%, 30%, 20%, 10%, or 5%) of the activity of the corresponding wild-type immunostimulatory cytokine in the free state.
In some embodiments, in the absence of binding of a second binding domain (e.g., ligand, receptor, VHH, scFv, or Fab) of an immunomodulatory molecule described herein to a second target molecule, the activity (binding affinity, and/or biological activity) of a first binding domain (e.g., cytokine variant) to a first target molecule, e.g., a corresponding wild-type or non-variant first binding domain (e.g., wild-type cytokine, or a corresponding recombinant "wild-type" cytokine expressed in the same form but comprising a wild-type subunit) is no more than about 80% (e.g., no more than about 70%, 60%, 50%, 40%, 30%, 20%, 10%, 9%, 8%, 7%, 6%, 5%, 3%, 2%, 3%, 0.0%, 3%, 0.0%, or any one of the second binding domain (e.g., ligand, receptor, VHH, scFv, or Fab) of the second binding domain (e.g., ligand, receptor, or Fab) of the immunomodulatory molecule to the second target molecule is no more than about 80% (e.g., binding affinity, and/or biological activity) of the corresponding wild-type or non-variant first binding domain (e.g., wild-type cytokine) of the same form but comprising the wild-type subunit. For example, in some embodiments, the IL-12 variant comprises, from N-terminus to C-terminus: variant p40 subunit-linker-wild-type p35 subunit, and the corresponding recombinant "wild-type" IL-12 comprises from N-terminus to C-terminus: wild-type p40 subunit-linker-wild-type p35 subunit. In some embodiments, the cytokine variant is an IL-2 variant, and the corresponding wild-type cytokine is "wild-type" IL-2.
In some embodiments, the activity (binding affinity, and/or biological activity) of a first binding domain (e.g., a cytokine variant) of a second binding domain (e.g., a ligand, receptor, VHH, scFv, or Fab) of an immunomodulatory molecule described herein, in the presence of binding to the second target molecule, is at least about 1% (e.g., at least about 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 140%, 120%, more, 120%, or more) of the activity of a corresponding wild-type or non-variant first binding domain (e.g., wild-type cytokine, or a corresponding recombinant "wild-type" cytokine expressed in the same form but comprising a wild-type subunit) located in the hinge region of the antigen binding polypeptide, or the hinge region located between the second binding domain (e.g., ligand, receptor, VHH, scFv, or Fab) and the Fc domain subunit (or portion thereof), is at least about 1% (e.g., at least about 2%, 3%, 4%, 5%, 6%, 7%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 140%, 120%, more than 120%, or more than any of the corresponding wild-type cytokine).
In some embodiments, in the absence of binding of a second binding domain (e.g., ligand, receptor, VHH, scFv, or Fab) of an immunomodulatory molecule described herein to a second target molecule, the activity (binding affinity to a first target molecule, such as a corresponding wild-type or non-variant first binding domain (e.g., wild-type cytokine, or a corresponding recombinant "wild-type" cytokine of the same form but comprising a wild-type subunit) of the antigen binding polypeptide is no more than about 80% (e.g., no more than about 70%, 60%, 50%, 40%, 30%, 20%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1.9%, 0.0%, or any of the following) of the activity of the first binding domain (e.g., ligand, receptor, VHH, scFv, or Fab) of the immunomodulatory molecule, or a second binding domain of the immunomodulatory molecule described herein is not present, such as a ligand, receptor, or Fab, to the second target molecule; and in the presence of binding of a second binding domain (e.g., ligand, receptor, VHH, scFv, or Fab) of an immunomodulatory molecule described herein to a second target molecule, activity of a first binding domain (e.g., a cytokine variant) located at the hinge region of the antigen binding polypeptide (e.g., at the heavy chain of an antibody (e.g., a full length antibody), or at the hinge region between the second binding domain (e.g., ligand, receptor, VHH, scFv, or Fab) and an Fc domain subunit (or portion thereof) (binding affinity with a first target molecule such as a corresponding cytokine receptor or subunit thereof), and/or biological activity) is at least about 1% (e.g., any of at least about 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200% or more) of the activity of a corresponding wild-type or non-variant first binding domain (e.g., a wild-type cytokine, or a corresponding recombinant "wild-type" cytokine of the same form but comprising a wild-type subunit) located in the same region.
Binding affinity
The binding affinity of a molecule (e.g., a cytokine moiety, an immunomodulatory molecule comprising a cytokine moiety, or a binding domain) and its binding partner (e.g., a cytokine receptor or subunit thereof, or a target molecule) can be determined experimentally by any suitable ligand binding assay or antibody/antigen binding assay known in the art, such as western blotting, sandwich enzyme-linked immunosorbent assay (ELISA), mesoscale discovery (MSD) electrochemiluminescence, microbead-based Multiplex Immunoassay (MIA), RIA, surface Plasmon Resonance (SPR), ECL, IRMA, FACS, EIA, biacore assay, octet assay, peptide scan, and the like. For example, by using cytokines or variants thereof, immunomodulatory molecules comprising cytokines or variants thereof or their corresponding receptors or subunits thereof (which are labeled with various labeling reagents) and using biacore x (manufactured by Amersham Biosciences), which is an over-the-counter drug, a measurement kit or similar kit according to the user manual and experimental protocols attached to the kit, it is possible to perform an easy analysis.
In some embodiments, protein microarrays are used to analyze on a large scale the interactions, functions, and activities of binding domains (e.g., cytokine moieties) described herein with their corresponding target molecules (e.g., cytokine receptors). The protein chip has a support surface to which a series of capture proteins (e.g., cytokine receptors or subunits thereof) are bound. Fluorescent-labeled probe molecules (e.g., cytokine moieties or immunomodulatory molecules as described herein) are then added to the array and, upon interaction with the bound capture proteins, fluorescent signals are released and read by a laser scanner.
In some embodiments, SPR (biacore t-200) is used to measure the binding affinity of a binding domain (e.g., cytokine moiety) or an immunomodulatory molecule and its corresponding target molecule (e.g., cytokine receptor or subunit thereof) described herein. For example, anti-human antibodies are coupled to the surface of a CM-5 sensor chip (e.g., using EDC/NHS chemistry). Human cytokine receptor-Fc fusion proteins (e.g., IL-2Rα -Fc, IL-2Rβ -Fc, IL-2Rγ -Fc) are then used on the surfaceCapturing the ligand. Serial dilutions of an immunomodulatory molecule comprising a cytokine moiety (e.g., an IL-2 variant) can be combined with the captured ligand (the free IL-2 variant is used as a control), and a Response Unit (RU) can be plotted against the concentration of the immunomodulatory molecule to determine EC50 values, or plotted against time to monitor the binding and dissociation of the immunomodulatory molecule to the cytokine receptor-Fc in real time. Equilibrium dissociation constant (K) D ) And dissociation rate constants, can be determined by kinetic analysis using Biacore evaluation software. The binding affinity of each test immunoregulatory molecule to a cytokine receptor can be calculated as a percentage of the cytokine moiety relative to the corresponding free state. In some embodiments, a cell line that expresses a cytokine receptor (e.g., IL-2R) on the cell surface is incubated with an immunomodulatory molecule comprising a cytokine moiety (e.g., IL-2 variant) described herein, the cells are washed after incubation, and then anti-IgG conjugated to a fluorescent protein (e.g., APC) is added to detect the binding affinity of the immunomodulatory molecule to the cells, such as by FACS.
In some embodiments, the binding K between a binding domain (e.g., cytokine or variant thereof) and its corresponding target molecule (e.g., cytokine receptor or subunit thereof) in a free state D Is about 10 or less -5 M、≤10 -6 M、≤10 -7 M、≤10 - 8 M、≤10 -9 M、≤10 -10 M、≤10 -11 M or less than or equal to 10 -12 M. In some embodiments, in the absence of binding of a second binding domain (e.g., ligand, receptor, VHH, scFv, or Fab) of an immunomodulatory molecule described herein to a second target molecule, K is located at the hinge region of an antigen binding polypeptide (e.g., at the hinge region of the heavy chain of an antibody (e.g., a full length antibody), or at the hinge region between the second binding domain (e.g., ligand, receptor, VHH, scFv, or Fab) and a first binding domain (e.g., cytokine or variant thereof) and a subunit of an Fc domain (or portion thereof) of a corresponding first target molecule (e.g., cytokine receptor or subunit thereof) D Undetectable (e.g., no binding), or the K D Higher than (i.e., weaker than in combination) in the presence ofK in the case of binding of a second binding domain (e.g., ligand, receptor, VHH, scFv or Fab) of an immunomodulatory molecule described herein to a second target molecule D 。
Biological activity
Various methods, such as bioassays, for determining the biological activity (biological activity/bioactivity) of a binding domain (e.g., a cytokine or variant thereof) or an immunomodulatory molecule described herein are described in the art. Any antigen/antibody binding, ligand/receptor binding or cytokine assay known in the art may be suitable for testing the biological activity of the binding domains (e.g., cytokine moieties) or immunomodulatory molecules described herein.
For example, bioassays focus on the biological activity of cytokines or ligands/receptors and use it as a readout. In bioassays, the activity of a sample is tested on sensitive cell lines (e.g., primary cell cultures or in vitro adapted cell lines, which rely on and/or are responsive to a test sample) and the results of this activity (e.g., cell proliferation) are compared to standard cytokine preparations. Other aspects of cytokine biological activity include induction of further cytokine secretion, induction of killing, antiviral activity, degranulation, cytotoxicity, chemotaxis, and promotion of colony formation. In vitro assays to measure all of these activities are available. See, e.g., from(http:// tools. Thermo folder. Com/content/sfs/manual/cytokine-Bioassays. Pdf) Bioassays-/>"cytokine bioassay (Cytokine Bioassays)", the contents of which are incorporated herein by reference in their entirety.
For example, in cytokine-induced proliferation assays, a sample (e.g., IL-2 moiety or IL-2 immunoregulatory molecule) and a standard (e.g., IL-2 in free form) are diluted by serial dilution in a medium-filled assay plate, washing the finger Cells (e.g., CTLL-2 or PBMCs stimulated with anti-CD 3 antibodies) are shown and resuspended in medium and then added to each well. The cells were incubated at 37℃with 5% CO 2 Is cultured in the humidified incubator for a sufficient time (e.g., 24 hours or more). Cell viability assay reagents (e.g., resazurin, MTT assay) can then be added to the plate and incubated well before reading with a spectrophotometer. EC50 values for cell proliferation (the concentration of test sample required to exhibit 50% of maximum response) can then be obtained from a non-linear regression analysis of the dose-response curve. The cell number can also be counted under a microscope and compared to the cell number of a standard or control treatment. For another example, in cytokine-induced cytokine production assays, samples (e.g., IL-12 or IL-23 moieties, or IL-12 or IL-23 immunoregulatory molecules) and standards (e.g., IL-12 or IL-23 in free form) are diluted by serial dilution in assay plates filled with medium, and indicator cells (e.g., splenocytes, activated CD4 are washed + T cell or activated CD8 + T cells) and resuspended in medium and then added to each well. Cells were incubated at 37℃with 5% CO 2 For a sufficient period of time (e.g., 24-48 hours) and then the supernatant is collected for use in determining cytokine expression by ELISA, following an ELISA protocol for the target cytokine of interest (e.g., interferon gamma). For another example, in cytokine-induced cell surface marker expression assays, a sample (e.g., IFN-gamma moiety, or IFN-gamma immunoregulatory molecule) and a standard (e.g., IFN-gamma in free form) are diluted in a medium-filled assay plate via serial dilution, washing indicator cells (e.g., HEK-Blue) TM IFN-gamma cells) and resuspended in medium and then added to each well. Cells were incubated at 37℃with 5% CO 2 For a sufficient period of time (e.g., 24-48 hours), then cell surface expression of the biomarker (e.g., PD-L1) can be detected (e.g., using an anti-human PD-L1APC conjugated antibody) and measured by ELISA or FACS. See also example 1 of the exemplary method.
The biological activity of a binding domain (e.g., cytokine moiety) or an immunomodulatory molecule described herein can also be reflected by in vivo or ex vivo experiments, for example, by measuring proliferation of an indicator cell (e.g., proliferation of cd8+ cells, NK cells, or Tregs after administration of an IL-2 moiety or IL-2 immunomodulatory molecule); by measuring induction or inhibition of cytokine secretion; by measuring the decrease in tumor volume in tumor xenograft mice after injection of the test cytokine moiety or immunomodulatory molecule described herein; or by measuring an autoimmune score.
Cell signaling assays can also be used to test the biological activity of binding domains (e.g., cytokine moieties) or immunomodulatory molecules described herein. There are a variety of cell signaling assay kits commercially available, for example, to detect analytes, such as ADP, AMP, UDP, GDP and growth factors, or phosphatase assays, generated during enzymatic reactions involving signaling to quantify total signaling protein and phosphorylated forms of signaling protein. For example, after incubating the cells with a cytokine moiety or immunomodulatory molecule described herein to determine if a particular kinase is active, the cell lysate is exposed to a known substrate for the enzyme in the presence of radioactive phosphate. The product was isolated by electrophoresis (with or without immunoprecipitation) and the gel was then exposed to X-ray film to determine whether the protein incorporated the isotope. In some embodiments, the biological activity of a binding domain (e.g., cytokine moiety) or an immunomodulatory molecule described herein on a cell is measured by immunohistochemistry to localize a signal transduction protein. For example, antibodies to the signaling protein itself or the signaling protein in an activated state may be used. These antibodies have recognition epitopes that include phosphate or other activated conformations. In some embodiments, movement of a particular signal transduction protein (e.g., nuclear translocation of a signal transduction molecule) can be tracked by incorporating a fluorescent protein gene (e.g., green Fluorescent Protein (GFP)) into a genetic carrier encoding the protein to be studied. In some embodiments, the binding domains (e.g., cytokine moieties) or immunomodulatory molecules described herein are tested for biological activity on cells by western blotting. For example, all tyrosine-phosphorylated proteins (or other phosphates The phosphorylated amino acid, e.g., serine or threonine, can be detected with an anti-phosphotyrosine antibody (or an antibody against other phosphorylated amino acids) on western blots of cell lysates obtained after time-series stimulation. In some embodiments, the biological activity of a binding domain (e.g., cytokine moiety) or immunomodulatory molecule described herein on a cell can be measured by immunoprecipitation. For example, a specific signal transduction protein or a primary antibody to all tyrosine-phosphorylated proteins is cross-linked to the beads. Cells after incubation with cytokine moieties or immunomodulatory molecules described herein are lysed in a buffer containing a protease inhibitor and then incubated with antibody coated beads. Proteins were separated using SDS electrophoresis, and then identified using the procedure described for western blotting. In some embodiments, a glutathione S-transferase (GST) binding or "pull down" assay may also be used that determines direct protein-protein (e.g., signal transduction protein) interactions. Cell-based signal transduction assays may also be used. Briefly, reporter cell lines (e.g., HEK-Blue TM ) Which stably express the respective receptor of the test cytokine moiety or immunoregulatory molecule, the respective signal transduction factor of the cytokine signal transduction pathway (e.g., STAT, JAK) and the cytokine signal pathway-induced reporter (e.g., fluorescent protein, or secreted embryonic alkaline phosphatase), CO at 37 ℃ in the presence of the test cytokine moiety or immunoregulatory molecule 2 Culturing in an incubator for a sufficient period of time (e.g., 24-48 hours), then the reporter can be detected, such as using a microscope or FACS for fluorescent protein, or determining alkaline phosphatase activity using a colorimetric enzyme (e.g., QUANTI-Blue TM ) To detect secreted embryonic alkaline phosphatase in cell culture medium.
Using IL-2 as an example of a first binding domain, STAT5 and ERK1/2 signaling can be measured to reflect IL-2 partial or immunomodulatory molecular bioactivity, for example, by measuring phosphorylation of STAT5 and ERK1/2 using any suitable method known in the art. For example, STAT5 and ERK1/2 phosphorylation, antibodies specific for the phosphorylated forms of these molecules can be used with flow cytometryThe combination was analyzed for measurement. For example, freshly isolated PBMC are incubated with IL-2 or a variant thereof or an IL-2 immunomodulatory molecule at 37 ℃. Immediately after incubation, cells were fixed (e.g., using Cytofix buffer) to maintain the phosphorylated state and permeabilized (e.g., using Phosflow Perm buffer III). Cells were stained with fluorophore labelled antibodies to phosphorylated STAT5 or ERK1/2 and analyzed by flow cytometry. Alternatively, a test sample (e.g., an IL-2 cytokine moiety or IL-2 immunomodulatory molecule as described herein) can be intraperitoneally injected into a mouse, and then the total spleen cells can be isolated and immediately fixed (e.g., phosphoflow) TM Lyse/Fix buffer), washed with ice-cold PBS, stained with anti-CD 4 and anti-CD 25 antibodies, and then permeabilized (e.g., phosFlow Perm buffer III). The cells were then washed with ice-cold FACS buffer, stained with anti-FoxP 3, washed with ice-cold FACS buffer, and stained with fluorophore-labeled anti-phosphoryl-STAT 5 at room temperature. The cells are washed with FACS buffer, and data can then be collected on a FACS cytometer and analyzed. PI 3-kinase signal transduction can also be measured using any suitable method known in the art to reflect IL-2 biological activity. For example, PI 3-kinase signaling can be measured using an antibody binding flow cytometry assay specific for phosphorylated-S6 ribosomal proteins.
In some embodiments, the first binding domain (e.g., immunostimulatory cytokine moiety) or immunomodulatory molecule described herein is capable of activating immune cells, such as inducing test cytokine (e.g., IL-2 moiety or IL-2 immunomodulatory molecule described herein) dependent immune cells (e.g., PBMC, NK cells, cd8+ T cells, th17 cells) to proliferate, differentiate and/or activate, cytokine secretion, activate signal transduction (e.g., inducing STAT5 phosphorylation, ERK1/2 phosphorylation, or stimulating PI 3-kinase signal transduction), and/or inducing immune cells to kill tumor cells or infected cells. In some embodiments, a second binding domain (e.g., an immunosuppressive cytokine moiety) or an immunomodulatory molecule described herein is capable of inhibiting immune cells, such as inhibiting cytokine (e.g., pro-inflammatory cytokine) production, antigen presentation, or immunization MHC molecule expression by cells, or to inhibit or improve signal transduction. In some embodiments, the immune cell is selected from the group consisting of: monocytes, dendritic cells, macrophages, B cells, killer T cells (T c Cytotoxic T lymphocytes or CTLs), helper T cells (T) h ) Regulatory T cells (tregs), γδ T cells, natural Killer T (NKT) cells and Natural Killer (NK) cells.
In some embodiments, the activity of the variant binding domain (e.g., cytokine variant) in the free state to activate/inhibit (or up/down regulate) the immune response is the same as or similar to (e.g., within about ±20% of) the activity of the corresponding wild-type or non-variant binding domain (e.g., wild-type cytokine) in the free state to activate/inhibit (or up/down regulate) the immune response. In some embodiments, a variant binding domain (e.g., cytokine variant) comprises a mutation or modification (e.g., post-translational modification) that reduces the activity of its activating/suppressing (or up/down regulating) an immune response (e.g., no more than any of about 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, or 0% of the biological activity of a wild-type or non-variant binding domain (e.g., wild-type cytokine) when bound in a free state or in the absence of the second target molecule-second binding domain of an immunomodulatory molecule described herein. In some embodiments, the activity of activating/inhibiting (or up/down regulating) the immune response of a variant binding domain (e.g., cytokine variant) in the presence of a second target molecule-second binding domain binding of an immunomodulatory molecule described herein is at least about 1% (e.g., at least about any of 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, or 200%) of the activity of the immune response of the corresponding wild-type or non-variant binding domain (e.g., wild-type cytokine).
Hinge
The Fd region (VH and CH1 domains) and the Fc region of the heavy chain of the immunoglobulin are hinged. In some embodiments, the hinge region connects the binding domain (e.g., ligand, receptor, VHH, scFv, or Fab) and the Fc domain subunit or portion thereof (e.g., ch2+ch3, or CH2 only). The hinge region found in the IgG, igA and IgD immunoglobulin classes acts as a flexible spacer that allows the Fab portion of the immunoglobulin to move freely in space relative to the Fc region. Hinge domains are structurally diverse, with sequence and length varying between immunoglobulin classes and subclasses. The heavy chains are interconnected via disulfide bonds of the hinge region. According to crystallographic studies, the immunoglobulin hinge region can be further subdivided in structure and function into three regions: an upper hinge, a core and a lower hinge. See Shin et al Immunological Reviews 130:87 (1992). The upper hinge includes these amino acids from the carboxy terminus of CH1 to the first residue in the hinge that limits movement, typically the first cysteine residue that forms an interchain disulfide bond between the two heavy chains. The length of the upper hinge region is related to the segment flexibility of the antibody. The core hinge region comprises inter-heavy chain disulfide bonds. The lower hinge region is attached to the amino terminus of the CH2 domain and includes residues therein. Id. The hinge region of human IgG1 antibodies corresponds to amino acid positions 216-230 according to EU numbering as specified in Kabat. Human IgG 1 The core hinge region of (a) contains the sequence Cys-Pro-Cys, which when dimerized by disulfide bond formation, produces a cyclic octapeptide, which is believed to act as a pivot, thereby imparting flexibility. Conformational changes allowed by the structure and flexibility of the immunoglobulin hinge region polypeptide sequence may affect the effector function of the Fc portion of the antibody.
In some embodiments, the hinge region may contain one or more glycosylation sites, which include many structurally different types of carbohydrate attachment sites. For example IgA 1 The inclusion of five glycosylation sites in the 17 amino acid segment of the hinge region confers specific resistance to intestinal proteases on the hinge region polypeptide, which is considered an advantageous property of secreted immunoglobulins.
In some embodiments, the avoidanceThe epidemic-modulating molecule comprises a hinge region present in a naturally occurring parent antibody. For example, the parent antibody is an IgG1 antibody and the hinge region of the antibodies or antigen binding fragments within the immunomodulatory molecules described herein is an IgG1 type hinge region. In some embodiments, the immunoregulatory molecule contains a modification of the heavy chain hinge region of the antibody. For example, the hinge region or portion thereof has been modified, e.g., by deletion, insertion, or substitution, e.g., having a different class (e.g., igG, igA, or IgE) and subclass (e.g., igG) than the naturally occurring class of antibodies 1 、IgG 2 、IgG 3 And IgG 4 Etc.) a hinge region or a portion thereof. For example, an IgG1, igG2, or IgG3 antibody can contain an IgG 4-type hinge region. In some embodiments, the hinge region or portion thereof comprises mutations (e.g., deletions, insertions, or substitutions) at one or more of the upper hinge, core, and lower hinge of the hinge region, so long as interchain disulfide bonds can still be formed, the immunomodulatory molecule has flexibility to ensure binding of the target antigen-antigen binding fragment, to mask cytokine activity in the absence of target antigen-antibody binding, to not mask cytokine activity in the presence of target antigen-antibody binding, to provide flexibility and/or sufficient space between two cytokine subunits or two cytokine portions to ensure proper cytokine activity (binding affinity and/or biological activity), and/or optionally to not eliminate effector function of the Fc portion. In some embodiments, the hinge region is or is derived from a human IgG1, igG2, igG3, or IgG4 hinge. In some embodiments, the hinge region is a mutated human IgG1, igG2, igG3, or IgG4 hinge. In some embodiments, one or more mutations (e.g., deletions, insertions, or substitutions) are introduced at one or more of the upper hinge, core, and lower hinge of the hinge region to reduce or eliminate effector functions (e.g., ADCC and/or CDC) of the Fc domain, such as L234 and/or L235 mutations in the IgG1 lower hinge region, e.g., one or both of L234A, L234K, L234D, L235E, L K and L235A mutations. In some embodiments, the hinge region comprises L234K and L235K mutations. In some embodiments, the hinge region comprises L234D and L235E mutations. In some embodiments, the hinge region is truncated or mutated to have fewer Cysteine to reduce disulfide mismatch during dimerization of the Fc domain. In some embodiments, one or more asymmetric charging mutations are introduced into the lower hinge to promote heterodimerization formation, e.g., one polypeptide comprises l234k+l235k in the IgG1 lower hinge region and a counterpart polypeptide comprises l234d+l235e in the IgG1 lower hinge region. In some embodiments, the hinge region comprises the amino acid sequence of EPKSCDKTHTCPPCPAPELLGGP (SEQ ID NO: 76). In some embodiments, the hinge region is an IgG1 hinge comprising L234K and L235K mutations. In some embodiments, the hinge region comprises the amino acid sequence of EPKSCDKTHTCPPCPAPEKKGGP (SEQ ID NO: 77). In some embodiments, the hinge region is an IgG1 hinge comprising L234D and L235E mutations. In some embodiments, the hinge region comprises the amino acid sequence of EPKSCDKTHTCPPCPAPEDEGGP (SEQ ID NO: 78). In some embodiments, the hinge region comprises the amino acid sequence of ERKCCVECPPCPAPPVAGP (SEQ ID NO: 82). In some embodiments, the hinge region comprises the amino acid sequence of ESKYGPPCPSSCPAPEFLGGP (SEQ ID NO: 83). In some embodiments, the hinge region comprises the amino acid sequence of ESKYGPPCPPCPAPEFLGGP (SEQ ID NO: 94). In some embodiments, the hinge region comprises the amino acid sequence of any of EPKSCDKDKTHTCPPCPAPELLGGP (SEQ ID NO: 79), EPKSCDKDKTHTCPPCPAPEKKGGP (SEQ ID NO: 80) or EPKSCDKDKTHTCPPCPAPEDEGGP (SEQ ID NO: 81). In some embodiments, the hinge region comprises the amino acid sequence of any of EPKSCDKPDKTHTCPPCPAPELLGGP (SEQ ID NO: 91), EPKSCDKPDKTHTCPPCPAPEKKGGP (SEQ ID NO: 92), EPKSCDKPDKTHTCPPCPAPEDEGGP (SEQ ID NO: 93) or EPPKSCDKTHTCPPCPAPELLGGP (SEQ ID NO: 95). In some embodiments, the hinge region, such as the hinge N' portion, comprises the amino acid sequence of any of EPKSCDKP (SEQ ID NO: 90), EPKSCDK (SEQ ID NO: 84) or EPKSC (SEQ ID NO: 85). In some embodiments, the hinge region comprises the amino acid sequence of DKTHT (SEQ ID NO: 89). In some embodiments, the hinge region, such as the hinge C' portion, comprises the amino acid sequence of any of DKTHTCPPCPAPELLGGP (SEQ ID NO: 86), DKTHTCPPCPAPEKKGGP (SEQ ID NO: 87) or DKTHTCPPCPAPEDEGGP (SEQ ID NO: 88). In some embodiments, the hinge comprises the sequence of any one of SEQ ID NOS.76-95.
In some embodiments, a first binding domain (e.g., a cytokine or variant thereof) described herein is located N-terminal to the heavy chain hinge region of a full length antibody comprising a second binding domain, i.e., between the C-terminal end of CH1 and the N-terminal end of the heavy chain hinge region of a full length antibody. In some embodiments, the heavy chain fusion polypeptide comprises, from N-terminus to C-terminus: VH-CH 1-first binding domain (e.g., cytokine moiety) -hinge-CH 2-CH3. In some embodiments, the first binding domain (e.g., a cytokine or variant thereof) is located N-terminal to the hinge region between the second binding domain (e.g., a ligand, receptor, VHH, scFv, or Fab) and the Fc domain subunit or portion thereof (e.g., CH2-CH3 or CH 2). For example, in some embodiments, the immunomodulatory molecule comprises from N-terminus to C-terminus a polypeptide of any one of: (1) VH-first binding domain (e.g., cytokine moiety) -hinge-CH 2-CH3; (2) VL-first binding domain (e.g., cytokine moiety) -hinge-CH 2-CH3; (3) VH-optional linker-VL-first binding domain (e.g., cytokine moiety) -hinge-CH 2-CH3; (4) VL-optional linker-VH-first binding domain (e.g., cytokine moiety) -hinge-CH 2-CH3; (5) VH-CH 1-first binding domain (e.g., cytokine moiety) -hinge-CH 2-CH3; (6) VH-first binding domain (e.g., cytokine moiety) -hinge-CH 2; (7) VL-cytokine moiety-hinge-CH 2; (8) VH-optional linker-VL-first binding domain (e.g., cytokine moiety) -hinge-CH 2; (9) VL-optional linker-VH-first binding domain (e.g., cytokine moiety) -hinge-CH 2; (10) VH-CH 1-first binding domain (e.g., cytokine moiety) -hinge-CH 2; (11) Ligand-optional linker-first binding domain (e.g., cytokine moiety) -hinge-CH 2-CH3; (12) Ligand-optional linker-first binding domain (e.g., cytokine moiety) -hinge-CH 2; (13) Receptor-optional linker-first binding domain (e.g., cytokine moiety) -hinge-CH 2-CH3; or (14) a receptor-optionally a linker-a first binding domain (e.g., cytokine moiety) -a hinge-CH 2.
In some embodiments, a first binding domain (e.g., a cytokine or variant thereof) described herein is located at the C-terminus of the heavy chain hinge region of a full-length antibody comprising a second binding domain, i.e., a heavy chain fusion polypeptide comprising, from N-terminus to C-terminus: VH-CH 1-hinge-first binding domain (e.g., cytokine moiety) -CH2-CH3. In some embodiments, the first binding domain (e.g., a cytokine or variant thereof) is located at the C-terminus of the hinge region between the second binding domain (e.g., a ligand, receptor, VHH, scFv, or Fab) and the Fc domain subunit or portion thereof (e.g., CH 2). For example, in some embodiments, the immunomodulatory molecule comprises from N-terminus to C-terminus a polypeptide of any one of: (1) VH-hinge-first binding domain (e.g., cytokine moiety) -CH2-CH3; (2) VL-hinge-first binding domain (e.g., cytokine moiety) -CH2-CH3; (3) VH-optional linker-VL-hinge-first binding domain (e.g., cytokine moiety) -CH2-CH3; (4) VL-optional linker-VH-hinge-first binding domain (e.g., cytokine moiety) -CH2-CH3; (5) VH-CH 1-hinge-first binding domain (e.g., cytokine moiety) -CH2-CH3; (6) VH-hinge-first binding domain (e.g., cytokine moiety) -CH2; (7) VL-hinge-first binding domain (e.g., cytokine moiety) -CH2; (8) VH-optional linker-VL-hinge-first binding domain (e.g., cytokine moiety) -CH2; (9) VL-optional linker-VH-hinge-first binding domain (e.g., cytokine moiety) -CH2; (10) VH-CH 1-hinge-first binding domain (e.g., cytokine moiety) -CH2; (11) Ligand-hinge-first binding domain (e.g., cytokine moiety) -CH2-CH3; (12) Ligand-hinge-first binding domain (e.g., cytokine moiety) -CH2; (13) Receptor-hinge-first binding domain (e.g., cytokine moiety) -CH2-CH3; or (14) a receptor-hinge-first binding domain (e.g., cytokine moiety) -CH2.
In some embodiments, a first binding domain (e.g., a cytokine or variant thereof) described herein is located within the hinge region of the heavy chain of a full-length antibody comprising a second binding domain, i.e., a heavy chain fusion polypeptide comprising, from N-terminus to C-terminus: VH-CH 1-hinge N 'moiety-first binding domain (e.g., cytokine moiety) -hinge C' moiety-CH 2-CH3. In some embodiments, the cytokine or variant thereof replaces a portion of the hinge region. In some embodiments, the cytokine or variant thereof is inserted into the hinge region without deleting any hinge amino acids. In some embodiments, a cytokine or variant thereof having a peptide linker fused to the N' of the cytokine or variant thereof is inserted into the hinge region. In some embodiments, a cytokine or variant thereof having a peptide linker fused to the C' of the cytokine or variant thereof is inserted into the hinge region. For example, in some embodiments, the hinge-cytokine moiety comprises the following structure from the N-terminus to the C-terminus: hinge N 'moiety-optional N' peptide linker-first binding domain (e.g., cytokine moiety) -optional C 'peptide linker-hinge C' moiety. In some embodiments, the hinge region is an IgG1 hinge and the cytokine or variant thereof is interposed between "EPKSC" (SEQ ID NO: 85) and "DKTHT" (SEQ ID NO: 89). In some embodiments, the N' peptide linker comprises the amino acid sequence of DKP (SEQ ID NO: 231) or P (SEQ ID NO: 242). Thus, in some embodiments, a cytokine or variant thereof is inserted between the otherwise introduced "DKP" and "DKTHT" sequences. In some embodiments, the N' peptide linker comprises the amino acid sequence of DKPGS (SEQ ID NO: 232), PGS (SEQ ID NO: 233), or GS (SEQ ID NO: 234). In some embodiments, the N' peptide linker comprises the amino acid sequence of DKGSG (SEQ ID NO: 235), PGSG (SEQ ID NO: 236), or GSG (SEQ ID NO: 203). In some embodiments, the N' peptide linker comprises the amino acid sequence of DKPGGSS (SEQ ID NO: 237), PGSGS (SEQ ID NO: 238), or GSGS (SEQ ID NO: 239). In some embodiments, the N' peptide linker comprises the amino acid sequence of DKPGSGGGGG (SEQ ID NO: 240), PGSGGGGG (SEQ ID NO: 241), GSGGGGG (SEQ ID NO: 206). In some embodiments, the cytokine or variant thereof is located within the hinge region between the antigen binding fragment (e.g., ligand, receptor, VHH, scFv, or Fab) and the Fc domain subunit or portion thereof (e.g., CH 2). For example, in some embodiments, the immunomodulatory molecule comprises from N-terminus to C-terminus a polypeptide of any one of: (1) VH-hinge N 'moiety-optional N' peptide linker-first binding domain (e.g., cytokine moiety) -optional C 'peptide linker-hinge C' moiety-CH 2-CH3; (2) VL-hinge N 'moiety-optionally N' peptide linker-first binding domain (e.g., cytokine moiety) -optionally C 'peptide linker-hinge C' moiety-CH 2-CH3; (3) VH-optional linker-VL-hinge N 'moiety-optional N' peptide linker-first binding domain (e.g., cytokine moiety) -optional C 'peptide linker-hinge C' moiety-CH 2-CH3; (4) VL-optional linker-VH-hinge N 'moiety-optional N' peptide linker-first binding domain (e.g., cytokine moiety) -optional C 'peptide linker-hinge C' moiety-CH 2-CH3; (5) VH-CH 1-hinge N 'moiety-optionally N' peptide linker-first binding domain (e.g., cytokine moiety) -optionally C 'peptide linker-hinge C' moiety-CH 2-CH3; (6) VH-hinge N 'moiety-optional N' peptide linker-first binding domain (e.g., cytokine moiety) -optional C 'peptide linker-hinge C' moiety-CH 2; (7) VL-hinge N 'moiety-optionally N' peptide linker-first binding domain (e.g., cytokine moiety) -optionally C 'peptide linker-hinge C' moiety-CH 2; (8) VH-optional linker-VL-hinge N 'moiety-optional N' peptide linker-first binding domain (e.g., cytokine moiety) -optional C 'peptide linker-hinge C' moiety-CH 2; (9) VL-optional linker-VH-hinge N 'moiety-optional N' peptide linker-first binding domain (e.g., cytokine moiety) -optional C 'peptide linker-hinge C' moiety-CH 2; (10) VH-CH 1-hinge N 'moiety-optionally N' peptide linker-first binding domain (e.g., cytokine moiety) -optionally C 'peptide linker-hinge C' moiety-CH 2; (11) Ligand-optional linker-hinge N 'moiety-optional N' peptide linker-first binding domain (e.g., cytokine moiety) -optional C 'peptide linker-hinge C' moiety-CH 2-CH3; (12) Ligand-optional linker-hinge N 'moiety-optional N' peptide linker-first binding domain (e.g., cytokine moiety) -optional C 'peptide linker-hinge C' moiety-CH 2; (13) Receptor-optional linker-hinge N 'moiety-optional N' peptide linker-first binding domain (e.g., cytokine moiety) -optional C 'peptide linker-hinge C' moiety-CH 2-CH3; or (14) a receptor-optionally a linker-hinge N 'moiety-optionally an N' peptide linker-a first binding domain (e.g., cytokine moiety) -optionally a C 'peptide linker-hinge C' moiety-CH 2.
Fc domain
In some embodiments, an immunomodulatory molecule described herein comprises an Fc domain, or portion thereof, at the C-terminus. The Fc domain comprises a CH2 domain and a CH3 domain. In some embodiments, the Fc domain portion comprises (consists essentially of, or consists of) a CH2 domain. In some embodiments, the Fc domain portion comprises (consists essentially of, or consists of) a CH3 domain.
In some embodiments, the Fc domain is derived from any of IgA, igD, igE, igG and IgM and subtypes thereof. In some embodiments, the Fc domain comprises CH2 and CH3. In some embodiments, the Fc domain is derived from IgG (e.g., igG1, igG2, igG3, or IgG 4). In some embodiments, the Fc domain is derived from human IgG. In some embodiments, the Fc domain is derived from human IgG1 or human IgG4. In some embodiments, two subunits of an Fc domain dimerize via one or more (e.g., 1, 2, 3, 4, or more) disulfide bonds. In some embodiments, each subunit of an Fc domain comprises a full-length Fc sequence. In some embodiments, each subunit of an Fc domain comprises an N-terminally truncated Fc sequence. In some embodiments, the Fc domain is truncated at the N-terminus, e.g., the first 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids of the complete immunoglobulin Fc domain are absent. In some embodiments, the Fc domain comprises the amino acid sequence of any of SEQ ID NOs 96-102.
Via the Fc domain, the immunoregulatory molecule can activate complement and interact with Fc receptors. This inherent immunoglobulin profile is considered disadvantageous because the immune modulatory molecules can target cells expressing Fc receptors, rather than the preferred antigen-bearing cells. Furthermore, the simultaneous activation of cytokine receptors and Fc receptor signaling pathways results in cytokine release, particularly in combination with the long half-life of immunoglobulin fusion proteins, making them difficult to use in therapeutic environments due to systemic toxicity. Thus, in some embodiments, the Fc domain is engineered to have altered binding to an Fc receptor (FcR), particularly to an fcγ receptor, and/or altered effector functions, such as altered (e.g., reduced or eliminated) antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell phagocytosis (ADCP), and/or complement-dependent cytotoxicity (CDC).
Although the presence of an Fc domain is critical to extending the half-life of an immunomodulatory molecule, in some cases it is beneficial to eliminate the effector functions associated with Fc receptor binding to the Fc domain. Thus, in some embodiments, the altered binding to Fc receptors and/or effector function is reduced binding to and/or effector function. In some embodiments, the Fc domain comprises one or more amino acid mutations that reduce binding of the Fc domain to an Fc receptor, particularly an fcγ receptor (responsible for ADCC). Preferably, such amino acid mutations do not reduce binding to FcRn receptor (responsible for half-life). In some embodiments, the Fc domain is derived from human IgG1 and comprises amino acid substitution N295A. In some embodiments, the Fc domain is derived from human IgG4 and comprises amino acid substitutions S228P and L235E in the hinge region. In some embodiments, the Fc domain is derived from human IgG1 and comprises amino acid substitutions L234A and L235A ("LALA") in the hinge region. In some embodiments, the Fc domain is derived from human IgG1 and comprises amino acid substitutions L234A and L235A in the hinge region, and, for example, P329G in each subunit thereof. See, e.g., lo m.et al j Biol chem.2017mar3; 292 (9) 3900-3908; schlothauer t.et al protein Eng Des sel.2016oct;29 (10):457-466.
In some embodiments, an Fc domain (e.g., human IgG 1) is mutated to remove one or more effector functions, such as ADCC, ADCP, or CDC, i.e., an Fc domain that is "null or" almost effector free. For example, in some embodiments, the Fc domain is an effector-free IgG1Fc comprising one or more of the following mutations (e.g., in each subunit thereof): L234A, L235E, G237A, A S and P331S. The combination of K322A, L234A and L235A in IgG1 was sufficient to almost completely eliminate FcgammaR and C1q binding (Hezareh et al JVirol 75,12161-12168,2001). The Medlmmune identified a set of three mutations L234F/L235E/P331S with very similar effects (oganesylan et al, acta Crystallographica 64,700-704,2008). In some embodiments, the Fc domain comprises a glycosylation modification on IgG1Fc domain N297, which is known to be necessary for optimal FcR interaction. The Fc domain modification may be any suitable IgG Fc engineering as described in Wang et al ("IgG Fc engineering to modulate antibody effector functions," Protein cell.2018Jan;9 (1): 63-73), the contents of which are incorporated herein by reference in their entirety.
In some embodiments, the Fc domain comprises two identical polypeptide chains (identical Fc subunits). Such Fc domains are also referred to herein as "homodimerized Fc domains". In some embodiments, each subunit of the homodimeric Fc domain comprises the amino acid sequence of any one of SEQ ID NOs 96 and 99-102.
In some embodiments, the Fc domain comprises a modification that promotes heterodimerization of two different polypeptide chains. Such Fc domains are also referred to herein as "heterodimeric Fc domains". In some embodiments, the Fc domain comprises a knob-to-hole (KIH) modification that includes a knob modification in one subunit of the Fc domain and a knob modification in the other subunit of the two subunits of the Fc domain. Any suitable knob-to-socket modification can be applied to the immunomodulatory molecules described herein, such as amino acid changes T22> Y in the B chain of the first CH3 domain (resulting in a knob), and Y86> T in the E chain of the chaperone CH3 domain (resulting in a socket). See also US20200087414, the contents of which are incorporated herein by reference in their entirety. In some embodiments, one subunit of the Fc domain comprises one or more of the T350V, L351Y, S400E, F405A and Y407V mutations relative to wild-type human IgG1 Fc; and the other subunit of the Fc domain comprises one or more of the T350V, T366L, N390R, K392M, T394W mutations relative to wild-type human IgG1 Fc. In some embodiments, one subunit of the Fc domain comprises the sequence of SEQ ID NO:97 and the other subunit of the Fc domain comprises the sequence of SEQ ID NO: 98.
In some embodiments, the Fc domain is a single chain Fc domain as described in WO2017134140 (the contents of which are incorporated herein by reference in their entirety).
Joint
In some embodiments, within an immunomodulatory molecule described herein, between two or more binding domains connected in series, a second binding domain (e.g., ligand, receptor, VHH, scFv, or Fab) and a first binding domain (e.g., cytokine moiety), a first binding domain (e.g., cytokine moiety) and CL, a first binding domain (e.g., cytokine moiety) and VH, a first binding domain (e.g., cytokine moiety) and VL, a CH1 domain and a first binding domain (e.g., cytokine moiety), two or more first binding domains connected in series (e.g., cytokine moiety), two or more subunits connected in series of cytokines or variants thereof, a first binding domain (e.g., cytokine moiety) and Fc domain subunit or portion thereof, a hinge region and CH1 domain, a hinge region and a first binding domain (e.g., cytokine moiety), an Fc domain subunit or portion thereof and antigen binding fragment, and/or a CH1 domain subunit and Fc domain subunit or portion thereof, are connected via a peptide, or more than one peptide, or more linker, such as a linker. In some embodiments, one or more of the linkers are the same. In some embodiments, one or more of the linkers are different (e.g., from one another). In some embodiments, one or more of the joints is a flexible joint. In some embodiments, one or more of the linkers is a stable linker. In some embodiments, some of the joints are flexible, while other joints are stable. In general, the linker does not affect or significantly affect the correct folding and conformation formed by the configuration of the immunomodulatory molecule. In some embodiments, the linker imparts flexibility and a broad space to each portion of the immunomodulatory molecule, such as allowing for target antigen-antigen binding fragment binding, allowing for ligand-receptor binding, masking of the first binding domain (e.g., cytokine) activity in the absence of second target molecule-second binding domain binding, while not masking of the first binding domain (e.g., cytokine) activity in the presence of second target molecule-second binding domain binding, providing flexibility and/or sufficient space between two binding domains or domain subunits (e.g., cytokine subunits or two cytokine portions) to ensure proper binding domain (e.g., cytokine) activity (binding affinity and/or biological activity), and the like.
The linker may be a peptide linker of any length. In some embodiments, the peptide linker is about 1 amino acid (aa) to about 10aa long, about 2aa to about 15aa long, about 3aa to about 12aa long, about 4aa to about 10aa long, about 5aa to about 9aa long, about 6aa to about 8aa long, about 1aa to about 20aa long, about 21aa to about 30aa long, about 1aa to about 30aa long, about 2aa to about 20aa long, about 10aa to about 30aa long, about 1aa to about 50aa long, about 2aa to about 19aa long, about 2aa to about 18aa long, about 2aa to about 17aa long, about 2aa to about 16aa long, about 2aa to about 10aa long, about 2aa to about 14aa long, about 2aa to about 13aa long, about 2aa to about 12aa long, about 2aa to about 11aa long, about 2aa to about 9aa long, about 2 to about 8aa long, about 2aa to about 7aa long, about 2aa to about 6aa to about 5aa long, or about 5aa long. In some embodiments, the peptide linker is any one of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids long. In some embodiments, the peptide linker is any of about 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids long. In some embodiments, the peptide linker is any of about 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 amino acids long. In some embodiments, the linker is about 10 to about 20 amino acids in length.
The peptide linker may have a naturally occurring sequence or a non-naturally occurring sequence. For example, sequences derived from the hinge region of heavy chain-only antibodies may be used as linkers. See, for example, WO1996/34103. In some embodiments, the peptide linker is a human IgG1, igG2, igG3, or IgG4 hinge or a portion thereof. In some embodiments, the peptide linker is a mutated human IgG1, igG2, igG3, or IgG4 hinge or a portion thereof. In some embodiments, the linker is a flexible linker. Exemplary flexible joints include, but are not limited to: glycine polymer (G) n (SEQ ID NO: 194), glycine-serine polymers (including, for example, (GS) n (SEQ ID NO:195)、(GGS) n (SEQ ID NO:196)、(GGGS) n (SEQ ID NO:197)、(GGS) n (GGGS) n (SEQ ID NO:198)、(GSGGS) n (SEQ ID NO:199)、(GGSGS) n (SEQ ID NO: 200) or (GGGGS) n (SEQ ID NO: 201), where n is an integer of at least 1), glycine-alanine polymers, alanine-serine polymers, and other flexible linkers known in the art. Glycine and glycine-serine polymers are relatively unstructured and thereforeCan act as a neutral tether (tether) between the components. Glycine accesses significantly more phi-psi space than even alanine and is less restricted than residues with longer side chains (see Scheraga, rev. Computational chem. 11-142 (1992)). Exemplary flexible joints include, but are not limited to: GG (SEQ ID NO: 202), GSG (SEQ ID NO: 203), GGSG (SEQ ID NO: 204), GGSGG (SEQ ID NO: 205), GSGGGGG (SEQ ID NO: 206), GSGSGSG (SEQ ID NO: 207), GSGGG (SEQ ID NO: 208), GGGSG (SEQ ID NO: 209), GSSSG (SEQ ID NO: 210), GGSGGS (SEQ ID NO: 211), SGGGGS (SEQ ID NO: 212), GGGGS (SEQ ID NO: 213), (GA) n (SEQ ID NO:214, n is an integer of at least 1), GRAGGGGAGGGG (SEQ ID NO: 215), GRAGGG (SEQ ID NO: 216), GSGGGSGGGGSGGGGS (SEQ ID NO: 217), GGGSGGGGSGGGGS (SEQ ID NO: 218), GGGSGGSGGS (SEQ ID NO: 219), GGSGGSGGSGGSGGG (SEQ ID NO: 220), GGSGGSGGGGSGGGGS (SEQ ID NO: 221), GGSGGSGGSGGSGGSGGS (SEQ ID NO: 222), GGGGSGGGGSGGS (SEQ ID NO: 229), GGGGGGSGGGGSGGGGSA (SEQ ID NO: 223), GSGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 224), KTGGGSGGGS (SEQ ID NO: 225), GGPGGGGSGGGSGGGGS (SEQ ID NO: 226), GGGSGGGGSGGGGSGGGGS (SEQ ID NO: 227), GGGGSGGGGSGGGGSGGGGSG (SEQ ID NO: 228), and the like. In some embodiments, the linker comprises the sequence of ASTMGP (SEQ ID NO: 230). In some embodiments, the linker comprises the sequence of any one of SEQ ID NOS 194-246. One of ordinary skill will recognize that the design of the immunomodulatory molecule may include a linker that is wholly or partially flexible, such that the linker may include a flexible linker moiety and one or more portions of a structure that imparts less flexibility to provide the desired structure and function of the immunomodulatory molecule (e.g., to mask cytokine activity in the absence of target antigen-antibody binding and not to mask cytokine activity in the presence of target antigen-antibody binding, or to provide flexibility and/or sufficient space between two cytokine subunits to ensure proper cytokine activity (binding affinity and/or biological activity)). In some embodiments, the peptide linker is rich in serine-glycine. In some embodiments, the cytokine moiety described herein comprises two cytokine subunits (wild-type or mutant) linked by a linker, e.g., comprising SEQ ID NOs 227-229, 245 and 246.
In some embodiments, the linker is a stable linker (e.g., is not cleavable by a protease, particularly MMP).
Any or all of the linkers described herein may retain the activity of the respective antigen binding, receptor binding, or binding of any of the antigen binding, receptor binding, or receptor binding, respectively, by being between a second binding domain (e.g., ligand, receptor, VHH, scFv, or Fab) and a first binding domain (e.g., cytokine moiety), between a first binding domain (e.g., cytokine moiety) and CL, a first binding domain (e.g., cytokine moiety) and VH, a first binding domain (e.g., cytokine moiety) and VL, CH1 domain and first binding domain (e.g., cytokine moiety), by being between two or more first binding domains (e.g., cytokine moieties) connected in series, by being between two or more subunits of a cytokine or variant thereof, by being between a first binding domain (e.g., cytokine moiety) and Fc domain subunit or portion thereof, by being between a hinge region and CH1 domain, hinge region and CH2 domain, a hinge region and first binding domain (e.g., cytokine moiety), by being between a first binding domain (e.g., cytokine moiety) and VL, CH1 domain and Fc domain, by being between two or portion of a cytokine domain connected in series, by being two or more subunits of a cytokine or variant thereof. Such attachment may include a number of chemical mechanisms, such as covalent binding, affinity binding, intercalation, coordination binding, and complexation. In some embodiments, the binding is covalent. Covalent binding may be achieved by direct condensation of existing side chains or by incorporation of external bridging molecules. Many bivalent or multivalent linkers can be used to couple protein molecules. For example, representative coupling agents may include organic compounds such as thioesters, carbodiimides, succinimidyl esters, diisocyanates, glutaraldehyde, diazobenzenes, and hexamethylenediamine. This list is not intended to be exhaustive of the various types of coupling agents known in the art, but rather examples of more common coupling agents (see Killen and Lindstrom, journal. Immun.133:1335-2549 (1984); jansen et al Immunological Reviews 62:185-216 (1982); and Vitetta et al Science 238:1098 (1987)).
The linkers applicable to the present application are described in the literature (see, e.g., ramakrishanan, S.et al, cancer Res.44:201-208 (1984), which describes the use of MBS (M-maleimidobenzoyl-N-hydroxysuccinimide ester )). In some embodiments, a non-peptide linker as used herein includes: (i) EDC (1-ethyl-3- (3-dimethylamino-propyl) carbodiimide hydrochloride, (ii) SMPT (4-succinimidyloxycarbonyl- α -methyl- α - (2-pyridinyl-dithio) -toluene (Pierce chemical Co., ltd.; cat. No. 21558G), (iii) SPDP (succinimidyl-6[3- (2-pyridinyl dithio) propionylamino ] hexanoate (Pierce chemical Co., ltd.; no. 21651G), (iv) sulfo-LC-SPDP (sulfosuccinimidyl-6[3- (2-pyridinyl dithio) -propionamide) hexanoate (Pierce chemical Co., ltd.; no. 2165-G), and (v) sulfo-NHS (N-hydroxysulfo-succinimide: pierce chemical Co., ltd., no. 24510) conjugated to EDC.
The above-described linkers may contain components with different properties, resulting in immunomodulatory molecules with different physicochemical properties. For example, the sulfo-NHS ester of an alkyl carboxylate is more stable than the sulfo-NHS ester of an aromatic carboxylate. The linker containing the NHS ester is less soluble than the sulfo-NHS ester. In addition, linker SMPT contains a sterically hindered disulfide bond, which can form a fusion protein with increased stability. In general, disulfide bonds are less stable than other bonds, as disulfide bonds are cleaved in vitro, resulting in fewer fusion proteins being available. In particular sulfo-NHS can improve the stability of carbodiimide coupling. Carbodiimide coupling (e.g., EDC) when used in combination with sulfo-NHS forms esters that are more resistant to hydrolysis than carbodiimide coupling reactions alone.
Other linker considerations include effects on the physical or pharmacokinetic properties of the resulting immunomodulatory molecule, such as solubility, lipophilicity, hydrophilicity, hydrophobicity, stability (more or less stable and planned degradation), rigidity, flexibility, immunogenicity, modulation of cytokine moiety/cytokine receptor binding, modulation of antigen binding domain/target antigen binding, modulation of ligand-receptor binding, ability to incorporate micelles or liposomes, and the like.
Immunomodulatory molecule variants
Glycosylation variants
In some embodiments, the immunoregulatory molecule is altered to increase or decrease the extent to which the construct is glycosylated. The addition or deletion of glycosylation sites of an Fc domain can be conveniently accomplished by altering the amino acid sequence to create or remove one or more glycosylation sites.
Natural antibodies produced by mammalian cells typically comprise branched, bi-branched oligosaccharides, which are typically attached to the Fc region C by N-linkages H Asn297 of domain 2. See, for example, wright et al TIBTECH 15:26-32 (1997). Oligosaccharides may include a variety of carbohydrates such as mannose, N-acetylglucosamine (GlcNAc), galactose and sialic acid, as well as fucose attached to GlcNAc in the "backbone" of a bi-branched oligosaccharide structure. In some embodiments, oligosaccharides in the Fc domain may be modified to produce certain improved properties.
In some embodiments, an immunomodulatory molecule described herein is provided having a carbohydrate structure that lacks fucose attached (directly or indirectly) to an Fc domain. For example, the amount of fucose in such an immunomodulatory molecule may be 1% to 80%, 1% to 65%, 5% to 65%, or 20% to 40%. The amount of fucose is determined by calculating the average amount of fucose in the sugar chain at Asn297 relative to the sum of all sugar structures attached to Asn297 (e.g. complex, hybrid and high mannose structures) as measured by MALDI-TOF mass spectrometry, e.g. as described in WO 2008/077546. Asn297 refers to an asparagine residue located at about position 297 in the Fc domain (EU numbering of Fc region residues); however, asn297 may also be located about ±3 amino acids upstream or downstream of position 297, i.e. between positions 294 and 300, due to minor sequence variations in the antibody. Such fucosylated variants may have improved ADCC function. See, for example, U.S. patent publication No. 2003/0157108 (Presta, l.); US2004/0093621 (Japanese Kogyo fermentation Co., ltd., kyowa Hakko Kogyo Co., ltd.). Examples of publications related to "defucosylation" or "fucose deficient" antibody variants include: US 2003/0157108; WO 2000/61739; WO 2001/29246; US 2003/015614; US2002/0164328; US2004/0093621; US 2004/013321; US 2004/010704; US2004/0110282; US2004/0109865; WO 2003/085119; WO 2003/084570; WO 2005/035586; WO 2005/035778; WO2005/053742; WO2002/031140; okazaki et al J.mol.biol.336:1239-1249 (2004); yamane-Ohnuki et al Biotech.Bioeng.87:614 (2004). Examples of cell lines capable of producing defucosylated antibodies include Lec13-CHO cells lacking protein fucosylation (Ripka et al, arch. Biochem. Biophys.249:533-545 (1986), U.S. patent application US2003/0157108 A1,Presta,L, and WO 2004/056312 A1,Adams et al, especially in example 11), and knockout cell lines such as alpha-1, 6-fucosyltransferase genes, FUT8, knockout CHO cells (see, e.g., yamane-Ohnuki et al, biotech. Bioeng.87:614 (2004), kanda, y. Et al, biotechnol. Bioeng.,94 (4): 533-688 (2006), and WO 2003/085107).
Effector function variants
In some embodiments, the present application contemplates immunomodulatory molecules with some, but not all, fc effector functions, which make them ideal candidates for use, i.e., where in vivo immunomodulatory molecule half-life is important, but some effector functions (such as CDC and ADCC) are unnecessary or detrimental. Some Fc domain variants have been discussed above. In vitro and/or in vivo cytotoxicity assays may be performed to confirm a reduction/depletion of CDC and/or ADCC activity. For example, an Fc receptor (FcR) binding assay may be performed to ensure that the antibody lacks fcγr binding (and thus may lack ADCC activity), but retains FcRn binding capacity. The primary cells mediating ADCC, NK cells, express fcyriii only, whereas monocytes express fcyri, fcyrii and fcyriii. FcR expression on hematopoietic cells is summarized in Table 2 at page 464 of Ravetch and Kinet, annu. Rev. Immunol.9:457-492 (1991). Non-limiting examples of in vitro assays for assessing ADCC activity of a target molecule are described in U.S. Pat. No. 5,500,362 (see, e.g., hellstrom, I.et al Proc.Nat 'l Acad.Sci.USA 83:7059-7063 (1986)) and Hellstrom, I.et al, proc.Nat' l Acad.Sci.USA 82:1499-1502 (198) 5) The method comprises the steps of carrying out a first treatment on the surface of the U.S. Pat. No. 5,821,337 (see Bruggemann, M.et al., J.exp. Med.166:1351-1361 (1987)). Alternatively, non-radioactive assay methods (see, e.g., ACTI for flow cytometry TM Non-radioactive cytotoxicity assay (CellTechnology, inc.Mountain View, CA; and Cytotox)Non-radioactive cytotoxicity assay (Promega, madison, wis.). Useful effector cells for such assays include Peripheral Blood Mononuclear Cells (PBMC) and Natural Killer (NK) cells. Alternatively, or in addition, ADCC activity of the target molecule can be assessed in vivo, e.g., in an animal model, as disclosed in Clynes et al Proc.Nat' l Acad.Sci.USA 95:652-656 (1998). A C1q binding assay may also be performed to determine that the antibody is unable to bind C1q and thus lacks CDC activity. See, e.g., C1q and C3C binding ELISA in WO2006/029879 and WO 2005/100402. To assess complement activation, CDC assays can be performed (see, e.g., gazzano-Santoro et al, J.Immunol. Methods 202:163 (1996); cragg, M.S. et al, blood 101:1045-1052 (2003); and Cragg, M.S. and M.J.Glennie, blood 103:2738-2743 (2004)). FcRn binding and in vivo clearance/half-life assays can also be performed using methods known in the art (see, e.g., petkova, s.b.et al, int' l.immunol.18 (12): 1759-1769 (2006)).
Fc domains with reduced effector function, including domains in which one or more of Fc region residues 238, 265, 269, 270, 297, 327 and 329 are replaced (U.S. Pat. No. 6,737,056). Such Fc mutants include substitutions at two or more of amino acids 265, 269, 270, 297 and 327, including so-called "DANA" Fc mutants in which residues 265 and 297 are replaced with alanine (U.S. Pat. No. 7,332,581). Certain antibody variants with improved or reduced binding to FcR are described (see, e.g., U.S. Pat. No. 6,737,056; WO2004/056312 and Shields et al, J.biol. Chem.9 (2): 6591-6604 (2001)). In some embodiments, the alteration in the Fc domain results in an alteration (i.e., an improvement or reduction) in C1q binding and/or CDC, e.g., as described in U.S. Pat. No. 6,194,551, WO99/51642, and Idusogie et al J.Immunol.164:4178-4184 (2000).
In some embodiments, the Fc domain comprises one or more amino acid substitutions that increase half-life and/or improve binding to neonatal Fc receptor (FcRn). Antibodies with increased half-life and improved binding to neonatal Fc receptor (FcRn) responsible for transfer of maternal IgG to the fetus (Guyer et al, J.Immunol.117:587 (1976) and Kim et al, J.Immunol.24:249 (1994)), are described in US2005/0014934A1 (Hinton et al). These antibodies comprise an Fc domain with one or more substitutions that improve the binding of the Fc region to FcRn. Such Fc variants include those having substitutions at one or more Fc region residues, e.g., substitution of Fc region residue 434 (US 7,371,826).
See also Duncan & Winter, nature 322:738-40 (1988); U.S. Pat. nos. 5,648,260; U.S. Pat. nos. 5,624,821; and WO94/29351, which relates to other examples of Fc domain variants.
Cysteine engineered variants
In some embodiments, it may be desirable to produce a cysteine engineered immunomodulatory molecule, such as a "ThioMAb," in which one or more residues of the immunomodulatory molecule are replaced with cysteine residues. In particular embodiments, the replacement residue occurs at an accessible site of the immunoregulatory molecule. By replacing those residues with cysteines, reactive thiol groups are therefore located at accessible sites of the immunomodulatory molecule and can be used to conjugate the immunomodulatory molecule to other moieties, such as drug moieties or linker-drug moieties, to produce an immunomodulatory molecule conjugate. In some embodiments, any one or more of the following residues may be replaced with a cysteine: a118 (EU numbering) of heavy chain; and S400 (EU numbering) of the heavy chain Fc domain. Cysteine engineered immunomodulatory molecules may be produced as described, for example, in U.S. patent No. 7,521,541.
Immunomodulatory molecule derivatives
In some embodiments, the immunomodulatory molecules provided herein may further comprise additional therapeutic compounds, such as any of the therapeutic compounds known in the art. For example, the parent antibody in some embodiments may be an Antibody Drug Conjugate (ADC). See, e.g., any ADC described in Shim h. (biomalects.2020mar; 10 (3): 360), and diamant n.and Banerji u.br J cancer.2016;114 (4) 362-367, the contents of which are incorporated herein by reference in their entirety. In some embodiments, the therapeutic compound is conjugated to an Fc domain or portion thereof. In some embodiments, the therapeutic compound is a cytotoxic agent, a chemotherapeutic agent, a growth inhibitory agent, or an antibiotic.
In some embodiments, the immunoregulatory molecule further comprises a label selected from the group consisting of: chromophores, fluorophores (e.g., coumarin, xanthene, anthocyanin, pyrene, boron polyazadazole, oxazine, and derivatives thereof), fluorescent proteins (e.g., GFP, phycobiliprotein, and derivatives thereof), phosphorescent dyes (e.g., dioxetane, xanthene or carbocyanine dyes, lanthanide chelates), tandem dyes (e.g., cyanine-phycobiliprotein derivatives and xanthene-phycobiliprotein derivatives), particles (e.g., gold clusters, colloidal gold, microspheres, quantum dots), haptens, enzymes (e.g., peroxidases, phosphatases, glycosidases, luciferases), and radioisotopes (e.g., 125 I、 3 H、 14 C、 32 P)。
vector encoding immunoregulatory molecule
The invention also provides isolated nucleic acids encoding any of the immunomodulatory molecules described herein (e.g., IL-2/anti-PD-1 agonist antibody immunomodulatory molecules, IL-12/anti-PD-1 agonist antibody immunomodulatory molecules, IL-2/PD-L1 immunomodulatory molecules, IL-12/PD-L1 immunomodulatory molecules, IL-2/PD-L2 immunomodulatory molecules, IL-12/PD-L2 immunomodulatory molecules, vectors comprising nucleic acids encoding any of the immunomodulatory molecules described herein, as described in any of figures 1A-1W and 11A-15D, examples, and sequence listings herein. Also provided are isolated host cells (e.g., CHO cells, HEK 293 cells, hela cells, COS cells) comprising a nucleic acid encoding any of the immunomodulatory molecules described herein, or vectors comprising a nucleic acid encoding any of the immunomodulatory molecules described herein.
In some embodiments, vectors comprising nucleic acids encoding any of the immunomodulatory molecules described herein are suitable for replication and integration in eukaryotic cells such as mammalian cells (e.g., CHO cells, HEK293 cells, hela cells, COS cells). In some embodiments, the vector is a viral vector. Examples of viral vectors include, but are not limited to: adenovirus vectors, adeno-associated virus vectors, lentiviral vectors, retrovirus vectors, herpes simplex virus vectors, and derivatives thereof. Viral vector technology is well known in the art and is described, for example, in Sambrook et al (2001,Molecular Cloning:A Laboratory Manual,Cold Spring Harbor Laboratory,New York), and other virology and molecular biology manuals.
Many virus-based systems have been developed for transferring genes into mammalian cells. For example, retroviruses provide a convenient platform for gene delivery systems. The heterologous nucleic acid may be inserted into a vector and packaged into retroviral particles using techniques known in the art. The recombinant virus can then be isolated and delivered to the engineered mammalian cells in vitro or ex vivo. Many retroviral systems are known in the art. In some embodiments, an adenovirus vector is used. Many adenoviral vectors are known in the art. In some embodiments, lentiviral vectors are used. In some embodiments, self-inactivating lentiviral vectors are used. For example, self-inactivating lentiviral vectors carrying an immune modulatory molecule coding sequence may be packaged using protocols known in the art. The resulting lentiviral vector may be used to transduce mammalian cells using methods known in the art. Vectors derived from retroviruses such as lentiviruses are suitable tools for achieving long-term gene transfer, as they allow long-term stable integration of transgenes and propagation in offspring cells. Lentiviral vectors also have low immunogenicity and can transduce non-proliferating cells.
In some embodiments, the vector is a non-viral vector. In some embodiments, the vector is a transposon, such as a Sleeping Beauty (SB) transposon system, or a PiggyBac transposon system. In some embodiments, the carrier is a polymer-based non-viral carrier, including, for example, poly (lactic-co-glycolic acid) (PLGA) and polylactic acid (PLA), poly (ethyleneimine) (PEI), and dendrimers. In some embodiments, the carrier is a non-viral carrier based on cationic lipids, such as cationic liposomes, lipid nanoemulsions, and Solid Lipid Nanoparticles (SLNs). In some embodiments, the vector is a peptide-based genetic non-viral vector, such as poly-L-lysine. Any known non-viral vector suitable for genome editing may be used to introduce nucleic acids encoding an immunomodulatory molecule into a host cell. See, e.g., yin H.et al Nature Rev.genetics (2014) 15:521-555; aronovich EL et al, "The Sleeping Beauty transposon system: a non-viral vector for gene treatment," hum. Mol. Genet. (2011) R1: R14-20; and Zhao s.et al, "PiggyBac transposon vectors: the tools of the human gene coding," trans.lung Cancer res (2016) 5 (1): 120-125, which is incorporated herein by reference. In some embodiments, any one or more nucleic acids or vectors encoding an immunomodulatory molecule described herein are introduced into a host cell (e.g., CHO, HEK293, hela, or COS) by physical methods, including, but not limited to, electroporation, acoustic electroporation, optical electroporation, magnetic infection, aqueous electroporation.
In some embodiments, the vector contains a selectable marker gene or reporter gene to select cells expressing the immunomodulatory molecules described herein from a population of host cells transfected with the vector (e.g., a lentiviral vector). Both the selectable marker and the reporter gene may be flanked by appropriate regulatory sequences to enable expression in the host cell. For example, the vector may comprise transcription and translation terminators, initiation sequences, and promoters useful for regulating expression of the nucleic acid sequences.
In some embodiments, a vector (e.g., a viral vector) comprises any of the nucleic acids encoding an immunomodulatory molecule described herein. The nucleic acid may be cloned into a vector using any molecular cloning method known in the art, including, for example, using restriction endonuclease sites and one or more selectable markers. In some embodiments, the nucleic acid is operably linked to a promoter. A variety of promoters for gene expression in mammalian cells have been explored and any promoter known in the art may be used in the present invention. Promoters can be broadly classified as constitutive or regulated, such as inducible.
In some embodiments, a nucleic acid encoding an immunomodulatory molecule described herein is operably linked to a constitutive promoter. Constitutive promoters allow a heterologous gene (also known as a transgene) to be expressed constitutively in a host cell. Exemplary promoters contemplated herein include, but are not limited to: the cytomegalovirus immediate early promoter (CMV), human elongation factor-1 alpha (hEFla), ubiquitin C promoter (Ubic), phosphoglycerate kinase Promoter (PGK), simian Virus 40 early promoter (SV 40), chicken beta-actin promoter coupled to CMV early enhancer (CAGG), rous Sarcoma Virus (RSV) promoter, polyoma enhancer/herpes simplex thymidine kinase (MCI) promoter, beta actin (beta-ACT) promoter, "myeloproliferative sarcoma Virus enhancer, negative control region deleted, d1587rev primer binding site replaced (MND)" promoter. The efficiency of such constitutive promoters in driving transgene expression has been widely compared in a number of studies. In some embodiments, a nucleic acid encoding an immunomodulatory molecule described herein is operably linked to a CMV promoter.
In some embodiments, a nucleic acid encoding an immunomodulatory molecule described herein is operably linked to an inducible promoter. Inducible promoters belong to the class of regulated promoters. The inducible promoter may be induced by one or more conditions, such as physical conditions, the microenvironment of the host cell or the physiological state of the host cell, an inducer (i.e., an inducer), or a combination thereof. In some embodiments, the induction conditions do not induce expression of an endogenous gene in the host cell. In some embodiments, the induction conditions are selected from the group consisting of: inducers, radiation (e.g., ionizing radiation, light), temperature (e.g., heat), redox state, and host cell activation state. In some embodiments, the inducible promoter may be the NFAT promoter, Promoters or nfkb promoters. In some embodiments, the inducible promoter is a tet-inducible promoter.
In some embodiments, the vector comprises more than one nucleic acid encoding an immunomodulatory molecule described herein, e.g., different polypeptides of the immunomodulatory molecule. In some embodiments, each vector comprises 2 nucleic acids encoding 2 polypeptides of the immunomodulatory molecules described herein.
In some embodiments, two or more nucleic acids encoding an immunomodulatory molecule described herein are operably regulated under the same promoter in a vector. In some embodiments, two or more nucleic acids are connected in series via a linking sequence (e.g., IRES) or a nucleic acid sequence encoding a self-cleaving 2A peptide (e.g., P2A, T2A, E2A, F2A, bmCPV2A, bmIFV a). In some embodiments, the nucleic acid encoding two or more polypeptides of the immunoregulatory molecule comprises a linking sequence (e.g., an IRES) between polypeptide coding sequences or a nucleic acid sequence encoding a self-cleaving 2A peptide (e.g., P2A, T2A, E2A, F2A, bmCPV2A, bmIFV 2A). In some embodiments, two or more nucleic acids encoding the immunomodulatory molecules described herein are operably regulated under different promoters in a vector. In some embodiments, the promoter operably linked to each nucleic acid is different. In some embodiments, the promoter operably linked to each nucleic acid is the same. In some embodiments, an immunomodulatory molecule described herein is encoded by two or more vectors, e.g., each vector encodes one heavy chain (or one polypeptide comprising VH and cytokine portions) and a pair of paired light chains, or each vector encodes one polypeptide of an immunomodulatory molecule.
IV preparation method
Also provided are methods of making any of the immunomodulatory molecules described herein (e.g., an IL-2/anti-PD-1 agonist antibody immunomodulatory molecule, an IL-12/anti-PD-1 agonist antibody immunomodulatory molecule, an IL-2/PD-L1 immunomodulatory molecule, an IL-12/PD-L1 immunomodulatory molecule, an IL-2/PD-L2 immunomodulatory molecule, an IL-12/PD-L2 immunomodulatory molecule, as described in any of figures 1A-1W and 11A-15D, examples, and sequence listings herein). Thus, in some embodiments, there is provided a method of producing an immunomodulatory molecule comprising:
(a) Culturing a host cell (e.g., CHO cell, HEK293 cell, hela cell, or COS cell) comprising any nucleic acid or vector encoding an immunomodulatory molecule described herein under conditions effective to express the encoded immunomodulatory molecule; and (b) obtaining the expressed immunomodulatory molecule from the host cell. In some embodiments, the method of step (a) further comprises producing a host cell comprising a nucleic acid or vector encoding an immunomodulatory molecule described herein. The immunomodulatory molecules described herein may be prepared using any method known in the art or as described herein. Exemplary methods are also described in examples 1, 4, 5, 7, 9, 10 and 12. In some embodiments, the immunoregulatory molecule is expressed by a eukaryotic cell, such as a mammalian cell. In some embodiments, the immunoregulatory molecule is expressed by a prokaryotic cell.
1. Recombinant production in prokaryotic cells
a) Vector construction
The polynucleotide sequences encoding the immunomodulatory molecules of the present application can be obtained using standard recombinant techniques. The desired polynucleic acid sequences may be isolated and sequenced from cells (e.g., hybridoma cells) that produce antibodies or immunoregulatory molecules. Alternatively, polynucleotides may be synthesized using nucleotide synthesizers or PCR techniques. Once obtained, the sequence encoding the polypeptide is inserted into a recombinant vector capable of replicating and expressing the heterologous polynucleotide in a prokaryotic host. Many vectors are available and known in the art for the purposes of the present invention. The choice of an appropriate vector will depend primarily on the size of the nucleic acid to be inserted into the vector and the particular host cell transformed with the vector. Each vector contains a number of components depending on its function (amplification or expression of the heterologous polynucleotide, or both) and its compatibility with the particular host cell in which it is located. Carrier components typically include, but are not limited to: an origin of replication, a selectable marker gene, a promoter, a Ribosome Binding Site (RBS), a signal sequence, a heterologous nucleic acid insertion and a transcription termination sequence.
In general, plasmid vectors containing replicon and control sequences which are derived from species compatible with the host cell are used in conjunction with these hosts. The vector will typically carry a replication site and a marker sequence capable of providing phenotypic selection in transformed cells. For example, E.coli is typically transformed with pBR322, a plasmid derived from E.coli species. pBR322 contains genes encoding ampicillin (Amp) and tetracycline (Tet) resistance and thus provides easy means for identifying transformed cells. pBR322, derivatives thereof, or other microbial plasmids or phages may also contain, or be modified to contain, promoters which can be used by the microorganism to express endogenous proteins. Examples of pBR322 derivatives for expressing specific antibodies are described in detail in Carter et al, U.S. Pat. No. 5,648,237.
In addition, phage vectors containing replicon and control sequences that are compatible with the host microorganism can be used as transformation vectors in connection with these hosts. For example, phages such as GEM TM -11 can be used to prepare recombinant vectors which can be used to transform susceptible host cells such as e.coli LE392.
The expression vectors of the present application may comprise two or more promoter-cistron pairs, encoding each polypeptide component. A promoter is an untranslated regulatory sequence located upstream (5') of a cistron that regulates its expression. Prokaryotic promoters are generally divided into two classes, inducible and constitutive. Inducible promoters are promoters that initiate an increased level of transcription of a cistron under their control in response to a change in culture conditions (e.g., the presence or absence of a nutrient, or a change in temperature).
A large number of promoters recognized by a variety of potential host cells are well known. The selected promoter may be operably linked to the cistron DNA encoding the polypeptide by digestion with a restriction enzyme to remove the promoter from the source DNA and insert the isolated promoter sequence into the vector of the present application. Both native promoter sequences and a number of heterologous promoters can be used to direct the amplification and/or expression of a target gene. In some embodiments, heterologous promoters are used because they generally allow for higher transcription and higher yields of expressed target genes than native target polypeptide promoters.
Promoters suitable for use in prokaryotic hosts include: phoA promoters, beta-galactosidase and lactose promoter systems, tryptophan (trp) promoter systems and hybrid promoters, such as tac or trc promoters. However, other promoters that are functional in bacteria (e.g., other known bacterial or phage promoters) are also suitable. Their nucleic acid sequences have been disclosed, thereby enabling the skilled artisan to operably link them to cistrons encoding the target light and heavy chains using linkers or adaptors (Siebenlist et al (1980) Cell 20:269) to provide any desired restriction sites.
In some embodiments, each cistron within the recombinant vector comprises a secretion signal sequence component that directs translocation of the expressed polypeptide across the membrane. In general, the signal sequence may be a component of the vector, or it may be part of the target polypeptide DNA inserted into the vector. The signal sequence selected for the purposes of the present invention should be one that is recognized and processed (i.e., cleaved by a signal peptidase) by the host cell. For prokaryotic host cells that do not recognize and process the native signal sequence of a heterologous polypeptide, the signal sequence is replaced with a prokaryotic signal sequence selected from, for example, the group consisting of: alkaline phosphatase, penicillinase, ipp or thermostable enterotoxin II (STII) leader molecule, lamB, phoE, pelB, ompA and MBP. In some embodiments of the present application, the signal sequences used in the two cistrons of the expression system are STII signal sequences or variants thereof.
In some embodiments, the production of the immunoregulatory molecule according to the present application may occur in the cytoplasm of the host cell, and thus the secretion signal sequence need not be present within each cistron. In some embodiments, the polypeptide component is expressed, folded, and assembled to form an immunomodulatory molecule (or a portion of an immunomodulatory molecule) within the cytoplasm. Certain host strains (e.g., E.coli trxB - Strains) provide cytoplasmic conditions that favor disulfide bond formation, thereby allowing for proper folding and assembly of the expressed protein subunits. See Proba and Pluckthun, gene,159:203 (1995).
The present invention provides an expression system in which the quantitative ratio of expressed polypeptide components can be adjusted to maximize the yield of secreted and correctly assembled immunomodulatory molecules of the present application. Such modulation is accomplished, at least in part, by simultaneously modulating the translational strength of the polypeptide components. One technique for adjusting translational strength is disclosed in U.S. Pat. No. 5,840,523 to Simmons et al. It utilizes variants of the Translation Initiation Region (TIR) within the cistron. For a given TIR, a series of amino acid or nucleic acid sequence variants can be created with a range of translational intensities, thereby providing a convenient way to adjust this factor to achieve the desired expression level for a particular strand. TIR variants can be produced by conventional mutagenesis techniques resulting in codon changes that can alter the amino acid sequence, although silent changes in the nucleic acid sequence are preferred. Alterations in TIR may include, for example, alterations in the number or spacing of Shine-Dalgarno sequences, as well as alterations in signal sequences. One way to generate mutant signal sequences is to generate a "codon bank" at the beginning of the coding sequence that does not alter the amino acid sequence of the signal sequence (i.e., the alteration is silent). This can be achieved by altering the third nucleotide position of each codon; in addition, some amino acids, such as leucine, serine, and arginine, have multiple first and second positions, which can increase the complexity of manufacturing the library. This mutagenesis method is described in detail in Yansura et al (1992) METHODS: A Companion to Methods in enzymol.4:151-158.
Preferably, a set of vectors is generated, wherein each cistron has a range of TIR intensities. This limited set provides a comparison of the expression level of each strand and the yield of the desired protein product at various combinations of TIR intensities. TIR strength can be determined by quantifying the expression level of the reporter gene as described in detail in Simmons et al, U.S. patent 5,840,523. Based on the translation strength comparison, the individual TIR required is selected for combination in the expression vector constructs of the present application.
b) Prokaryotic host cell
Prokaryotic host cells suitable for expressing the immunomodulatory molecules of the present application include archaebacteriaAnd eubacteria, such as gram-negative or gram-positive microorganisms. Examples of useful bacteria include: escherichia (e.g., escherichia coli), bacillus (e.g., bacillus subtilis), enterobacter, pseudomonas (e.g., pseudomonas aeruginosa), salmonella typhimurium, serratia marcescens, klebsiella, proteus, shigella, rhizobium, vitreoscilla, or paracoccus. In some embodiments, gram negative cells are used. In some embodiments, E.coli cells are used as hosts in the present invention. Examples of E.coli strains include strain W3110 (Bachmann, cellular and Molecular Biology, vol.2 (Washington, D.C.: american Society for Microbiology, 1987), pp.1190-1219; ATCC accession No. 27,325) and derivatives thereof, including those having genotype W3110 AfhuA (AtonA) ptr3 lac Iq lacL8 AompT A (nmpc-fepE) degP41 kan R Is described (U.S. Pat. No. 5, 5,639,635). Other strains and derivatives thereof, such as E.coli 294 (ATCC 31,446), E.coli B, E.coli 1776 (ATCC 31,537) and E.coli RV308 (ATCC 31,608), are also suitable. These examples are illustrative and not limiting. Methods of constructing derivatives of any of the above bacteria having defined genotypes are known in the art and are described, for example, in Bass et al, proteins,8:309-314 (1990). It is often necessary to select a suitable bacterium taking into account the replicative capacity of the replicon in the bacterial cell. For example, when a known plasmid such as pBR322, pBR325, pACYC177 or pKN410 is used to provide a copy, escherichia coli, serratia or Salmonella may be suitably used as the host.
In general, the host cell should secrete minimal amounts of proteolytic enzymes, and additional protease inhibitors may desirably be incorporated into the cell culture.
c) Protein production
The host cells are transformed with the above-described expression vectors and cultured in conventional nutrient media which are appropriately modified to induce promoters, select transformants or amplify the genes encoding the desired sequences. Transformation means introducing DNA into a prokaryotic host such that the DNA is replicable, either as an extrachromosomal element or by chromosomal integrant. Depending on the host cell used, transformation is accomplished using standard techniques suitable for such cells. Calcium treatment with calcium chloride is commonly used for bacterial cells containing a large number of cell wall barriers. Another transformation method uses polyethylene glycol/DMSO. Yet another technique used is electroporation.
The host cells are transformed with the above-described expression vectors and cultured in conventional nutrient media which are appropriately modified to induce promoters, select transformants or amplify the genes encoding the desired sequences. Transformation means introducing DNA into a prokaryotic host such that the DNA is replicable, either as an extrachromosomal element or by chromosomal integrant. Depending on the host cell used, transformation is accomplished using standard techniques suitable for such cells. Calcium treatment with calcium chloride is commonly used for bacterial cells containing a large number of cell wall barriers. Another transformation method uses polyethylene glycol/DMSO. Yet another technique used is electroporation.
Prokaryotic cells used to produce the immunomodulatory molecules of the present application are grown in media known in the art and are suitable for culture of the host cell of choice. Examples of suitable media include Luria Broth (LB) plus the necessary nutritional supplements. In some embodiments, the medium further contains a selection agent selected based on the construction of the expression vector to selectively allow growth of the prokaryotic cells containing the expression vector. For example, ampicillin is added to the medium for growth of cells expressing the ampicillin resistance gene.
In addition to the carbon source, nitrogen source and inorganic phosphate source, any necessary supplements may be included in suitable concentrations, either alone or as a mixture with another supplement or medium (e.g., a complex nitrogen source). Optionally, the medium may contain one or more reducing agents selected from the group consisting of: glutathione, cysteine, cystamine, thioglycolate, dithioerythritol and dithiothreitol. The prokaryotic host cell is cultured at a suitable temperature. For example, for E.coli growth, a preferred temperature range is from about 20℃to about 39 ℃, more preferably from about 25℃to about 37 ℃, and even more preferably at about 30 ℃. The pH of the medium may be any pH in the range of about 5 to about 9, depending primarily on the host microorganism. For E.coli, the pH is preferably from about 6.8 to about 7.4, more preferably about 7.0.
If an inducible promoter is used in the expression vector of the present application, protein expression is induced under conditions suitable for promoter activation. In one aspect of the application, the PhoA promoter is used to control transcription of a polypeptide. Thus, the transformed host cells are cultured in phosphate-limiting medium for induction. Preferably, the phosphate limiting medium is a C.R.A.P medium (see, e.g., simmons et al, J.Immunol. Methods (2002), 263:133-147). Depending on the vector construct used, a variety of other inducers may be used, as known in the art.
The expressed immunomodulatory molecules of the present application are secreted into and recovered from the periplasm of the host cell. Protein recovery typically involves destruction of the microorganism, typically by such means as osmotic shock, sonication, or lysis. Once the cells are destroyed, cell debris or whole cells can be removed by centrifugation or filtration. The protein may be further purified, for example, by affinity resin chromatography. Alternatively, the protein may be transported to the culture medium and isolated therein. Cells may be removed from the culture, and the culture supernatant filtered and concentrated to further purify the produced protein. Expressed polypeptides may be further isolated and identified using commonly known methods such as polyacrylamide gel electrophoresis (PAGE) and western blot assays.
Alternatively, protein production is carried out in large quantities by fermentation methods. A variety of large-scale fed-batch fermentation procedures can be used to produce recombinant proteins. Large scale fermentation has a capacity of at least 1000 liters, preferably about 1,000 to 100,000 liters. These fermentors use agitator impellers to dispense oxygen and nutrients, especially glucose (preferably carbon/energy sources). Small scale fermentation generally refers to fermentation in a fermenter having a volume of no more than about 100 liters and may range from about 1 liter to about 100 liters.
In fermentation processes, induction of protein expression is typically effected by growing the cells under appropriate conditionsStarting after a desired density, e.g. an OD of about 180-220 550 At this stage the cells are in early stationary phase. Depending on the vector construct used, a variety of inducers may be used, as known in the art and described above. Cells may be grown for a short period of time prior to induction. Cells are typically induced for about 12-50 hours, although longer or shorter induction times may be used.
To improve the yield and quality of the immunomodulatory molecules of the present application, a variety of fermentation conditions can be varied. For example, to improve the correct assembly and folding of the secreted polypeptide, other vectors that overexpress chaperones such as Dsb protein (DsbA, dsbB, dsbC, dsbD or DsbG) or FkpA (a peptide-based prolyl cis, trans-isomerase with chaperone activity) may be used to co-transform host prokaryotic cells. Chaperones have been shown to facilitate the correct folding and solubilization of heterologous proteins produced in bacterial host cells. Chen et al (1999) J Bio Chem 274:19601-19605; georgiou et al, U.S. patent US6,083,715; georgiou et al, U.S. patent US6,027,888; bothmann and Pluckthun (2000) J.biol.chem.275:17100-17105; ramm and Pluckthun (2000) J.biol.chem.275:17106-17113; arie et al (2001) mol. Microbiol.39:199-210.
In order to minimize proteolysis of expressed heterologous proteins (especially those susceptible to proteolysis), certain protease deficient host strains may be used in the present invention. For example, the host cell strain may be modified to affect gene mutations in genes encoding known bacterial proteases (e.g., protease III, ompT, degP, tsp, protease I, protease Mi, protease V, protease VI, and combinations thereof). Some E.coli protease deficient strains are available, which are described, for example, in Joly et al (1998), supra; georgiou et al, U.S. patent No. 5,264,365; georgiou et al, U.S. patent No. 5,508,192; hara et al Microbial Drug Resistance,2:63-72 (1996).
Protease deficiency and E.coli strains transformed with plasmids that overexpress one or more chaperones can be used as host cells in expression systems encoding the immunomodulatory molecules of the present application.
d) ProteinsPurification of the substances
The immunomodulatory molecules produced herein are further purified to obtain a substantially homogeneous formulation for further assay and use. Standard protein purification methods known in the art may be used. The following procedure is an example of a suitable purification procedure: fractionation on an immunoaffinity column or ion exchange column, ethanol precipitation, reverse phase HPLC, silica gel chromatography or cation exchange resins such as DEAE chromatography, chromatography focusing, SDS-PAGE, ammonium sulfate precipitation and gel filtration, for example using SephadexG-75.
In some embodiments, protein a immobilized on a solid phase is used for immunoaffinity purification of immunomodulatory molecules comprising the Fc region of the present application. Protein a is a 42kDa surface protein from staphylococcus aureus (Staphylococcus aureas) that binds with high affinity to Fc-containing constructs, e.g., antigen binding fragment-hinge-Fc fusion proteins, antibodies, or immunomodulatory molecules described herein. Lindmark et al (1983) J.Immunol. Meth.62:1-13. The solid phase to which protein a is immobilized is preferably a column comprising a glass or silica surface, more preferably a controlled pore glass column or a silicic acid column. In some applications, the column is coated with an agent such as glycerol in an attempt to prevent non-specific adhesion of contaminants. The solid phase is then washed to remove contaminants that bind non-specifically to the solid phase. Finally, the target immunomodulatory molecule is recovered from the solid phase by elution.
2. Recombinant production in eukaryotic cells
For eukaryotic expression, the vector components generally include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter, a transcription termination sequence.
a) Signal sequence components
The vector for eukaryotic hosts may also be an insert encoding a signal sequence or other polypeptide having a specific cleavage site at the N-terminus of the mature protein or polypeptide. The heterologous signal sequence selected is preferably one that is recognized and processed (i.e., cleaved by a signal peptidase) by the host cell. In mammalian cell expression, mammalian signal sequences and viral secretion leader sequences, such as the gD signal of herpes simplex virus, are useful. The DNA of such a precursor region is linked in-frame to DNA encoding an immunomodulatory molecule of the present application.
b) Origin of replication
In general, mammalian expression vectors do not require an origin of replication component (typically only the SV40 origin can be used because it contains an early promoter).
c) Selection of genome Components
Expression and cloning vectors may contain a selection gene, also known as a selectable marker. Typical selection genes encode proteins that: (a) confers resistance to antibiotics or other toxins (e.g., ampicillin, neomycin, methotrexate, or tetracycline), (b) supplements auxotrophs, or (c) provides critical nutrients not available from complex media, e.g., genes encoding D-alanine racemase for Bacillus.
An example of an alternative is to use a drug to prevent growth of the host cell. Those cells that are successfully transformed with the heterologous gene can produce a protein that confers resistance and thus survive the selection regimen. Examples of such dominant selection use the drugs neomycin, mycophenolic acid and hygromycin.
Another example of suitable selectable markers for mammalian cells are those that are capable of identifying cells that are capable of taking up nucleic acids encoding the immunomodulatory molecules of the present application, such as DHFR, thymidine kinase, metallothionein-I and-II, preferably primate metallothionein genes, adenosine deaminase, ornithine decarboxylase, and the like.
For example, cells transformed with the DHFR selection gene are first identified by culturing all transformants in medium containing methotrexate (Mtx) (DHFR competitive antagonist). When wild-type DHFR is used, a suitable host cell is a Chinese Hamster Ovary (CHO) cell line deficient in DHFR activity (e.g., ATCC CRL-9096).
Alternatively, host cells transformed or co-transformed with a DNA sequence encoding a polypeptide, a wild-type DHFR protein, and another selectable marker such as aminoglycoside 3' -phosphotransferase (APH), particularly wild-type hosts containing endogenous DHFR, may be selected by cell growth in a medium containing a selectable marker such as a selection agent for an aminoglycoside antibiotic (e.g., kanamycin, neomycin, or G418). See U.S. Pat. No. 4,965,199.
d) Promoter component
Expression and cloning vectors typically contain a promoter that is recognized by the host organism and is operably linked to a nucleic acid encoding a desired polypeptide sequence. Virtually all eukaryotic genes have an AT-rich region located about 25 to 30 bases upstream of the site where transcription is initiated. Another sequence found 70 to 80 bases upstream of the start of transcription of many genes is the CNCAAT region, where N may be any nucleotide. The 3 'end of most eukaryotic organisms is an AATAAA sequence, which can add a poly-A tail to the signal 3' of the coding sequence. All of these sequences can be inserted into eukaryotic expression vectors. See also section "promoter" under the heading "vector encoding an immunomodulatory molecule" above.
Polypeptide transcription of the vector in a mammalian host cell is controlled, for example, by a promoter obtained from the genome of a virus, such as polyoma virus, chicken pox virus, adenovirus (e.g., adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, retrovirus, hepatitis b virus, and most preferably simian virus 40 (SV 40), from a heterologous mammalian promoter, such as an actin promoter or an immunoglobulin promoter, from a heat shock promoter, provided that such a promoter is compatible with the host cell system.
The early and late promoters of SV40 virus are conveniently obtained as SV 40-restricted fragments which also contain the SV40 viral origin of replication. The direct early promoter of human cytomegalovirus is conveniently available as a HindIII E restriction fragment. A system for expressing DNA in a mammalian host using bovine papilloma virus as a vector is disclosed in U.S. Pat. No. 4,419,446. A modification of this system is described in U.S. Pat. No. 4,601,978. See also Reyes et al, nature 297:598-601 (1982), for expression of human interferon cDNA in mouse cells under the control of the thymidine kinase promoter from herpes simplex virus. Alternatively, the Rous (Rous) sarcoma virus long terminal repeat can be used as a promoter.
e) Enhancer element component
Transcription of DNA encoding the immunomodulatory molecules of the present application by higher eukaryotes is typically increased by inserting an enhancer sequence into the vector. Many enhancer sequences are now known from mammalian genes (globin, elastase, albumin, alpha fetoprotein, and insulin). However, in general, one will use enhancers from eukaryotic viruses. Examples include: the SV40 enhancer (100-270 bp) posterior to the replication origin, the cytomegalovirus early promoter enhancer, the polyoma enhancer posterior to the replication origin, and the adenovirus enhancer. See also Yaniv, nature 297:17-18 (1982), for enhancement elements of eukaryotic promoter activation. Enhancers may be spliced into the vector 5' or 3' to the polypeptide coding sequence, but are preferably located 5' to the promoter.
f) Transcription termination component
Expression vectors used in eukaryotic host cells (yeast, fungi, insect, plant, animal, human or nucleated cells from other multicellular organisms) will also contain sequences necessary for stopping transcription and stabilizing the mRNA. Such sequences are generally available from 5 'and occasionally 3' untranslated regions of eukaryotic or viral DNA or cDNA. These regions contain nucleotide fragments transcribed as polyadenylation fragments in the untranslated portion of the mRNA encoding the polypeptide. One useful transcription termination component is the bovine growth hormone polyadenylation region. See WO94/11026 and expression vectors disclosed therein.
g) Selection and transformation of host cells
Suitable host cells for cloning or expressing the DNA in the vectors herein include the higher eukaryotic cells described herein, including vertebrate host cells. Propagation of vertebrate cells in culture (tissue culture) has become a routine procedure. Examples of useful mammalian host cell lines are: monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney (293 or 293 cell subclones for suspension culture, graham et al, J.Gen. Virol.36:59 (1977)); baby hamster kidney cells (BHK, ATCC CCL 10); chinese hamster ovary cells/-DHFR (CHO, urlaub et al, proc.Natl. Acad.sci.usa 77:4216 (1980)); mouse support cells (TM 4, mather, biol. Reprod.23:243-251 (1980)); monkey kidney cells (CV 1 ATCC CCL 70); african green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical cancer cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat hepatocytes (BRL 3a, atcc CRL 1442); human lung cells (W138, ATCC CCL 75); human hepatocytes (Hep G2, HB 8065); mouse mammary tumor (MMT 060562,ATCC CCL 51); TR1 cells (Mather et al, annals N.Y. Acad. Sci.383:44-68 (1982)); MRC 5 cells; FS4 cells; and human liver cancer cell line (Hep G2).
Host cells are transformed with the expression or cloning vectors described above for the production of the immunoregulatory molecule and cultured in conventional nutrient media which are suitably modified to induce promoters, select transformants or amplify the genes encoding the desired sequences.
h) Culturing host cells
Host cells for producing the immunomodulatory molecules of the present application can be cultured in a variety of media. Commercially available media such as Ham's Fl0 (Sigma), minimal essential media ((MEM), (Sigma), RPMI-1640 (Sigma) and Du Erba family Modified Eagle's Medium (DMEM), sigma) are suitable for culturing host cells in addition, in Ham et al, meth.Enz.58:44 (1979), barnes et al, anal.biochem.102:255 (1980), U.S. Pat. No. 4,767,704, U.S. Pat. No. 4,657,866, U.S. Pat. No. 4,927,762, U.S. Pat. No. 4,560,655, or U.S. Pat. No. 5,122,469, WO 90/03430, WO 87/00195, or any of the media described in U.S. Pat. No. 5,122,469, WO 30,985 may be used as a Medium for host cells, any of these media may be supplemented with hormones and/or other growth factors (such as insulin, transferrin or epidermal growth factors), salts (such as sodium chloride, calcium, magnesium and phosphate), buffers (such as ES), nucleotides (such as adenosine and thymidine (such as GENTAMYCIN) as desired TM Drugs), trace elements (defined as inorganic compounds, typically present in final concentrations in the micromolar range), and glucose or equivalent energy sources. Any other necessary supplements may also be included at appropriate concentrations known to those skilled in the art. Culture conditions, such as temperature, pH, etc., are conditions previously used to select host cells for expression and will be apparent to one of ordinary skill.
i) Protein purification
When recombinant techniques are used, the immunoregulatory molecules may be produced in the intracellular, periplasmic space, or secreted directly into the culture medium. If the immunoregulatory molecule is produced intracellularly, as a first step, particulate debris, whether host cells or lysed fragments, is removed, for example, by centrifugation or ultrafiltration. Carter et al, bio/Technology 10:163-167 (1992) describes a procedure for isolating antibodies secreted into the periplasmic space of E.coli. Briefly, the cell paste was thawed in the presence of sodium acetate (ph 3.5), EDTA and phenylmethylsulfonyl fluoride (PMSF) for about 30 minutes. Cell debris can be removed by centrifugation. When an immunomodulatory molecule is secreted into the culture medium, the supernatant from such an expression system is typically first concentrated using a commercially available protein concentration filter (e.g., an Amicon or Millipore Pellicon ultrafiltration device). Protease inhibitors such as PMSF may be included in any of the foregoing steps to inhibit proteolysis, and antibiotics may be included to prevent the growth of foreign contaminants.
Protein compositions prepared from cells can be purified using, for example, hydroxyapatite chromatography, gel electrophoresis, dialysis, and affinity chromatography, with affinity chromatography being the preferred purification technique. The suitability of protein a as an affinity ligand depends on the type and isotype of any immunoglobulin Fc domain present in the immunoregulatory molecule. Protein A can be used to purify immunoregulatory molecules, antigen binding fragment-Fc fusion proteins or antibodies based on human immunoglobulins containing 1, 2 or 4 heavy chains (Lindmark et al, J.Immunol. Meth.62:1-13 (1983)). Protein G was recommended for all mouse isoforms and human 3 (Guss et al, EMBO J.5:15671575 (1986)). The matrix to which the affinity ligand is attached is most commonly agarose,other substrates are also useful. Mechanically stable matrices such as controlled pore glass or poly (styrene-divinylbenzene) allow for faster flow rates and shorter processing times than are achieved using agarose. When the immunoregulatory molecule comprises C H 3 domain, bakerbond ABXTM resin (j.t. baker, philips burg, n.j.) is useful for purification. Other techniques for protein purification, such as fractionation on ion exchange columns, ethanol precipitation, reverse phase HPLC, silica chromatography, heparin SEPHAROSE, can also be used depending on the immunoregulatory molecule to be recovered TM Chromatography, anion or cation exchange resin (e.g., polyaspartic acid column) chromatography, chromatography focusing, SDS-PAGE, and ammonium sulfate precipitation.
After any preliminary purification steps, the mixture comprising the target immunomodulatory molecule and the contaminant may be subjected to low pH hydrophobic interaction chromatography using an elution buffer at a pH of about 2.5-4.5, preferably at a low salt concentration (e.g., about 0-0.25M salt).
V, pharmaceutical composition
Also provided are pharmaceutical compositions comprising any of the immunomodulatory molecules described herein (e.g., an IL-2/anti-PD-1 agonist antibody immunomodulatory molecule, an IL-12/anti-PD-1 agonist antibody immunomodulatory molecule, an IL-2/PD-L1 immunomodulatory molecule, an IL-12/PD-L1 immunomodulatory molecule, an IL-2/PD-L2 immunomodulatory molecule, an IL-12/PD-L2 immunomodulatory molecule, as described in any of figures 1A-1W and 11A-15D, examples, and sequence listings herein), and optionally a pharmaceutically acceptable carrier. Pharmaceutical compositions may be prepared by mixing an immunomodulatory molecule of desired purity as described herein with an optional pharmaceutically acceptable carrier, excipient or stabilizer (Remington's Pharmaceutical Sciences 16th edition,Osol,A.Ed (1980)) in the form of a lyophilized formulation or aqueous solution.
Reconstituted formulations can be prepared by dissolving the lyophilized immunomodulatory molecules described herein in a diluent to disperse the protein throughout. Exemplary pharmaceutically acceptable (safe and non-toxic for human administration) diluents suitable for use in the present application include, but are not limited to: sterile water, bacteriostatic water for injection (BWFI), pH buffered solutions (e.g., phosphate buffered saline), sterile saline solutions, ringer's solution or dextrose solution, or aqueous solutions of salts and/or buffers.
In some embodiments, the pharmaceutical composition comprises a homogeneous population of the immunomodulatory molecules described herein. By homogeneous population is meant that the immunomodulatory molecules are identical to each other, e.g., identical immunomodulatory molecule configurations, identical first binding domains (e.g., cytokine moieties), identical second binding domains (e.g., ligands, receptors, VHHs, scFv, or Fab), identical linkers (if any), identical hinge regions, and identical Fc domains. In some embodiments, at least about 70% (e.g., at least about any of 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) of the immunomodulatory molecules in the pharmaceutical composition are homogeneous.
The pharmaceutical composition is preferably stable, wherein the immunomodulatory molecule substantially retains its physical and chemical stability and integrity upon storage. A variety of analytical techniques for measuring protein stability are available in the art and are reviewed in Peptide and Protein Drug Delivery,247-301,Vincent Lee Ed, marcel Dekker, inc., new York, N.Y., pubs. (1991) and Jones, A.adv. Drug Delivery Rev.10:29-90 (1993). Stability may be measured at a selected temperature for a selected period of time. For rapid screening, the formulation may be stored at 40 ℃ for 2 weeks to 1 month, at which time stability is measured. When the formulation is to be stored at 2-8 ℃, the formulation should generally be stable for at least 1 month at 30 ℃ or 40 ℃ and/or at least 2 years at 2-8 ℃. When the formulation is to be stored at 30 ℃, the formulation should generally be stable for at least 2 years at 30 ℃ and/or for at least 6 months at 40 ℃. For example, the extent of aggregation during storage can be used as an indicator of protein stability. In some embodiments, a stable formulation of an immunomodulatory molecule described herein may comprise less than about 10% (preferably less than about 5%) of the immunomodulatory molecule present as aggregates in the formulation.
Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers, antioxidants (including ascorbic acid, methionine, vitamin E, sodium metabisulfite); preservatives, isotonic agents (e.g., sodium chloride), stabilizers, metal complexes (e.g., zinc-protein complexes); chelating agents such as EDTA and/or nonionic surfactants.
Examples of physiologically acceptable carriers include: buffers such as phosphates, citrates and other organic acids; antioxidants include ascorbic acid and methionine; preservatives (e.g., octadecyldimethylbenzyl ammonium chloride, hexamethylammonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butanol or benzyl alcohol, alkyl p-hydroxybenzoates such as methyl or propyl p-hydroxybenzoate, catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol); a low molecular weight (less than about 10 residues) polypeptide; proteins, such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., zinc-protein complexes); and/or nonionic surfactants such as TWEEN TM Polyethylene glycol (PEG) and PLURONICS TM Or polyethylene glycol (PEG).
Buffers are used to control the pH within a range that optimizes the therapeutic effect, especially if the stability is pH dependent. The buffer is preferably present at a concentration in the range of about 50mM to about 250 mM. Buffer agents suitable for use in the present application include organic and inorganic acids and salts thereof. For example, citrate, phosphate, succinate, tartrate, fumarate, gluconate, oxalate, lactate, acetate. In addition, the buffer may comprise histidine and trimethylamine salts such as Tris.
Preservatives are added to retard microbial growth and are typically present in the range of 0.2% to 1.0% (w/v). The addition of preservatives may, for example, facilitate the production of multiple use (multi-dose) formulations. Preservatives suitable for use in the present application include: octadecyldimethylbenzyl ammonium chloride; hexamine chloride; benzalkonium chloride halides (e.g., chloride, bromide, iodide), benzethonium chloride; merthiolate, phenol, butanol, or benzyl alcohol; alkyl parahydroxybenzoates (e.g., methyl parahydroxybenzoate or propyl parahydroxybenzoate); catechol; resorcinol; cyclohexanol, 3-pentanol and m-cresol.
Tonicity agents, sometimes referred to as "stabilizers," are present to regulate or maintain the tonicity of the liquid in the composition. When used with charged large biomolecules (such as proteins and antibodies), they are often referred to as "stabilizers" because they can interact with the charged groups of the amino acid side chains, thereby reducing the likelihood of intermolecular and intramolecular interactions. The tonicity agent may be present in any amount between 0.1% and 25% by weight, preferably 1% to 5% by weight, taking into account the relative amounts of the other ingredients. Preferred tonicity agents include polyols, preferably tri-or higher sugar alcohols, such as glycerol, erythritol, arabitol, xylitol, sorbitol, and mannitol.
Other excipients include agents that may be used as one or more of the following: (1) a filler, (2) a solubility enhancer, (3) a stabilizer, and (4) an agent that prevents denaturation or adhesion to the container wall. Such excipients include: a polyhydric sugar alcohol (listed above); amino acids such as alanine, glycine, glutamine, asparagine, histidine, arginine, lysine, ornithine, leucine, 2-phenylalanine, glutamic acid, threonine, etc.; organic sugars or sugar alcohols such as sucrose, lactose, lactitol, trehalose, stachyose, mannose, sorbose, xylose, ribose, ribitol, myo-inositol, galactose, galactitol, glycerol, cyclic sugar alcohols (e.g., inositol), polyethylene glycol; sulfur-containing reducing agents such as urea, glutathione, lipoic acid, sodium thioglycolate, thioglycerol, alpha-monothioglycerol, and sodium thiosulfate; low molecular weight proteins such as human serum albumin, bovine serum albumin, gelatin or other immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; monosaccharides (e.g., xylose, mannose, fructose, glucose, disaccharides (e.g., lactose, maltose, sucrose), trisaccharides such as raffinose, and polysaccharides such as dextrin or dextran.
Nonionic surfactants or detergents (also referred to as "wetting agents") are present to help solubilize the immunoregulatory molecules and protect the immunoregulatory molecules from agitation-induced aggregation, which also allows the formulation to be exposed to shear surface stresses without causing denaturation of the active immunoregulatory molecules. The nonionic surfactant is present in a range of about 0.05mg/ml to about 1.0mg/ml, preferably about 0.07mg/ml to about 0.2 mg/ml.
Suitable nonionic surfactants include: polysorbates (20, 40, 60, 65, 80, etc.), poloxamers (184, 188, etc.),polyol(s)>Polyoxyethylene sorbitan monoether ]-20、/>-80, etc.), laurol 400, polyoxyl 40 stearate, polyoxyethylene hydrogenated castor oil 10, 50 and 60, glyceryl monostearate, sucrose fatty acid ester, methylcellulose and carboxymethylcellulose. Anionic detergents that may be used include: sodium lauryl sulfate, sodium dioctyl sulfosuccinate and sodium dioctyl sulfonate. Cationic detergents include benzalkonium chloride or benzethonium chloride.
In order for pharmaceutical compositions to be useful for in vivo administration, they must be sterile. The pharmaceutical composition may be rendered sterile by filtration through a sterile filtration membrane. The pharmaceutical compositions herein are typically placed in a container having a sterile access port, such as an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle.
Sustained release formulations can be prepared. Suitable examples of sustained-release formulations include semipermeable matrices of solid hydrophobic polymers containing the antagonist, which matrices are in the form of shaped articles, e.g., films, or microcapsules. Examples of sustained release matrices include: polyesters, hydrogels (e.gPoly (2-hydroxyethyl-methacrylate) or poly (vinyl alcohol)), polylactide (U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and ethyl L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers, such as LUPRON DEPOT TM (injectable microspheres consisting of lactic acid-glycolic acid copolymer and leuprorelin acetate) and poly-D- (-) -3-hydroxybutyric acid.
The pharmaceutical compositions herein may also contain more than one active compound necessary for the particular indication being treated, preferably those active compounds having complementary activities that do not adversely affect each other. Alternatively, or in addition, the composition may comprise a cytotoxic agent, a chemotherapeutic agent, a cytokine, an immunosuppressant, or a growth inhibitory agent. Such molecules are suitably present in combination in an amount effective for the intended purpose.
The active ingredient may also be embedded in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, such as hydroxymethylcellulose or gelatin-microcapsules and poly- (methylmesylate) microcapsules, respectively, in colloidal drug delivery systems (e.g. liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences, 18 th edition.
In some embodiments, the pharmaceutical composition is contained in a single-use vial, such as a single-use sealed vial. In some embodiments, the pharmaceutical composition is contained in a multi-use vial. In some embodiments, the pharmaceutical composition is in bulk in a container. In some embodiments, the pharmaceutical composition is cryopreserved.
Methods of treating diseases or directing cytokine activity
The immunomodulatory molecules described herein (e.g., as described in any of figures 1A-1W and 11A-15D, examples, and sequence listing herein, e.g., IL-2/anti-PD-1 agonist antibody immunomodulatory molecules, IL-12/anti-PD-1 agonist antibody immunomodulatory molecules, IL-2/PD-L1 immunomodulatory molecules, IL-12/PD-L1 immunomodulatory molecules, IL-2/PD-L2 immunomodulatory molecules, IL-12/PD-L2 immunomodulatory molecules), and compositions thereof (e.g., pharmaceutical compositions) can be used in a variety of applications, such as in diagnostics, molecular assays, and therapeutics. In some embodiments, a method of treating a disease (e.g., cancer (e.g., PD-l1+ and/or PD-l2+ cancer), an infection (e.g., a viral infection), an autoimmune disease, allergy, graft rejection, or GvHD) in an individual (e.g., a human) is provided that includes administering to the individual an effective amount of any of the immunomodulatory molecules described herein or a pharmaceutical composition thereof. In some embodiments, there is also provided a method of modulating an immune response in an individual (e.g., a human) comprising administering to the individual an effective amount of any of the immunomodulatory molecules described herein or a pharmaceutical composition thereof. In some embodiments, the activity of the first binding domain (e.g., a cytokine or variant thereof) is selectively activated upon binding of the immunomodulatory molecule to the second target molecule when the first binding domain is located at the hinge region between the second binding domain and the Fc domain or portion thereof. In some embodiments, the immunoregulatory molecule or pharmaceutical composition thereof is administered intravenously, subcutaneously, or intratumorally. In some embodiments, the immunomodulatory molecule, or a pharmaceutical composition thereof, is administered in an amount of about 1 μg/kg to about 10 mg/kg. In some embodiments, the immunomodulatory molecule, or a pharmaceutical composition thereof, is administered once every three weeks. In some embodiments, the cancer is selected from the group consisting of: lung cancer, liver cancer, renal disease, colorectal cancer, ovarian cancer, breast cancer, pancreatic cancer, gastric cancer, bile duct cancer, squamous cell carcinoma, bladder cancer, esophageal cancer, mesothelioma, melanoma, head and neck cancer, thyroid cancer, sarcoma, prostate cancer, glioblastoma, cervical cancer, thymus cancer, leukemia, lymphoma, myeloma, mycosis fungoides, and merck cell carcinoma.
In some embodiments, the method of treating cancer has one or more of the following biological activities: (1) killing cancer cells; (2) inhibiting proliferation of cancer cells; (3) Inducing an immune response in the tumor (e.g., inducing infiltration of immune effector cells to the tumor site, inducing proliferation, differentiation, and/or activation of immune cells, and/or inducing secretion of pro-inflammatory cytokines by immune cells); (4) reducing the tumor size; (5) Alleviating one or more symptoms in an individual suffering from cancer; (6) inhibiting tumor metastasis; (7) prolonged survival; (8) extending the time to progression of cancer; and (9) preventing, inhibiting or reducing the likelihood of cancer recurrence. In some embodiments, methods of killing cancer cells mediated by an immunomodulatory molecule or pharmaceutical composition described herein can achieve a tumor cell death rate of at least about any one of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more. In some embodiments, a method of reducing tumor size mediated by an immunomodulatory molecule or pharmaceutical composition described herein can reduce tumor size by at least about 10% (including, for example, at least about any of 20%, 30%, 40%, 60%, 70%, 80%, 90%, or 100%). In some embodiments, a method of inhibiting tumor metastasis mediated by an immunomodulatory molecule or pharmaceutical composition described herein can inhibit metastasis by at least about 10% (including, for example, at least about any one of 20%, 30%, 40%, 60%, 70%, 80%, 90%, or 100%). In some embodiments, a method of prolonging survival of an individual (e.g., a human) mediated by an immunomodulatory molecule or pharmaceutical composition described herein can prolong survival of the individual by at least any one of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 18, or 24 months. In some embodiments, the method of prolonging the progression of cancer mediated by an immunomodulatory molecule or pharmaceutical composition described herein can extend the progression of cancer by at least any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 weeks. In some embodiments, the method of inducing an immune response to a tumor may increase, enhance or stimulate an immune response or function in a subject. In some embodiments, the immune response or function is increased, enhanced, or stimulated by activating effector cells (e.g., T cells, such as cd8+ and/or cd4+ T cells), expanding (increasing) effector cell populations, and/or killing target cells (e.g., target tumor cells) in the subject. In some embodiments, CD4 and/or CD 8T cells in the subject have increased or enhanced priming, activation, proliferation, cytokine release, and/or cytolytic activity relative to prior to administration of the immunomodulatory molecule or pharmaceutical composition described herein.
The methods described herein are useful for treating a variety of cancers, including solid and liquid cancers. The method is applicable to all stages of cancer including early stage cancer, non-metastatic cancer, primary cancer, advanced cancer, locally advanced cancer, metastatic cancer, or remission stage cancer. The methods described herein may be used as a first therapy, a second therapy, a third therapy, or a combination therapy with other types of cancer therapies known in the art such as surgery, radiation, chemotherapy, immunotherapy, hormonal therapy, or a combination thereof. In some embodiments, the methods are used to treat an individual having previously received treatment. In some embodiments, the cancer is refractory to prior treatment. In some embodiments, the method is for treating an individual who has not previously been treated. In some embodiments, the cancer is partially resistant to immune checkpoint inhibitor monotherapy (e.g., partially resistant to anti-PD-1 or anti-PD-L1 antibody monotherapy treatment).
In some embodiments, the cancer is a PD-L1 expressing cancer. In some embodiments, the methods are useful for treating cancers having aberrant PD-1 or PD-L1/PD-L2 expression (e.g., her2+ cancer), activity, and/or signaling, including hematological cancers and/or solid tumors, as non-limiting examples. Some cancers whose growth may be inhibited using the immunomodulatory molecules of the invention include cancers that are generally responsive to immunotherapy. Non-limiting examples of other cancers for treatment include: melanoma (e.g., metastatic malignant melanoma), renal cancer (e.g., clear cell carcinoma), prostate cancer (e.g., hormone refractory prostate cancer), breast cancer, colon cancer, and lung cancer (e.g., non-small cell lung cancer). In addition, the invention includes refractory or recurrent malignancies whose growth can be inhibited using the immunomodulatory molecules of the invention. The invention is also useful for the treatment of metastatic cancers, particularly those that express PD-L1 (Iwai et al (2005) int. Immunol. 17:133-144). In some embodiments, cancers with aberrant PD-1 or PD-L1/PD-L2 expression, activity, and/or signaling are partially resistant to PD-1 or PD-L1 blockade (e.g., partially resistant to anti-PD-1 antibodies or anti-PD-L1 antibody therapies).
In some embodiments, the methods described herein are suitable for treating a solid cancer selected from the group consisting of: colon cancer, rectal cancer, renal cell carcinoma, liver cancer, non-small cell lung cancer, small intestine cancer, esophagus cancer, melanoma, bone cancer, pancreas cancer, skin cancer, head and neck cancer, malignant melanoma of the skin or eye, uterine cancer, ovarian cancer, rectal cancer, anal cancer, stomach cancer, testicular cancer, uterine cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulval cancer, hodgkin's disease, non-hodgkin's lymphoma (NHL), cutaneous T-cell lymphoma (CTCL), cancer of the endocrine system, thyroid cancer, parathyroid cancer, adrenal gland cancer, soft tissue sarcoma, urinary tract cancer, penile cancer, childhood solid tumor, bladder cancer, kidney or ureter cancer, renal pelvis cancer, neoplasms of the Central Nervous System (CNS), primary CNS lymphoma, tumor angiogenesis, spinal axis tumor, brain stem glioma, pituitary adenoma, kaposi sarcoma, epidermoid carcinoma, squamous cell carcinoma, T-cell lymphoma, environmentally induced cancer, combinations of said cancers, and metastatic lesions of said cancers.
In some embodiments, the methods described herein are suitable for treating hematological cancers selected from one or more of the following: acute Myelogenous Leukemia (AML), chronic Lymphocytic Leukemia (CLL), acute leukemia, acute Lymphoblastic Leukemia (ALL), B-cell acute lymphoblastic leukemia (B-ALL), T-cell acute lymphoblastic leukemia (T-ALL), chronic Myelogenous Leukemia (CML), B-cell prolymphocytic leukemia, blast plasmacytoid dendritic cell neoplasm, burkitt lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, hairy cell leukemia, small or large cell follicular lymphoma, malignant lymphoproliferative condition, MALT lymphoma, mantle cell lymphoma, marginal zone lymphoma, multiple myeloma, myelodysplastic and myelodysplastic syndrome, non-hodgkin lymphoma, plasmablastoid lymphoma, plasmacytoid dendritic cell neoplasm, fahrenheit macroglobulinemia or pre-leukemia. 387
In some embodiments, the methods described herein are used to treat an infection, such as a fungal, viral, bacterial, protozoal or other parasitic infection. In some embodiments, the methods of treating an infection described herein prevent exacerbation of at least one symptom of a pathogen infection, prevent and/or ameliorate at least one symptom of a pathogen infection, reduce or eliminate a pathogen, prevent damage to the individual or an organ or tissue of the individual, and/or prevent death in an individual in need thereof. In some embodiments, the methods described herein may implement one or more of the following: (a) Control, amelioration and/or prevention of tissue and/or organ damage or failure, as caused by viral infection; (b) Control, reduce, and/or inhibit cell necrosis (e.g., reduce cell necrosis by at least about 10% (including, e.g., at least about any of 20%, 30%, 40%, 60%, 70%, 80%, 90%, or 100%), such as necrosis in infected and/or uninfected tissues and/or organs; (c) Controlling and/or increasing infiltration of inflammatory cells (e.g., NK cells, cytotoxic T cells, neutrophils) in the infected tissue and/or organ, such as increasing inflammatory cell infiltration by at least about 10% (including, for example, at least about any of 20%, 30%, 40%, 60%, 70%, 80%, 90%, or 100%); (d) Controlling, ameliorating and/or preventing inflammation, systemic inflammation and/or cytokine storm of uninfected tissues and/or organs, down-regulating at least about 10% (including, for example, at least about any of 20%, 30%, 40%, 60%, 70%, 80%, 90% or 100%); (e) Reducing mortality associated with pathogen infection, and/or preventing mortality, such as reducing mortality by at least about 10% (including, for example, at least about any of 20%, 30%, 40%, 60%, 70%, 80%, 90%, or 100%); and (f) reducing or eliminating pathogens by at least about 10% (including, for example, at least about any of 20%, 30%, 40%, 60%, 70%, 80%, 90%, or 100%).
In some embodiments, the methods described herein are used to treat an immune disorder, such as an autoimmune disorder or immunosuppression.
In some embodiments, the methods described herein are used to treat immunosuppression. Immunosuppression is a decrease or complete loss of activation or efficacy of the immune system, resulting in an immune system that is weak against a disease, such as an infectious disease or cancer. Immunosuppression may be the result of a disease or may be the result of a drug or infection, resulting in increased susceptibility to secondary infections by pathogens such as bacteria and viruses. Many diseases are characterized by the occurrence of progressive immunosuppression in patients. Patients with malignant tumors (e.g., leukemia, lymphoma, multiple myeloma) are well documented for an impaired immune response. Progressive immunosuppression is also observed in certain chronic infections such as aids, sepsis, leprosy, cytomegalovirus infection, malaria, lupus, and the like. Immunodeficiency is also a potential adverse effect of many therapeutic approaches (e.g., radiation therapy or chemotherapy). For example, but not limited to, diseases and conditions associated with immunodeficiency or immunosuppression include: human Immunodeficiency Virus (HIV) infection and acquired immunodeficiency syndrome (AIDS), hypogammaglobulinemia, hematological cancers such as leukemia and lymphoma, lymphopenia of any origin (lymphopenia), lupus erythematosus, cachexia, opioid abuse, mastocytosis, rheumatic fever, trypanosomiasis, and alcohol abuse. In some embodiments, immunosuppression is associated with immune checkpoint signaling (e.g., PD-1 or CTLA-4 signaling). In such unintended immunosuppressive situations, the patient is typically treated with an immunostimulant (e.g., cytokine) to enhance the immune system. However, due to lack of specificity, such immunostimulants typically activate the immune system and may trigger excessive activation of the immune system.
In some embodiments, the methods of treating immunosuppression described herein activate or enhance an immune response, increase the ratio of CD8 to CD4, promote immune cell proliferation and/or differentiation, induce or enhance cytokine release (e.g., IL-2, IL-6, IFN- γ), prevent worsening of at least one symptom of immunosuppression, prevent and/or ameliorate at least one symptom of immunosuppression, and/or prevent death in an individual in need thereof.
In some embodiments, the methods described herein are used to treat autoimmune diseases. Autoimmune diseases are diseases caused by an immune response against self-tissues or tissue components, including autoantibody responses and cell-mediated responses. As used herein, the term "autoimmune disease" encompasses organ-specific autoimmune diseases, wherein the autoimmune response is against a single tissue, such as type I diabetes (T1D), crohn's disease, ulcerative colitis, myasthenia gravis, white spot disease, graves' disease, hashimoto's disease, ai Disen's disease, and autoimmune gastritis and autoimmune hepatitis. The term "autoimmune disease" also encompasses non-organ specific autoimmune diseases, wherein the autoimmune response is directed against components present in several or more organs throughout the body. Such autoimmune diseases include, for example, rheumatoid disease, systemic lupus erythematosus, progressive systemic sclerosis and variants, polymyositis and dermatomyositis. Other autoimmune diseases include pernicious anaemia, including some autoimmune gastritis, primary biliary cirrhosis, autoimmune thrombocytopenia, sjogren's syndrome, multiple sclerosis, and psoriasis. In some embodiments, the autoimmune disease is selected from the group consisting of: polyuria, diabetes, arthritis (including rheumatoid arthritis, juvenile rheumatoid arthritis, osteoarthritis, psoriatic arthritis), multiple sclerosis, myasthenia gravis, systemic lupus erythematosus, autoimmune thyroiditis, dermatitis (including atopic dermatitis and eczematous dermatitis), psoriasis, sjogren's syndrome, including keratoconjunctivitis sicca secondary to sjogren's syndrome, alopecia areata, allergic reactions to arthropod biting reactions, crohn's disease, aphthous ulcers, iritis, conjunctivitis, keratoconjunctivitis, ulcerative colitis, asthma, allergic asthma, cutaneous lupus erythematosus, scleroderma, vaginitis, proctitis, drug eruptions, leprosy reversal reactions, leprosy erythema, autoimmune uveitis, allergic encephalomyelitis, acute necrotizing hemorrhagic encephalopathy, idiopathic bilateral progressive sensorineural deafness, aplastic anemia, pure erythrocyte anemia, idiopathic thrombocytopenia, polychondritis, wegener's granulomatosis, chronic active hepatitis, stevens-johnson syndrome, idiopathic sprue diarrhea, lichen planus, inflammatory Bowel Disease (IBD), crohn's disease, graves' eye disease, sarcoidosis, primary biliary cirrhosis, post-uveitis and interstitial pulmonary fibrosis. Those skilled in the art will appreciate that the methods of the present invention may be applied to these or other autoimmune diseases as desired.
In some embodiments, the methods of treating an autoimmune disease described herein are capable of preventing, halting and/or ameliorating at least one symptom of an autoimmune disease, preventing injury to healthy self-tissues or organs, and controlling, ameliorating and/or preventing infiltration of immune cells into healthy self-tissues and/or organs, systemic inflammation, and/or cytokine storm, and/or preventing death in an individual in need thereof.
In some embodiments, the methods of treating graft rejection described herein prevent exacerbation of at least one symptom of graft rejection in an individual in need thereof, prevent and/or ameliorate at least one symptom of graft rejection in an individual in need thereof; preventing damage to donor/foreign tissues or organs; controlling, ameliorating and/or preventing immune cell infiltration into donor/foreign tissues or organs, systemic inflammation and/or cytokine storms; reducing Th17 cell activation; improving graft survival; prolonging survival, improving survival, and/or preventing death. In some embodiments, the methods of treating GvHD described herein prevent exacerbation of at least one symptom of GvHD in an individual in need thereof, prevent and/or ameliorate at least one symptom of GvHD in an individual in need thereof; reducing Th17 cell activation; preventing damage to self/healthy tissue or organs; controls, improves and/or prevents infiltration of immune cells into self/healthy tissues or organs, systemic inflammation, and/or cytokine storms; improving graft survival; prolonging survival, improving survival, and/or preventing death; and/or to improve disease activity scores (see, e.g., P.J. Martin, biol Blood Marrow Transplant.2009Jul;15 (7): 777-784).
In some embodiments, a method of selectively activating the activity of a cytokine or variant thereof (e.g., IL-2, IFN- α (e.g., IFN- α2b), IFN- γ, IL-10, IL-12, or IL-23) on a cell expressing a target antigen (e.g., CTLA-4, PD-L1, PD-L2, CD123, CD25, HER2, PD-1, CD3, CD4, or CD 8) in an individual (e.g., a human) is provided, comprising administering to the individual an effective amount of an immunomodulatory molecule (or pharmaceutical composition thereof), wherein the immunomodulatory molecule comprises: a) An antigen binding protein (e.g., an antibody such as a full-length antibody, or an antigen binding fragment-hinge-Fc fusion protein such as a ligand/receptor-hinge-Fc fusion protein) that specifically recognizes a target antigen (e.g., CTLA-4, PD-L1, PD-L2, CD123, CD25, HER2, PD-1, CD3, CD4, or CD 8); and b) a cytokine (e.g., IL-2, IFN- α (e.g., IFN- α2b), IFN- γ, IL-10, IL-12, or IL-23), or a variant thereof, wherein the antigen binding protein comprises an antigen binding polypeptide (e.g., an antibody heavy chain, or an antigen binding fragment-hinge-Fc fusion polypeptide such as a ligand/receptor-hinge-Fc fusion polypeptide) comprising from N-terminus to C-terminus: an antigen-binding fragment (e.g., ligand, receptor, VHH, scFv, or VH), a hinge region, and an Fc domain subunit or portion thereof (e.g., ch2+ch3, or CH2 only), wherein the cytokine or variant thereof is located at the hinge region (e.g., N ', C', or within); and wherein the activity of the cytokine or variant thereof is selectively activated upon binding of the antigen binding protein to the target antigen. In some embodiments, a method of selectively activating the activity of a cytokine or variant thereof (e.g., IL-2, IFN- α (e.g., IFN- α2b), IFN- γ, IL-10, IL-12, or IL-23) on a cell expressing a target antigen (e.g., CTLA-4, PD-L1, PD-L2, CD123, CD25, HER2, PD-1, CD3, CD4, or CD 8) in an individual (e.g., a human) is provided, comprising administering to the individual an effective amount of an immunomodulatory molecule (or pharmaceutical composition thereof), wherein the immunomodulatory molecule comprises: a) An antibody (e.g., a full length antibody, a heavy chain-only antibody, or an antigen-binding fragment fused to an Fc domain subunit or portion thereof via a hinge region) that specifically recognizes a target antigen (e.g., CTLA-4, PD-L1, PD-L2, CD25, CD123, HER2, PD-1, CD3, CD4, or CD 8); and b) a cytokine (e.g., IL-2, IFN- α (e.g., IFN- α2b), IFN- γ, IL-10, IL-12, or IL-23) or variant thereof, wherein the antibody comprises a heavy chain comprising a hinge region, and wherein the cytokine or variant thereof is located in the hinge region of the heavy chain (e.g., within the hinge region, or between the C-terminus of CH1 and the N-terminus of the hinge region); and wherein the activity of the cytokine or variant thereof is selectively activated upon binding of the antibody to the target antigen. In some embodiments, a method of selectively activating the activity of a cytokine or variant thereof (e.g., IL-2, IFN- α (e.g., IFN- α2b), IFN- γ, IL-10, IL-12, or IL-23) on a cell expressing a target antigen (e.g., CTLA-4, PD-L1, PD-L2, CD123, CD25, HER2, PD-1, CD3, CD4, or CD 8) in an individual (e.g., a human) is provided, comprising administering to the individual an effective amount of an immunomodulatory molecule (or pharmaceutical composition thereof), wherein the immunomodulatory molecule comprises: a) An antibody (e.g., a full length antibody, or an antigen binding fragment fused to an Fc domain subunit or portion thereof via a hinge region) that specifically recognizes a target antigen (e.g., CTLA-4, PD-L1, PD-L2, CD25, CD123, HER2, PD-1, CD3, CD4, or CD 8); and b) a cytokine (e.g., IL-2, IFN- α (e.g., IFN- α2b), IFN- γ, IL-10, IL-12, or IL-23), or a variant thereof, wherein the antibody comprises a heavy chain comprising from N-terminus to C-terminus: a chain of a VH domain, optionally a CH1 domain, a cytokine or variant thereof at the hinge region, a CH2 domain, and optionally a CH3 domain; and wherein the activity of the cytokine or variant thereof is selectively activated upon binding of the antibody to the target antigen. In some embodiments, a method of selectively activating the activity of a cytokine or variant thereof (e.g., IL-2, IFN- α (e.g., IFN- α2b), IFN- γ, IL-10, IL-12, or IL-23) on a cell expressing a target antigen (e.g., CTLA-4, PD-L1, PD-L2, CD123, CD25, HER2, PD-1, CD3, CD4, or CD 8) in an individual (e.g., a human) is provided, comprising administering to the individual an effective amount of an immunomodulatory molecule (or pharmaceutical composition thereof), wherein the immunomodulatory molecule comprises: a) Full length antibodies that specifically recognize a target antigen (e.g., CTLA-4, PD-L1, PD-L2, CD25, CD123, HER2, PD-1, CD3, CD4, or CD 8); and b) a cytokine (e.g., IL-2, IFN- α (e.g., IFN- α2b), IFN- γ, IL-10, IL-12, or IL-23) or variant thereof, wherein the cytokine or variant thereof is located in the hinge region (e.g., within the hinge region, or between the C-terminus of CH1 and the N-terminus of the hinge region) of the heavy chain of the full-length antibody; and wherein the activity of the cytokine or variant thereof is selectively activated upon binding of the full length antibody to the target antigen. In some embodiments, the activity (binding affinity to a corresponding cytokine receptor or subunit thereof, and/or biological activity) of the cytokine is increased by at least about 20% (e.g., at least about any of 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500% or more) in the presence of binding of the antigen binding protein (e.g., an antibody such as a full-length antibody, or an antigen binding fragment-hinge-Fc fusion protein such as a ligand/receptor-hinge-Fc fusion protein) or antigen binding fragment (e.g., a ligand, receptor, VHH, scFv, fab) to the target antigen, as compared to the absence of binding of the antigen binding protein (e.g., an antibody such as a full-length antibody, or an antigen binding fragment-hinge-Fc fusion protein such as a ligand/receptor-hinge-Fc fusion protein) or antigen binding fragment (e.g., ligand, receptor, VHH, scFv, fab) to the target antigen. In some embodiments, the activity (binding affinity to the corresponding cytokine receptor or subunit thereof, and/or biological activity) of the cytokine or variant thereof at the hinge region of the heavy chain in the absence of binding of the antigen binding protein (e.g., an antibody such as a full length antibody, or an antigen binding fragment-hinge-Fc fusion protein such as a ligand/receptor-hinge-Fc fusion protein) or antigen binding fragment (e.g., a ligand, receptor, VHH, scFv, fab) to the target antigen is no more than about 70% (e.g., no more than about any of 60%, 50%, 40%, 30%, 20%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1% or 0%) of the activity of the corresponding cytokine or variant thereof in the free state. In some embodiments, the cytokine or variant thereof is a cytokine variant, and wherein the activity (binding affinity to the corresponding cytokine receptor or subunit thereof, and/or biological activity) of the cytokine variant in the free state is no more than about 80% (e.g., no more than any of 70%, 60%, 50%, 40%, 30%, 20%, 10%, or 5%) of the activity of the corresponding wild-type cytokine in the free state.
Administration of the immunomodulatory molecules described herein, or pharmaceutical compositions thereof, may be performed in any convenient manner, including by injection or transfusion. Routes of administration are according to known and accepted methods, such as by single or multiple bolus or infusion over a prolonged period of time in a suitable manner. The immunoregulatory molecule or pharmaceutical composition thereof may be administered to the patient arterially, subcutaneously, intradermally, intratumorally, intranodal, intramedullary, intramuscularly, intravenously or intraperitoneally. In some embodiments, the immunomodulatory molecule, or a pharmaceutical composition thereof, is administered systemically. In some embodiments, the immunomodulatory molecule, or a pharmaceutical composition thereof, is administered to the individual by infusion (e.g., intravenous infusion). Infusion techniques for immunotherapy are known in the art (see, e.g., rosenberg et al, new Eng. J. Of Med.319:1676 (1988)). In some embodiments, the immunomodulatory molecule, or a pharmaceutical composition thereof, is administered to the individual by intradermal or subcutaneous (i.e., under the skin) injection. For subcutaneous injection, a syringe may be used to inject the immunomodulatory molecule or pharmaceutical composition. However, other devices for administering an immunomodulatory molecule or pharmaceutical composition are available, such as injection devices; an injection pen; an automatic injector device, a needleless device; subcutaneous patch delivery systems. In some embodiments, the immunomodulatory molecule, or a pharmaceutical composition thereof, is administered by intravenous injection. In some embodiments, the immunoregulatory molecule or pharmaceutical composition thereof is injected directly into the tumor or lymph node. In some embodiments, the immunomodulatory molecule, or a pharmaceutical composition thereof, is administered topically to a tumor site, such as directly to tumor cells, or to tissue with tumor cells. In some embodiments, the immunomodulatory molecule, or a pharmaceutical composition thereof, is administered by sustained release or extended release.
The dosage and desired concentration of the pharmaceutical composition of the present invention may vary depending upon the particular use contemplated. Determination of the appropriate dosage or route of administration is well within the skill of the ordinarily skilled artisan. Animal experiments provide reliable guidance for determining effective dosages for human therapy. Effective dose interspecific scaling may be performed according to the principles established by Mordinti, J.and Chappell, W. "The Use of Interspecies Scaling in Toxicokinetics," In Toxicokinetics and New Drug Development, yacobi et al, eds, pergamon Press, new York 1989, pp.42-46. Within the scope of the present application, different formulations may be effective for different treatments and different conditions, and administration intended to treat a particular organ or tissue may need to be delivered in a different manner than to another organ or tissue.
When the immunomodulatory molecules described herein, or a pharmaceutical composition thereof, are administered in vivo, the normal dose may vary from about 1 μg/kg to about 10mg/kg of mammalian body weight, depending on the route of administration and the type of mammal. Within the scope of the present application, different formulations may be effective for different treatments and different conditions, and administration intended to treat a particular organ or tissue may need to be delivered in a different manner than to another organ or tissue. Furthermore, the dose may be administered by one or more separate administrations or by continuous infusion. For repeated administrations over several days or longer, depending on the condition, the treatment continues until the desired inhibition of disease symptoms occurs. However, other dosage regimens may be useful. The progress of this treatment is readily monitored by conventional techniques and assays. In some embodiments, an immunomodulatory molecule described herein, or a pharmaceutical composition thereof, is administered in an amount of about 1 μg/kg to about 10mg/kg, such as any one of the following: about 1 μg/kg to about 500 μg/kg, about 500 μg/kg to about 1mg/kg, about 1mg/kg to about 10mg/kg, about 1 μg/kg to about 1mg/kg, about 1 μg/kg to about 200 μg/kg, about 100 μg/kg to about 500 μg/kg, about 100 μg/kg to about 1mg/kg, or about 500 μg/kg to about 1mg/kg.
In some embodiments, an immunomodulatory molecule, or a pharmaceutical composition thereof, described herein is administered to an individual (e.g., a human) for a period of no more than about any one of 24 hours, 20 hours, 15 hours, 10 hours, 8 hours, 6 hours, 3 hours, 2 hours, 1 hour, 30 minutes, or less. In some embodiments, an immunomodulatory molecule, or a pharmaceutical composition thereof, described herein is administered to an individual (e.g., a human) over a period of any of about 30 minutes to about 1 hour, about 1 hour to about 2 hours, about 2 hours to about 4 hours, about 4 hours to about 6 hours, about 6 hours to about 8 hours, about 8 hours to about 10 hours, about 10 hours to about 12 hours, about 12 hours to about 18 hours, about 18 hours to about 24 hours, about 30 minutes to about 2 hours, about 2 hours to about 5 hours, about 5 hours to about 10 hours, about 10 hours to about 20 hours, about 30 minutes to about 10 hours, or about 30 minutes to about 24 hours.
In some embodiments, an immunomodulatory molecule described herein or a pharmaceutical composition thereof is administered in a single administration (e.g., bolus injection). In some embodiments, an immunomodulatory molecule described herein, or a pharmaceutical composition thereof, is administered multiple times (e.g., any of 2, 3, 4, 5, 6, or more times). If administered multiple times, they may be carried out by the same or different routes, and may be carried out at the same site or at alternative sites. The immunomodulatory molecules described herein, or a pharmaceutical composition thereof, may be administered daily to once a year. The interval between administrations may be any of about 24 hours to one year. The spacing may also be irregular (e.g., after tumor progression). In some embodiments, the dosing schedule is not interrupted. The optimal dosage and treatment regimen for a particular patient can be readily determined by one skilled in the medical arts by monitoring the patient for signs of disease and adjusting the treatment accordingly. In some embodiments, an immunomodulatory molecule, or a pharmaceutical composition thereof, described herein is administered once daily (daily), once every 2 days, once every 3 days, once every 4 days, once every 5 days, once every 6 days, once every week, once every 10 days, once every 2 weeks, once every 3 weeks, once every 4 weeks, once every month, once every 2 months, once every 3 months, once every 4 months, once every 5 months, once every 6 months, once every 7 months, once every 8 months, once every 9 months, or once every year. In some embodiments, the interval between administrations is any of about 1 week to 2 weeks, 2 weeks to 1 month, 2 weeks to 2 months, 1 month to 3 months, 3 months to 6 months, or 6 months to a year. In some embodiments, an immunomodulatory molecule described herein, or a pharmaceutical composition thereof, is administered once every 3 weeks.
In some embodiments, the pharmaceutical composition is administered in divided doses, such as any of about 2, 3, 4, 5, or more doses. In some embodiments, the divided doses are administered over about a week, a month, 2 months, 3 months, or longer. In some embodiments, the dose is aliquoted. In some embodiments, the divided doses are about 20%, about 30% and about 50% of the total dose. In some embodiments, the interval between consecutive divided doses is about 1 day, 2 days, 3 days, 1 week, 2 weeks, 3 weeks, one month or more. For repeated administrations over several days or longer, depending on the condition, the treatment continues until the desired inhibition of disease symptoms occurs. However, other dosage regimens may be useful. The progress of this treatment is readily monitored by conventional techniques and assays.
VII articles and kits
Kits, unit doses, and articles of manufacture comprising any of the immunomodulatory molecules described herein (as described in any of figures 1A-1W and 11A-15D, examples, and sequence listings herein) are also provided. In some embodiments, kits are provided that comprise any of the pharmaceutical compositions described herein, and preferably instructions for their use, as used in treating a disorder described herein (e.g., cancer, infection, or autoimmune disease).
Kits of the invention include one or more containers comprising an immunomodulatory molecule described herein for use in treating a disease. For example, the instructions include descriptions of administration of an immunomodulatory molecule to treat a disease such as cancer. The kit may also include a description of selecting an individual (e.g., a human) suitable for treatment based on identifying whether the individual has the disease and the stage of the disease. Instructions regarding the use of the immunoregulatory molecule generally include information regarding the dosage, dosing regimen, and route of administration of the intended treatment. The container may be a unit dose, a bulk package (e.g., a multi-dose package), or a subunit dose. The instructions provided in the kits of the invention are typically written instructions on a label or package insert (e.g., a piece of paper contained in the kit), but machine readable instructions (e.g., instructions on a magnetic or optical storage disc) are also acceptable. The kits of the present application are in suitable packaging. Suitable packages include, but are not limited to: vials, bottles, jars, flexible packaging (e.g., sealed mylar or plastic bags), and the like. Packages such as infusion devices, such as micropumps, are also contemplated for use in combination with certain devices. The kit may have a sterile inlet (for example, the container may be an intravenous solution bag or a vial with a stopper pierceable by a hypodermic injection needle). At least one active agent in the composition is an immunomodulatory molecule described herein. The container may also contain a second pharmaceutically active agent. The kit may optionally provide additional components such as buffers and interpretation information. Generally, a kit includes a container and a label or package insert on or associated with the container.
Thus, the present application also provides articles of manufacture including vials (e.g., sealed vials), bottles, jars, flexible packaging, and the like. The article of manufacture may comprise a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, and the like. The container may be formed from a variety of materials such as glass or plastic. In general, the container is filled with a composition effective to treat a disease or disorder described herein (e.g., cancer), and may have a sterile access port (e.g., the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). The label or package insert indicates that the composition is used to treat a particular condition in an individual. The label or package insert will further include instructions for applying the composition to an individual. The tag may indicate instructions for reconstruction and/or use. The container containing the pharmaceutical composition may be a multi-use vial that allows for repeated administration (e.g., from 2-6 administrations) of the reconstituted formulation. Package inserts refer to instructions that are typically contained in commercial packages of therapeutic products that contain information regarding the indication, usage, dosage, administration, contraindications, and/or warnings of such therapeutic products. In addition, the article of manufacture may further comprise a second container comprising a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate buffered saline, ringer's solution, and dextrose solution. It may also include other materials, including other buffers, diluents, filters, needles and syringes, as desired from a commercial and user perspective.
The kit or article of manufacture may comprise a plurality of unit doses of the pharmaceutical composition and instructions for use, packaged in amounts sufficient for storage and use in a pharmacy (e.g., hospital pharmacy and compound pharmacy).
Exemplary embodiments
Embodiment 1. An immunomodulatory molecule comprising a first binding domain that specifically recognizes a first target molecule and a second binding domain that specifically recognizes a second target molecule, wherein the first binding domain upregulates an immune response upon binding to the first target molecule, and wherein the second binding domain downregulates the immune response upon binding to the second target molecule.
Embodiment 2. The immunomodulatory molecule of embodiment 1, wherein the first binding domain, when bound to the first target molecule, upregulates an immune response by one or more activities ("upregulation activities") selected from the group consisting of: up-regulating the release of immunostimulatory cytokines, down-regulating the release of immunosuppressive cytokines, up-regulating immune cell proliferation, up-regulating immune cell differentiation, up-regulating immune cell activation, up-regulating cytotoxicity to tumor cells, and up-regulating elimination of infectious pathogens.
Embodiment 3. The immunomodulatory molecule of embodiment 1 or 2, wherein the second binding domain down-regulates the immune response upon binding to the second target molecule by one or more activities ("down-regulating activities") selected from the group consisting of: down-regulating the release of immunostimulatory cytokines, up-regulating the release of immunosuppressive cytokines, down-regulating immune cell proliferation, down-regulating immune cell differentiation, down-regulating immune cell activation, down-regulating cytotoxicity to tumor cells, and down-regulating elimination of infectious pathogens.
Embodiment 4. The immunoregulatory molecule of any one of embodiments 1-3, wherein said immunostimulatory cytokine is selected from the group consisting of: IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-12, IL-15, IL-17, IL-18, IL-21, IL-22, IL-23, IL-27, IFN-alpha, IFN-beta, IFN-gamma, TNF-alpha, erythropoietin, thrombopoietin, G-CSF, M-CSF, SCF and GM-CSF.
Embodiment 5. The immunoregulatory molecule of any one of embodiments 1-4, wherein said immunosuppressive cytokine is selected from the group consisting of: IL-1Ra, IL-4, IL-5, IL-6, IL-10, IL-11, IL-13, IL-27, IL-33, IL-35, IL-37, IL-39, IFN-alpha, LIF and TGF-beta.
Embodiment 6. The immunoregulatory molecule of any one of embodiments 1-5, wherein said first target molecule and/or said second target molecule is a stimulatory checkpoint molecule.
Embodiment 7. The immunoregulatory molecule of embodiment 6 wherein said stimulatory checkpoint molecule is selected from the group consisting of: CD27, CD28, CD40, CD122, CD137, OX40, GITR and ICOS.
Embodiment 8. The immunomodulatory molecule of embodiment 6 or 7, wherein the first binding domain is an agonist antibody or antigen-binding fragment thereof.
Embodiment 9. The immunomodulatory molecule of embodiment 6 or 7, wherein the first binding domain is an agonist ligand or variant thereof.
Embodiment 10. The immunomodulatory molecule of embodiment 9, wherein the agonist ligand is selected from the group consisting of: CD27L (TNFSF 7, CD 70), CD40L (CD 154), CD80, CD86, CD137L, OX L (CD 252), GITRL and ICOSLG (CD 275).
Embodiment 11. The immunomodulatory molecule of embodiment 9 or 10, wherein the first binding domain is a variant of an agonist ligand, and wherein the variant of an agonist ligand has increased or decreased binding affinity for the first target molecule as compared to the agonist ligand.
Embodiment 12. The immunoregulatory molecule of any one of embodiments 6-11, wherein said second binding domain is an antagonist antibody or antigen-binding fragment thereof.
Embodiment 13. The immunoregulatory molecule of any one of embodiments 6-11, wherein said second binding domain is an antagonist ligand or variant thereof.
Embodiment 14. The immunomodulatory molecule of embodiment 13, wherein the second binding domain is a variant of an antagonist ligand, and wherein the variant of an antagonist ligand has increased or decreased binding affinity for the second target molecule compared to the antagonist ligand.
Embodiment 15. The immunoregulatory molecule of any one of embodiments 1-5, wherein said first target molecule and/or said second target molecule is a receptor for an immunostimulatory cytokine.
Embodiment 16. The immunomodulatory molecule of embodiment 15, wherein the immunostimulatory cytokine is selected from the group consisting of: IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-12, IL-15, IL-17, IL-18, IL-21, IL-22, IL-23, IL-27, IFN-alpha, IFN-beta, IFN-gamma, TNF-alpha, erythropoietin, thrombopoietin, G-CSF, M-CSF, SCF and GM-CSF.
Embodiment 17. The immunomodulatory molecule of embodiment 15 or 16, wherein the first binding domain is the immunostimulatory cytokine or variant thereof.
Embodiment 18. The immunomodulatory molecule of embodiment 17, wherein the first binding domain is a variant of an immunostimulatory cytokine, and wherein the variant of an immunostimulatory cytokine has increased or decreased binding affinity for the first target molecule as compared to the immunostimulatory cytokine.
Embodiment 19. The immunoregulatory molecule of embodiment 17 or 18, wherein said first binding domain is IL-12, IL-2, or a variant thereof.
Embodiment 20. The immunomodulatory molecule of embodiment 15 or 16, wherein the first binding domain is an agonist antibody or antigen-binding fragment thereof.
Embodiment 21. The immunomodulatory molecule of any of embodiments 15-20, wherein the second binding domain is an antagonist antibody or antigen-binding fragment thereof.
Embodiment 22. The immunoregulatory molecule of any one of embodiments 15-20, wherein said second binding domain is an antagonist ligand or variant thereof.
Embodiment 23. The immunomodulatory molecule of embodiment 22, wherein the second binding domain is a variant of an antagonist ligand, and wherein the variant of an antagonist ligand has increased or decreased binding affinity for the second target molecule as compared to the antagonist ligand.
Embodiment 24. The immunoregulatory molecule of any one of embodiments 1-5, wherein said first target molecule and/or said second target molecule is an activated immune cell surface receptor.
Embodiment 25. The immunoregulatory molecule of embodiment 24, wherein said activated immune cell surface receptor is selected from the group consisting of: CD2, CD3, CD4, CD8, CD16, CD56, CD96, CD161, CD226, NKG2C, NKG2D, NKG2E, NKG2F, NKG2H, NKp, NKp44, NKp46, CD11c, CD11b, CD13, CD45RO, CD33, CD123, CD62L, CD45RA, CD36, CD163 and CD206.
Embodiment 26. The immunomodulatory molecule of embodiment 24 or 25, wherein the first binding domain is an agonist antibody or antigen-binding fragment thereof.
Embodiment 27. The immunomodulatory molecule of embodiment 24 or 25, wherein the first binding domain is an agonist ligand or variant thereof.
Embodiment 28. The immunomodulatory molecule of embodiment 27, wherein the first binding domain is a variant of an agonist ligand, and wherein the variant of an agonist ligand has increased or decreased binding affinity for the first target molecule compared to the agonist ligand.
Embodiment 29. The immunomodulatory molecule of any of embodiments 24-28, wherein the second binding domain is an antagonist antibody or antigen-binding fragment thereof.
Embodiment 30. The immunomodulatory molecule of any of embodiments 24-28, wherein the second binding domain is an antagonist ligand or variant thereof.
Embodiment 31. The immunomodulatory molecule of embodiment 30, wherein the second binding domain is a variant of an antagonist ligand, and wherein the variant of an antagonist ligand has increased or decreased binding affinity for the second target molecule compared to the antagonist ligand.
Embodiment 32. The immunoregulatory molecule of any one of embodiments 1-5, wherein said first target molecule and/or said second target molecule is an inhibitory checkpoint molecule.
Embodiment 33. The immunoregulatory molecule of embodiment 32, wherein said inhibitory checkpoint molecule is selected from the group consisting of: PD-1, PD-L2, CTLA-4, LAG-3, TIM-3, HHA 2, CD47, CXCR4, CD160, CD73, BLTA, B7-H4, TIGIT, siglec7, siglec9 and VISTA.
Embodiment 34. The immunomodulatory molecule of embodiment 32 or 33, wherein the first binding domain is an antagonist ligand or variant thereof.
Embodiment 35. The immunomodulatory molecule of embodiment 34, wherein the first binding domain is a variant of an antagonist ligand, and wherein the variant of an antagonist ligand has increased or decreased binding affinity for the first target molecule as compared to the antagonist ligand.
Embodiment 36. The immunomodulatory molecule of embodiment 32 or 33, wherein the first binding domain is an antagonist antibody or antigen-binding fragment thereof.
Embodiment 37 the immunomodulatory molecule of any of embodiments 32-36, wherein the second binding domain is an agonist antibody or antigen-binding fragment thereof.
Embodiment 38. The immunomodulatory molecule of embodiment 37, wherein the agonist antibody or antigen-binding fragment thereof specifically recognizes PD-1, TIGIT, LAG-3, TIM-3, or CTLA-4.
Embodiment 39. The immunomodulatory molecule of any of embodiments 32-36, wherein the second binding domain is an agonist ligand or variant thereof.
Embodiment 40 the immunomodulatory molecule of embodiment 39, (i) wherein the second target molecule is PD-1, and wherein the second binding domain is PD-L1, PD-L2, or a variant thereof; (ii) Wherein the second target molecule is TIGIT, and wherein the second binding domain is CD112, CD155, or a variant thereof; (iii) Wherein the second target molecule is LAG-3, and wherein the second binding domain is MHC II, lseclin, or a variant thereof; (iv) Wherein the second target molecule is TIM-3, and wherein the second binding domain is galectin-9, caecam-1, HMGB-1, phosphatidylserine or a variant thereof; or (v) wherein the second target molecule is CTLA-4, and wherein the second binding domain is CD80, CD86, or variant thereof.
Embodiment 41. The immunomodulatory molecule of embodiment 40, wherein the second binding domain is a variant of an agonist ligand, and wherein the variant of an agonist ligand has increased or decreased binding affinity for the second target molecule compared to the agonist ligand.
Embodiment 42. The immunoregulatory molecule of any one of embodiments 39-41, wherein said second binding domain comprises an extracellular domain of said agonist ligand or variant thereof.
Embodiment 43. The immunoregulatory molecule of any one of embodiments 1-5, wherein said first target molecule and/or said second target molecule is a receptor for an immunosuppressive cytokine.
Embodiment 44. The immunoregulatory molecule of embodiment 43, wherein said immunosuppressive cytokine is selected from the group consisting of: IL-1Ra, IL-4, IL-5, IL-6, IL-10, IL-11, IL-13, IL-27, IL-33, IL-35, IFN-alpha, LIF and TGF-beta.
Embodiment 45. The immunomodulatory molecule of embodiment 43 or 44, wherein the second binding domain is an immunosuppressive cytokine or variant thereof.
Embodiment 46. The immunoregulatory molecule of embodiment 45, wherein said second binding domain is a variant of said immunosuppressive cytokine, wherein said variant of immunosuppressive cytokine has increased or decreased binding affinity to said second target molecule as compared to said immunosuppressive cytokine.
Embodiment 47. The immunoregulatory molecule of embodiment 45 or 46, wherein said second binding domain is IL-10 or a variant thereof.
Embodiment 48. The immunoregulatory molecule of embodiment 45 or 46, wherein said second binding domain is TGF- β or a variant thereof.
Embodiment 49 the immunomodulatory molecule of embodiment 43 or 44, wherein the second binding domain is an agonist antibody or antigen-binding fragment thereof.
Embodiment 50. The immunomodulatory molecule of any of embodiments 43-49, wherein the first binding domain is an antagonist antibody or antigen-binding fragment thereof.
Embodiment 51. The immunomodulatory molecule of any of embodiments 43-49, wherein the first binding domain is an antagonist ligand or variant thereof.
Embodiment 52. The immunomodulatory molecule of embodiment 51, wherein the first binding domain is a variant of an antagonist ligand, and wherein the variant of an antagonist ligand has increased or decreased binding affinity for the first target molecule as compared to the antagonist ligand.
Embodiment 53. The immunoregulatory molecule of any one of embodiments 1-5, wherein said first target molecule and/or said second target molecule is an inhibitory immune cell surface receptor.
Embodiment 54. The immunoregulatory molecule of embodiment 53, wherein said inhibitory immune cell surface receptor is selected from the group consisting of: CD5, NKG2A, NKG2B, KLRG, FCRL4, siglec2, CD72, CD244, GP49B, lair-1, pirB, PECAM-1, CD200R, ILT2 and KIR2DL.
Embodiment 55. The immunomodulatory molecule of embodiment 53 or 54, wherein the second binding domain is an agonist antibody or antigen-binding fragment thereof.
Embodiment 56. The immunomodulatory molecule of embodiment 53 or 54, wherein the second binding domain is an agonist ligand or variant thereof.
Embodiment 57. The immunomodulatory molecule of embodiment 56, wherein the second binding domain is a variant of an agonist ligand, wherein the variant of an agonist ligand has increased or decreased binding affinity for the second target molecule compared to the agonist ligand.
Embodiment 58 the immunomodulatory molecule of any of embodiments 53-57, wherein the first binding domain is an antagonist antibody or antigen-binding fragment thereof.
Embodiment 59. The immunomodulatory molecule of any of embodiments 53-57, wherein the first binding domain is an antagonist ligand or variant thereof.
Embodiment 60. The immunomodulatory molecule of embodiment 59, wherein the first binding domain is a variant of an antagonist ligand, and wherein the variant of an antagonist ligand has increased or decreased binding affinity for the first target molecule compared to the antagonist ligand.
Embodiment 61. The immunomodulatory molecule of any of embodiments 1-5, wherein the first binding domain is IL-12 or a variant thereof, and wherein the second binding domain is an agonist antibody or antigen-binding fragment thereof that specifically recognizes PD-1.
Embodiment 62. The immunoregulatory molecule of any one of embodiments 1-5, wherein said first binding domain is IL-12 or a variant thereof, and wherein said second binding domain is PD-L1 or a variant thereof.
Embodiment 63. The immunomodulatory molecule of embodiment 62, wherein the second binding domain is a variant of PD-L1, and wherein the variant of PD-L1 has increased or decreased binding affinity for the second target molecule compared to PD-L1.
Embodiment 64. The immunoregulatory molecule of any one of embodiments 1-5, wherein said first binding domain is IL-12 or a variant thereof, and wherein said second binding domain is PD-L2 or a variant thereof.
Embodiment 65. The immunomodulatory molecule of embodiment 64, wherein the second binding domain is a variant of PD-L2, and wherein the variant of PD-L2 has increased or decreased binding affinity for the second target molecule compared to PD-L2.
Embodiment 66. The immunoregulatory molecule of any one of embodiments 61-65, wherein said first binding domain is a variant of IL-12, and wherein said variant of IL-12 has increased or decreased binding affinity for said first target molecule as compared to IL-12.
Embodiment 67. The immunomodulatory molecule of any of embodiments 1-5, wherein the first binding domain is IL-2 or a variant thereof, and wherein the second binding domain is an agonist antibody or antigen-binding fragment thereof that specifically recognizes PD-1.
Embodiment 68. The immunomodulatory molecule of any of embodiments 1-5, wherein the first binding domain is IL-2 or a variant thereof, and wherein the second binding domain is PD-L1 or a variant thereof.
Embodiment 69. The immunomodulatory molecule of embodiment 68, wherein the second binding domain is a variant of PD-L1, and wherein the variant of PD-L1 has increased or decreased binding affinity for the second target molecule compared to PD-L1.
Embodiment 70. The immunomodulatory molecule of any of embodiments 1-5, wherein the first binding domain is IL-2 or a variant thereof, and wherein the second binding domain is PD-L2 or a variant thereof.
Embodiment 71. The immunomodulatory molecule of embodiment 70, wherein the second binding domain is a variant of PD-L2, and wherein the variant of PD-L2 has increased or decreased binding affinity for the second target molecule compared to PD-L2.
Embodiment 72. The immunomodulatory molecule of any of embodiments 67-71, wherein the first binding domain is a variant of IL-2, and wherein the variant of IL-2 has increased or decreased binding affinity for the first target molecule compared to IL-2.
Embodiment 73. The immunoregulatory molecule of any one of embodiments 1-72, wherein the immunoregulatory molecule comprises: i) An antigen binding protein comprising an antigen binding polypeptide; and ii) the first binding domain, wherein the antigen binding polypeptide comprises, from N-terminus to C-terminus: the second binding domain or portion thereof, a hinge region, and an Fc domain subunit or portion thereof, and wherein the first binding domain is located at the hinge region.
Embodiment 74. The immunomodulatory molecule of embodiment 73, wherein the activity of the first binding domain is increased by at least about 20% in the presence of binding of the second binding domain to the second target molecule as compared to the absence of binding of the second binding domain to the second target molecule.
Embodiment 75. The immunomodulatory molecule of embodiment 73 or 74, wherein the activity of the first binding domain at the hinge region in the absence of binding of the second binding domain to the second target molecule is no more than about 70% of the activity of the corresponding first binding domain in the free state.
Embodiment 76. The immunomodulatory molecule of any of embodiments 73-75, wherein the antigen-binding protein comprises two antigen-binding polypeptides, each antigen-binding polypeptide comprising a hinge region, and wherein only one antigen-binding peptide comprises a first binding domain located at the hinge region.
Embodiment 77. The immunomodulatory molecule of any of embodiments 73-75, wherein the antigen-binding protein comprises two antigen-binding polypeptides, each antigen-binding polypeptide comprising a hinge region, and wherein each antigen-binding peptide comprises a first binding domain located at the hinge region.
Embodiment 78 the immunomodulatory molecule of any of embodiments 73-77, wherein the immunomodulatory molecule comprises two or more first binding domains, wherein the two or more first binding domains are positioned in tandem at the hinge region of the antigen binding polypeptide.
Embodiment 79. The immunomodulatory molecule of any of embodiments 73-78, wherein the first binding domain is an immunostimulatory cytokine or variant thereof.
Embodiment 80. The immunoregulatory molecule of embodiment 79, wherein said immunostimulatory cytokine is selected from the group consisting of: IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-12, IL-15, IL-17, IL-18, IL-21, IL-22, IL-23, IL-27, IFN-alpha, IFN-beta, IFN-gamma, TNF-alpha, erythropoietin, thrombopoietin, G-CSF, M-CSF, SCF and GM-CSF.
Embodiment 81. The immunomodulatory molecule of embodiment 79 or 80, wherein the first binding domain is an immunostimulatory cytokine variant, and wherein the activity of the immunostimulatory cytokine variant in the free state is no more than about 80% of the activity of a corresponding wild-type immunostimulatory cytokine in the free state.
Embodiment 82. The immunoregulatory molecule of any one of embodiments 79-81, wherein said immunostimulatory cytokine or variant thereof is a monomeric immunostimulatory cytokine or variant thereof.
Embodiment 83. The immunoregulatory molecule of any one of embodiments 79-81, wherein said immunostimulatory cytokine or variant thereof is a dimeric immunostimulatory cytokine or variant thereof.
Embodiment 84. The immunoregulatory molecule of embodiment 83, wherein the two subunits of the dimeric immunostimulatory cytokine or variant thereof are located in tandem at the hinge region of the antigen binding polypeptide.
Embodiment 85. The immunomodulatory molecule of embodiment 83, wherein the antigen-binding protein comprises two antigen-binding polypeptides, each antigen-binding polypeptide comprising a hinge region, wherein one subunit of the dimeric immunostimulatory cytokine or variant thereof is located in the hinge region of one antigen-binding polypeptide, and wherein the other subunit of the dimeric immunostimulatory cytokine or variant thereof is located in the hinge region of another antigen-binding polypeptide.
Embodiment 86. The immunoregulatory molecule of any one of embodiments 79-82, wherein said immunostimulatory cytokine or variant thereof is IL-2 or a variant thereof.
Embodiment 87. The immunoregulatory molecule of embodiment 86, wherein said IL-2 variant comprises one or more mutations at a position selected from the group consisting of: f24, K35, R38, F42, K43, E61 and P65.
Embodiment 88. The immunoregulatory molecule of embodiment 86 or 87, wherein said IL-2 variant comprises one or more mutations relative to wild-type IL-2 selected from the group consisting of: F24A, R D, K3543, E, E R and P65L.
Embodiment 89. The immunoregulatory molecule of any one of embodiments 86-88, wherein said IL-2 variant comprises an R38D/K43E/E61R mutation relative to wild-type IL-2.
Embodiment 90. The immunoregulatory molecule of any one of embodiments 79-81 and 83-85, wherein said immunostimulatory cytokine or variant thereof is IL-12 or a variant thereof.
Embodiment 91. The immunomodulatory molecule of embodiment 90, wherein said IL-12 variant comprises one or more mutations within said p40 subunit at a position selected from the group consisting of: e45, Q56, V57, K58, E59, F60, G61, D62, A63, G64, Q65 and C177.
Embodiment 92. The immunoregulatory molecule of embodiment 90 or 91, wherein said IL-12 variant comprises one or more mutations within said p40 subunit, relative to a wild-type p40 subunit, selected from the group consisting of: Q56A, V57A, K A, E3559A, F60A, G61A, D A, A63S, G a and Q65A.
Embodiment 93. The immunoregulatory molecule of any one of embodiments 90-92, wherein said IL-12 variant comprises an E59A/F60A mutation within said p40 subunit relative to a wild-type p40 subunit.
Embodiment 94. The immunoregulatory molecule of any one of embodiments 90-92, wherein said IL-12 variant comprises an F60A mutation within said p40 subunit relative to a wild-type p40 subunit.
Embodiment 95. The immunoregulatory molecule of any one of embodiments 90-94, wherein the p40 subunit and the p35 subunit of IL-12 or a variant thereof are linked by a linker.
Embodiment 96. The immunomodulatory molecule of any of embodiments 77-95, wherein the two or more first binding domains are the same.
Embodiment 97. The immunomodulatory molecule of any of embodiments 77-95, wherein the two or more first binding domains are different.
Embodiment 98 the immunoregulatory molecule of any one of embodiments 73-97, wherein said second binding domain is an agonist ligand of an inhibitory checkpoint molecule or a variant thereof.
Embodiment 99. The immunoregulatory molecule of embodiment 98, wherein said inhibitory checkpoint molecule is selected from the group consisting of: PD-1, PD-L2, CTLA-4, LAG-3, TIM-3, HHA 2, CD47, CXCR4, CD160, CD73, BLTA, B7-H4, TIGIT, siglec7, siglec9 and VISTA.
Embodiment 100. The immunomodulatory molecule of embodiment 98 or 99, wherein the second binding domain is PD-L1 or a variant thereof.
Embodiment 101. The immunomodulatory molecule of embodiment 100, wherein the PD-L1 variant has increased binding affinity for PD-1 as compared to wild-type PD-L1.
Embodiment 102. The immunomodulatory molecule of embodiment 100 or 101, wherein the PD-L1 variant comprises one or more mutations at a position selected from the group consisting of: i54, Y56, E58, R113, M115, S117, and G119.
Embodiment 103. The immunomodulatory molecule of any of embodiments 100-102, wherein the PD-L1 variant comprises one or more mutations relative to wild-type PD-L1 selected from the group consisting of: I54Q, Y56F, E58M, R113T, M115L, S a and G119K.
Embodiment 104. The immunoregulatory molecule of any one of embodiments 100-103, wherein said PD-L1 variant comprises an I54Q/Y56F/E58M/R113T/M115L/S117A/G119K mutation relative to wild-type PD-L1.
Embodiment 105. The immunomodulatory molecule of embodiment 98 or 99, wherein the second binding domain is PD-L2 or a variant thereof.
Embodiment 106. The immunomodulatory molecule of embodiment 105, wherein the PD-L2 variant has increased binding affinity for PD-1 as compared to wild-type PD-L2.
Embodiment 107. The immunoregulatory molecule of any one of embodiments 73-97, wherein said second binding domain is an agonist antibody or antigen binding fragment thereof of an inhibitory checkpoint molecule.
Embodiment 108. The immunoregulatory molecule of embodiment 107, wherein said inhibitory checkpoint molecule is selected from the group consisting of: PD-1, PD-L2, CTLA-4, LAG-3, TIM-3, HHA 2, CD47, CXCR4, CD160, CD73, BLTA, B7-H4, TIGIT, siglec7, siglec9 and VISTA.
Embodiment 109. The immunomodulatory molecule of embodiment 107 or 108, wherein the agonist antibody or antigen-binding fragment thereof specifically recognizes PD-1 ("anti-PD-1 agonist antibody or antigen-binding fragment thereof").
Embodiment 110. The immunomodulatory molecule of any of embodiments 107-109, wherein the agonist antibody or antigen binding fragment thereof is a Fab.
Embodiment 111 the immunomodulatory molecule of any of embodiments 107-109, wherein the agonist antibody or antigen-binding fragment thereof is a scFv.
Embodiment 112 the immunomodulatory molecule of any of embodiments 73-111, wherein the antigen-binding protein comprises two or more second binding domains.
Embodiment 113 the immunomodulatory molecule of embodiment 112, wherein said two or more second binding domains, or portions thereof, are located in tandem at the N-terminus of said antigen binding polypeptide.
Embodiment 114. The immunomodulatory molecule of embodiment 112 or 113, wherein the antigen-binding protein comprises two antigen-binding polypeptides, each antigen-binding polypeptide comprising a hinge region, and wherein only one antigen-binding peptide comprises two or more second binding domains, or portions thereof, in tandem at the N-terminus of the antigen-binding polypeptide.
Embodiment 115. The immunomodulatory molecule of embodiment 112 or 113, wherein the antigen-binding protein comprises two antigen-binding polypeptides, each antigen-binding polypeptide comprising a hinge region, and wherein each antigen-binding polypeptide comprises one or more second binding domains, or portions thereof, at the N-terminus of each antigen-binding polypeptide.
Embodiment 116. The immunomodulatory molecule of any of embodiments 73-114, wherein the antigen-binding protein comprises two antigen-binding polypeptides, each antigen-binding polypeptide comprising a hinge region, wherein the first antigen-binding polypeptide comprises one or more second binding domains, or portions thereof, at the N-terminus of the first antigen-binding polypeptide, wherein the second antigen-binding polypeptide comprises a third binding domain, or portions thereof, at the N-terminus of the second antigen-binding polypeptide, and wherein the first binding domain specifically recognizes a third target molecule.
Embodiment 117 the immunomodulatory molecule of embodiment 116, wherein the third binding domain and the second binding domain are the same.
Embodiment 118 the immunomodulatory molecule of embodiment 116, wherein said third binding domain and said second binding domain are different.
Embodiment 119. The immunomodulatory molecule of any of embodiments 116-118, wherein the third target molecule and the second target molecule are the same.
Embodiment 120. The immunomodulatory molecule of embodiments 116 or 118, wherein the third target molecule and the second target molecule are different.
Embodiment 121. The immunomodulatory molecule of any of embodiments 73-120, comprising: i) A first antigen binding polypeptide comprising, from N-terminus to C-terminus: a first PD-L2 or PD-L1 or variant thereof, a second PD-L2 or PD-L1 or variant thereof, a p35 subunit and a p40 subunit in tandem located at IL-12 or variant thereof at the first hinge region, and a first subunit of an Fc domain or portion thereof; ii) a second antigen binding polypeptide comprising, from N-terminus to C-terminus: VH, optionally CH1, a second hinge region, and a second subunit of an Fc domain, or portion thereof; and iii) a third antigen binding polypeptide comprising, from N-terminus to C-terminus: VL and optionally CL; wherein said VH and said VL, and optionally said CH1 and said CL, form a third binding domain that specifically recognizes a third target molecule.
Embodiment 122. The immunomodulatory molecule of embodiment 121, wherein the third binding domain is an agonist antigen-binding fragment that specifically recognizes PD-1.
Embodiment 123 the immunomodulatory molecule of any of embodiments 73-120, comprising: i) A first antigen binding polypeptide comprising, from N-terminus to C-terminus: a first VH, optionally a first CH1, a p35 subunit and a p40 subunit in tandem with IL-12 or variant thereof at a first hinge region, and a first subunit of an Fc domain or portion thereof; ii) a second antigen binding polypeptide comprising, from N-terminus to C-terminus: a second VH, optionally a second CH1, a second hinge region, and a second subunit of an Fc domain, or portion thereof; iii) A third antigen binding polypeptide comprising, from N-terminus to C-terminus: a first VL and optionally a first CL; and iv) a fourth antigen binding polypeptide comprising, from N-terminus to C-terminus: a second VL and optionally a second CL, wherein the first VH and the first VL, and optionally the first CH1 and the first CL, form the second binding domain that is an agonist antigen-binding fragment that specifically recognizes PD-1, and wherein the second VH and the second VL, and optionally the second CH1 and the second CL, form a third binding domain that specifically recognizes a third target molecule.
Embodiment 124. The immunomodulatory molecule of embodiment 123, wherein the third binding domain is an agonist antigen-binding fragment that specifically recognizes PD-1.
Embodiment 125 the immunomodulatory molecule of any of embodiments 73-120, comprising: i) A first antigen binding polypeptide comprising, from N-terminus to C-terminus: a first PD-L2 or PD-L1 or variant thereof, a p35 subunit and a p40 subunit in tandem with the first subunit of the Fc domain, or a portion thereof, of IL-12 or variant thereof at the first hinge region; and ii) a second antigen binding polypeptide comprising, from N-terminus to C-terminus: a second PD-L2 or PD-L1 or variant thereof, a second hinge region, and a second subunit of an Fc domain, or a portion thereof.
Embodiment 126 the immunomodulatory molecule of any of embodiments 73-120, comprising: i) A first antigen binding polypeptide comprising, from N-terminus to C-terminus: a first PD-L2 or PD-L1 or variant thereof, a second PD-L2 or PD-L1 or variant thereof, a p35 subunit and a p40 subunit in tandem of IL-12 or variant thereof at the first hinge region, and a first subunit of an Fc domain or portion thereof; and ii) a second antigen binding polypeptide comprising, from N-terminus to C-terminus: a third PD-L2 or PD-L1 or variant thereof, a fourth PD-L2 or PD-L1 or variant thereof, a second hinge region, and a second subunit of an Fc domain or portion thereof.
Embodiment 127 the immunomodulatory molecule of any of embodiments 73-120, comprising: i) A first antigen binding polypeptide comprising, from N-terminus to C-terminus: a first PD-L2 or PD-L1 or variant thereof, a p35 subunit of IL-12 or variant thereof located at the first hinge region, and a first subunit of an Fc domain or portion thereof; and ii) a second antigen binding polypeptide comprising, from N-terminus to C-terminus: a second PD-L2 or PD-L1 or variant thereof, a p40 subunit of IL-12 or variant thereof located in a second hinge region, and a second subunit of an Fc domain or portion thereof.
Embodiment 128 the immunomodulatory molecule of any of embodiments 73-120, comprising: i) A first antigen binding polypeptide comprising, from N-terminus to C-terminus: a p35 subunit or p40 subunit of IL-12 or a variant thereof located in the first hinge region and a first subunit of an Fc domain or a portion thereof; and ii) a second antigen binding polypeptide comprising, from N-terminus to C-terminus: a first PD-L2 or PD-L1 or variant thereof, a second PD-L2 or PD-L1 or variant thereof, a p40 subunit or p35 subunit of IL-12 or variant thereof located at the second hinge region, and a second subunit of an Fc domain or portion thereof.
Embodiment 129 the immunomodulatory molecule of any of embodiments 73-120, comprising: i) A first antigen binding polypeptide comprising, from N-terminus to C-terminus: a first VH, optionally a first CH1, a p35 subunit or a p40 subunit of IL-12 or a variant thereof located at a first hinge region, and a first subunit of an Fc domain or portion thereof; ii) a second antigen binding polypeptide comprising, from N-terminus to C-terminus: a second VH, an optional second CH1, a p40 subunit or a p35 subunit of IL-12 or a variant thereof located in a second hinge region, and a second subunit of an Fc domain or portion thereof; iii) A third antigen binding polypeptide comprising, from N-terminus to C-terminus: a first VL and optionally a first CL; and iv) a fourth antigen binding polypeptide comprising, from N-terminus to C-terminus: a second VL and optionally a second CL, wherein the first VH and the first VL, and optionally the first CH1 and the first CL, form the second binding domain that is an agonist antigen-binding fragment that specifically recognizes PD-1, and wherein the second VH and the second VL, and optionally the second CH1 and the second CL, form a third binding domain that specifically recognizes a third target molecule.
Embodiment 130. The immunomodulatory molecule of embodiment 129, wherein the third binding domain is an agonist antigen-binding fragment that specifically recognizes PD-1.
Embodiment 131. The immunomodulatory molecule of any of embodiments 1-72, wherein the immunomodulatory molecule comprises an antigen-binding protein comprising an antigen-binding polypeptide, wherein the antigen-binding polypeptide comprises from N-terminus to C-terminus: the first binding domain or portion thereof, the second binding domain or portion thereof, an optional hinge region, and an Fc domain subunit or portion thereof.
Embodiment 132. The immunomodulatory molecule of embodiment 131, wherein the second binding domain is an agonist Fab or agonist scFv that specifically recognizes an inhibitory checkpoint molecule.
Embodiment 133. The immunomodulatory molecule of embodiment 131, wherein the second binding domain is an agonist ligand of an inhibitory checkpoint molecule or a variant thereof.
Embodiment 134. The immunoregulatory molecule of embodiment 133, wherein said second binding domain is PD-L1 or PD-L2 or a variant thereof.
Embodiment 135 the immunomodulatory molecule of any of embodiments 131-134, wherein the first binding domain is an immunostimulatory cytokine or variant thereof.
Embodiment 136. The immunoregulatory molecule of embodiment 135, wherein said immunostimulatory cytokine or variant thereof is IL-2 or IL-12 or variant thereof.
Embodiment 137 the immunomodulatory molecule of any of embodiments 131-136, wherein the antigen-binding protein comprises two antigen-binding polypeptides, each comprising a hinge region, wherein the first antigen-binding polypeptide comprises from N-terminus to C-terminus: the first binding domain or portion thereof, the second binding domain or portion thereof, the first hinge region, and the first subunit of the Fc domain or portion thereof; wherein the second antigen binding polypeptide comprises, from N-terminus to C-terminus: a third binding domain or portion thereof, a second hinge region, and a second subunit of an Fc domain or portion thereof; and wherein the third binding domain specifically recognizes a third target molecule.
Embodiment 138 the immunomodulatory molecule of embodiment 137, wherein said third binding domain and said second binding domain are the same.
Embodiment 139. The immunoregulatory molecule of embodiment 137, wherein the third binding domain and the second binding domain are different.
Embodiment 140 the immunomodulatory molecule of any of embodiments 137-139, wherein the third target molecule and the second target molecule are the same.
Embodiment 141. The immunomodulatory molecule of embodiment 137 or 139, wherein the third target molecule and the second target molecule are different.
Embodiment 142 the immunomodulatory molecule of any of embodiments 131-141, comprising: i) A first antigen binding polypeptide comprising, from N-terminus to C-terminus: a p35 subunit and a p40 subunit of IL-12 or a variant thereof, optionally a first CH1, a first hinge region, and a first subunit of an Fc domain, or a portion thereof, fused in tandem; ii) a second antigen binding polypeptide comprising, from N-terminus to C-terminus: a second VH, optionally a second CH1, a second hinge region, and a second subunit of an Fc domain, or portion thereof; iii) A third antigen binding polypeptide comprising, from N-terminus to C-terminus: a first VL and optionally a first CL; and iv) a fourth antigen binding polypeptide comprising, from N-terminus to C-terminus: a second VL and optionally a second CL, wherein the first VH and the first VL, and optionally the first CH1 and the first CL, form the second binding domain that is an agonist antigen-binding fragment that specifically recognizes PD-1, and wherein the second VH and the second VL, and optionally the second CH1 and the second CL, form a third binding domain that specifically recognizes a third target molecule.
Embodiment 143. The immunomodulatory molecule of embodiment 142, wherein the third binding domain is an agonist antigen-binding fragment that specifically recognizes PD-1.
Embodiment 144 the immunomodulatory molecule of any of embodiments 131-141, comprising: i) A first antigen binding polypeptide comprising, from N-terminus to C-terminus: a p35 subunit and a p40 subunit of IL-12 or a variant thereof, a first PD-L2 or PD-L1 or variant thereof, a second PD-L2 or PD-L1 or variant thereof, a first hinge region, and a first subunit of an Fc domain or portion thereof fused in tandem; and ii) a second antigen binding polypeptide comprising, from N-terminus to C-terminus: a third PD-L2 or PD-L1 or variant thereof, a fourth PD-L2 or PD-L1 or variant thereof, a second hinge region, and a second subunit of an Fc domain, or a portion thereof.
Embodiment 145 the immunomodulatory molecule of any of embodiments 131-141, comprising: i) A first antigen binding polypeptide comprising, from N-terminus to C-terminus: a p35 subunit and a p40 subunit of IL-12 or a variant thereof, a first PD-L2 or PD-L1 or variant thereof, a second PD-L2 or PD-L1 or variant thereof, a first hinge region, and a first subunit of an Fc domain or portion thereof fused in tandem; ii) a second antigen binding polypeptide comprising, from N-terminus to C-terminus: VH, optionally CH1, a second hinge region, and a second subunit of an Fc domain, or portion thereof; and iii) a third antigen binding polypeptide comprising, from N-terminus to C-terminus: VL and optionally CL, wherein the VH and the VL, and optionally the CH1 and the CL, form a third binding domain that specifically recognizes a third target molecule.
Embodiment 146. The immunomodulatory molecule of embodiment 145, wherein the third binding domain is an agonist antigen-binding fragment that specifically recognizes PD-1.
Embodiment 147 the immunomodulatory molecule of any of embodiments 131-141, comprising: i) A first antigen binding polypeptide comprising, from N-terminus to C-terminus: a p35 subunit and a p40 subunit of IL-12 or variant thereof, VH, optionally CH1, a first hinge region, and a first subunit of an Fc domain, or portion thereof, fused in tandem; ii) a second antigen binding polypeptide comprising, from N-terminus to C-terminus: a first PD-L2 or PD-L1 or variant thereof, a second hinge region, and a second subunit of an Fc domain or portion thereof; and iii) a third antigen binding polypeptide comprising, from N-terminus to C-terminus: VL and optionally CL, wherein VH and VL, and optionally CH1 and CL, form a second binding domain that is an agonist antigen-binding fragment that specifically recognizes PD-1.
Embodiment 148 the immunomodulatory molecule of any of embodiments 1-72, comprising: i) A first antigen binding polypeptide comprising, from N-terminus to C-terminus: a first VH, optionally a first CH1, a first hinge region, and a first subunit of an Fc domain, or portion thereof; ii) a second antigen binding polypeptide comprising, from N-terminus to C-terminus: a second VH, optionally a second CH1, a second hinge region, and a second subunit of an Fc domain, or portion thereof; iii) A third antigen binding polypeptide comprising, from N-terminus to C-terminus: a p35 subunit and a p40 subunit of IL-12, or a variant thereof, fused in tandem, a first VL, and optionally a first CL; and iv) a fourth antigen binding polypeptide comprising, from N-terminus to C-terminus: a second VL and optionally a second CL, wherein the first VH and the first VL, and optionally the first CH1 and the first CL, form the second binding domain that is an agonist antigen-binding fragment that specifically recognizes PD-1, and wherein the second VH and the second VL, and optionally the second CH1 and the second CL, form a third binding domain that specifically recognizes a third target molecule.
Embodiment 149. The immunomodulatory molecule of embodiment 148, wherein the third binding domain is an agonist antigen-binding fragment that specifically recognizes PD-1.
Embodiment 150 the immunomodulatory molecule of any of embodiments 1-72, comprising: i) A first antigen binding polypeptide comprising, from N-terminus to C-terminus: VH, optionally CH1, a first hinge region, and a first subunit of an Fc domain, or portion thereof; ii) a second antigen binding polypeptide comprising, from N-terminus to C-terminus: a first PD-L2 or PD-L1 or variant thereof, a second hinge region, and a second subunit of an Fc domain or portion thereof; and iii) a third antigen binding polypeptide comprising, from N-terminus to C-terminus: in a tandem fusion of the p35 subunit and the p40 subunit of IL-12 or a variant thereof, VL and optionally CL, wherein said VH and said VL and optionally said CH1 and said CL form said second binding domain, which is an agonist antigen binding fragment specifically recognizing PD-1.
Embodiment 151. The immunomodulatory molecule of any of embodiments 1-72, wherein the immunomodulatory molecule comprises an antigen-binding protein comprising a first antigen-binding polypeptide and a second antigen-binding polypeptide, wherein the first antigen-binding polypeptide comprises from N-terminus to C-terminus: the second antigen binding domain or portion thereof, the first hinge domain, and the first subunit of the Fc domain or portion thereof; wherein the second antigen binding polypeptide comprises, from N-terminus to C-terminus: the first antigen binding domain or portion thereof, a second hinge domain, and a second subunit of the Fc domain or portion thereof.
Embodiment 152. The immunomodulatory molecule of embodiment 151, wherein the second binding domain is an agonist Fab or agonist scFv that specifically recognizes an inhibitory checkpoint molecule.
Embodiment 153. The immunomodulatory molecule of embodiment 151, wherein the second binding domain is an agonist ligand of an inhibitory checkpoint molecule or a variant thereof.
Embodiment 154. The immunomodulatory molecule of embodiment 153, wherein the second binding domain is PD-L1 or PD-L2 or a variant thereof.
Embodiment 155 the immunomodulatory molecule of any of embodiments 151-154, wherein the first binding domain is an immunostimulatory cytokine or variant thereof.
Embodiment 156. The immunoregulatory molecule of embodiment 155, wherein said immunostimulatory cytokine or variant thereof is IL-2 or IL-12 or a variant thereof.
Embodiment 157 the immunomodulatory molecule of any of embodiments 151-156, comprising: i) A first antigen binding polypeptide comprising, from N-terminus to C-terminus: VH, optionally CH1, a first hinge region, and a first subunit of an Fc domain, or portion thereof; ii) a second antigen binding polypeptide comprising, from N-terminus to C-terminus: a p35 subunit and a p40 subunit of IL-12 or a variant thereof, a second hinge region, and a second subunit of an Fc domain, or a portion thereof, fused in tandem; and iii) a third antigen binding polypeptide comprising, from N-terminus to C-terminus: VL and optionally CL, wherein the VH and the VL, and optionally the CH1 and the CL, form the second binding domain, which is an agonist antigen-binding fragment that specifically recognizes PD-1.
Embodiment 158 the immunomodulatory molecule of any of embodiments 151-156, comprising: i) A first antigen binding polypeptide comprising, from N-terminus to C-terminus: a first PD-L2 or PD-L1 or variant thereof, a second PD-L2 or PD-L1 or variant thereof, a first hinge region, and a first subunit of an Fc domain or portion thereof; and ii) a second antigen binding polypeptide comprising, from N-terminus to C-terminus: the p35 subunit and the p40 subunit of IL-12 or variant thereof, the second hinge region, and the second subunit of the Fc domain, or a portion thereof, are fused in tandem.
Embodiment 159 the immunomodulatory molecule of any of embodiments 1-72, wherein the immunomodulatory molecule comprises an antigen-binding protein comprising an antigen-binding polypeptide, wherein the antigen-binding polypeptide comprises from N-terminus to C-terminus: the second binding domain or portion thereof, an optional hinge region, an Fc domain subunit or portion thereof, and the first binding domain or portion thereof.
Embodiment 160. The immunomodulatory molecule of embodiment 159, wherein the second binding domain is an agonist Fab or agonist scFv that specifically recognizes an inhibitory checkpoint molecule.
Embodiment 161. The immunomodulatory molecule of embodiment 159, wherein the second binding domain is an agonist ligand of an inhibitory checkpoint molecule or a variant thereof.
Embodiment 162. The immunoregulatory molecule of embodiment 161, wherein said second binding domain is PD-L1 or PD-L2 or a variant thereof.
The immunomodulatory molecule of any of embodiments 159-162, wherein the first binding domain is an immunostimulatory cytokine or variant thereof.
Embodiment 164. The immunoregulatory molecule of embodiment 163, wherein said immunostimulatory cytokine or variant thereof is IL-2 or IL-12 or variant thereof.
Embodiment 165. The immunomodulatory molecule of embodiment 163 or 164, wherein the immunostimulatory cytokine or variant thereof is a monomeric immunostimulatory cytokine or variant thereof.
Embodiment 166. The immunomodulatory molecule of embodiment 163 or 164, wherein the immunostimulatory cytokine or variant thereof is a dimeric immunostimulatory cytokine or variant thereof.
Embodiment 167. The immunomodulatory molecule of embodiment 166, wherein the two subunits of the dimeric immunostimulatory cytokine or variant thereof are located in tandem at the C-terminus of the antigen binding polypeptide.
The immunoregulatory molecule of embodiment 168, wherein said antigen binding protein comprises two antigen binding polypeptides, each antigen binding polypeptide comprising a hinge region and an Fc domain subunit or portion thereof, wherein one subunit of said dimeric immunostimulatory cytokine or variant thereof is fused to the C-terminus of the Fc domain subunit or portion thereof of one antigen binding polypeptide, and wherein the other subunit of said dimeric immunostimulatory cytokine or variant thereof is fused to the C-terminus of the Fc domain subunit or portion thereof of said other antigen binding polypeptide.
Embodiment 169. The immunomodulatory molecule of embodiment 168, wherein the antigen-binding polypeptide that does not comprise the second binding domain or portion thereof comprises, from N-terminus to C-terminus: specifically recognizes a third binding domain of a third target molecule or portion thereof, a hinge region, a subunit of an Fc domain or portion thereof, and a subunit of a dimeric immunostimulatory cytokine or variant thereof.
The immunomodulatory molecule of any of embodiments 159-168, wherein the antigen-binding protein comprises a first antigen-binding polypeptide and a second antigen-binding polypeptide, wherein the first antigen-binding peptide comprises from N-terminus to C-terminus: a second binding domain or portion thereof, a first hinge region, a first subunit of an Fc domain or portion thereof, and the first binding domain or portion thereof; wherein the second antigen binding polypeptide comprises, from N-terminus to C-terminus: specifically recognizes a third binding domain of a third target molecule or portion thereof, a second hinge region, and a second subunit of an Fc domain or portion thereof.
Embodiment 171 the immunomodulatory molecule of embodiment 169 or 170, wherein said third binding domain and said second binding domain are the same.
Embodiment 172. The immunomodulatory molecule of embodiment 169 or 170, wherein the third binding domain and the second binding domain are different.
Embodiment 173 the immunomodulatory molecule of any of embodiments 169-172, wherein the third target molecule and the second target molecule are the same.
Embodiment 174. The immunomodulatory molecule of any of embodiments 169, 170, and 172, wherein the third target molecule and the two target molecules are different.
Embodiment 175 the immunomodulatory molecule of any of embodiments 159-174, comprising: i) A first antigen binding polypeptide comprising, from N-terminus to C-terminus: a first PD-L2 or PD-L1 or variant thereof, a first hinge region, a first subunit of an Fc domain or portion thereof, and a p35 subunit and a p40 subunit of IL-12 or variant thereof fused in tandem; and ii) a second antigen binding polypeptide comprising, from N-terminus to C-terminus: a second PD-L2 or PD-L1 or variant thereof, a second hinge region, and a second subunit of an Fc domain or portion thereof.
Embodiment 176 the immunomodulatory molecule of any of embodiments 159-174, comprising: i) A first antigen binding polypeptide comprising, from N-terminus to C-terminus: a first VH, optionally a first CH1, a first hinge region, a first subunit of an Fc domain or portion thereof, and a p35 subunit and a p40 subunit of IL-12 or variant thereof fused in tandem; ii) a second antigen binding polypeptide comprising, from N-terminus to C-terminus: a second VH, optionally a second CH1, a second hinge region, and a second subunit of an Fc domain, or portion thereof; iii) A third antigen binding polypeptide comprising, from N-terminus to C-terminus: a first VL and optionally a first CL; and iv) a fourth antigen binding polypeptide comprising, from N-terminus to C-terminus: a second VL and optionally a second CL, wherein the first VH and the first VL, and optionally the first CH1 and the first CL, form the second binding domain that is an agonist antigen-binding fragment that specifically recognizes PD-1, and wherein the second VH and the second VL, and optionally the second CH1 and the second CL, form a third binding domain that specifically recognizes a third target molecule.
Embodiment 177. The immunomodulatory molecule of embodiment 176, wherein the third binding domain is an agonist antigen-binding fragment that specifically recognizes PD-1.
Embodiment 178 the immunomodulatory molecule of any of embodiments 159-174, comprising: i) A first antigen binding polypeptide comprising, from N-terminus to C-terminus: VH, optionally CH1, a first hinge region, a first subunit of an Fc domain or portion thereof, and a p35 subunit and a p40 subunit of IL-12 or variant thereof fused in tandem; ii) a second antigen binding polypeptide comprising, from N-terminus to C-terminus: a first PD-L2 or PD-L1 or variant thereof, a second hinge region, and a second subunit of an Fc domain or portion thereof; and iii) a third antigen binding polypeptide comprising, from N-terminus to C-terminus: VL and optionally CL, wherein the VH and the VL, and optionally the CH1 and the CL, form the second binding domain, which is an agonist antigen-binding fragment that specifically recognizes PD-1.
Embodiment 179 the immunomodulatory molecule of any of embodiments 159-174, comprising: i) A first antigen binding polypeptide comprising, from N-terminus to C-terminus: a first PD-L2 or PD-L1 or variant thereof, a first hinge region, a first subunit of an Fc domain or portion thereof, and a p35 subunit or p40 subunit of IL-12 or variant thereof; and ii) a second antigen binding polypeptide comprising, from N-terminus to C-terminus: a second PD-L2 or PD-L1 or variant thereof, a second hinge region, and a second subunit of an Fc domain or portion thereof, and a p40 subunit or p35 subunit of IL-12 or variant thereof.
Embodiment 180 the immunomodulatory molecule of any of embodiments 159-174, comprising: i) A first antigen binding polypeptide comprising, from N-terminus to C-terminus: a first VH, optionally a first CH1, a first hinge region, a first subunit of an Fc domain or portion thereof, and a p35 subunit or p40 subunit of IL-12 or variant thereof; ii) a second antigen binding polypeptide comprising, from N-terminus to C-terminus: a second VH, optionally a second CH1, a second hinge region, a second subunit of an Fc domain or portion thereof, and a p40 subunit or p35 subunit of IL-12 or variant thereof; iii) A third antigen binding polypeptide comprising, from N-terminus to C-terminus: a first VL and optionally a first CL; and iv) a fourth antigen binding polypeptide comprising, from N-terminus to C-terminus: a second VL and optionally a second CL, wherein the first VH and the first VL, and optionally the first CH1 and the first CL, form the second binding domain that is an agonist antigen-binding fragment that specifically recognizes PD-1, and wherein the second VH and the second VL, and optionally the second CH1 and the second CL, form a third binding domain that specifically recognizes a third target molecule.
Embodiment 181. The immunomodulatory molecule of embodiment 180, wherein the third binding domain is an agonist antigen-binding fragment that specifically recognizes PD-1.
Embodiment 182 the immunomodulatory molecule of any of embodiments 1-72, wherein the immunomodulatory molecule comprises an antigen-binding protein comprising a first antigen-binding polypeptide and a second antigen-binding polypeptide, wherein the first antigen-binding peptide comprises from N-terminus to C-terminus: VH, CH1, optional hinge region, and Fc domain subunit or portion thereof; wherein the second antigen binding polypeptide comprises, from N-terminus to C-terminus: VL, CL and the first binding domain or a portion thereof; and wherein said VH and said VL, and optionally said CH1 and said CL, form said second binding domain.
Embodiment 183 the immunomodulatory molecule of embodiment 182, wherein the first antigen binding polypeptide comprises, from N-terminus to C-terminus: VH, CH1, first hinge region, first subunit of Fc domain, or portion thereof; wherein the antigen binding protein further comprises a third antigen binding polypeptide comprising, from N-terminus to C-terminus: specifically recognizes a third binding domain of a third target molecule or portion thereof, a second hinge region, and a second subunit of an Fc domain or portion thereof.
Embodiment 184. The immunomodulatory molecule of embodiment 183, wherein said third binding domain and said second binding domain are the same.
Embodiment 185. The immunoregulatory molecule of embodiment 183, wherein said third binding domain and said second binding domain are different.
The immunomodulatory molecule of any of embodiments 183-185, wherein the third target molecule and the second target molecule are the same.
Embodiment 187 the immunomodulatory molecule of embodiment 183 or 185, wherein the third target molecule and the second target molecule are different.
The immunomodulatory molecule of any of embodiments 183-187, wherein the immunomodulatory molecule comprises an antigen-binding protein comprising four antigen-binding polypeptides, wherein the first antigen-binding polypeptide comprises, from N-terminus to C-terminus: a first VH, a first CH1, a first hinge region, a first subunit of an Fc domain, or a portion thereof; wherein the second antigen binding polypeptide comprises, from N-terminus to C-terminus: a first VL, a first CL, and the first binding domain or a portion thereof; wherein the third antigen binding polypeptide comprises, from N-terminus to C-terminus: a second VH, a second CH1, a second hinge region, and a second subunit of an Fc domain, or portion thereof; wherein the fourth antigen binding polypeptide comprises, from N-terminus to C-terminus: a second VL and a second CL; wherein the first VH and the first VL and the first CH1 and the first CL form the second binding domain; and wherein the second VH and the second VL and the second CH1 and the second CL form a third binding domain that specifically recognizes a third target molecule.
Embodiment 189 the immunomodulatory molecule of any one of embodiments 182-188, wherein the first binding domain is an immunostimulatory cytokine or variant thereof.
Embodiment 190. The immunoregulatory molecule of embodiment 189, wherein said immunostimulatory cytokine or variant thereof is IL-2 or IL-12 or a variant thereof.
Embodiment 191. The immunomodulatory molecule of embodiment 189 or 190, wherein the immunostimulatory cytokine or variant thereof is a monomeric immunostimulatory cytokine or variant thereof.
Embodiment 192. The immunomodulatory molecule of embodiment 189 or 190, wherein the immunostimulatory cytokine or variant thereof is a dimeric immunostimulatory cytokine or variant thereof.
Embodiment 193 the immunomodulatory molecule of embodiment 192, wherein the two subunits of said dimeric immunostimulatory cytokine or variant thereof are located in tandem at the C-terminus of said second antigen binding polypeptide and/or said fourth antigen binding polypeptide.
Embodiment 194. The immunomodulatory molecule of embodiment 192, wherein one subunit of the dimeric immunostimulatory cytokine or variant thereof is fused to the C-terminus of the first CL of the second antigen binding polypeptide, and wherein the other subunit of the dimeric immunostimulatory cytokine or variant thereof is fused to the second CL of the fourth antigen binding polypeptide.
Embodiment 195. The immunomodulatory molecule of any of embodiments 182-194, comprising: i) A first antigen binding polypeptide comprising, from N-terminus to C-terminus: a first VH, a first CH1, a first hinge region, and a first subunit of an Fc domain, or portion thereof; ii) a second antigen binding polypeptide comprising, from N-terminus to C-terminus: a first VL, a first CL, and a p35 subunit and a p40 subunit of IL-12 or a variant thereof fused in tandem; iii) A third antigen binding polypeptide comprising, from N-terminus to C-terminus: a second VH, a second CH1, a second hinge region, and a second subunit of an Fc domain, or portion thereof; and iv) a fourth antigen binding polypeptide comprising, from N-terminus to C-terminus: a second VL, a second CL, and a p35 subunit and a p40 subunit of IL-12 or a variant thereof fused in tandem; wherein the first VH and the first VL and the first CH1 and the first CL form the second binding domain that is an agonist antigen-binding fragment that specifically recognizes PD-1, and wherein the second VH and the second VL and the second CH1 and the second CL form a third binding domain that specifically recognizes a third target molecule.
Embodiment 196. The immunomodulatory molecule of embodiment 195, wherein the third binding domain is an agonist antigen-binding fragment that specifically recognizes PD-1.
Embodiment 197 an isolated nucleic acid encoding the immunomodulatory molecule of any of embodiments 1-196.
Embodiment 198. A vector comprising the nucleic acid of embodiment 197.
Embodiment 199 an isolated host cell comprising the nucleic acid of embodiment 197 or the vector of embodiment 198.
Embodiment 200 the host cell of embodiment 199, which is a Chinese Hamster Ovary (CHO) cell.
Embodiment 201. A method of producing an immunomodulatory molecule comprising: (a) Culturing a host cell comprising the nucleic acid of embodiment 197 or the vector of embodiment 198, or the host cell of embodiment 199 or 200, under conditions effective to express the encoded immunoregulatory molecule; and (b) obtaining the expressed immunomodulatory molecule from the host cell.
Embodiment 202. A pharmaceutical composition comprising the immunomodulatory molecule of any of embodiments 1-196 and optionally a pharmaceutically acceptable carrier.
Embodiment 203. A method of treating a disease or disorder in a subject comprising administering to the subject an effective amount of the immunomodulatory molecule of any of embodiments 1-196 or the pharmaceutical composition of embodiment 202.
Embodiment 204 the method of embodiment 203, wherein said immunomodulatory molecule or pharmaceutical composition is administered intravenously or subcutaneously.
Embodiment 205 the method of embodiment 203 or 204, wherein the immunoregulatory molecule or pharmaceutical composition is administered in an amount of about 1 μg/kg to about 10 mg/kg.
Embodiment 206 the method of any one of embodiments 203-205, wherein the immunomodulatory molecule or pharmaceutical composition is administered once every three weeks.
Embodiment 207. The method of any of embodiments 203-206, wherein the disease or disorder is cancer.
Embodiment 208 the method of embodiment 207, wherein said cancer is selected from the group consisting of: lung cancer, liver cancer, kidney cancer, colorectal cancer, ovarian cancer, breast cancer, pancreatic cancer, gastric cancer, bile duct cancer, squamous cell carcinoma, bladder cancer, esophageal cancer, mesothelioma, melanoma, head and neck cancer, thyroid cancer, sarcoma, prostate cancer, glioblastoma, cervical cancer, thymus cancer, leukemia, lymphoma, myeloma, mycosis fungoides, and merck cell cancer.
Embodiment 209 the method of any one of embodiments 203-206, wherein the disease or disorder is an infection, an autoimmune disease, allergy, graft rejection, or graft versus host disease (GvHD).
Examples
The following examples are intended purely as examples of the invention and should therefore not be considered as limiting the invention in any way. The following examples and detailed description are provided by way of illustration and not by way of limitation.
EXAMPLE 1 in vitro analysis of IL-12 bioactivity in IL-12/PD-L1-Fc, PD-L1-Fc/IL-12, IL-12/anti-PD-1, CTLA-4-Fc/IL-12 and IL-12/CTLA-4-Fc immunomodulatory molecules
IL-12/PD-L1-Fc, PD-L1-Fc/IL-12, IL-12/anti-PD-1, CTLA-4-Fc/IL-12 and IL-12- Construction of CTLA-4-Fc immunomodulatory molecules
IL-12 is a heterodimeric cytokine consisting of covalently linked p35 and p40 subunits. By substituting alanine or serine for amino acids 56 to 65 of the p40 subunit, an IL-12 variant comprising an amino acid substitution in the p40 subunit was constructed (see table 1), and single-chain IL-12 variants were prepared, with from N-terminus to C-terminus: the p40 variant subunit-linker (SEQ ID NO: 228) -p35 wild-type subunit (SEQ ID NO: 61). Single-stranded "wild-type" IL-12 was also constructed as a control (SEQ ID NO: 67), from N-terminus to C-terminus: the p40 wild-type subunit (SEQ ID NO: 62) -linker (SEQ ID NO: 228) -p35 wild-type subunit is referred to as "WT" in Table 1. The linker may also be changed to SEQ ID NO. 226, and the single-stranded "wild-type" IL-12 may also comprise SEQ ID NO. 253.
IL-12/anti-PD-1 (hinge) immunoregulatory molecules
Will contain sodium Wu Liyou mab (oldiewok,) Anti-human PD-1 antibodies of the sequences VH (SEQ ID NO: 48) and VL (SEQ ID NO: 49) were used as parent full-length antibodies comprising two light chains, each comprising the amino acid sequence of SEQ ID NO: 50. To construct the heterodimer, one heavy chain comprises: a hinge region comprising SEQ ID NO. 78 and an Fc domain subunit comprising SEQ ID NO. 97; the other heavy chain comprises: a hinge region comprising SEQ ID NO. 77 and an Fc domain subunit comprising SEQ ID NO. 98. Various single chain IL-12 variants (or single chain "wild-type" IL-12 controls) are located within the hinge region of the heavy chain of an anti-PD-1 antibody (see the exemplary structure of fig. 1C, anti-PD-1 na Wu Liyou mab is an antagonist antibody) to construct an IL-12/anti-PD-1 immunoregulatory molecule "Fab-IL-12-Fc-PD-1Ab". For example, a Fab-IL-12 (E59A/F60A) -Fc-PD-1 antibody immunoregulatory molecule (or "IL-12 (E59A/F60A)/anti-PD-1 immunoregulatory molecule", "IW- #48", or "construct # 48") comprising a single chain IL-12 variant IL-12B (p 40E 59A/F60A) -linker-IL-12A (wt p 35) located at the hinge region comprises: two light chains and two heavy chains, wherein each light chain comprises the amino acid sequence SEQ ID NO. 50, one heavy chain comprises the amino acid sequence of SEQ ID NO. 21, and the other heavy chain has a single chain IL-12 (E59A/F60A) variant (SEQ ID NO. 68) located at the hinge region and comprises the amino acid sequence of SEQ ID NO. 22. IL-12 (G64A)/anti-PD-1 immunoregulatory molecule comprising a single chain IL-12 variant IL-12B (p 40G 64A) -linker-IL-12A (wt p 35) located in the hinge region ("construct #47" or "IW- # 47") comprises: two light chains and two heavy chains, wherein each light chain comprises the amino acid sequence of SEQ ID NO. 50, one heavy chain comprises the amino acid sequence of SEQ ID NO. 21, and the other heavy chain has a single chain IL-12 (G64A) variant at the hinge region (SEQ ID NO: 70). IL-12 (E59A)/anti-PD-1 immunoregulatory molecule comprising a single chain IL-12 variant IL-12B (p 40E 59A) -linker-IL-12A (wt p 35) located in the hinge region comprises: two light chains and two heavy chains, wherein each light chain comprises the amino acid sequence of SEQ ID NO. 50, one heavy chain comprises the amino acid sequence of SEQ ID NO. 21 and the other heavy chain has a single chain IL-12 (E59A) variant (SEQ ID NO. 69) located at the hinge region. IL-12 (F60A)/anti-PD-1 immunoregulatory molecule comprising a single chain IL-12 variant IL-12B (p 40F 60A) -linker-IL-12A (wt p 35) located in the hinge region ("construct #46" or "IW- # 46") comprises: two light chains and two heavy chains, wherein each light chain comprises the amino acid sequence of SEQ ID NO. 50, one heavy chain comprises the amino acid sequence of SEQ ID NO. 21 and the other heavy chain has a single chain IL-12 (F60A) variant (SEQ ID NO. 71) located at the hinge region and comprises the amino acid sequence of SEQ ID NO. 23. The heavy chain comprising the amino acid sequence of SEQ ID NO. 21 may also be substituted with a heavy chain comprising the amino acid sequence of SEQ ID NO. 51. The linker within a single chain IL-12 variant (e.g., a single chain IL-12 (E59A/F60A) variant) may also be changed to SEQ ID NO:246, and a single chain IL-12 (E59A/F60A) variant may comprise SEQ ID NO:254.
● IL-12/PD-L1-Fc (hinge) and PD-L1-Fc/IL-12 (C-terminal) immunoregulatory molecules
PD-L1-hinge-Fc fusion proteins (two PD-L1 ectodomain-hinge-Fc polypeptides) were used as parent antigen binding proteins to construct immunoregulatory molecules that bind to PD-1. To construct a heterodimeric PD-L1-hinge-Fc fusion protein, a PD-L1-hinge-Fc fusion polypeptide comprises: a hinge region comprising SEQ ID NO. 88 and an Fc domain subunit comprising SEQ ID NO. 97; another PD-L2-Fc fusion polypeptide comprises: a hinge region comprising SEQ ID NO. 87 and an Fc domain subunit comprising SEQ ID NO. 98. The single chain IL-12 variant is located in the hinge region of a PD-L1-hinge-Fc polypeptide (hereinafter referred to as "IL-12/PD-L1-Fc immunoregulatory molecule") or is fused to the C-terminus of a PD-L1-hinge-Fc polypeptide (hereinafter referred to as "PD-L1-Fc/IL-12 immunoregulatory molecule").
For example, an IL-12 (E59A/F60A)/PD-L1 (wt) -Fc immunoregulatory molecule comprises an IL-12 fusion polypeptide and a partner polypeptide, wherein the IL-12 fusion polypeptide is: PD-L1 (wt) ectodomain (SEQ ID NO: 121) -GGGGGGSGGG linker (SEQ ID NO: 244) -single chain IL-12 (E69A/F60A) variant-GGGGSGGG linker (SEQ ID NO: 244) -hinge (SEQ ID NO: 88) -Fc domain subunit (SEQ ID NO: 97); and the pairing polypeptide is from N-terminal to C-terminal: PD-L1 (wt) ectodomain (SEQ ID NO: 121) -GGGGSGGG linker (SEQ ID NO: 244) -hinge (SEQ ID NO: 87) -Fc domain subunit (SEQ ID NO: 98). The IL-12 (E59A/F60A)/PD-L1 (mutant) -Fc immunoregulatory molecule comprises an IL-12 fusion polypeptide and a mating polypeptide, wherein the IL-12 fusion polypeptide comprises, from N-terminus to C-terminus: PD-L1 (mutant) extracellular domain (e.g., SEQ ID NO: 129) -GGGGGGSGGG linker (SEQ ID NO: 244) -single chain IL-12 (E69A/F60A) variant-GGGGSGGG linker (SEQ ID NO: 244) -hinge (SEQ ID NO: 88) -Fc domain subunit (SEQ ID NO: 97); and the pairing polypeptide is from N-terminal to C-terminal: PD-L1 (mutant) ectodomain-GGGGSGGG linker (SEQ ID NO: 244) -hinge (SEQ ID NO: 87) -Fc domain subunit (SEQ ID NO: 98). PD-L1 (wt) -Fc/IL-12 (E59A/F60A) (C-terminal) immunoregulatory molecule comprises an IL-12 fusion polypeptide and a partner polypeptide, wherein the IL-12 fusion polypeptide comprises from N-terminus to C-terminus: PD-L1 (wt) extracellular domain (SEQ ID NO: 121) -GSG linker (SEQ ID NO: 203) -hinge (SEQ ID NO: 88) -Fc domain subunit (SEQ ID NO: 97) -GGGGSGGGGSGGGGS linker (SEQ ID NO: 229) -single-chain IL-12 (E59A/F60A) variant (SEQ ID NO:68 or 254); and the mating polypeptide comprises, from N-terminus to C-terminus: PD-L1 (wt) ectodomain (SEQ ID NO: 121) -GSG linker (SEQ ID NO: 203) -hinge (SEQ ID NO: 87) -Fc domain subunit (SEQ ID NO: 98)). The linker may be changed to other linkers (e.g., GSG linker; SEQ ID NO: 203), or may be optional.
IL-12/CTLA-4-Fc (hinge) and CTLA-4-Fc/IL-12 (C-terminal) immunoregulatory molecules were similarly constructed. See table 2 for sequences.
Nucleic acids encoding various forms of IL-12/PD-L1-Fc, IL-12/anti-PD-1 and IL-12/CTLA-4-Fc immunoregulatory molecules (amino acid sequences per polypeptide chain are shown in Table 2) were chemically synthesized, cloned into lentiviral vectors, and transfected into CHO cells for expression. Expressed immunoregulatory molecules were collected from the supernatant, purified by protein a chromatography and purity verified on SDS-PAGE.
IL-12 signaling assay
HEK-BLUE was prepared following InvivoGen user Manual (InvivoGen accession number hkb-il 12) TM IL-12 cells (InvivoGen accession number hkb-IL 12) and HEK-PD-1-IL-12 cells (by use of lentiviral vectors in HEK-Blue TM The overexpression of human PD-1 in IL-12 cells and generated internally) was used to evaluate the IL-12 signaling activity of a variety of IL-12 immunoregulatory molecules including different IL-12 moieties, also referred to below as "HEK-IL-12 reporter assays" or "HEK-PD-1-IL-12 reporter assays". HEK-Blue TM IL-12 reporter cells and HEK-PD-1IL-12 reporter cells stably express the human IL-12 receptor complex consisting of IL-12 receptor beta 1 (IL-12R beta 1) and IL-12R beta 2, as well as the human STAT4 gene, to obtain a fully functional IL-12 signaling pathway (TyK/JAK 2/STAT 4). In addition, these reporter cells also carry STAT 4-inducible SEAP reporter genes. HEK-Blue after IL-12 stimulation TM IL-12 reporter cells and HEK-PD-1-IL-12 reporter cells trigger STAT4 activation and subsequent SEAP secretion at levels that may be achieved using QUANTI-Blue TM (InvivoGen accession number rep-qbs) colorimetric enzyme assay of alkaline phosphatase activity.
Briefly, HEK-Blue TM IL-12 cells were added to a variety of IL-12 containing immunomodulatory molecules in each plate well (or recombinant human IL-12 (rIL-12) was added to the well as a positive control) and CO at 37 ℃C 2 Incubate in incubator for 20-24 hours or overnight. After incubation, the supernatant was transferred to fresh plate wells and QUANTI-Blue was added TM The solution was incubated in an incubator at 37℃for 30 minutes to 3 hours. SEAP levels were then measured using a spectrophotometer at 620-655 nm. In activating IL-12 signal into the pathway, recombinant human IL-12 (positive control) activity was measured as 10 units/ng and used as a reference. The IL-12 signaling percentage of various IL-12 immunoregulatory molecules was calculated by dividing the immunoregulatory molecule reading by the recombinant human IL-12 reading.
In the HEK-PD-1-IL-12 reporter assay, the IL-12/anti-PD-1 immunoregulatory molecule can only bind to HEK-IL-12 cells by binding to PD-1 or IL-12 moiety/IL-12 receptor interactions. In the absence of PD-1 binding, localization of the wild-type p40 subunit comprising IL-12 to the hinge region of an anti-PD-1 antibody ("IL-12 (WT)/anti-PD-1") reduced IL-12 activity to 50.0%. As can be seen from Table 1, positions 59 and 60 of the p40 subunit are critical to the biological activity of IL-12. IL-12/anti-PD-1 immunoregulatory molecules comprising the E59A/F60A double mutation in the IL-12p40 subunit ("IL-12 (E59A/F40A)/anti-PD-1", "IW- # 48") showed almost complete loss of IL-12 activity as measured by IL-12 signaling (0.1%).
In HEK-PD-1-IL-12 reporter assays, IL-12/anti-PD-1 immunoregulatory molecules are capable of binding to HEK-PD-1-IL-12 cells through IL-12 moiety/IL-12 receptor interactions and anti-PD-1 antigen binding fragment/PD-1 interactions. As can be seen from Table 1, the biological activity of all IL-12 variants (and "WT" IL-12) in the IL-12/anti-PD-1 immunoregulatory molecule increases with the presence of PD-1 binding. In particular, IL-12 activity of IL-12 (E59A/F60A)/anti-PD-1 immunoregulatory molecule (IW- # 48) was rescued to 5.9% by PD-1/anti-PD-1 antibody binding, which was 59-fold greater than in the absence of PD-1/anti-PD-1 antibody binding (0.1%).
The E59A/F60A double mutation in the IL-12p40 subunit showed excellent effects compared to other mutations in the IL-12p40 subunit. By locating such IL-12 (E59A/F60A) variants at the hinge region of the heavy chain of an anti-PD-1 full length antibody, the resulting IL-12 (E69A/F60A)/anti-PD-1 immunoregulatory molecule (IW- # 48) only exhibits IL-12 bioactivity in the presence of target antigen (PD-1) -antibody binding, but not in the absence of target antigen (PD-1) -antibody binding, indicating targeting specificity.
TABLE 1 IL-12 biological Activity of IL-12/anti-PD-1 immunomodulators comprising different IL-12 moieties
In the HEK-PD-1-IL-12 reporter assay, the IL-12 immunoregulatory molecule can only bind to HEK-IL-12 cells by binding to PD-1 or IL-12 moiety/IL-12 receptor interactions. CTLA-4/IL-12 immunomodulatory molecules cannot bind to PD-1+HEK cells that do not express receptors such as CD80 or CD 86. In the absence of receptor (CD 80 or CD 86) -ligand (CTLA-4) binding from these CLTA-4 immunomodulatory molecules (Table 2; SEQ ID NOs: 1-5), the biological activity of IL-12 is absent: 0.2-2.3%. Notably, single mutant IL-12 (F60A) at the C-terminus without receptor (CD 80 or CD 86) -ligand (CTLA-4) binding showed some activity (2.3%), whereas single mutant IL-12 (F6 0A) at the hinge lost any activity entirely (0.2%). The wild type PD-L1 ligand (wt ectodomain SEQ ID NO: 121) that binds PD-1 has a low affinity (Kd about 8.2. Mu.M). Mutation of the PD-L1 ligand (I54Q/Y56F/E58M/R113T/M115L/S117A/G119K; mutant 8 ectodomain SEQ ID NO: 129) increased the binding affinity by about 200-fold (Kd about 0.041. Mu.M). Wild-type PD-L1 ligands (Table 2; SEQ ID NO: 6-10) that bind to PD-1 from these wild-type constructs rescue the biological activity of mutant IL-12 (greater than 100%) except for the double mutant IL12 located in the hinge region (E59A/F60A) (8.2%). In contrast, the high affinity PD-L1 ligands (Table 2; SEQ ID NO: 11-15) from these mutant PD-L1 constructs rescue all the biological activity of mutant IL-12, including mutant IL-12 at the hinge (E59A/F60A) (125.6%). These data indicate that the activity of mutant IL-12 (E59A/F60A) can be rescued by increasing the binding affinity of PD-L1 ligands in the same construct. Rescue of biological activity depends on the affinity of the PD-L1 ligand for PD-1. In addition, in the presence of PD-L1/PD-1 and IL-12/IL-12R binding, IL-12 activity is greater than that of the positive control (rIL-12 alone). This suggests that the presence of a second domain that binds to a target cell (e.g., PD-L1/PD-1 that binds to a T cell) promotes binding of an IL-12 immunomodulatory molecule to the same target cell (e.g., IL-12/IL-12R binding to a T cell).
TABLE 2 IL-12 biological Activity of various IL-12 immunomodulatory molecules
EXAMPLE 2 in vivo efficacy of IL-12/PD-L2-Fc and PD-L2-Fc/IL-12 immunomodulatory molecules in established CT26 syngeneic tumor mouse models
Construction of IL-12/PD-L2-Fc (hinge) and PD-L2-Fc/IL-12 (C-terminal) immunoregulatory molecules
PD-L2-hinge-Fc fusion proteins (two PD-L2 hinge-Fc polypeptides each comprising SEQ ID NO: 111) were used as parent antigen binding proteins to construct immunoregulatory molecules that bind to PD-1. To construct a heterodimeric PD-L2-hinge-Fc fusion protein, a PD-L2-hinge-Fc fusion polypeptide comprises: a hinge region comprising SEQ ID NO. 88 and an Fc domain subunit comprising SEQ ID NO. 97; another PD-L2-Fc fusion polypeptide comprises: a hinge region comprising SEQ ID NO. 87 and an Fc domain subunit comprising SEQ ID NO. 98. The single chain IL-12 variant is located in the hinge region of a PD-L2-hinge-Fc polypeptide (hereinafter referred to as "IL-12/PD-L2-Fc immunoregulatory molecule") or fused to the C-terminus of a PD-L2-hinge-Fc polypeptide (hereinafter referred to as "PD-L2-Fc/IL-12 immunoregulatory molecule"). For example, IL-12 (E59A/F60A)/PD-L2-Fc immunomodulatory molecule ("construct #29" or "IW- # 29") comprises an IL-12 fusion polypeptide and a mating polypeptide, wherein the IL-12 fusion polypeptide comprises SEQ ID NO. 17 (from N-terminus to C-terminus: PD-L2 extracellular domain (SEQ ID NO. 106) -GSG linker (SEQ ID NO. 203) -single chain IL-12 (E69A/F60A) variant (SEQ ID NO. 68) -hinge (SEQ ID NO. 88) -Fc domain subunit (SEQ ID N0: 97)); and the mating polypeptide comprises SEQ ID NO. 16 (from N-terminus to C-terminus: PD-L2 extracellular domain (SEQ ID NO. 106) -GSG linker (SEQ ID NO. 203) -hinge (SEQ ID NO. 87) -Fc domain subunit (SEQ ID NO. 98)). Single-chain IL-12 (E69A/F60B) variants within IL-12 (E59A/F60A)/PD-L2-Fc immunoregulatory molecules were replaced with single-chain IL-2 (F60A) variants (SEQ ID NO: 71) or single-chain IL-12 (G64A) variants (SEQ ID NO: 70), respectively, to construct IL-12 (F60A)/PD-L2-Fc immunoregulatory molecules ("construct #30" or "IW- # 30") and IL-12 (G64A)/PD-L2-Fc immunoregulatory molecules. For example, the IL-12 (F60A)/PD-L2-Fc immunoregulatory molecule comprises an IL-12 fusion polypeptide and a mating polypeptide, wherein the IL-12 fusion polypeptide comprises SEQ ID NO. 18 or 142 (from N-terminus to C-terminus: PD-L2 extracellular domain (SEQ ID NO. 106) -GSG linker (SEQ ID NO. 203) -single chain IL-12 (F6 0A) variant (SEQ ID NO. 71) -hinge (SEQ ID NO. 88) -Fc domain subunit (SEQ ID NO. 97)); and the mating polypeptide comprises SEQ ID NO. 16 or 115 (from N-terminus to C-terminus: PD-L2 extracellular domain (SEQ ID NO. 106) -GSG linker (SEQ ID NO. 203) -hinge (SEQ ID NO. 87) -Fc domain subunit (SEQ ID NO. 98)). PD-L2-Fc/IL-12 (F60A) immunoregulatory molecules ("construct #34" or "IW- # 34") comprise an IL-12 fusion polypeptide and a partner polypeptide, wherein the IL-12 fusion polypeptide comprises SEQ ID NO:20 or 143 (from N-terminus to C-terminus: PD-L2 extracellular domain (SEQ ID NO: 106) -GSG linker (SEQ ID NO: 203) -hinge (SEQ ID NO: 88) -Fc domain subunit (SEQ ID NO: 97) -GGGGSGGGGSGGGGS linker (SEQ ID NO: 229) -single chain IL-12 (F60A)) and the partner polypeptide comprises SEQ ID NO:16 or 115 (from N-terminus to C-terminus: PD-L2 extracellular domain (SEQ ID NO: 106) -GSG linker (SEQ ID NO: 203) -hinge (SEQ ID NO: 87) -Fc domain subunit (SEQ ID NO: 98))IL-12 variants (e.g., single chain IL-12 (E: 59A) may also comprise variants such as single chain IL-12A: 59A) variants, such as single chain variants of SEQ ID NO: 59A).
Nucleic acid encoding an immunomodulatory molecule was chemically synthesized, cloned into a lentiviral vector, and transfected into CHO cells for expression. Expressed immunoregulatory molecules were collected from the supernatant, purified by protein a chromatography and purity verified on SDS-PAGE.
Mice (about 20g body weight) were vaccinated with 0.25X10 6 Individual CT26 mouse colon cancer cells. The tumor size was measured to be about 100-200mm 11 days after tumor inoculation 3 . After measuring tumor size, mice were injected with 200. Mu.g (10 mg/kg) of IL-12 (F60A)/PD-L2-Fc, hinge (IW- # 30) immunoregulatory molecule (structure see FIG. 1G (first polypeptide chain SEQ ID NO:16 or 115, second polypeptide chain SEQ ID NO:18 or 142), wherein the cytokine is variant IL-12F 60A), 200. Mu.g (10 mg/kg) of IL-12 (F60A)/PD-L2-Fc, HCC-terminal (IW- # 34) immunoregulatory molecules (structure shown in FIG. 1I (first polypeptide chain SEQ ID NO:16 or 115, second polypeptide chain SEQ ID NO:20 or 143), wherein the cytokine is variant IL-12F60A; also referred to herein as PD-L2-Fc/IL-12 (F60A)), or PBS (negative control). Five mice per group. A total of three injections (indicated by black arrows in fig. 2A-2C) were made on days 11, 14 and 18 post inoculation. Tumor size was measured every 3 days from the first injection. The mean initial (pre-injection) tumor volume plus or minus one standard deviation is given in brackets in the legend. Once the tumor size reached 2000mm 3 Mice were sacrificed as above. Figure 2A depicts the mean tumor volume (±standard deviation) in each treatment group. Individual mouse patterns for each group are also provided in fig. 2B and 2C, with fig. 2B showing IL-12 (F60A)/PD-L2-Fc, hinge (IW- # 30) immunomodulatory molecules, and fig. 2C showing IL-12 (F60A)/PD-L2-Fc, C-terminal (IW- # 34) immunomodulatory molecules of HC. IL-12 (F60A)/PD-L2-Fc, hinge (IW- # 30) immunoregulatory molecules cured 4/5 mice (80% cure rate), IL-12 (F60A)/PD-L2-Fc, C-terminal (IW- # 34) immunoregulatory molecules cured 5/5 mice (100% cure rate). In cured mice, the tumor inhibition efficacy of both IL-12/PD-L2-Fc immunoregulatory molecules was similar.
CT26 mouse models are highly responsive to current immunotherapy, including anti-PD-1, anti-CTLA-4, and combination therapies of anti-PD-1 and anti-CTLA 4 antibodies. The data herein show that both IL-12/PD-L2-Fc immunoregulatory molecules are capable of resolving CT26 tumors in 80-100% of mice, showing promising in vivo efficacy.
Example 3 IL-12/PD-L2-Fc and PD-L2-Fc/IL-12 immunoregulatory molecules capable of inducing specific anti-tumor memory in a cured CT26 syngeneic tumor mouse model
To investigate whether the immunomodulatory molecules described herein can function as cancer vaccines, or prevent cancer recurrence, all cured mice in example 2 were tumor re-challenged. 30 days after the final injection of the immunoregulatory molecule, the right flank of the cured CT26 mice was inoculated with 0.25X10 6 Colon cancer cells of CT26 mice, left flank inoculation of 0.25x10 6 Fine breast cancer in individual EMT6 miceCells (as control). Tumor size was recorded every 4 days after tumor inoculation was re-stimulated. Once the tumor size reached 1000mm 3 Mice were sacrificed as above.
As shown in fig. 3A-3B, all cured mice previously treated with anti-CT 26 tumor immunoregulatory molecules were protected from the effects of re-challenge of CT26 tumors, but from the effects of EMT 6. IL-12 (F60A)/PD-L2-Fc immunoregulatory molecule (IW- #30; hinge) and PD-L2-Fc/IL-12 (F60A) immunoregulatory molecule (IW- #34, C-terminal of HC) exhibited similar protective efficacy against re-excitation of CT-26 tumors. These results indicate successful generation of anti-CT 26 tumor memory, indicating that both immunomodulatory molecules described herein, such as PD-L2-Fc/IL-12 (F60A) immunomodulatory molecule (IW- # 34) and IL-12 (F60A)/PD-L2-Fc immunomodulatory molecule (IW- #30; hinge), can be used as cancer vaccines in mice (e.g., anti-CT 26 colon cancer), and/or can prevent cancer recurrence, can induce specific anti-tumor memory.
EXAMPLE 4 IL-12/PD-L2-Fc immunoregulatory molecule was able to regress very large CT26 tumors [ ]>250mm 3 ) Or advanced CT26 tumor
Successful treatment of advanced cancers remains a great unmet clinical need. To investigate whether the immunomodulatory molecules described herein were effective against advanced cancers, mice were vaccinated with cancer cells to grow tumors to 250mm 3 Above, this is considered as not treatable in mice by immunotherapy. Such mouse tumor volumes may mimic the tumor burden of patients with advanced human cancers.
Briefly, 0.25X10 6 Individual CT26 mice colon cancer cells were inoculated into mice (about 20g body weight). The tumor size was measured to be greater than 250mm 14 days after tumor inoculation 3 . The mean initial tumor volume plus or minus one standard deviation is given in brackets in the legend of fig. 4A. After measuring tumor size, mice were injected with 200. Mu.g (10 mg/kg) of IL-12 (E59A/F60A)/PD-L2-Fc, hinge (constructed in example 2, IW- # 29) immunoregulatory molecule (structure shown in FIG. 1G (first polypeptide chain SEQ ID NO:16, second polypeptide chain SEQ ID NO: 17), wherein the cytokine was variant IL-12E 59A/F60A), 200. Mu.g (10 mg/kg)) IL-12 (F60A)/PD-L2-Fc, hinge (constructed in example 2, IW- # 30) immunoregulatory molecule (structure see FIG. 1G (first polypeptide chain SEQ ID NO:16, second polypeptide chain SEQ ID NO: 18), wherein the cytokine is variant IL-12F 60A). Seven mice per group. Three injections (indicated by black arrows) were made on days 14, 17 and 21 total after inoculation. Tumor size was measured every 4 days since the first injection. Once the tumor size reached 1000mm 3 Mice were sacrificed as above. Figure 4A depicts the average tumor volume in each treatment group.
As shown in FIG. 4A, the difference in tumor regression efficacy observed between IL-12 (E59A/F60A)/PD-L2-Fc, hinge (IW- # 29) immunoregulatory molecule and IL-12 (F60A)/PD-LN-Fc, hinge (IW- # 30) immunoregulatory molecule may be due to the lower potency (e.g., receptor binding and/or signaling ability) of double mutant IL-12 (E59A/F60A) compared to single mutant IL-12 (F6 0A). Such differences in efficacy can be compensated for by injecting a higher dose (e.g., 20mg/kg versus 10 mg/kg) or more injections (e.g., from 3 injections to 5 injections) of IL-12 (E59A/F60A) -based immunomodulatory molecules.
FIG. 4B depicts photographs of mice during treatment with IL-12 (F60A)/PD-L2-Fc, hinge (IW- # 30) immunoregulatory molecules. Initial tumor volume was 290.4mm 3 . The structural integrity of the tumor rapidly regresses within one week after the first injection and forms a scab. Two weeks after the initial injection, the tumor had completely resolved.
Immunotherapy (monotherapy or combination therapy) is typically greater than 150mm 3 Is non-responsive in isogenic tumor volumes. These conditions in the mouse model may correspond to tumor burden in patients with advanced cancer. Our data indicate that our IL-12/PD-L2-Fc immunomodulatory molecules can successfully treat very large syngeneic tumors (equivalent to advanced human cancers), suggesting their promising application in clinical settings.
EXAMPLE 5 in vivo efficacy of IL-12/PD-L2-Fc and IL-12/anti-PD-1 immunomodulatory molecules in established EMT6 syngeneic tumor mouse models
Mice (about 20g body weight) were vaccinated with 0.25X10 6 Individual EMT6 mice breast cancer cells. At 11 days after tumor inoculation, a tumor size of about 100-150mm was measured 3 . Average initial tumor volume plus or minus one standard deviation is given in brackets in the legend (fig. 5A). After measuring tumor size, mice were injected with 200 μg (10 mg/kg) of IL-12 (E59A/F60A)/PD-L2-Fc, hinge (constructed in example 2, IW- # 29) immunoregulatory molecule (structure see FIG. 1G (first polypeptide chain SEQ ID NO:16, second polypeptide chain SEQ ID NO: 17)), 200 μg (10 mg/kg) of IL-12 (F60A)/PD-L2-Fc, hinge (constructed in example 2, IW- # 30) immunoregulatory molecule (structure see FIG. 1G (first polypeptide chain SEQ ID NO:16, second polypeptide chain SEQ ID NO: 18)), 200 μg (10 mg/kg) of IL-12 (E59A/F60A)/anti-PD-1, hinge (constructed in example 1, IW- # 48) immunoregulatory molecule (structure see FIG. 1C (two light chains, each comprising the amino acid sequence of SEQ ID NO: 50; one heavy chain comprising the amino acid sequence of SEQ ID NO:21, and the other comprising the amino acid sequence of SEQ ID NO:22, the other hinge 1F 60A) and the single chain of PBS, the hinge (10 mg/kg) or the variant thereof, the single chain variant being located in the PBS domain, but not comprising the amino acid sequence of the F-60A/F or the single chain region of the variant. Five mice per group. Three injections (indicated by black arrows) were made on days 7, 12 and 16 total after inoculation. Tumor size was measured every 3 days from the first injection. Once the tumor size reaches 1500mm 3 Mice were sacrificed as above. Figure 5A depicts the average tumor volume in each treatment group. Also provided in figures 5B, 5C and 5D are individual mouse plots for each group, with figure 5B showing IL-12 (E59A/F60A)/PD-L2-Fc, hinge (IW- # 29) immunoregulatory molecules, figure 5C showing IL-12 (F60A)/PD-L2-Fc, hinge (IW- # 30) immunoregulatory molecules, and figure 5D showing IL-12 (E59 AF 60A)/anti-PD-1, hinge (IW- # 48) immunoregulatory molecules. IL-12 (E59A/F60A)/PD-L2-Fc, hinge (IW- # 29) immunoregulatory molecules successfully inhibited tumor growth in 3/5 mice (60% cure rate), whereas IL-12 (F60A)/PD-L2-Fc, hinge (IW- # 30) immunoregulatory molecules and IL-12 (E69A/F68A)/anti-PD-1, hinge (IW- # 48) immunoregulatory molecules all successfully inhibited tumor growth in 5/5 mice (100% cure rate).
The initial average tumor size of the mice treated with IL-12 (E59A/F60A)/anti-PD-1 immunoregulatory molecule (IW- # 48) was more than twice that of the other two test groups. These results indicate that all three IL-12 immunomodulatory molecules tested completely reverse EMT6 isogenic mammary tumors in mice, with anti-PD-1-based immunomodulatory molecules having optimal efficacy.
As shown in FIG. 5A, the difference in tumor regression efficacy observed between IL-12 (E59A/F60A)/PD-L2-Fc, hinge (IW- # 29) immunoregulatory molecule and IL-12 (F60A)/PD-LN-Fc, hinge (IW- # 30) immunoregulatory molecule may be due to the lower potency (e.g., receptor binding and/or signaling ability) of double mutant IL-12 (E59A/F60A) compared to single mutant IL-12 (F6 0A). Such differences in efficacy can be compensated for by injecting a higher dose (e.g., 20mg/kg versus 10 mg/kg) or more (e.g., from 3 injections to 5 injections) of IL-12 (E59A/F60A) -based immunomodulatory molecules per injection.
The EMT6 mouse model was moderately responsive to current immunotherapy. Combination therapy with anti-PD-1 and anti-CTLA-4 antibodies can significantly inhibit tumor growth, but does not completely regress the tumor. As can be seen in FIG. 5A, all IL-12/PD-L2-Fc and IL-12/anti-PD-1 immunoregulatory molecules tested were able to regress EMT6 tumors in 60-100% of mice, showing promising in vivo efficacy.
Example 6 IL-12/PD-L2-Fc and IL-12/anti-PD-1 immunoregulatory molecules capable of inducing specific anti-tumor memory in a mouse model of a cured EMT6 syngeneic tumor
To investigate whether the immunomodulatory molecules described herein can function as cancer vaccines, or prevent cancer recurrence, all cured mice in example 5 were tumor re-challenged. The right abdomen of the cured mice was inoculated with 0.25X10 at 30 days after the last injection of the immunoregulatory molecule 6 Breast cancer cells of each EMT6 mouse were inoculated with 0.25X10 on the left flank 6 Individual CT26 mouse colon cancer cells (as control). Tumor size was recorded every 4 days after re-tumor inoculation. Once the tumor size reached 1000mm 3 Mice were sacrificed as above.
As shown in fig. 6A-6C, all cured mice previously treated with anti-EMT 6 tumor immunoregulatory molecules were protected from EMT6 tumor re-challenge but from CT 26. In addition, all three IL-12 immunoregulatory molecules showed similar protective effects against EMT6 tumor re-challenge. These results indicate successful generation of anti-EMT 6 tumor memory, indicating that immunomodulatory molecules described herein, such as IL-12 (E59A/F60A)/PD-L2-Fc immunomodulatory molecule (IW- # 29), IL-12 (F60A)/PD-LN-Fc immunomodulatory molecule (IW- # 30), and IL-12 (E59A/F60A)/anti-PD-1 immunomodulatory molecule (IW- # 48), can be used as cancer vaccines (e.g., anti-breast cancer (e.g., EMT 6) tumors) in mice, and/or can prevent cancer recurrence, capable of inducing specific anti-tumor memory.
Example 7 in vivo efficacy of IL-12/PD-L2-Fc and IL-12/anti-PD-1 immunomodulatory molecules showed that they could significantly inhibit tumor growth in established 4T1 Triple Negative Breast Cancer (TNBC) syngeneic tumor models
4T1 is a standard murine breast tumor model for preclinical studies of breast cancer metastasis. 4T1 is a refractory model for immunotherapy and is not responsive to anti-PD-1, anti-CTLA-4, or a combination of anti-PD-1 and anti-CTLA-4 antibody therapies.
To test the therapeutic efficacy of the immunomodulatory molecules described herein against immunotherapeutic resistant cancer types, 0.25X10 were used 6 The 4T1 mice were vaccinated with breast cancer cells (-20 g body weight). 7 days after tumor inoculation, the tumor size was measured to be about 100mm 3 . The mean initial tumor volume plus or minus one standard deviation is given below the title of the graph (fig. 7A-7D). After measuring tumor size, mice were injected with increasing concentrations of IL-12 (F60A)/PD-L2-Fc, hinge (constructed in example 2, IW- # 30) (first polypeptide chain SEQ ID NO:16, second polypeptide chain SEQ ID NO: 18) immunoregulatory molecule (structure see FIG. 1G), IL-12 (E59A/F60A)/PD-L2-Fc, hinge (constructed in example 2, IW- # 29) (first polypeptide chain SEQ ID NO:16, second polypeptide chain SEQ ID NO: 17) immunoregulatory molecule (structure see FIG. 1G), IL-12 (F60A)/anti-PD-1, hinge (constructed in example 1, IW- # 46) immunoregulatory molecule (structure see FIG. 1C (two light chains, each light chain comprising the amino acid sequence of SEQ ID NO: 50; one heavy chain comprising the amino acid sequence of SEQ ID NO:21, the other heavy chain having the amino acid sequence located at the hinge region) A single chain IL-12 (F60A) variant (SEQ ID NO: 71) and comprising the amino acid sequence of SEQ ID NO:23, wherein the Fab binds PD-1 but is not an agonist), and IL-12 (E59A/F60A)/anti-PD-1, hinge (constructed in example 1, IW- # 48) (two light chains, each light chain comprising the amino acid sequence of SEQ ID NO: 50; one heavy chain comprises the amino acid sequence of SEQ ID NO. 21 and the other heavy chain has a single chain IL-12 (E59A/F60A) variant (SEQ ID NO. 68) located in the hinge region and comprising the amino acid sequence of SEQ ID NO. 22) an immunoregulatory molecule (structure see FIG. 1C, wherein Fab binds PD-1 but is not an agonist): 0. 1, 3, 10 and 50mg/kg. Five mice per group. This range is based on the maximum tolerated dose of mul 12 (0.5 mg/kg) and the reported IL-12 immunomodulator @<2.5 mg/kg). Our IL-12/PD-L2-Fc immunoregulatory molecules can reach doses up to 50mg/kg without significant toxic symptoms. A total of three injections (indicated by black arrows) were given on days 8, 11 and 14 post inoculation. Tumor size was measured every 3 days from the first injection. Once the tumor size exceeds 1500mm 3 Mice were sacrificed.
From FIGS. 7A-7D, it can be seen that all IL-12 (mutant)/PD-L2-Fc, PD-L2-Fc/IL-12 (mutant) and IL-12 (mutant)/anti-PD-1 immunoregulatory molecules significantly inhibited 4T1 tumor growth after a dose-dependent response. This demonstrates promising in vivo efficacy, particularly considering that 4T1 is a refractory model for the non-response of anti-PD-1, anti-CTLA-4, or a combination of anti-PD-1 and anti-CTLA-4 antibody therapies.
Example 8 in vivo efficacy of IL-12/PD-L2-Fc immunomodulatory molecules showed that they significantly inhibited tumor growth in established B16-F10 syngeneic tumor models
B16 is a murine melanoma cell line used to study as a model of human skin cancer. B16 is a refractory model for immunotherapy and is not responsive to anti-PD-1, anti-CTLA-4, or a combination of anti-PD-1 and anti-CTLA-4 antibody therapies.
To test the therapeutic efficacy of the immunomodulatory molecules described herein against more immunotherapeutic resistant cancer types, 0.25X10 were used 6 Individual B16 murine melanoma cells were inoculated into mice (-20 g body weight). When the tumor size reaches about 50-100mm 3 In the time-course of which the first and second contact surfaces,mice were injected with 200 μg (10 mg/kg) of IL-12 (F60A)/PD-L2-Fc immunoregulatory molecule (constructed in example 2; construct IW- #30; see FIG. 1G structure), 200 μg (10 mg/kg) of PD-L2-Fc/IL-12 (F60A) immunoregulatory molecule (constructed in example 2; construct IW- # 34), or PBS (negative control). A total of three injections (10 mg/kg per injection) were given on days 10, 13 and 16 post-inoculation (indicated by the black arrows in fig. 8A-8C). Tumor size was recorded over time. Once the tumor size exceeds 1000mm 3 Mice were sacrificed. The tumor sizes in brackets in fig. 8A represent the average tumor size (±standard deviation) for each group when the first treatment was administered.
As shown in fig. 8A-8C, PD-L2-Fc/IL-12 (F60A) immunoregulatory molecule (IW- # 34) and IL-12 (F60A)/PD-L2-Fc immunoregulatory molecule (IW- # 30) significantly inhibited B16 tumor growth until day 24 post-inoculation compared to PBS-treated group with abrupt growth of B16 tumor at day 13 post-inoculation, indicating that both IL-12 immunoregulatory molecules may slow tumor progression and/or extend life in immunotherapy-resistant cancer (e.g., melanoma) individuals, indicating promising in vivo efficacy.
Example 9 in vivo efficacy of IL-12/PD-L2-Fc and PD-L2-Fc/IL-12 immunomodulatory molecules showed that they significantly inhibited tumor growth in established LL2 syngeneic tumor models
The LL2 murine lung cancer model is a refractory model to immunotherapy that is non-responsive to anti-PD-1, anti-CTLA-4, or a combination of anti-PD-1 and anti-CTLA-4 antibody therapies.
To test the therapeutic efficacy of the immunomodulatory molecules described herein against more immunotherapeutic resistant cancer types, 0.25X10 were used 6 Mice (. About.20 g body weight) were vaccinated with LL2 murine lung cancer cells. About 16 days after tumor inoculation, the tumor size was measured to be about 50-100mm 3 . The mean initial tumor volume plus or minus one standard deviation is given in brackets (fig. 9A). After measuring tumor size, mice were injected with 200 μg (10 mg/kg) of IL-12 (F60A)/PD-L2-Fc, hinge (constructed in example 2, IW- # 30) immunoregulatory molecule (see FIG. 1G structure), or 200 μg (10 mg/kg) of IL-12 (F60A)/PD-L2-Fc, C-terminal of HC (constructed in example 2, IW- # 34) immunoregulatory molecule (see FIG. 1I structure). PBS injection as negative And (3) controlling. A total of three injections (10 mg/kg per injection) were given on days 13, 16 and 20 post-inoculation (indicated by black arrows). Tumor size was measured every 4 days from the first injection. Once the tumor size exceeds 1000mm 3 Mice were sacrificed.
As shown in fig. 9A-9C, PD-L2-Fc/IL-12 (F60A) and IL-12 (F60A)/PD-L2-Fc immunomodulatory molecule (#iw-34) significantly inhibited LL2 tumor growth until day 32-35 post-inoculation compared to PBS-treated group with significant growth of LL2 tumor about 20 days post-inoculation, indicating that both IL-12 immunomodulatory molecules may slow tumor progression and/or extend life in immunotherapy-resistant cancer (e.g., lung cancer) individuals, exhibiting promising in vivo efficacy, particularly considering their lack of response to anti-PD-1, anti-CTLA-4, or a combination of anti-PD-1 and anti-CTLA-4 antibody therapies.
In summary, the data described herein (e.g., see examples 7, 8, and 11) demonstrate the promising in vivo efficacy of the immunomodulatory molecules described herein (e.g., IL-12/PD-L2-Fc based immunomodulatory molecules) in: treating a variety of advanced and/or refractory cancer types (e.g., TNBC, melanoma, lung cancer), inhibiting cancer metastasis, treating or delaying tumor progression of cancer types that are resistant to current immunotherapy (e.g., anti-PD-1 therapy, anti-CTLA-4 therapy, or a combination thereof), and/or extending the life of such patients.
Example 10 replacement of the anti-PD-1 parent antibody with PD-L2-hinge-Fc fusion protein significantly reduces toxicity of IL-12 immunomodulatory molecules
40 BALB/c mice were randomly divided into 16 groups (5 mice per group) and were intraperitoneally injected with 200. Mu.g or 1000. Mu.g of the following: i) IL-12 (F60A)/PD-L2-Fc immunoregulatory molecule (IW- # 30), ii) IL-12 (G64A)/PD-L2-Fc immunoregulatory molecule (constructed in example 2), iii) IL-12 (E59A/F60A)/PD-L2-Fc immunoregulatory molecule (IW- # 29), iv) PD-L2-Fc/IL-12 (F60A) immunocytokine (IL-12 (F60A) moiety (IW-34), v) PD-L2-Fc/IL-12 (E59A/F60A) immunocytokine (IL-12 (E59A/F60A) moiety) located in one Fc fragment C', vi) IL-12 (F60A)/anti-PD-1 immunoregulatory molecule (IW- # 46), vii) IL-12 (F60A)/anti-PD-1 immunoregulatory molecule (IW-3535), and viii) IL-12 (E59A/F60A)/anti-PD-1 immunoregulatory molecule (IW- #48 "), and (G64A)/anti-PD- # 47"). These were constructed in examples 1 and 2. Each group received intraperitoneal injections on days 1 and 5. Four parameters of mice were monitored daily: i) Fur wrinkling, ii) reduced activity, iii) morbidity, and iv) weight loss of greater than 10%.
As can be seen from Table 3, the IL-12/anti-PD-1 immunomodulator comprising the IL-12 (G64A) variant (IW- # 47) showed the highest toxicity, as indicated by the death of 4/5 mice in the low dose group and 5/5 mice in the high dose group. In contrast, treatment with IL-12/anti-PD-1 immunoregulatory molecules comprising IL-12 (F60A) variant (IW- # 46) induced only one death in the high dose group (1000. Mu.g) and no death in the low dose group (200. Mu.g); treatment with IL-12/anti-PD-1 immunomodulator molecules comprising IL-12 (E59A/F60A) variant (IW- # 48) did not induce death at either dose. IL-12/anti-PD-1 immunoregulatory molecules comprising IL-12 double mutation E59A/F60A (IW- # 48) also demonstrated less toxicity as compared to IL-12/anti-PD-1 immunoregulatory molecules comprising IL-12 single F60A mutation (IW- # 46), as indicated by the difference in severity of toxic symptoms.
Among the immunoregulatory molecules having IL-12 variants located in the hinge region, IL-12/PD-L2-Fc immunoregulatory molecules comprising IL-12 (G64A) variants showed the highest toxicity, as indicated by the death of 3/5 mice in the low dose group and 5/5 mice in the high dose group. This is consistent with the highest toxicity results for IL-12 (G64A) in all IL-12 variants in the IL-12/anti-PD-1 immunoregulatory molecule, as well as the IL-12 (G64A) bioactivity shown in example 1. When the IL-12 (F60A) variant was placed at the C-terminus of the PD-L2-hinge-Fc polypeptide (IW- # 34), 2 of 5 mice died in the high dose group. In contrast, when the IL-12 (F60A) variant is located in the hinge region of the PD-L2-hinge-Fc polypeptide (IW- # 30), all mice survive even when a high dose of the immunomodulatory molecule (1000. Mu.g) is administered.
IL-12/PD-L2-Fc immunoregulatory molecules comprising IL-12 double mutation E59A/F60A exhibit less toxicity compared to IL-12/PD-L2-Fc immunoregulatory molecules comprising IL-12 single F60A mutation, whether the IL-12 (E59A/F60A) variant is located at the hinge region or C-terminus of the Fc, as indicated by the difference in severity of toxic symptoms. Dose-dependent toxicity was observed for most immunoregulatory molecules, such as when the dose was increased from 200 μg to 1000 μg, the severity of toxic symptoms increased, such as poor cortex, weight loss and/or more reduced activity. IL-12 (E59A/F60A)/PD-L2-Fc immunoregulatory molecules comprising the IL-12 variant located in the hinge region (IW- # 29) exhibit minimal in vivo toxicity, with 0 mortality and no toxic symptoms even when administered at high doses, among virtually all IL-12/PD-L2-Fc, IL-12/anti-PD-1 and PD-L2-Fc/IL-12 immunoregulatory molecules.
As can be seen from table 3, our results indicate that replacement of the anti-PD-1 antigen-binding fragment with a PD-L2 ligand in an IL-12 based immunomodulatory molecule can further reduce overall toxicity. For example, the 0 mortality of the high dose group IL-12 (F60A)/PD-L2-Fc (IW- # 30) immunomodulatory molecule is compared to the 1/5 mortality of the high dose group IL-12 (F60A)/anti-PD-1 (IW- # 46) immunomodulatory molecule; the 3/5 mortality of the low dose group IL-12 (G64A)/PD-L2-Fc immunoregulatory molecule was compared to the 4/5 mortality of the low dose group IL-12 (G64A)/anti-PD-1 immunoregulatory molecule (IW- # 47). The lower toxicity of PD-L2-Fc based immunomodulatory molecules is even more pronounced when comparing the symptoms of toxicity between individual IL-12 variant immunomodulatory molecules. For example, in comparison to an IL-12 (E59A/F60A)/anti-PD-1 (IW- # 48) immunoregulatory molecule, the IL-12 (E59A/F60A)/PD-L2-Fc (IW- # 29) immunoregulatory molecule completely eliminates toxic symptoms, whether administered at low or high doses; IL-12 (F60A)/PD-L2-Fc (IW- # 30) immunoregulatory molecules showed fewer toxic symptoms (coat wrinkling alone) than IL-12 (F60A)/anti-PD-1 (IW- # 46) immunoregulatory molecules, either in the low dose or high dose groups. The reduced toxicity observed in PD-L2-Fc based IL-12 immunomodulatory molecules may be due to stimulated PD-1 inhibitory immune checkpoint signaling following PD-L2/PD-1 binding, which produces an immunosuppressive signal that "balances" the immunostimulatory/pro-inflammatory activity of IL-12. In contrast, anti-PD-1 antibody (non-agonist antibody) -based IL-12 immunoregulatory molecules lack such immunosuppressive signals because they bind only to PD-1 and are not agonists.
TABLE 3 in vivo toxicity of IL-12/PD-L2-Fc immunoregulatory molecules
Example 11 in vivo efficacy of IL-12-based immunomodulatory molecules in 4T1 Triple Negative Breast Cancer (TNBC) in situ tumor mouse models
4T1 is a standard murine breast tumor model for preclinical studies of breast cancer metastasis. 4T1 is a refractory model for immunotherapy and is not responsive to anti-PD-1, anti-CTLA-4, or a combination of anti-PD-1 and anti-CTLA-4 antibody therapies. Breast fat pad injection of 4T1 can reproducibly generate 4T1 breast cancer-derived lung metastases.
To test the therapeutic efficacy of the immunomodulatory molecules described herein for the type of cancer resistant to immunotherapy and cancer metastasis, 0.25X10 s in #3 mammary fat pad was used 6 The 4T1 mice were vaccinated with breast cancer cells (-20 g body weight). Tumor progression was monitored for about 21-30 days. Mice were injected 4 days after tumor inoculation with 20mg/kg (per injection) of IL-12 (E59A/F60A)/PD-L2-Fc immunoregulatory molecule (constructed in example 2; construct IW- # 29), 20mg/kg (per injection) of IL-12 (F60A)/PD-L2-Fc immunoregulatory molecule (constructed in example 2; construct IW- # 30), 10mg/kg of anti-PD-1 antibody in combination with 10mg/kg of anti-CTLA-4 antibody (per injection), or PBS (negative control). A total of five injections were administered every four days. Four weeks later mice were sacrificed and primary tumors were extracted from mammary fat pads.
As shown in FIG. 16, IL-12 (F60A)/PD-L2-Fc immunoregulatory molecule (IW- # 30) inhibited 4T1 growth in the mammary glands of all mice tested, IL-12 (E59A/F60A)/PD-L2-Fc immunoregulatory molecule (IW- # 29) inhibited 4T1 growth in the mammary glands in 1 out of 3 mice tested, and anti-PD-1+ anti-CTLA-4 combination therapy failed to inhibit 4T1 growth in the mammary glands in all 3 mice tested, as compared to PBS control. As noted above, the difference in efficacy may be due to the lower potency (e.g., receptor binding and/or signaling ability) of double mutant IL-12 (E59A/F60A) compared to single mutant IL-12 (F60A). Such differences in efficacy can be compensated for by injecting a higher dose (e.g., 40mg/kg versus 20 mg/kg) or more injections (e.g., increasing from 5 injections to 7 injections) of IL-12 (E59A/F60A) -based immunomodulatory molecules.
To investigate the efficacy of treatment for cancer metastasis, lungs were removed from sacrificed mice. Lung tissue was resuspended in collagenase/dnase solution and filtered through a 70 μm cell filter. Cells were washed with PBS and resuspended in medium. Four 1:10 serial dilutions were performed. Cells were incubated at 7% CO 2 The cells were cultured in an incubator at 37℃for 14 days to form 4T1 cell colonies.
As shown in FIG. 17, both IL-12 (E59A/F60A)/PD-L2-Fc immunoregulatory molecule (IW- # 29) and IL-12 (F60A)/PD-L2-Fc immunoregulatory molecule (IW- # 30) significantly inhibited 4T1 metastasis in the lung compared to the combination of anti-PD-1 and anti-CTLA-4 antibodies or PBS (negative control). These findings were statistically significantly different (p-value < 0.001).
These data demonstrate the promising in vivo efficacy of IL-12 immunomodulatory molecules in: treatment of advanced and/or refractory breast cancers (e.g., TNBC), inhibition of cancer metastasis, and possibly treatment of other cancer types that are resistant to current immunotherapy.
Example 12 immunomodulatory molecules with cytokine in hinge region first facilitate target antigen-antibody (or ligand-receptor) binding followed by cytokine-cytokine receptor binding
IL-2 variants and construction of immunomodulatory molecules
IL-2/anti-PD-1 immunoregulatory molecules ("Fab-IL-2 mutant-Fc-PD-1 antibodies") were constructed similarly to example 1. Will contain sodium Wu Liyou mab (oldiewok,) Anti-human PD-1 antibodies of the VH (SEQ ID NO: 48) and VL (SEQ ID NO: 49) sequences were used as parent full-length antibodies. IL-2 variants comprising the triple mutation of R38D/K43E/E61R (SEQ ID NO: 26) were located within the hinge region of the heavy chain of an anti-PD-1 antibody (see FIG. 1C for an exemplary structure, anti-PD-1 is an antagonist antibody). Fab-IL-2 mutant Fc-PD-1 antibody immunomodulator molecules (or's') " IL-2 (R38D/K43E/E61R)/anti-PD-1 immunoregulatory molecule ") is composed of two light chains and two heavy chains, wherein each light chain comprises the amino acid sequence of SEQ ID NO. 50, one heavy chain comprises the amino acid sequence of SEQ ID NO. 51, and the other heavy chain has the IL-2 variant (SEQ ID NO. 26) located in the hinge region and comprises the amino acid sequence of SEQ ID NO. 144.
IL-2/PD-L2-Fc immunoregulatory molecule "ligand-IL-2 mutant-PD-L2-Fc" or "IL-2 (R38D/K43E/E61R)/PD-L2-Fc immunoregulatory molecule" ("IW- #11" or "construct # 11") was constructed similarly to example 2. It comprises a fusion polypeptide (SEQ ID NO: 24) and a pair of partner polypeptides (SEQ ID NO: 113), wherein the fusion polypeptide comprises, from N-terminus to C-terminus: PD-L2 ectodomain-GGGGS linker (SEQ ID NO: 213) -IL-2 variant (SEQ ID NO: 26) -N' -truncated IgG1 hinge (SEQ ID NO: 88) -Fc fragment (SEQ ID NO: 97), and the pairing polypeptide comprises from N-terminus to C-terminus: PD-L2 ectodomain-GGGGS linker (SEQ ID NO: 213) -N' -truncated IgG1 hinge (SEQ ID NO: 87) -paired Fc fragment (SEQ ID NO: 98).
Fab-IL-2 mutant-Fc-PD-1 antibodies and ligand-IL-2 mutant-PD-L2-Fc were constructed, expressed and purified as described in example 1. FACS was used to confirm that Fab-IL-2 mutant-Fc-PD-1 antibodies and ligand-IL-2 mutant PD-L2-Fc bind to HEK-PD-1-IL-2 cells (see below), but to HEK-Blue TM IL-2 cells (InvivoGen accession number hkb-IL 2) did not bind.
HEK-BLUE TM IL-2 cells and HEK-PD-1-IL-2 cells were used to evaluate the IL-2 signaling activity of Fab-IL-2 mutant-Fc-PD-1 antibodies and ligand-IL-2 mutant-PD-L2-Fc, see below. Recombinant human IL-2 (rIL-2) was used as a positive control. anti-PD-1 antibody na Wu Liyou mab (addiowa,) And the parent PD-L2-Fc fusion protein (each polypeptide chain comprising SEQ ID NO: 111) were used as negative controls.
IL-2 signaling assay
HEK-BLUE was used according to InvivoGen user Manual (InvivoGen accession number hkb-il 2) TM IL-2 cells (InvivoGen accession number hkb-IL 2) and HEK-PD-1-IL-2 cells (using lentiviral vectors, by growth on HEK-Blue TM Overexpression of human PD-1 in IL-2 cells is produced internally), and the IL-2 signaling activity of a variety of IL-2-based immunomodulatory molecules, also referred to below as "HEK-IL-2 reporter assays" or "HEK-PD-1-IL-2 reporter assays", is assessed. HEK-Blue TM IL-2 reporter cells and HEK-PD-1-IL-2 reporter cells stably express human IL-2 receptors (human IL-2Rα, IL-2Rβ, and IL-2Rγ), as well as human JAK3 and STAT5 genes, to obtain a fully functional IL-2 signaling pathway. In addition, these reporter cells also carry STAT 5-inducible Secreted Embryonic Alkaline Phosphatase (SEAP) reporter genes. HEK-Blue under IL-2 stimulation TM IL-2 reporter cells and HEK-PD-1-IL-2 reporter cells trigger STAT5 activation and subsequent SEAP secretion at levels that may be achieved using QUANTI-Blue TM (InvivoGen cat. Rep-qbs) colorimetric enzyme assay monitoring alkaline phosphatase activity.
Briefly, HEK-Blue TM IL-2 cells were added to a variety of IL-2-based immunomodulatory molecules in each plate well (or recombinant human IL-2 was added to positive control wells, anti-PD-1 antibody sodium Wu Liyou mab (European,) CO) at 37 °c 2 Incubate in incubator for 20-24 hours or overnight. After incubation, the supernatant was transferred to fresh plate wells and QUANTI-Blue was added TM The solution was incubated in an incubator at 37℃for 30 minutes to 3 hours. SEAP levels were then measured using a spectrophotometer at 620-655 nm. Recombinant human IL-2 (positive control) was tested for 10 units/ng of activity in activating the IL-2 signaling pathway and was used as a reference. The IL-2 signaling percentage of various IL-2-based immunomodulatory molecules was calculated by dividing the IL-2-based immunomodulatory molecule reading by the recombinant human IL-2 reading.
TABLE 4 biological Activity of Fab-IL-2 mutant-Fc-PD-1 antibodies and ligand-IL-2 mutant-PD-L2-Fc
In agreement with the data shown above, IL-2 located at the hinge region of the immunoregulatory molecule showed little biological activity (2.1% or 2.4%) compared to rIL-2 in the free state (100.0%) in the absence of target antigen (PD-1) binding, as measured by the HEK-IL-2 reporter assay. Comparing the results of the HEK-IL-2 reporter assay and the HEK-PD-1-IL-2 reporter assay in Table 4, the binding of the anti-PD-1 antigen binding fragment or PD-L2 extracellular domain to PD-1 on the cell surface greatly facilitated the binding of the IL-2 variant to the IL-2 receptor. In other words, the immunoregulatory molecule advantageously binds to the target antigen-antibody (Fab-IL-2 mutant-Fc-PD-1 antibody, 35.2% versus 2.1%) or ligand-receptor (ligand-IL-2 mutant-PD-L2-Fc, 43.5% versus 2.4%) followed by cytokine-cytokine receptor binding.
By targeting the mutant IL-2 moiety to a trastuzumab-containing regionOne hinge region of the VH and VL anti-HER 2 full length antibodies or anti-CD 3 epsilon full length antibodies (prepared internally) similarly constructed IL-2/anti-HER 2 and IL-2/anti-CD 3 immunomodulatory molecules. anti-HER 2/IL-2 and anti-CD 3/IL-2 immune cell molecules were constructed by locating the same mutant IL-2 moiety at the C-terminus of one heavy chain of an anti-HER 2 full length antibody or an anti-CD 3 epsilon full length antibody. IL-2 mutant-Fc-HER 2 antibodies and IL-2 mutant-Fc-CD 3 antibodies were constructed by fusing the same IL-2 moiety to the N-terminus of one subunit of the Fc fragment of an anti-HER 2 antibody or an anti-CD 3 epsilon antibody.
Using IL-2 signaling assay (HEK-Blue TM IL-2 cells, which do not express CD3 or HER 2), or PBMC proliferation assays, the activity of the immunomodulatory molecules. The results (data not shown) show that cytokines (e.g., IL-2 variants) located at the hinge region of the heavy chain of a full-length antibody (e.g., anti-HER 2 or anti-CD 3 antibody) in the absence of binding of the antibody to a target antigen (e.g., HER2 or CD 3) are displayed compared to when such cytokines are located at the N-terminus of the subunits of the Fc fragment or the C-terminus of the heavy chain of the full-length antibody Showing more limited biological activity. IL-2/anti-CD 3 immunoregulatory molecules are capable of binding to T cells via CD3, revealing the biological activity of cytokines located in the hinge region of one heavy chain of an anti-CD 3 full-length antibody.
PBMC proliferation assay
The biological activity of IL-2 can also be tested by Peripheral Blood Mononuclear Cell (PBMC) proliferation/survival assays. IL-2 is essential for proliferation and survival of activated T cells. Human PBMC (80,000 cells/well) were stimulated with anti-CD 3 antibody (OKT 3, 0.5. Mu.g/mL) in the presence of increasing concentrations of recombinant human IL-2 ("rIL-2"; 0, 0.04, 0.2, 1.0 or 5.0 ng/mL). Based on the number of PBMC cells (< 80,000 cells/well) and viability after 6 days of culture, 1ng/mL of rIL-2 was determined to be the minimum concentration required for T cell proliferation. To determine the minimum concentration of IL-2-based immunomodulatory molecules required for T cell proliferation, PBMCs (80,000 cells/well) were stimulated with anti-CD 3 antibody (OKT 3,0.5 μg/mL) in the presence of increasing concentrations (0, 0.32, 1.6, 8, 40, 200 or 1000 ng/mL) of various forms of IL-2-based immunomodulatory molecules. Free rIL-2 was used as a positive control (0.2 or 1 ng/mL). The percent T cell proliferation of IL-2-based immunomodulatory molecules relative to rIL-2 was calculated by normalization to the corresponding molecular weights. For example, the molecular weights of IL-2/anti-HER 2 immunoregulatory molecule and rIL-2 are about 162kDa and 12kDa, respectively, so that about 13ng of IL-2/anti-HER 2 immunoregulatory molecule corresponds to about 1ng of rIL-2 for the same molar concentration of IL-2.
Example 13 production of IL-23/anti-PD-1 immunoregulatory molecule (Fab-IL-23-Fc-PD-1 Ab) with IL-23 biological Activity against PD-1 positive cells
IL-23 variants and construction of immunomodulatory molecules
IL-23 is a heterodimeric cytokine consisting of a p19 subunit and a p40 subunit. The p40 subunit is shared with IL-12. IL-23 variants were constructed by generating amino acid substitutions in the shared p40 subunit, similar to that described in example 1 (see Table 5). Single-chain IL-23 variants were prepared, which were: p40 variant subunits (SEQ ID NOS: 63-66 and 140) -linker (SEQ ID NO: 229) -p19 wild type subunit (SEQ ID NO: 73). Single-stranded "wild-type" IL-23 was also constructed as a control (SEQ ID NO: 74), which was: the p40 wild-type subunit-linker (SEQ ID NO: 229) -p19 wild-type subunit is referred to as "WT" in Table 5.
The construction of IL-23/anti-PD-1 immunoregulatory molecules ("Fab-IL-23 (mutant) -Fc-PD-1 antibodies" or "Fab-IL-23 (wt) -Fc-PD-1 antibodies") was similar to example 1. The inclusion of nal Wu Liyou mab (addiow,) Anti-human PD-1 antibodies of VH (SEQ ID NO: 48) and VL (SEQ ID NO: 49) sequences were used as parent full-length antibodies. Various single-chain IL-23 variants (or single-chain "wild-type" IL-23 controls) were positioned within the hinge region of the heavy chain of an anti-PD-1 antibody (see fig. 1C for an exemplary structure, anti-PD-1 is an antagonist antibody). For example, a Fab-IL-23 (E59A/F68A) -Fc-PD-1 antibody immunomodulator comprising a single chain IL-23 variant IL-12B (p 40E 59A/F60A) -linker-IL-23A (wt p 19) (SEQ ID NO: 75) at the hinge region comprises two light chains and two heavy chains, wherein each light chain comprises the amino acid sequence of SEQ ID NO: 50; one heavy chain comprises the amino acid sequence of SEQ ID NO. 51 and the other heavy chain has a single-chain IL-23 variant (SEQ ID NO. 75) located in the hinge region and comprises the amino acid sequence of SEQ ID NO. 145. Immunomodulatory molecules were constructed, expressed, and purified as described in example 1. The heavy chain comprising the amino acid sequence of SEQ ID NO. 51 may also be substituted with a heavy chain comprising the amino acid sequence of SEQ ID NO. 21.
IL-23 signaling assay
HEK-Blue was performed following InvivoGen user Manual (InvivoGen accession number hkb-il 23) TM IL-23 cells (InvivoGen accession number hkb-IL 23) and HEK-PD-1-IL-23 cells (using lentiviral vectors, by growth on HEK Blue TM Overexpression of human PD-1 in IL-23 cells prepared internally), for assessing IL-23 signaling activity of a variety of Fab-IL-23-Fc-PD-1 antibody immunomodulatory molecules including different IL-23 moieties, also referred to below as "HEK-IL-23 reporter assays" or "HEK-PD-1-IL-23 reporter assays". HEK-Blue TM IL-23 reporter cells and HEKPD-1-IL-23 reporter cells stably express the receptor complex consisting of IL-12Rβ1 and IL-23 receptor (IL-23R), as well as the human STAT3 gene, to obtain a fully functional IL-23 signaling pathway (TyK/JAK 2/STAT 3). In addition, these reporter cells also carry STAT 3-induced SEAP reporter genes. HEK-Blue after IL-2 stimulation TM IL-23 reporter cells and HEK-PD-1-IL-23 reporter cells trigger STAT3 activation and subsequent SEAP secretion at levels that may be achieved using QUANTI-Blue TM (InvivoGen accession number rep-qbs) colorimetric enzyme assay alkaline phosphatase activity. The experimental procedure was similar to the IL-2 signaling assay described in example 12. Recombinant human IL-23 (rIL-23) in the free state served as a reference for positive control and activity percentage calculation.
TABLE 5 IL-23 biological Activity of Fab-IL-23-Fc-PD-1 antibodies comprising different IL-23 moieties
rIL-23 “WT” G64A E59A F60A E59A/F60A
HEK-IL-23 cells 100.0％ 70.0％ 56.0％ 6.9％ 8.2％ 0.0％
HEK-PD-1-IL-23 cells 100.0％ 150.0％ 180.0％ 39.0％ 46.0％ 4.8％
As can be seen from Table 5, localization of IL-23 comprising the wild-type p40 subunit at the hinge region retains about 70.0% of IL-23 activity even in the absence of target antigen (PD-1) -antibody binding in HEK-IL-23 reporter cells. In the absence of PD-1/anti-PD-1 antibody binding, the E59A and F60A mutations in the p40 subunit significantly reduced IL-23 activity to about 6.9% or 8.2%, whereas IL-23 activity was rescued to about 39.0% or 46.0% in HEK-PD-1-IL-23 cells in the presence of PD-1/anti-PD-1 antibody binding. Fab-IL-23-Fc-PD-1 antibodies ("Fab-IL-23 (E59A/F40A) -Fc-PD-1 antibodies") comprising the E59A/F60A double mutation in the IL-23p40 subunit showed PD-1 positive cell-specific IL-23 bioactivity (4.8%) and no cross-reactivity (0.0%) with PD-1 negative cells. These data indicate that anti-PD-1 antibody-based immunomodulatory molecules were successfully generated that can specifically target cytokine (e.g., IL-23) biological activity against PD-1+ cells.
Use of a pharmaceutical composition comprising ibalizumabanti-CD 4 antibodies with VH and VL as parent antibodies and placing the IL-23 portion in one hinge region of the full length anti-CD 4 antibody similarly produced IL-23/anti-CD 4 immunomodulatory molecules. A variety of assays were performed using IFN-gamma release assays IL-23 bioactivity of Fab-IL-23-Fc-CD 4-antibody. rIL-23 served as a positive control and as a percentage of activity reference. Localization of IL-23 comprising the wild-type p40 subunit at the hinge region still retains about 21.0% of IL-23 activity, even in the absence of target antigen (CD 4) -antibody binding in cd8+ T cells; in the presence of CD 4/anti-CD 4 antibody binding, the activity was 34.0%. The E59A and F60A mutations in the p40 subunit significantly reduced IL-23 activity to about 2.0% or 1.5% in the absence of CD 4/anti-CD 4 antibody binding, and the IL-23 activity was rescued to about 24.0% or 29.0% in the presence of cd4+ T cell/anti-CD 4 antibody binding. Fab-IL-23-Fc-CD4 antibodies ("Fab-IL-23 (E59A/F40A) -Fc-CD 4-antibodies") comprising the E59A/F60A double mutation in the IL-23p40 subunit showed CD4+ T cell-specific IL-23 bioactivity (6.8%) and no cross-reactivity (0.0%) with CD8+ T cells. These demonstrate successful generation of anti-CD 4 antibody-based immunomodulatory molecules that can specifically target cytokine (e.g., IL-23) biological activity against CD4 positive cells.
Interferon-gamma release assay (IGRA) for measuring IL-12 or IL-23 bioactivity
IL-23 and IL-12 may stimulate IFN-gamma release from activated CD4+ or CD8+ T cells. The biological activity of IL-12 or IL-23 can be measured by the amount of IFN-gamma released from activated T cells. Binding of IL-12 to its receptor (a heterodimeric receptor consisting of IL-12R-. Beta.1 and IL-12R-. Beta.2 subunits) triggers a signaling pathway involving TyK (tyrosine kinase 2), JAK2 (Janus kinase 2) and STAT4 (Signal transduction and transcriptional activator 4), leading to IFN-gamma production. CD4+ T cells or CD8+ T cells were isolated from PBMC and stimulated with anti-CD 3 antibody (1. Mu.g/mL OKT 3) in the presence of recombinant human IL-2 (30 units/mL) for 5 days. After 5 days, activated CD4+ or CD8+ T cells (80,000 cells/well) were cultured in the presence of an increased concentration of recombinant human IL-12 (rIL-12) or recombinant human IL-23 (rIL-23) (0, 0.62, 1.25, 2.5, 5, 10 or 20 ng/mL). The following day, the amount of IFN-gamma released into the cell culture medium was measured by ELISA assay. The IL-12 or IL-23 biological activity percentage by dividing the IL-12-based immunoregulatory molecule or IL-23-based immunoregulatory molecule reading by the rIL-12 or rIL-23 reading.
The minimum concentration of rIL-12 required to stimulate IFN-gamma release from activated T cells was 2.5ng/ml, while the minimum concentration of rIL-23 required was 5ng/ml. To determine the minimum concentration of IL-12-based or IL-23-based immunomodulatory molecules at which a positive biological response is observed, activated cd4+ or cd8+ T cells (80,000 cells/well) are cultured overnight in the presence of an increased concentration of IL-12-based or IL-23-based immunomodulatory molecules (0, 0.32, 1.6, 8, 40, 200, or 1000 ng/mL). rIL-12 (2.5 ng/mL) or rIL-23 (5 ng/mL) served as positive controls. The percent biological activity of the IL-12-based immunoregulatory molecule or IL-23-based immunoregulatory molecule relative to the corresponding free state cytokine (rIL-12 or rIL-23) is calculated by normalization to the corresponding molecular weight. The molecular weights of the IL-12-based immunoregulatory molecule and rIL-12 are about 220kDa and about 70kDa, respectively. The molecular weights of the IL-23-based immunoregulatory molecule and rIL-23 are about 215kDa and about 65kDa, respectively. Thus, for the same molar concentration of IL-12 or IL-23, about 3ng of IL-12-based or IL-23-based immunoregulatory molecule corresponds to about 1ng of rIL-12 or rIL-23.
Example 14 production of IL-10/anti-PD-1 immunoregulatory molecule (Fab-IL-10-Fc-PD-1 Ab) with IL-10 biological Activity against PD-1 positive cells
IL-10 variants and construction of immunomodulatory molecules therefor
IL-10 is naturally expressed as a non-covalently linked homodimer. IL-10 variants were constructed by substituting amino acids at positions 24 to 32 with alanine or serine (see Table 6), and single chain IL-10 variants were prepared which were N-terminal to C-terminal: IL-10 variant subunit (SEQ ID NO: 53-58) -linker (SEQ ID NO: 227) -IL-10 variant subunit (SEQ ID NO: 53-58). Single-stranded "wild-type" IL-10 was also constructed as a control (SEQ ID NO: 59), which was: IL-10 wild-type subunit (SEQ ID NO: 52) -linker (SEQ ID NO: 227) -IL-10 wild-type subunit is referred to as "WT" in Table 6.
IL-10/anti-PD-1 immunoregulatory molecule ("Fab-IL-10 (mutant) -Fc-PD-1 antibody" or "was constructed analogously to example 1"Fab-IIL-10 (wt) -Fc-PD-1-antibody "). Will contain sodium Wu Liyou mab (oldiewok,) Anti-human PD-1 antibodies of the VH (SEQ ID NO: 48) and VL (SEQ ID NO: 49) sequences were used as parent full-length antibodies. A variety of single chain IL-10 variants (or single chain "wild-type" IL-10 controls) are located within the hinge region of the heavy chain of an anti-PD-1 antibody (see FIG. 1C for exemplary structures, anti-PD-1 is an antagonist antibody). For example, a Fab-IL-10 (R27A) -Fc-PD-1 antibody immunomodulatory molecule comprising a single chain IL-10 variant IL-10 (R27A) -linker-IL-10 (R27A) (SEQ ID NO: 60) at the hinge region comprises: two light chains and two heavy chains, wherein each light chain comprises the amino acid sequence of SEQ ID NO. 50, one heavy chain comprises the amino acid sequence of SEQ ID NO. 51 and the other heavy chain has a single chain IL-10 variant (SEQ ID NO. 60) located at the hinge region and comprises the amino acid sequence of SEQ ID NO. 146. The heavy chain comprising the amino acid sequence of SEQ ID NO. 51 may also be substituted with a heavy chain comprising the amino acid sequence of SEQ ID NO. 21. Immunomodulatory molecules were constructed, expressed, and purified as described in example 1.
IL-10 signaling assay
HEK-Blue was used according to InvivoGen user Manual (InvivoGen accession number hkb-110) TM IL-10 cells (InvivoGen accession number hkb-illO) and HEK-PD-1-IL-10 cells (by use of lentiviral vectors in HEK Blue TM Human PD-1 is overexpressed in IL-10 cells and is prepared internally), and IL-10 signaling activity of various Fab-IL-10-Fc-PD-1 Abs comprising different IL-10 moieties is assessed, also referred to hereinafter as "HEK-IL-10 reporter assay" or "HEK-PD-1-IL-10 reporter assay". HEK-Blue TM IL-10 reporter cells and HEK-PD-1-IL-10 reporter cells stably express the IL-10 receptor hIL-10Rα and hIL-10Rβ chains, human STAT3 and STAT 3-inducible SEAP. IL-10 and its use in HEK-Blue TM Binding of IL-10 cells or HEK-PD-1-IL-23 reporter cell surface receptors triggers JAK1/STAT3 signaling and subsequent SEAP production at levels in cell culture supernatants that may be used with QUANTLBue TM (InvivoGen accession number rep-qbs) monitoring. Experimental procedure and examples12 for IL-2 signaling assay. Recombinant human IL-10 (rIL-10) in the free state was used as a reference for positive control and percent activity calculation.
TABLE 6 IL-10 biological Activity of Fab-IL-10-Fc-PD-1Ab comprising different IL-10 moieties
rIL-10 “WT” R24A D25A/L26A
HEK-IL-10 cells 100.0％ 26.0％ 15.0％ 4.0％
HEK-PD-1-IL-10 cells 100.0％ 200.0％ 150.0％ 56.0％
R27A D28A/A29S F30A/S31A R32A
HEK-IL-10 cells <0.1％ 17.0％ 13.0％ 10.0％
HEK-PD-1-IL-10 cells 21.0％ 70.0％ 67.0％ 42.0％
As can be seen from Table 6, IL-10 comprising the wild-type IL-10 subunit retained about 26.0% of IL-10 activity at the hinge region even in the absence of target antigen (PD-1) -antibody binding in HEK-IL-10 cells. All tested IL-10 variants reduced IL-10 activity in the absence of PD-1/anti-PD-1 antibody binding compared to "wild-type" IL-10, and their IL-10 activity was rescued in the presence of PD-1/anti-PD-1 antibody binding in HEK-PD-1-IL-10 cells. Fab-IL-10-Fc-PD-1Ab containing the R27A mutation in IL-10 ("Fab-IL-10 (R27A) -Fc-PD-1 Ab") showed IL-10 bioactivity specific for PD-1 positive cells (21.0%) with minimal cross-reactivity (< 0.1%) with PD-1 negative cells. These data indicate successful generation of anti-PD-1 antibody-based immunomodulatory molecules that can specifically target cytokine (e.g., IL-10) bioactivity to PD-1 positive cells.
Example 15 production of IFN- γ/anti-PD-1 immunoregulatory molecule (Fab-IFN- γ -Fc-PD-1 Ab) with IFN- γ biological Activity against PD-1 positive cells
IFN-gamma variants and construction of immunoregulatory molecules therefor
IFN-gamma is naturally expressed as a symmetrical homodimer. Construction of IFN-gamma variants by substitution of amino acids at positions 20 to 25 with A, K, S, E, Q or V (see Table 7), and preparation of single-chain IFN-gamma variants which are N-terminal to C-terminal: IFN-gamma variant subunit (SEQ ID NO: 39-45) -linker (SEQ ID NO: 227) -IFN-gamma variant subunit (SEQ ID NO: 39-45). A single-stranded "wild-type" IFN-gamma was also constructed as a control (SEQ ID NO: 46), which was: IFN-. Gamma.wild-type subunit (SEQ ID NO: 38) -linker (SEQ ID NO: 227) -IFN-. Gamma.wild-type subunit (SEQ ID NO: 38), referred to as "WT" in Table 7.
IFN- γ/anti-PD-1 immunoregulatory molecules ("Fab-FIFN- γ (mutant) -Fc-PD-1Ab" or "Fab-FN- γ (wt) -Fc-PD-1 Ab") were constructed similarly to example 1. Will contain sodium Wu Liyou mab (oldiewok,) Anti-human PD-1 antibodies of the VH (SEQ ID NO: 48) and VL (SEQ ID NO: 49) sequences were used as parent full-length antibodies. A number of single-chain IFN- γ variants (or single-chain "wild-type" IFN- γ controls) are located within the hinge region of the heavy chain of an anti-PD-1 antibody (see fig. 1C for exemplary structures, anti-PD-1 is an antagonist antibody). For example, a Fab-IFN-gamma (A23V) -Fc-PD-1 antibody immunomodulator ("IFN-gamma (A23V)/anti-PD-1") comprising a single-chain IFN-gamma variant IFN-gamma (A23V) -linker-IFN-gamma (A23V) (SEQ ID NO: 47) located at the hinge region comprises two light chains and two heavy chains, wherein each light chain comprises the amino acid sequence of SEQ ID NO:50, one heavy chain comprises the amino acid sequence of SEQ ID NO:51, and one heavy chain has a single-chain IFN-gamma variant (SEQ ID NO: 47) located at the hinge region and comprises the amino acid sequence of SEQ ID NO: 147. The heavy chain comprising the amino acid sequence of SEQ ID NO. 51 may also be substituted with a heavy chain comprising the amino acid sequence of SEQ ID NO. 21. Single chain homodimeric IFN-. Gamma.variants (A23V/A23V) may comprise the sequence of SEQ ID NO. 47 or 252. Immunomodulatory molecules were constructed, expressed, and purified as described in example 1.
IFN-gamma signaling assays
HEK-Blue was used according to InvivoGen user Manual (InvivoGen accession number hkb-ifng) TM IFN-gamma cells (InvivoGen accession number hkb-ifng) and HEK-PD-1-IFN-gamma cells (byUse of lentiviral vectors in HEK Blue TM Human PD-1 is overexpressed in IL-10 cells and is prepared internally), and various Fab-IFN- γ -Fc-PD-1 antibodies comprising different IFN- γ moieties are evaluated for IFN- γ signaling activity, hereinafter also referred to as "HEK-IFN- γ reporter assay" or "HEK-PD-1-IFN- γ reporter assay". HEK-Blue TM IFN-gamma reporter cells and HEK-PD-1-IFN-gamma reporter cells stably express the human STAT1 gene and STAT 1-inducible SEAP. Other genes of this pathway are naturally expressed in the reporter cell in sufficient amounts. IFN-gamma and its use in HEK-Blue TM Binding of heterodimeric receptor consisting of IFNGR1 and IFNGR2 chains on the surface of IFN-gamma cells or HEK-PD-1-IFN-gamma reporter cells triggers JAK1/JAK2/STAT1 signaling and subsequent SEAP production at levels in cell culture supernatants that may use QUANTI-Blue TM (InvivoGen accession number rep-qbs) monitoring. The experimental procedure was similar to that described in example 12 for the IL-2 signaling assay. Recombinant human IFN-gamma (rIFN-gamma) in the free state was used as a positive control and reference for the calculated percentage of activity.
TABLE 7 IFN-gamma biological Activity of Fab-IFN-gamma-Fc-PD-1 antibodies comprising different IFN-gamma moieties
As can be seen from Table 7, locating IFN-gamma comprising the wild-type IFN-gamma subunit at the hinge region maintained about 36.0% IFN-gamma activity even in the absence of target antigen (PD-1) -antibody binding in HEK-IFN-gamma cells. The a23 residues show crucial for the biological activity of IFN- γ, since all IFN- γ variants comprising the a23 mutation reduce IFN- γ activity in the absence of PD-1/anti-PD-1 antibody binding and their IFN- γ activity is rescued in the presence of PD-1/anti-PD-1 antibody binding in HEK-PD-1-IFN- γ cells compared to "wild-type" IFN- γ. Fab-IFN-gamma-Fc-PD-1 Ab containing the A23V mutation in IFN-gamma ("Fab-IFN-gamma (A23V) -Fc-PD-1 Ab") showed specific IFN-gamma bioactivity (27.0%) for PD-1 positive cells and minimal cross-reactivity (0.6%) with PD-1 negative cells. Fab IFN-gamma-Fc-PD-1 Ab containing the A23E/D24E/N25K triple mutation in IFN-gamma ("Fab-IFN-gamma (A23E/D24E/N25K) -Fc-PD-1 Ab") showed IFN-gamma bioactivity specific for PD-1 positive cells (23.0%) and minimal cross-reactivity (0.2%) with PD-1 negative cells. These data indicate successful generation of anti-PD-1 antibody-based immunomodulatory molecules that can specifically target cytokine (e.g., IFN- γ) biological activity to PD-1 positive cells.
IFN-gamma/anti-CD 4 immunomodulatory molecules were similarly generated and displayed IFN-gamma activity similar to IFN-gamma/anti-PD-1 immunomodulatory molecules (data now shown). IFN-gamma can induce PD-L1 expression on the cell surface. In comparison to "WT" IFN- γ, all IFN- γ variants containing the a23 mutation greatly reduced IFN- γ activity and approached baseline levels in the absence of CD 4/anti-CD 4 antibody binding, and their IFN- γ activity was recovered in the presence of CD 4/anti-CD 4 antibody binding. Fab ifnγ -Fc-CD4 antibodies comprising the a23E/D24E/N25K triple mutation in IFN- γ ("Fab IFN- γ (a 23E/D224E/N25K) -Fc-CD4 antibody") or the a23V mutation ("Fab IFN- γ (a 23V) -Fc-CD4 antibody") show cd4+ cell specific IFN- γ biological activity with little or no cross-reactivity with CD4 negative cells. These demonstrate successful generation of anti-CD 4 antibody-based immunomodulatory molecules that can specifically target cytokine (e.g., IFN- γ) bioactivity of CD4 positive cells.
Example 16 production of IFN-. Alpha.2b/anti-PD-1 immunoregulatory molecules (Fab-IFN-. Alpha.2b-Fc-PD-1 Ab) with IFN-. Alpha.2b biological Activity against PD-1 positive cells
IFN-alpha 2b variants and construction of immunoregulatory molecules therefor
IFN-α2bIs an antiviral or antineoplastic drug. It is a recombinant form of IFN- α2. IFN-. Alpha.2b variants were constructed by substituting alanine for the amino acids at positions 30 and 32-34 (SEQ ID NOs: 32, 34, 35 and 36; see Table 8).
IFN-. Alpha.2b/anti-PD-1 immunoregulatory molecules ("Fab ]IFN-. Alpha.2b (mutant) -Fc-PD-1 antibody "or" Fab-IIFN-. Alpha.2b (wt) -Fc-PD-1 antibody "). Will contain sodium Wu Liyou mab (oldiewok,) Anti-human PD-1 antibodies of the VH (SEQ ID NO: 48) and VL (SEQ ID NO: 49) sequences were used as parent full-length antibodies. A number of IFN-. Alpha.2b variants (or wild-type IFN-. Alpha.2b control "WT") are located within the hinge region of the heavy chain of an anti-PD-1 antibody (see FIG. 1C for exemplary structure, anti-PD-1 is an antagonist antibody). For example, a Fab-IFN-. Alpha.2b (L30A) -Fc-PD-1 antibody immunomodulator ("IFN-. Alpha.2b (L30A)/anti-PD-1") comprises two light chains and two heavy chains, wherein each light chain comprises the amino acid sequence of SEQ ID NO:50, one heavy chain comprises the amino acid sequence of SEQ ID NO:51, and one heavy chain has an IFN-. Alpha.2b (L30A) variant (SEQ ID NO: 32) at the hinge region and comprises the amino acid sequence of SEQ ID NO: 148. The heavy chain comprising the amino acid sequence of SEQ ID NO. 51 may also be replaced by a heavy chain comprising the amino acid sequence of SEQ ID NO. 21 or comprising a different linker (e.g.GSGGGGG; SEQ ID NO. 206) in the hinge region. Immunomodulatory molecules were constructed, expressed, and purified as described in example 1.
IFN-alpha/beta signaling assays
HEK-Blue was used according to InvivoGen user Manual (InvivoGen accession number hkb-ifnab) TM IFN-alpha/beta cells (InvivoGen accession number hkb-ifnab) and HEK-PD-1-IFN-alpha/beta cells (in HEK-Blue by use of lentiviral vectors) TM Overexpression of human PD-1 in IFN- α/β cells but prepared internally), various Fab-IFN- α2b-Fc-PD-1 abs comprising different IFN- α2b moieties were evaluated for IFN- α2b signaling activity, hereinafter also referred to as "HEK-IFN- α/β reporter assay" or "HEK-PD-1-IFN- α/β reporter assay". HEK-Blue TM IFN-alpha/beta reporter cells and HEK-PD-1-IFN-alpha/beta reporter cells are generated by stable transfection of HEK293 cells with human STAT2 and IRF9 genes to obtain fully active type I IFN signaling pathways and inducible SEAP under the control of the IFN-alpha/beta inducible ISG54 promoter. Other genes of this pathway (IFNAR 1, IFNAR2, JAK1, tyK and STAT 1) are naturally expressed by these cells. IFN-alpha or IFN-betaIts binding to heterodimeric receptor consisting of IFNAR1 and IFNAR2 chains triggers JAK/STAT/ISGF3 signaling and subsequent SEAP production at levels in cell culture supernatants that can be achieved using QUANTI-Blue TM (InvivoGen accession number rep-qbs) monitoring. The experimental procedure was similar to that described in example 12 for the IL-2 signaling assay. IFN-. Alpha.2b in the free state served as a reference for positive control and calculation of percentage of activity.
TABLE 8 IFN-alpha 2b biological Activity of Fab-IFN-alpha 2b-Fc-PD-1Ab comprising different IFN-alpha 2b moieties
As can be seen from Table 8, locating wild-type IFN-. Alpha.2b at the hinge region of the anti-PD-1 antibody retains about 54.0% IFN-. Alpha.2b activity even in the absence of target antigen (PD-1) -antibody binding in HEK-IFN-. Alpha.p cells. The L30, D32, R33 and H34 residues all appear to be critical for IFN- α2b biological activity because all IFN- α2b variants greatly reduce IFN- α2b activity in the absence of PD-1/anti-PD-1 antibody binding compared to wild-type IFN- α2b, and their IFN- α2b activity is rescued in the presence of PD-1/anti-PD-1 antibody binding in HEK-PD-1-IFN- α/β cells. Fab-IFN- α2b-Fc-PD-1Ab containing the L30A mutation in IFN- α2b ("Fab-IFN- α2b (L30A) -Fc-PD-1 Ab") showed specific IFN- α2b bioactivity (110.0%) for PD-1 positive cells and greatly reduced cross-reactivity (15.0%) with PD-1 negative cells. Fab-IFN- α2b-Fc-PD-1Ab containing the R33A mutation in IFN- α2b ("Fab-IFN- α2b (R33A) -Fc-PD-1 Ab") showed specific IFN- α2b bioactivity (25.0%) for PD-1 positive cells, greatly reducing cross-reactivity (5.0%) with PD-1 negative cells. These data indicate successful generation of anti-PD-1 antibody-based immune cytokines, which can specifically target cytokine (e.g., IFN- α2b) biological activity to PD-1 positive cells, while reducing cytokine biological activity to PD-1 negative cells.
Example 17 locating IL-2 variants at the hinge region of IL-2/PD-L2-Fc immunoregulatory molecules significantly reduced toxicity in mice IL-2/PD-L2-Fc (hinge) and PD-L2-Fc/IL-2 (C-terminal) immunoregulatory molecules were constructed similarly to those in examples 2 and 12.
25 BALB/C mice were randomly divided into 5 groups (5 per group) and either 200 μg or 1000 μg of IL-2 (R38D/K43E/E61R)/PD-L2-Fc immunoregulatory molecule ("IW- #11" or "construct #11"; constructed in example 12), PD-L2-Fc/IL-2 (R38D/K43E/E61R) immunoregulatory molecule (IL-2 (R38D/K43E/E61R) portion fused to C' of one Fc fragment of the parent PD-L2-hinge-Fc fusion protein (SEQ ID NO: 26)), or the parent PD-L2-hinge-Fc fusion protein (two PD-L2-hinge-Fc polypeptides, each comprising SEQ ID NO: 111) was injected intraperitoneally as control. Each group received intraperitoneal injections on days 1 and 5. Mice were monitored daily for four parameters: i) Fur wrinkling, ii) reduced activity, iii) morbidity, and iv) weight loss of more than 10%.
As can be seen from table 9, when the IL-2 variant was located at the C-terminus of the PD-L2-hinge-Fc polypeptide, five of the five mice died 4-6 days after injection in the high dose group and the low dose group. In contrast, when the IL-2 variant is located in the hinge region, all mice survive even with high doses of immunomodulatory molecule (1000. Mu.g). The same survival was observed in the control group (PD-L2-Fc without IL-2 fusion). For immunomodulatory molecules with IL-2 variants located in the hinge region, toxicity appears to be dose dependent, as indicated by greater weight loss and activity loss when the dose is increased from 200 μg to 1000 μg.
TABLE 9 in vivo toxicity of different forms of IL-2/PD-L2-Fc immunocytokines
EXAMPLE 18 in vivo efficacy of IL-12 immunomodulatory molecules in 4T1 syngeneic tumor mouse models
Construction of IL-12 (E59A/F60A)/IL-2 (R38D/K43E/E61R)/anti-PD-1 immunoregulatory molecules
As described in example 1, the use of a containing sodium Wu LiEspecially monoclonal antibody (the one of the oldham,) As parent full-length antibody. The IL-2R38D/K43E/E61R variant (SEQ ID NO: 26) is located within the hinge region of one heavy chain of the heterodimeric anti-PD-1 antibody, and the single chain IL-12E59A/F60A variant (SEQ ID NO: 68) is located within the hinge region of the other heavy chain of the heterodimeric anti-PD-1 antibody to construct an "IL-12 (E59A/F60A)/IL-2 (R38D/K43E/E61R)/anti-PD-1 immunoregulatory molecule" ("IW- #54" or "construct # 54"). The linker within the single chain IL-12 (E59A/F60A) variant may also be changed to SEQ ID NO:246, and the (E59 AF 60A) variant of single chain IL-12 may comprise SEQ ID NO:254. The construct was expressed and purified as described in example 1.
Mice (20 g body weight) were vaccinated with 0.25x10 6 Breast cancer cells from 4T1 mice. 7 days after tumor inoculation, the tumor size was measured to be about 50-150mm 3 . After measuring tumor size, mice were injected with 10mg/kg (-200. Mu.g) IL-12 (E59A/F60A)/anti-PD-1 immunoregulatory molecule (constructed in example 1; IW- # 48), 10mg/kg (-200. Mu.g) IL-12 (E59A/F60A)/PD-L2-Fc immunoregulatory molecule (constructed in example 2; IW- # 29), 5mg/kg (-100. Mu.g) IL-12 (E59A/F60A)/IL-2 (R38D/K43E/E61R)/anti-PD-1 immunoregulatory molecule (IW- # 54), or PBS (negative control). Three injections (10 mg/kg or 5mg/kg each, respectively) were given in total on days 7, 13 and 19 after tumor inoculation (indicated by black arrows in fig. 18). Tumor size was measured every 3 days from the first injection. Once the tumor size reaches over 2000mm 3 Mice were sacrificed.
The 4T1 mouse model reflects a high degree of resistance to current immunotherapy, including anti-PD-1, anti-CTLA-4, and combination therapy of anti-PD-1 and anti-CTLA-4 antibodies. As can be seen from fig. 18, all three IL-12-based immunomodulatory molecules significantly inhibited 4T1 tumor growth, demonstrating promising in vivo efficacy.
In addition, IL-12 (E59A/F60A)/anti-PD-1 (IW- # 48) and IL-12 (E59A/F60A)/PD-L2-Fc (IW- # 29) showed similar cytotoxicity against 4T1 tumors when administered at the same dose (FIG. 18). In conjunction with the results of example 10, these data further demonstrate that the use of the PD-L2 ectodomain in an immunomodulatory molecule construct not only achieves a similar anti-tumor effect, but also reduces unwanted toxicity, as compared to an anti-PD-1 antigen binding domain (non-agonist antibody) that blocks (or does not induce) PD-1 immunosuppressive signaling, possibly balancing the immunostimulatory/pro-inflammatory activity of cytokines (e.g., IL-12) by immunosuppressive signaling from PD-L2/PD-1 signaling.
Example 19 in vivo efficacy of IL-12 immunomodulatory molecules in EMT6 syngeneic tumor mouse model
With 0.25X10 6 Individual EMT6 murine breast cancer cells were inoculated into mice (-20 g body weight). 7 days after tumor inoculation, the tumor size was measured to be about 50-150mm 3 . After measuring tumor size, mice were injected with 10mg/kg (-200. Mu.g) IL-12 (E59A/F60A)/anti-PD-1 immunoregulatory molecule (constructed in example 1; IW- # 48), 10mg/kg (-200. Mu.g) IL-12 (E59A/F60A)/PD-L2-Fc immunocytokine (constructed in example 2; IW- # 29), 10mg/kg (-200. Mu.g) IL-2 (R38D/K43E/E61R)/PD-L2-Fc immunocytokine (constructed in example 11; IW- # 11), or PBS (negative control). A total of 3 injections (10 mg/kg per injection) were made on days 7, 13 and 19 after tumor inoculation (indicated by black arrows in fig. 19). Tumor size was measured every 3 days from the first injection. Once the tumor size reaches over 2000mm 3 Mice were sacrificed.
EMT6 tumor growth is resistant to anti-PD-1 immunotherapy. As can be seen from FIG. 19, all immunoregulatory molecules significantly inhibited EMT6 tumor growth, with IL-12 (E59A/F60A)/PD-L2-Fc immunocytokines, where IL-12 (E59A/F60A)/anti-PD-L (IW- # 48) and IL-2 (R38D/K43E/E61R)/PD-L2-Fc (IW- # 11) immunoregulatory molecules exhibited better efficacy. The slightly lower efficacy of PD-L2-Fc based IL-12 immunomodulatory molecules may be due to stimulation of PD-1 inhibitory immune checkpoint signaling upon PD-L2-PD-1 binding, which results in an immunosuppressive signal that "balances" the immunostimulatory/pro-inflammatory activity of IL-12.
Example 20 location of cytokines or variants thereof in immunocytokines affects nonspecific Activity of immunocytokines
Two immunomodulatory molecule designs were generated to test whether placement of a cytokine or variant thereof in the hinge region (between the antigen binding domain and the Fc fragment; in hidden form) or in the C-terminus of the Fc fragment (e.g., C' of the antibody heavy chain; in exposed form) could affect the targeted activity of the cytokine or variant thereof. The first design incorporates cytokines in the hinge region of one heavy chain of an anti-PD-1 antibody (non-agonist): the hinge region between CH1 and CH2 (the immunoregulatory molecule is named "IL-12/anti-PD-1"). The second design fuses the cytokine to the C-terminus of the heavy chain of an anti-PD-1 antibody (non-agonist) (the immunoregulatory molecule is named in the form of "anti-PD-1/IL-12") via a linker, which is a common design in immunocytokines today.
IL-12 (E59A/F60A)/anti-PD-1 immunocytokine (IW- #48 or construct # 48), IL-12 (E59A)/anti-PD-1 immunocytokine (IW- #46 or construct # 46), and IL-12 (G64A)/anti-PD-1 immunocytokine (IW- #47 or construct # 47) were constructed as described in example 1 with an IL-12 variant (single chain from N-terminus to C-terminus: IL-12B (p 40 variant) -linker-IL-12A (wt p 35)) located within the hinge region of one heavy chain of a heterodimeric anti-PD-1 antibody (nal Wu Liyou mab).
To prepare heavy chain C 'cytokine fusion constructs, single chain IL-12 (E59A) variants (SEQ ID NO: 69), single chain IL-12 (G64A) variants (SEQ ID NO: 70), or single chain IL-12 (E59A/F60A) variants (SEQ ID NO: 68) were fused via a G/S containing peptide linker to the C' of a heavy chain of a heterodimeric anti-PD-1 antibody (nano Wu Liyou mab). These constructs are hereinafter referred to as anti-PD-1/IL-12 (E59A) (construct #46HC '), anti-PD-1/IL-12 (G64A) (construct #47HC '), and anti-PD-1/IL-12 (E59A/F60A) (construct #48HC '), respectively. The heavy chain non-fusion polypeptide of the heterodimeric anti-PD-1 antibody has the sequence of SEQ ID NO. 51. The linker within a single chain IL-12 variant (e.g., a single chain IL-12 (E59A/F60A) variant) may also be changed to SEQ ID NO:246, e.g., a single chain IL-12 (E59 AF 60A) variant may include SEQ ID NO:254.
HEK-Blue was used as described in example 1 using the two immunocytokine configurations described above and rIL-12 (positive control) TM IL-12 and HEK-PD-1-IL-12 (in HEK-Blue by use of lentiviral vectors) TM Overexpression of human PD-1 in IL-12 cells and preparation internally) cells, IL-12 signaling assays were similarly performed.
TABLE 10 biological Activity of IL-12 in different forms of IL-12 immunocytokines
In the HEK-IL-12 reporter assay, if the IL-12 moiety is available (e.g., heavy chain C' fusion form), both IL-12 immunoregulatory molecular forms can only bind to HEK-IL-12 cells via IL-12 moiety/IL-12 receptor interactions. In the HEK-PD-1-IL-12 reporter assay, both IL-12 immunoregulatory molecular formats are capable of binding to HEK-PD-1-IL-12 cells via IL-12 moiety/IL-12 receptor interactions and anti-PD-1 antigen binding fragment/PD-1 interactions.
As shown in table 10, the hinge fusion design has significantly reduced non-specific activity (i.e., cytokine activity in the absence of PD-1 binding) compared to the heavy chain C-terminal fusion design. Construct #48 showed almost no detectable IL-12 activity level (< 0.2%) in PD-1 negative cells (HEK-IL-12) compared to 4% of construct #48 HC'. Similar results were observed for construct #47 and construct #47HC' (5% versus 15%, respectively). IL-12 double mutant E59A/F60A also significantly reduced nonspecific activity compared to single mutant E59A or G64A. In PD-1 positive cells (HEK-PD-1-IL-12), IL-12 targeting activity is similar between the corresponding hinge fusion form and heavy chain C-terminal fusion form, suggesting that the hinge fusion design does not significantly inhibit IL-12 activity in the presence of antigen positive cells (or antigen binding). In summary, in the absence of binding of antigen binding domains, the hinge position of cytokines (especially certain cytokine variants) can greatly reduce nonspecific IL-12 activity.
Example 21 production of IL-12/anti-PD-1 immunoregulatory molecules with reduced affinity for PD-1
Generation of anti-PD-1 antibody variants with reduced PD-1 binding affinity
Because of the high binding affinity of nal Wu Liyou mab to PD-1, IL-12 activity can be directed to all PD-1 positive cells, regardless of PD-1 expression level, using wild-type nal Wu Liyou mab as an IL-12/anti-PD-1 immunomodulatory molecule of the parent antibody. Targeting such a large number of PD-1 positive cells may result in cytokine storms or other adverse side effects due to the partial activation of any PD-1 positive immune cells (e.g., T cells) by the immunostimulatory cytokine.
To generate anti-PD-1 mutants (non-agonist antibodies) with reduced binding affinity for PD-1, which target only highly expressing PD-1 cells (e.g., T cells), mutations were introduced into HC-CDR3 at the D100 or N99 positions of the nal Wu Liyou mab: HC-CDR3 (D100N), HC-CDR3 (D100G), HC-CDR3 (D100R), HC-CDR3 (N99G), HC-CDR3 (N99A) and HC-CDR3 (N99M). The affinity of these anti-PD-1 antibodies (non-agonist antibodies) was measured by Biacore and cell-based assays, calibrated by wild-type nalv Wu Liyou mab binding affinity (see table 11). "N/A" means that PD-1 binding is not detected.
To construct an anti-PD-1 heterodimer, one heavy chain comprises: a hinge region comprising SEQ ID NO. 78 and an Fc domain subunit comprising SEQ ID NO. 97; the other heavy chain comprises: comprising a hinge region comprising SEQ ID NO:77 and an Fc domain subunit comprising SEQ ID NO: 98. Each of the two light chains comprises the amino acid sequence of SEQ ID NO. 50.
TABLE 11 PD-1 binding affinities of various anti-PD-1 heavy chain mutants (non-agonists)
Heavy chain mutation Affinity for PD-1 (Kd)
WT 2.6nM
D100N 25nM
D100G 130nM
D100R 910nM
N99G 2300nM
N99A N/A
N99M N/A
Construction of IL-12/anti-PD-1 immunomodulatory molecules with reduced PD-1 affinity
A variety of IL-12/anti-PD-1 immunoregulatory molecules were generated by placing a single chain IL-12 (E59A/F60A) variant (SEQ ID NO:68 or 354) in the hinge region of one heavy chain of the various heterodimeric anti-PD-1 mutants (non-agonists) described above, as described in example 1. The sequences of the heavy chain cytokine fusion polypeptides for each construct are provided in table 12. Corresponding paired non-fusion heavy chains comprising, from N-terminus to C-terminus: VH (with corresponding HC-CDR3 mutation) -CH 1-hinge (SEQ ID NO: 77) -Fc domain subunit (SEQ ID NO: 98).
IL-12 signaling assay
UsingIL-12/anti-PD-1 (mutant) immunoregulatory molecules (IL-12 (E59A/F60A)/anti-PD-1 (wild-type) and rIL-12 as controls) with reduced PD-1 binding affinity as described in example 1, in HEK-Blue TM IL-12 signaling assays were performed similarly on IL-12 cells and HEK-PD-1-IL-12 cells. Two variants of HEK-PD-1-IL-12 cells were used: one with high PD-1 expression "HEK-IL-12-PD-1 (high)" (over-expressing PD-1 as described in example 1), and one with 30-fold lower PD-1 expression "HEK-IL-12-PD-1 (low)" (in HEK-Blue by using lentiviral vectors) TM Lower amounts of human PD-1 are expressed in IL-12 cells and are produced internally). Cells were incubated with 20ng/mL of various IL-12/anti-PD-1 immunoregulatory molecules (or controls) for 24 hours.
IFN-gamma release assay
IFN- γ release assays were performed similarly as described in example 13 using IL-12 (E59A/F60A)/anti-PD-1 (mutant) immunomodulatory molecules with reduced PD-1 binding affinity. IL-12 (E59A/F60A)/anti-PD-1 (wild-type) and rIL-12 were used as controls. Briefly, T cells were activated by incubating PBMCs with anti-CD 3 antibody (OKT 3, 100 ng/mL) for three days. PBMCs were washed to remove anti-CD 3 antibodies and incubated with 200ng/mL of various IL-12 (E59A/F60A)/anti-PD-1 (mutant) immunomodulatory molecules (or controls) for 24 hours. After one day, the amount of IFN-gamma released into the cell culture medium was measured.
TABLE 12 IL-12 biological Activity (reduced PD-1 binding affinity) of IL-12 (E59A/F60A)/anti-PD-1 immunomodulatory molecules comprising various anti-PD-1 heavy chain mutations
As can be seen from Table 12, no nonspecific IL-12 activity was observed in the absence of anti-PD-1 binding for all the immunomodulatory molecules tested (see HEK-IL-12 column). Their ability to transduce IL-12 signals in the presence of PD-1 binding and their ability to induce IFN- γ release decreases with decreasing anti-PD-1 binding affinity, indicating antigen binding dependent cytokine activity of the hinge fusion design. IL-12 (E59A/F60A)/anti-PD-1 immunoregulatory molecules having a D100G, D R or N99G mutation in the anti-PD-1 heavy chain showed significant differences in binding between cells with high and low PD-1 expression. These results indicate that cells expressing higher levels of PD-1 can be specifically targeted using IL-12 (E59A/F60A)/anti-PD-1 (mutant) immunomodulatory molecules with reduced affinity for PD-1. These constructs also induced much lower IFN-gamma secretion compared to IL-12/anti-PD-1 (wild-type) immunoregulatory molecules and rIL-12 controls.
Thus, the IL-12 (E59A/F60A)/anti-PD-1 (mutant) immunomodulatory molecules described herein, as well as other immunomodulatory molecules that may be constructed based on antigen binding domains with reduced antigen binding affinity, can be used to specifically target cells of interest with high antigen expression, with reduced off-target effects and/or cytokine storms.
Example 22 reduction of PD-1 binding affinity in IL-12/anti-PD-1 immunoregulatory molecules toxicity in mice
Humanized PD-1 mice derived from strain C57 (5-6 weeks old, 20g female) were injected (by inserting chimeric PD-1 having a human extracellular domain, a murine transmembrane domain and a murine intracellular domain into the mouse PD-1 locus) with 10mg/kg or 50mg/kg (per injection) of the various IL-12 (E59A/F60A)/anti-PD-1 (mutant) immunomodulatory molecules described in example 21. IL-12 (E59A/F60A)/anti-PD-1 (wild-type) immunoregulatory molecule (IW- #48 or construct # 48) was used as a control. Four injections were made on days 0, 4, 8 and 12 in total. Mice were monitored daily for mortality and four toxic symptoms: i) Fur wrinkling, ii) reduced activity, iii) morbidity, and iv) weight loss.
Mice injected with IL-12 (E59A/F60A)/anti-PD-1 (wild-type) immunoregulatory molecule (IW- # 48) comprising wild-type nals Wu Liyou exhibited the greatest toxicity, and all mice in this group died after receiving the second or third injection, even at lower doses. In contrast, mice injected with IL-12 (E59A/F60A)/anti-PD-1 (mutant) immunomodulatory molecules comprising anti-PD-1 having reduced PD-1 binding affinity exhibited reduced toxicity, with death only observed in the group treated with IL-12 (E59A/F60A)/anti-PD-1 (D100N). As can be seen from table 13, in the constructs, the severity of the toxic symptoms decreased with decreased PD-1 binding affinity and/or decreased dose.
TABLE 13 in vivo toxicity of IL-12/anti-PD-1 immunomodulating molecules
Due to the high binding affinity (K) of wild-type NA Wu Liyou mab (non-agonist) to PD-1 d ≈10 -8 -10 -9 M), IL-12 (E59/F60A)/anti-PD-1 (wild type) is most likely to bind to and stimulate (via cytokine activity) any PD-1 positive cells. This would involve activated T cells and NK cells, leading to cytokine release syndrome. In contrast, IL-12/anti-PD-1 based immunomodulatory molecules with reduced binding affinity for hPD-1 can bind only to smaller populations of PD-1 positive cells, particularly cells with very high PD-1 expression levels, such as depleted cell T cells. The data presented here are consistent with the data from the in vitro PBMC IFN-gamma release assay in example 21. These findings indicate that the PD-1 binding affinity of the anti-PD-1 antigen binding domain (non-agonist anti-PD-1) is reduced to K d About 10 -6 -10 -7 M (see, e.g., D100G, D100R, N G in the anti-PD-1 heavy chain) can greatly enhance the safety of IL-12/anti-PD-1 immunomodulatory molecules while retaining therapeutic efficacy.
Example 23 increasing the binding affinity of PD-L1 and PD-L2 does not increase the toxicity of IL-12/PD-L1-Fc and IL-12/PD-L2-Fc immunomodulatory molecules
As shown in examples 10, 18 and 19, replacement of the anti-PD-1 antigen-binding fragment (non-agonist antibody) with the PD-L2 extracellular domain in an IL-12-based immunomodulatory molecule may significantly reduce toxicity, possibly due to stimulated PD-1 inhibitory immune checkpoint signaling upon PD-L2-PD-1 binding, which produces an immunosuppressive signal that "balances" the immunostimulatory/pro-inflammatory activity of IL-12. To investigate whether the safety of these "balanced" constructs could be further improved, IL-12 immunomodulatory molecules comprising PD-L1 or PD-L2 extracellular domains and having increased PD-1 binding affinity were constructed to enhance PD-1 immunosuppressive signaling.
Generation of PD-L1 variants with increased PD-1 binding affinity
Wild type PD-L1 has a binding affinity for PD-1 of about 10 -5 -10 -6 M, binding affinity lower than that of the Na Wu Liyou mab (Kd. Apprxeq.10 -8 -10 -9 M). To increase affinity for PD-1, PD-L1 mutants were generated. Mutations were introduced into the extracellular domain of wild-type PD-L1, the amino acid position of which was related to SEQ ID NO. 120. These mutant PD-L1 ectodomains are then fused to an Fc fragment via a hinge region to construct the parent PD-L1-Fc construct. To construct a PD-L1-Fc heterodimer, one polypeptide chain comprises: a hinge region comprising SEQ ID NO. 88 and an Fc domain subunit comprising SEQ ID NO. 97; and the other polypeptide chain comprises: a hinge region comprising SEQ ID NO. 87 and an Fc domain subunit comprising SEQ ID NO. 98. The mutant constructs were named in the form of PD-L1 (mutant) -Fc.
A description of the mutations generated and the PD-1 binding affinity (measured as PD-L1-Fc form) is shown in table 14. The binding affinity of each PD-L1 (mutant) -Fc was calibrated based on PD-L1 (wild-type) -Fc binding affinity. N/A indicates that PD-1 binding cannot be detected. These results indicate that all PD-L1 (mutants) achieved about a 4-60 fold increase in PD-1 binding affinity over wild-type PD-L1. Wherein, compared to wild type PD-L1, PD-L1 (I54Q/E58M/R113T/M115L/S117A/G119K) (PD-L1 (mutant 2)), PD-L1 (I54Q/E58M/R113T/M115L/G119K) (PD-L1 (mutant 6)), and PD-L1 (I54Q/E58M/R113T/M115L/S117A) (PD-L1 (mutant 7)) show the highest fold increase in affinity for PD-1.
TABLE 14 PD-1 binding affinities of various PD-L1 mutants
Generation of PD-L2 variants with increased PD-1 binding affinity
PD-L2 has a binding affinity for PD-1 of about 10 -6 -10 -7 M, which is lower than the binding affinity of the nal Wu Liyou mab (Kd. Apprxeq.10 -8 -10 -9 M). To increase affinity for PD-1, PD-L2 mutants were generated. Mutations were introduced into the extracellular domain of wild-type PD-L2, the amino acid position of which correlates with SEQ ID NO. 105. These mutated PD-L2 extracellular domains are then fused to an Fc fragment via a hinge region to construct a parent PD-L2-Fc construct. To construct a PD-L2-Fc heterodimer, one polypeptide chain comprises: a hinge region comprising SEQ ID NO. 88 and an Fc domain subunit comprising SEQ ID NO. 97; and the other polypeptide chain comprises: a hinge region comprising SEQ ID NO. 87 and an Fc domain subunit comprising SEQ ID NO. 98. The mutant constructs were named in the form of PD-L2 (mutant) -Fc.
A description of the mutations generated and the PD-1 binding affinity (measured as PD-L2-Fc form) is shown in table 15. The binding affinity of each PD-L2 (mutant) -Fc was calibrated based on PD-L2 (wild-type) -Fc binding affinity. These results indicate that all PD-L2 (mutants) achieved about a 2-5 fold increase in PD-1 binding affinity over wild-type PD-L2. Wherein the affinity of PD-L2 (S58V) (PD-L2 (mutant 2)) and PD-L2 (T56V/S58V/Q60L) (PD-L2 (mutant 4)) for PD-1 is increased by the highest fold compared to wild-type PD-L2.
TABLE 15 PD-1 binding affinities of various PD-L2 mutants
IL-12/PD-L1-Fc and IL-12/PD-L2-Fc immunomodulatory molecules with increased affinity for PD-1 Construction
The heterodimeric PD-L1 (mutant) -Fc or PD-L2 (mutant) -Fc produced herein was used as a parent antigen binding protein to construct an IL-12 immunomodulatory molecule that binds PD-1, similar to that described in example 10. A single chain IL-12 (E59A/F60A) variant (e.g., SEQ ID NO:68 or 254) is located at the N' of the hinge of one polypeptide chain in the parent PD-L1 (mutant) -Fc or PD-L2 (mutant) -Fc heterodimer.
IL-12 (E59A/F60A)/PD-L2 (wt) -Fc immunoregulatory molecules were constructed using the wild-type PD-L2 ectodomain as in example 2 ("construct #29" or "IW- # 29"). IL-12 (E59A/F60A)/PD-L2 (mutant) -Fc immunocytokines comprise: an IL-12 fusion polypeptide (from N-terminus to C-terminus: PD-L2 (mutant) ectodomain-GGGGSGGG linker (SEQ ID NO: 244) -single chain IL-12 (E59A/F60A) variant (e.g., SEQ ID NO:68 or 254) -GGGGGGSGGG linker (SEQ ID NO: 244) -hinge (SEQ ID NO: 88) -Fc domain subunit (SEQ ID NO: 97)); and a partner polypeptide (PD-L2 (mutant) ectodomain-GGGGSGGG linker (SEQ ID NO: 244) -hinge (SEQ ID NO: 87) -Fc domain subunit (SEQ ID NO: 98)) from N-terminus to C-terminus. Exemplary IL-12 cytokine fusion chains of IL-12 (E59A/F60A)/PD-L2 (mutant) -Fc immunoregulatory molecules may include SEQ ID NO 167 or 168.
IL-12 (E59A/F60A)/PD-L1 (wild-type) -Fc immunoregulatory molecules comprise: an IL-12 fusion polypeptide (from N-terminus to C-terminus: PD-L1 (wild-type) ectodomain (SEQ ID NO: 121) -GGGGSGGG linker (SEQ ID NO: 244) -single chain IL-12 (E59A/F60A) variant-GGGGSGGG linker (SEQ ID NO: 244) -hinge (SEQ ID NO: 88) -Fc domain subunit (SEQ ID NO: 97)); and a partner polypeptide (from N-terminus to C-terminus: PD-L1 (wild-type) extracellular domain (SEQ ID NO: 121) -GGGGSGGG linker (SEQ ID NO: 244) -hinge (SEQ ID NO: 87) -Fc domain subunit (SEQ ID NO: 98)). IL-12 (E59A/F60A)/PD-L1 (mutant) -Fc immunoregulatory molecules comprise: an IL-12 fusion polypeptide (from N-terminus to C-terminus: PD-L1 (mutant) ectodomain (e.g., SEQ ID NO: 129) -GGGGSGGG linker (SEQ ID NO: 244) -single chain IL-12 (E59A/F60A) variant-GGGGSGGG linker (SEQ ID NO: 244) -hinge (SEQ ID NO: 88) -Fc domain subunit (SEQ ID NO: 97)); and a partner polypeptide (PD-L1 (mutant) ectodomain-GGGGSGGG linker (SEQ ID NO: 244) -hinge (SEQ ID NO: 87) -Fc domain subunit (SEQ ID NO: 98)) from N-terminus to C-terminus. The linker may be changed to other linkers (e.g., GSG linker; SEQ ID NO: 203), or may be optional. Exemplary IL-12 cytokine fusion chains of IL-12 (E59A/F60A)/PD-L1 (mutant) -Fc immunoregulatory molecules may include SEQ ID NO 155 or 156.
To test the safety profile of IL-12-based immunomodulatory molecules constructed to have increased affinity for PD-1, wild-type C57 mice (5-6 weeks old, 20g females) were injected with 10mg/kg or 50mg/kg (per injection) of IL-12 (E59A/F60A)/PD-L1 (mutant 2) -Fc immunomodulatory molecules or IL-12 (E59A/F60A)/PD-L2 (mutant 2) -Fc immunomodulatory molecules, as both constructs showed a potential for PD-1 (-10) in humans and mice -7 M) similar PD-1 binding affinity. IL-12 (E59A/F60A)/PD-L1 (wild-type) -Fc immunoregulatory molecules or IL-12 (E59A/F60A)/PD-L2 (wild-type) -Fc immunoregulatory molecules were used as controls. Four injections were given on days 0, 4, 8 and 12 in total. Mice were monitored daily for mortality and four toxic symptoms: i) Fur wrinkling, ii) reduced activity, iii) morbidity, and iv) weight loss.
As can be seen from Table 16, increasing the binding affinity to PD-1 did not significantly affect the safety profile of either the IL-12 (E59A/F60A)/PD-L1-Fc immunoregulatory molecule or the IL-12 (E59A/F60A)/PD-L2-Fc immunoregulatory molecule. These results indicate that the mutant forms of IL-12 immunomodulatory molecules comprising PD-L1 and PD-L2 with increased binding affinity for PD-1 retain the safety profile of wild-type IL-12/PD-L1-Fc and IL-12/PD-L2-Fc immunomodulatory molecules. This can also be applied to other PD-L1-Fc or PD-L2-Fc based immunomodulatory molecules to construct other immunomodulatory molecules (e.g., IL-2 immunomodulatory molecules).
TABLE 16 in vivo toxicity of IL-12/PD-L2 immunomodulating molecules
Example 24 production of IL-2/PD-L1 immunoregulatory molecules with IL-2 biological Activity against PD-1-positive cells
Certain cytokines have synergistic effects such as IL-12 and IL-2, IL-12 and IFN-gamma. To reduce the toxicity of IL-2 and its immunomodulatory molecules, two groups of IL-2 mutations were generated: mutations within the IL-2 domain that interact with IL2Rα (CD 25) (R38D/K43E/E61R; SEQ ID NO: 26), and mutations within the IL-2 domain that interact with IL2Rγ (CD 132) (L18R, Q22E, Q T, S130R or any combination thereof). See table 17.
Heterodimeric PD-L1 (mutant 2) -Fc immunomodulatory molecules were used as parent PD-1 binding proteins. The first polypeptide chain comprises SEQ ID NO. 132 (from N-terminus to C-terminus: PD-L1 (mutant 2) ectodomain (SEQ ID NO. 123) -GGGGSGGG linker (SEQ ID NO. 244) -hinge (SEQ ID NO. 88) -Fc domain subunit (SEQ ID NO. 97)), and the second polypeptide chain comprises SEQ ID NO. 134 (from N-terminus to C-terminus: PD-L1 (mutant 2) ectodomain (SEQ ID NO. 123) -GGGGSGGG linker (SEQ ID NO. 244) -hinge (SEQ ID NO. 87) -Fc domain subunit (SEQ ID NO. 98)). To construct IL-2 immunomodulatory molecules, IL-2 variants are placed between the PD-L1 (mutant 2) extracellular domain and the hinge. Briefly, the IL-2 (mutant)/PD-L1 (mutant 2) -Fc immunocytokine comprises: an IL-2 fusion polypeptide (from N-terminus to C-terminus: PD-L1 (mutant 2) ectodomain (SEQ ID NO: 123) -GGGSG linker (SEQ ID NO: 209) -IL-2 (mutant) variant-GGGGSGGG linker (SEQ ID NO: 244) -hinge (SEQ ID NO: 87) -Fc domain subunit (SEQ ID NO: 98)); and a mating polypeptide SEQ ID NO. 132.
PD-L1 (mutant) -Fc immunomodulatory molecules comprising other PD-L1 (mutant) ectodomains and/or cytokine moieties (e.g., other IL-2 variants) can be similarly constructed. For example, a PD-L1 (mutant 7) -Fc immunoregulatory molecule can be constructed by replacing the PD-L1 (mutant 2) ectodomain with the PD-L1 (mutant 7) ectodomain (SEQ ID NO: 128). The parent heterodimeric PD-L1 (mutant 7) -Fc immunomodulatory molecule may comprise one strand of SEQ ID NO:133 and the other strand of SEQ ID NO: 335. An exemplary IL-2 cytokine fusion chain of IL-2 (mutant)/PD-L1 (mutant 7) -Fc immunoregulatory molecule may comprise any of SEQ ID NOs 163-166; the paired non-cytokine fusion chain may include 133.
HEK-Blue as described in example 12 TM IL-2 cells and HEK-PD-1-IL-2 cells were used to evaluate the IL-2 signaling activity of the constructs. As can be seen from Table 17, IL-2 (R38D/K43E/E61R)/PD-L1 (mutant 2) -Fc having IL-2 mutation in the CD25 binding domain (R38D/K43E/E61R) alone (see construct comprising the chain of SEQ ID NO: 179) retained about 16% of IL-2 activity (NO PD-1 binding) as determined on the basis of HEK-IL-2, whereas IL-2 immunoregulatory molecules carrying IL-2 mutation in the CD132 binding domain significantly reduced IL-2 activity as determined on the basis of HEK-IL-2 in the absence of PD-1 binding. Notably, the IL-2 activity of some IL-2 immunomodulatory molecules that also carry mutations in the CD132 binding domain (see constructs comprising the strand of SEQ ID NO:159, SEQ ID NO:160, or SEQ ID NO: 161) can be partially rescued when binding to PD-1 (determined based on HEK-IL-2-PD-1). S130 may be critical for IL-2 activity because IL-2 immunomodulatory molecules that also carry the S130R mutation in the CD132 binding domain (see constructs comprising the chain of SEQ ID NO: 162) bind to other IL-2 mutations and do not exhibit any IL-2 activity even in the presence of PD-1 binding.
TABLE 17 IL-2 biological Activity of IL-2/PD-1-Fc immunoregulatory molecules
/>
Example 25 production of IL-2/IL-12/PD-L1 immunoregulatory molecules with IL-2 and IL-12 biological Activity on PD-1-positive cells
Certain cytokines have synergistic effects such as IL-12 and IL-2. As shown in the above examples, IL-12 (E59A/F60A)/PD-L1-Fc immunoregulatory molecules (hinge region) showed PD-1 binding-dependent IL-12 activity. To investigate whether immunomodulatory molecules can be constructed with synergistic IL-12 and IL-2 activity while retaining PD-1 binding dependent cytokine activity, immunomodulatory molecules of different configurations were constructed. Heterodimeric PD-L1 (mutant 2) -Fc was used as the parent PD-1 binding fusion protein (constructed in example 23). Group I: one polypeptide chain comprises a single chain IL-12 (E59A/F60A) polypeptide located in the hinge region of PD-L1 (mutant 2) -Fc (see SEQ ID NO:155 constructed in example 23); the partner polypeptide chain does not comprise an IL-2 part (control; SEQ ID NO:134 constructed in examples 23 and 24), or comprises an IL-2 variant (with the L18R/Q22E/R38D/K43E/E61R mutation (SEQ ID NO: 27), or with the R38D/K43E/E61R/Q126T mutation (SEQ ID NO: 28)) located in the hinge region of PD-L1 (mutant 2) -Fc. These immunoregulatory molecules are named in the form of IL-2/IL-12 (E59A/F60A)/PD-L1 (mutant 2) -Fc. An exemplary structure is shown in fig. 14A. Group II: one polypeptide chain comprises IL-12 (E59A/F60A) fused via a GGGGSGGG linker to the C' of PD-L1 (mutant 2) -Fc (see SEQ ID NO: 157); the partner polypeptide chain does not comprise an IL-2 part (control; SEQ ID NO:134 constructed in examples 23 and 24), or comprises an IL-2 variant (with the L18R/Q22E/R38D/K43E/E61R mutation (SEQ ID NO: 27), or with the R38D/K43E/E61R/Q126T mutation (SEQ ID NO: 28)) located in the hinge region of PD-L1 (mutant 2) -Fc. These immunoregulatory molecules were named in the form of IL-2/PD-L1 (mutant 2) -Fc/IL-12 (E59A/F60A), indicating that the IL-12 moiety is located at C' of Fc. An exemplary structure is shown in fig. 15A. See table 18 for the construct sequences.
HEK-Blue as described in example 12 TM IL-2 cells and HEK-PD-1-IL-2 cells were used to evaluate the IL-2 signaling activity of the constructs. HEK-Blue as described in example 1 TM IL-12 cells and HEK-PD-1-IL-12 cells were used to evaluate the IL-12 signaling activity of the constructs.
As can be seen from Table 18, in the absence of PD-1 binding, IL-12 (E59A/F60A)/PD-L1 (mutant 2) -Fc (hinge fusion) had no detectable IL-12 activity, whereas PD-1 binding rescued IL-12 activity to 24%. When IL-12 (E59A/F60A) is placed as PD-L1 (mutant 2) -Fc/IL-12 (E59A/F60A) at Fc C', IL-12 activity is about 1% -2% in the absence of PD-1 binding, and IL-12 activity is further salvaged (25%) by PD-1 binding to a similar degree of fusion with the IL-12 hinge.
By adding IL-2 to the mating chain of the hinge region, IL-2 activity is about 2% -4% in the absence of PD-1 binding for the IL-12 hinge fusion and C' fusion forms, but is saved to about 20% -35% by PD-1 binding.
These data indicate that both IL-12 and IL-2 can retain PD-1 binding-dependent activity when constructed in the form of trispecific immunomodulatory molecules. Further, IL-12 and IL-2 portions have no significant negative effect on each other's activity.
TABLE 18 IL-2/IL-12/PD-L1-Fc immunoregulatory molecules and IL-2/PD-L1-Fc/IL-12 immunoregulatory molecule IL-2 and IL-12 biological Activity
EXAMPLE 26 positioning of IL-12 moiety in the hinge region can greatly enhance the safety profile of IL-12/PD-L1-Fc immunomodulatory molecules and IL-2/IL-12/PD-L1-Fc immunomodulatory molecules
The PD-L1 (mutant 2) -Fc constructed in examples 23 and 24 was used as a parent PD-1 binding fusion protein because it shows similar PD-1 binding affinity in humans and mice.
To test safety profiles in vivo, a mouse single-chain IL-12 variant (SEQ ID NO: 72) with E59A/F60A mutations in the p40 subunit and the p35 wild-type subunit was similarly constructed as described herein: from N-terminus to C-terminus is the p40 (E59A/F60A) -GGPGGGGSGGGSGGGG linker (SEQ ID NO: 245) -p35 (wild type). Two sets of IL-12 fusion polypeptides were constructed, similar to example 25. Group I: one polypeptide chain comprises a single chain mIL-12 (E59A/F60A) polypeptide located in the hinge region of PD-L1 (mutant 2) -Fc (see SEQ ID NO: 180); the paired polypeptide chains do not contain an IL-2 moiety (control; SEQ ID NO:134 constructed in examples 23 and 24), or comprise IL-2 variants (with R38D/K43E/E61R mutation (SEQ ID NO: 26), L18R/Q22E/R38D/K43E/E61R mutation (SEQ ID NO: 27), or with R38D/K43E/E61R/Q126T mutation (SEQ ID NO: 28)) located in the hinge region of PD-L1 (mutant 2) -Fc. These immunoregulatory molecules are named in the form of IL-2/IL-12 (E59A/F60A)/PD-L1 (mutant 2) -Fc. An exemplary structure is shown in fig. 14A. Group II: one polypeptide chain comprises single-chain mIL-12 (E59A/F60A) fused via a GGGGSGGG linker to the C' of PD-L1 (mutant 2) -Fc (see SEQ ID NO: 157); the paired polypeptide chains do not contain an IL-2 moiety (control; SEQ ID NO:134 constructed in examples 23 and 24), or comprise IL-2 variants (with R38D/K43E/E61R mutation (SEQ ID NO: 26), L18R/Q22E/R38D/K43E/E61R mutation (SEQ ID NO: 27), or R38D/K43E/E61R/Q126T mutation (SEQ ID NO: 28)) located in the hinge region of PD-L1 (mutant 2) -Fc. These immunocytokines were named in the form of IL-2/PD-L1 (mutant 2) -Fc/IL-12 (E59A/F60A), indicating that the IL-12 moiety is located at C' of Fc. An exemplary structure is shown in fig. 15A. The construct sequences are shown in table 7. The control group did not have any IL-12 moiety fused to PD-L1 (mutant 2) -Fc (SEQ ID NO: 132).
To test the safety profile of these constructs, wild type C57 mice (5-6 weeks old, body weight 20g, female) were injected with PBS (control) or the immunomodulatory molecules described herein (10 mg/kg per injection). Injections were given once every 4 days for a total of 4 times. Mice were monitored for mortality and toxic symptoms such as coat wrinkling, reduced activity, and weight loss. 48 hours after the second injection, blood was collected and the serum concentration of IFN-gamma was measured.
As shown in Table 19, in addition to R38D/K43E/E61R in the CD25 binding domain, the immunoregulatory molecules comprising the IL-2 additional mutation (L18R/Q22E or Q126T) in the CD132 binding domain showed higher safety profiles (see construct comprising SEQ ID NO:179 chain) compared to the absence of the CD132 binding domain mutation, whether the IL-12 moiety was at C' or at the hinge.
As shown in Table 19, IL-2 (mutant)/PD-L1 (mutant 2) -Fc/mIL-12 (E59A/F60A) having an immunomodulatory molecule of IL-12 at C' of Fc showed higher toxicity than IL-12 (IL-2 (mutant)/mIL-12 (E59A/F6 OA)/PD-L1 (mutant 2) -Fc located in the hinge region. IL-2 (mutant)/PD-L1 (mutant 2) -Fc/mIL-12 (E59A/F60A) also induced higher (20-30 fold) cytokine release (see IFN-gamma levels) than the IL-12 hinge fusion design.
In summary, the in vivo and in vitro data presented herein indicate that an immunomodulatory molecule with a cytokine (e.g., an immunostimulatory molecule such as IL-12 or a variant thereof) located in the hinge region can significantly improve the safety profile even if more cytokines (e.g., IL-2/IL-12/PD-L1-Fc) with a synergistic effect are present in the same construct. Mutations in the cytokine that reduce its immunostimulatory activity, and/or mutations in the antigen binding domain (e.g., anti-PD-1 or PD-L1, or PD-L2), may further improve the safety and/or therapeutic efficacy of the construct.
TABLE 19 IL-2/IL-12/PD-L1-Fc immunoregulatory molecules and IL-2/PD-L1-Fc/IL-12 immunoregulatory molecule IL-2 and IL-12 biological Activity
Example 27 production of IL-12/IFN- γ/PD-L1-Fc immunomodulatory molecules with IL-12 and IFN- γ biological Activity against PD-1 positive cells
Certain cytokines have synergistic effects such as IL-12 and IFN-gamma. To investigate whether immunomodulatory molecules can be constructed that have synergistic IL-12 and IFN-gamma activity while retaining PD-1 binding dependent cytokine activity, different configurations of immunomodulatory molecules were constructed using heterodimeric PD-L1-Fc or heterodimeric PD-L2-Fc as the parent PD-1 binding protein.
Construction of IL-12/IFN-gamma/PD-L1-Fc immunoregulatory molecules
Heterodimeric PD-L1 (mutant 2) -Fc and PD-L1 (mutant 7) -Fc immunoregulatory molecules were used as parent PD-1 binding proteins (constructed in examples 23 and 24). Heterodimeric PD-L1 (mutant 2) -Fc has a first polypeptide chain comprising SEQ ID NO:132 and a second polypeptide chain comprising SEQ ID NO: 134. Heterodimeric PD-L1 (mutant 7) -Fc has a first polypeptide chain comprising SEQ ID NO:133 and a second polypeptide chain comprising SEQ ID NO: 135.
To construct an IL-12/IFN-gamma/PD-L1-Fc immunoregulatory molecule, one polypeptide chain does not comprise IL-12 (as a control; SEQ ID NO: 132), or comprises a single chain IL-12 (E59A/F60A) polypeptide located in the hinge region of PD-L1 (mutant) -Fc (see, e.g., SEQ ID NO: 155); the paired polypeptide chains comprise a single-chain IFN-gamma (A23V/A23V) homodimer located in the hinge region of PD-L1 (mutant) -Fc (from N-terminus to C-terminus: PD-L1 (mutant) ectodomain-GGGSG linker (SEQ ID NO: 209) -single-chain IFN-gamma (A23V/A23V) homodimer (SEQ ID NO:47 or 252) -GGGGGGSGGG linker (SEQ ID NO: 244) -hinge (SEQ ID NO: 87) -Fc domain subunit (SEQ ID NO: 98)).
Construction of IL-12/IFN-gamma/PD-L2-Fc immunoregulatory molecules
Heterodimeric PD-L2 (mutant 2) -Fc and PD-L2 (mutant 4) -Fc immunomodulatory molecules were used as parent PD-1 binding proteins (constructed in example 23). Heterodimeric PD-L2 (mutant 2) -Fc has a first polypeptide chain comprising SEQ ID NO:116 and a second polypeptide chain comprising SEQ ID NO: 118. Heterodimeric PD-L2 (mutant 4) -Fc has a first polypeptide chain comprising SEQ ID NO:117 and a second polypeptide chain comprising SEQ ID NO: 119.
To construct an IL-12/IFN-gamma/PD-L2-Fc immunoregulatory molecule, one polypeptide chain comprises from N-terminus to C-terminus: PD-L2 (mutant) extracellular domain (e.g., SEQ ID NO:108 or 110) -GGGGSGGG linker (SEQ ID NO: 244) -single chain IL-12 (E59A/F60A) variant (e.g., SEQ ID NO:68 or 254) -GGGGSGGG linker (SEQ ID NO: 244) -hinge (SEQ ID NO: 88) -Fc domain subunit (SEQ ID NO: 97); and one paired polypeptide chain comprises, from N-terminus to C-terminus: PD-L2 (mutant) extracellular domain (e.g., SEQ ID NO:108 or 110) -GGGSG linker (SEQ ID NO: 209) -single chain IFN-gamma (A23V/A23V) homodimer variant (SEQ ID NO:47 or 252) -GGGGSGGG linker (SEQ ID NO: 244) -hinge (SEQ ID NO: 87) -Fc domain subunit (SEQ ID NO: 98).
IL-12 and IFN-gamma signaling assays
As described in example 1, HEK-Blue was used TM IL-12 cells and HEK-PD-1-IL-12 cells to evaluate immunityModulating IL-12 signaling activity of the molecule. To assess the biological activity of various IFN-gamma moieties in immunoregulatory molecules, the antibodies were prepared by the method described in HEK-Blue TM Overexpression of human PD-1 in IFN-gamma cells HEK-IFN-gamma-PD-1 cells were prepared internally. The HEK-IFN-gamma reporter assay and HEK-PD-1-IFN-gamma reporter assay were performed in analogy to example 15.
As can be seen from Table 20, IL-12/IFN-gamma/PD-L1-Fc and IL-12/IFN-gamma/PD-L2-Fc immunoregulatory molecules exhibited IL-12 and IFN-gamma activity only in the presence of PD-1 binding. Furthermore, IL-12 and IFN-gamma activity appear not to be strongly affected by the type of PD-1 binding protein used, since the action of the immunomodulatory molecules comprising the PD-L1 extracellular domain and the PD-L2 extracellular domain is about the same.
These data indicate that both the IL-12 and IFN-gamma moieties at the hinge retain PD-1 binding-dependent activity when constructed as trispecific immunomodulatory molecules. Furthermore, the IL-12 and IFN-gamma moieties did not have a significant negative effect on each other's activity (compare HEK-IFN-gamma-PD-1 and HEK-IL-12-PD-1 columns).
TABLE 20 IL-12 and IFN-gamma biological Activity of IL-12/IFN-gamma/PD-L1-Fc and IL-12/IFN-gamma/PD-L2-Fc immunoregulatory molecules
Example 28 production of IL-2/IL-12/CD155-Fc and IL-12/IFN-gamma/CD 155-Fc immunoregulatory molecules with IL-2, IL-12 and IFN-gamma biological Activity against TIGIT-positive cells
The TIGIT/CD155 pathway plays a similar role in inhibiting T cell function as PD-1/PD-L. Like PD-1, TIGIT is highly expressed in T cells within tumors, such as depleted T cells. To investigate whether an immunomodulatory molecule may be constructed from IL-12, IL-2 and/or IFN-gamma activity that is dependent on binding to cells expressing TIGIT (e.g., T cells), a different configuration of immunomodulatory molecule was constructed using the CD155 ectodomain (SEQ ID NO: 137) as the TIGIT binding protein.
A heterodimeric CD155-Fc was used as a parent CD155-Fc protein comprising a first polypeptide chain (SEQ ID NO: 138) and a second polypeptide chain (SEQ ID NO: 139), the first polypeptide chain being N-terminal to C-terminal: CD155 ectodomain (SEQ ID NO: 137) -GGGGSGGG linker (SEQ ID NO: 244) -hinge (SEQ ID NO: 88) -Fc domain subunit 1 (SEQ ID NO: 97)); and the second polypeptide chain is from N-terminus to C-terminus: CD155 ectodomain (SEQ ID NO: 137) -GGGGSGGG linker (SEQ ID NO: 244) -hinge (SEQ ID NO: 87) -Fc domain subunit 2 (SEQ ID NO: 98).
Construction of IL-12/CD155-Fc (IL-12 hinge) and CD155-Fc/IL-12 (IL-12C') immunomodulatory molecules
To generate IL-12/CD155-Fc and CD155Fc/IL-12 immunoregulatory molecules, one polypeptide chain comprises: i) NO IL-12 fusion (as a control), or ii) IL-12 at the hinge region (SEQ ID NO:190; from the N end to the C end: CD155 ectodomain (SEQ ID NO: 137) -linker (SEQ ID NO: 244) -single chain IL-12 (E59A/F60A) variants (e.g., SEQ ID NO:68 or 254) -linker (SEQ ID NO: 244) -hinge (SEQ ID NO: 88) -Fc domain subunit 1 (SEQ ID NO: 97)), or iii) IL-12 (SEQ ID NO: 191) located at the C-terminus of one Fc domain subunit; from the N end to the C end: CD155 ectodomain (SEQ ID NO: 137) -linker (SEQ ID NO: 244) -hinge (SEQ ID NO: 88) -Fc domain subunit 1 (SEQ ID NO: 97) -linker (SEQ ID N0: 244) -single chain IL-12 (E59A/F60A) variants (e.g., SEQ ID NO:68 or 254)). The mating polypeptide chain comprises the sequence of SEQ ID NO. 139.
Construction of IL-2/CD155-Fc immunoregulatory molecules
To generate IL-2/CD155-Fc immunomodulatory molecules, a polypeptide chain comprises IL-12 or a mutant variant located in the hinge region (from N-terminus to C-terminus: CD155 ectodomain (SEQ ID NO: 137) -GGGSG linker (SEQ ID NO: 209) -IL-2 (mutant) (e.g., any of SEQ ID NO: 26-30) -GGGGSGGG linker (SEQ ID NO: 244) -hinge (SEQ ID NO: 87) -Fc domain subunit 2 (SEQ ID NO: 98)). Thus, a polypeptide chain having an IL-2 portion at the hinge may comprise the sequence of any one of SEQ ID NOS 247-250. The paired polypeptide chain without IL-2 fusion comprises the sequence of SEQ ID NO. 138.
Construction of IFN-gamma/CD 155-Fc immunoregulatory molecules
To generate IFN-gamma/CD 155-Fc immunoregulatory molecules, one polypeptide chain comprises: i) No IFN- γ (as control; 139) or ii) the single chain homodimer IFN-gamma (A23V/A23V) located in the hinge region (N-terminal to C-terminal: CD155 ectodomain (SEQ ID NO: 137) -linker (SEQ ID NO: 244) -single-chain IFN-gamma (A23V/A23V) homodimer variant (SEQ ID NO:47 or 252) -linker (SEQ ID NO: 244) -hinge (SEQ ID NO: 87) -Fc domain subunit 2 (SEQ ID NO: 98)). Thus, a polypeptide chain having an IFN-gamma moiety at the hinge may comprise the sequence of SEQ ID NO: 193. The paired polypeptide chain without IFN-gamma fusion comprises the sequence of SEQ ID NO. 138.
IL-12/IL-2/CD155-Fc (IL-12 hinge) and IL-2/CD155-Fc/IL-12 (IL-12 at C') immunization Construction of regulatory molecules
The IL-2/CD155-Fc (IL-2 at the hinge) heterodimeric immunoregulatory molecules constructed as described above can be used as parent constructs for preparing IL-12/IL-2/CD155-Fc (IL-12 at the hinge) or IL-2/CD155-Fc/IL-12 (IL-12 at C' of one of the Fc subunits) immunoregulatory molecules. The polypeptide chain having an IL-2 portion at the hinge may comprise the sequence of any one of SEQ ID NOS 247-250. The mating polypeptide having a single chain IL-12 (E59A/F60A) variant at the hinge region may comprise a sequence of 190; or a mating polypeptide having a single chain IL-12 (E59A/F60A) variant located at C' of the Fc subunit may comprise the sequence of 191 (see above).
IL-12/IFN-gamma/CD 155-Fc (IL-12 hinge) and IFN-gamma-CD 155-Fc/IL-12 (IL-12 at C') exempt Construction of epidemic-regulating molecules
The IFN-gamma/CD 155-Fc (IFN-gamma at the hinge) heterodimeric immunoregulatory molecules constructed as described above can be used as parent constructs for the preparation of IL-12/IFN-gamma/CD 155-Fc (IL-12 at the hinge) or IFN-gamma CD155-Fc/IL-12 (IL-12 at C' of one of the Fc subunits) immunoregulatory molecules. The polypeptide chain having a single chain IFN-gamma (A23V/A23V) homodimer located at the hinge may comprise the sequence of SEQ ID NO: 193. The mating polypeptide having a single chain IL-12 (E59A/F60A) variant at the hinge region may include a sequence of 190; or a mating polypeptide having a single chain IL-12 (E59A/F60A) variant located at C' of the Fc subunit may comprise the sequence of 191 (see above).
IL-12, IL-2 and IFN-gamma signaling assays
To assess the biological activity of the IL-12 moiety in IL-12 containing immunomodulatory molecules, the antibodies were prepared by the method described in HEK-Blue TM Overexpression of TIGIT in IL-12 cells, internal preparation of HEK Blue TM IL-12-TIGIT cells (see example 1). To assess the biological activity of the IL-2 moiety in IL-2 containing immunomodulatory molecules, the activity was demonstrated by the reaction in HEK-Blue TM Overexpression of TIGIT in IL-2 cells, internal preparation of HEK Blue TM IL-2-TIGIT cells (see example 12). To assess the biological activity of various IFN-gamma moieties in IFN-gamma containing immunomodulatory molecules, the antibodies were prepared by the method described in HEK-Blue TM Human TIGIT was overexpressed in IFN- γ cells, HEK-IFN- γ -TIGIT cells were prepared internally (see example 15).
As can be seen from Table 21, bispecific and trispecific immunomodulatory molecules comprising an IL-12 moiety at the hinge, exhibit minimal IL-12 activity in the absence of CD155/TIGIT binding; in the presence of TIGIT binding, IL-12 activity is rescued. Similarly, bispecific and trispecific immunomodulatory molecules comprising an IL-2 or IFN-gamma moiety at the hinge region exhibit minimal IL-2 and IFN-gamma activity in the absence of CD155/TIGIT binding; in the presence of TIGIT binding, the activity of IL-2 or IFN- γ is rescued. These data indicate that when constructed as bispecific or trispecific immunomodulatory molecules, IL-12, IL-2 and IFN-gamma all retain TIGIT binding-dependent activity when located in the hinge region. Furthermore, these data indicate that IL-12, IL-2 and IFN-gamma moieties do not have a significant negative effect on each other's activity when constructed as bispecific or trispecific immunomodulatory molecules.
TABLE 21 IL-2, IL-2 and IFN-gamma biological Activity of an immunomodulatory molecule comprising IL-2, IL-12 and/or IFN-gamma against TIGIT positive cells
/>
/>
Sequence listing
SEQ ID NO. 1 (wild type human CTLA-4 ectodomain-hinge-IgG 1 Fc mutant 2; CTLA-4 ectodomain underlined; hinge bold; linker bold and underlined)
SEQ ID NO. 2 (wild type human CTLA-4 ectodomain-linker-hinge-IgG 1 Fc mutant 1-linker-single chain IL-12 mutant heterodimeric IL-12B (p 40E 59A/F60A) -linker-IL-12A (wt p 35); CTLA-4 ectodomain underlined; linker bold and underlined; hinge bold; IL-12 subunit italics)
SEQ ID NO. 3 (wild type human CTLA-4 ectodomain-linker-hinge-IgG 1 Fc mutant 1-linker-Single chain IL-12 mutant heterodimeric IL-12B (p 40F 60A) -linker-IL-12A (wt p 35); CTLA-4 ectodomain underlined; linker bold and underlined; hinge bold; IL-12 subunit italics)
SEQ ID NO. 4 (wild type human CTLA-4 ectodomain-linker-Single chain IL-12 mutant heterodimeric IL-12B (p 40E 59A/F60A) -linker-IL-12A (wt p 35) -hinge-IgG 1 Fc mutant 1; CTLA-4 ectodomain underlined; linker bold and underlined; hinge bold; IL-12 subunit italics)
SEQ ID NO. 5 (wild type human CTLA-4 ectodomain-linker-Single chain IL-12 mutant heterodimeric IL-12B (p 40F 60A) -linker-IL-12A (wt p 35) -hinge-IgG 1 Fc mutant 1; CTLA-4 ectodomain underlined; linker bold and underlined; hinge bold; IL-12 subunit italics)
SEQ ID NO. 6 (wild type human PD-L1 WT ectodomain-hinge-IgG 1 Fc mutant 2; PD-L1 ectodomain underlined; hinge bold; linker bold and underlined)
SEQ ID NO. 7 (wild type human PD-L1 WT ectodomain-linker-hinge-IgG 1 Fc mutant 1-linker-single chain IL-12 mutant heterodimeric IL-12B (p 40E 59A/F60A) -linker-IL-12A (WT p 35); PD-L1 ectodomain underlined; hinge bold; linker bold and underlined)
SEQ ID NO. 8 (wild type human PD-L1 WT ectodomain-linker-hinge-IgG 1 Fc mutant 1-linker-Single chain IL-12 mutant heterodimeric IL-12B (p 40F 60A) -linker-IL-12A (WT p 35); PD-L1 ectodomain underlined; hinge bold; linker bold and underlined)
/>
SEQ ID NO. 9 (wild type human PD-L1 WT ectodomain-linker-Single chain IL-12 mutant heterodimeric IL-12B (p 40E 59A/F60A) -linker-IL-12A (WT p 35) -hinge-IgG 1 Fc mutant 1; PD-L1 ectodomain underlined; linker bold and underlined; hinge bold; IL-12 subunit italics)
SEQ ID NO. 10 (wild type human PD-L1 WT ectodomain-linker-Single chain IL-12 mutant heterodimeric IL-12B (p 40F 60A) -linker-IL-12A (WT p 35) -hinge-IgG 1 Fc mutant 1; PD-L1 ectodomain underlined; linker bold and underlined; hinge bold; IL-12 subunit italics)
SEQ ID NO. 11 (human PD-L1 mutant ectodomain-hinge-IgG 1 Fc mutant 2; PD-L1 ectodomain underlined; hinge bold; linker bold and underlined)
/>
SEQ ID NO. 12 (human PD-L1 mutant ectodomain-linker-hinge-IgG 1 Fc mutant 1-linker-Single chain IL-12 mutant heterodimeric IL-12B (p 40E 59A/F60A) -linker-IL-12A (wt p 35); PD-L1 ectodomain underlined; linker bold and underlined; hinge bold; IL-12 subunit italics)
SEQ ID NO. 13 (human PD-L1 mutant ectodomain-linker-hinge-IgG 1 Fc mutant 1-linker-Single chain IL-12 mutant heterodimeric IL-12B (p 40F 60A) -linker-IL-12A (wt p 35); PD-L1 ectodomain underlined; linker bold and underlined; hinge bold; IL-12 subunit italics)
SEQ ID NO. 14 (human PD-L1 mutant ectodomain-linker-Single chain IL-12 mutant heterodimeric IL-12B (p 40E 59A/F60A) -linker-IL-12A (wt p 35) -hinge-IgG 1 Fc mutant 1; PD-L1 ectodomain underlined; linker bold and underlined; hinge bold; IL-12 subunit italics)
/>
SEQ ID NO. 15 (human PD-L1 mutant ectodomain-linker-Single chain IL-12 mutant heterodimeric IL-12B (p 40F 60A) -linker-IL-12A (wt p 35) -hinge-IgG 1 Fc mutant 1; PD-L1 ectodomain underlined; linker bold and underlined; hinge bold; IL-12 subunit italics)
SEQ ID NO. 16 (wild type human PD-L2 ectodomain-hinge-IgG 1 Fc mutant 2; PD-L2 ectodomain underlined; hinge bold; linker bold and underlined)
SEQ ID NO. 17 (wild type human PD-L2 ectodomain-linker-Single chain IL-12 mutant heterodimeric IL-12B (p 40E 59A/F60A) -linker-IL-12A (wt p 35) -hinge-IgG 1 Fc mutant 1; PD-L2 ectodomain underlined; linker bold and underlined; hinge bold; IL-12 subunit italics)
SEQ ID NO. 18 (wild type human PD-L2 ectodomain-linker-Single chain IL-12 mutant heterodimeric IL-12B (p 40F 60A) -linker-IL-12A (wt p 35) -hinge-IgG 1 Fc mutant 1; PD-L2 ectodomain underlined; linker bold and underlined; hinge bold; IL-12 subunit italics)
SEQ ID NO. 19 (wild type human PD-L2 ectodomain-linker-hinge-IgG 1 Fc mutant 1-linker-Single chain IL-12 mutant heterodimeric IL-12B (p 40E 59A/F60A) -linker-IL-12A (wt p 35); PD-L2 ectodomain underlined; linker bold and underlined; hinge bold; IL-12 subunit italics)
SEQ ID NO. 20 (wild type human PD-L2 ectodomain-linker-hinge-IgG 1 Fc mutant 1-linker-Single chain IL-12 mutant heterodimeric IL-12B (p 40F 60A) -linker-IL-12A (wt p 35); PD-L2 ectodomain underlined; linker bold and underlined; hinge bold; IL-12 subunit italics)
SEQ ID NO. 21 (anti-PD-1 antibody VH-CH1-IgG1 Fc mutant 2; VH underlined; hinge is bold; linker is bold and underlined)
/>
SEQ ID NO. 22 (anti-PD-1 antibody VH-CH1-N' hinge portion-linker-single chain IL-12 mutant heterodimeric IL-12B (p 40)
E59A/F60A) -linker-IL-12A (wt p 35) -C' hinge portion-IgG 1 Fc mutant 1; VH bands underlined; the hinge is bold; the joints are bold and underlined; IL-12 subunit is italic
SEQ ID NO. 23 (anti-PD-1 antibody VH-CH1-N' hinge portion-linker-single chain IL-12 mutant heterodimeric IL-12B (p 40)
F60A) -linker-IL-12A (wt p 35) -C' hinge moiety-IgG 1 Fc mutant 1; VH bands underlined; the hinge is bold; the joints are bold and underlined; IL-12 subunit is italic
SEQ ID NO. 24 (wild type human PD-L2 ectodomain-linker-IL-2 mutant R38D/K43E/E61R-hinge-IgG 1 Fc mutant 1; PD-L2 underlined; linker bold and underlined; hinge bold; IL-2 mutant italic)
SEQ ID NO. 25 (wild type mature human IL-2)
APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT
SEQ ID NO. 26 (IL-2 mutant 1R 38D/K43E/E61R)
SEQ ID NO. 27 (IL-2 mutant 2L 18R/Q22E/R38D/K43E/E61R)
SEQ ID NO. 28 (IL-2 mutant 3R 38D/K43E/E61R/Q126T)
SEQ ID NO. 29 (IL-2 mutant 4L 18R/Q22E/R38D/K43E/E61R/Q126T)
SEQ ID NO. 30 (IL-2 mutant 5L 18R/Q22E/R38D/K43E/E61R/Q126T/S130R)
SEQ ID NO. 31 (wild type mature human IFN-. Alpha.2b)
CDLPQTHSLGSRRTLMLLAQMRKISLFSCLKDRHDFGFPQEEFGNQFQKAETIPVLHEMIQQIFNLFSTKDSSAAWDETLLDKFYTELYQQLNDLEACVIQGVGVTETPLMKEDSILAVRKYFQRITLYLKEKKYSPCAWEVVRAEIMRSFSLSTNLQESLRSKE
SEQ ID NO. 32 (IFN-. Alpha.2b mutant L30A)
SEQ ID NO. 33 (IFN-. Alpha.2b mutant K31A)
SEQ ID NO. 34 (IFN-. Alpha.2b mutant D32A)
SEQ ID NO. 35 (IFN-. Alpha.2b mutant R33A)
SEQ ID NO. 36 (IFN-. Alpha.2b mutant H34A)
SEQ ID NO. 37 (IFN-. Alpha.2b mutant D35A)
SEQ ID NO. 38 (wild type mature human IFN-. Gamma.monomer)
QDPYVKEAENLKKYFNAGHSDVADNGTLFLGILKNWKEESDRKIMQSQIVSFYFKLFKNFKDDQSIQKSVETIKEDMNVKFFNSNKKKRDDFEKLTNYSVTDLNVQRKAIHELIQVMAELSPAAKTGKRKRSQMLFRG
SEQ ID NO. 39 (IFN-. Gamma.mutant S20A/D21A monomer)
SEQ ID NO. 40 (IFN-. Gamma.mutant V22A/A23S monomer)
SEQ ID NO. 41 (IFN-. Gamma.mutant A23V monomer)
SEQ ID NO. 42 (IFN-. Gamma.mutant D24A/N25A monomer)
SEQ ID NO. 43 (IFN-. Gamma.mutant A23E/D24E/N25K monomer)
SEQ ID NO. 44 (IFN-. Gamma.mutant A23Q monomer)
SEQ ID NO. 45 (IFN-. Gamma.mutant D21K monomer)
SEQ ID NO. 46 (Single chain "wild type" IFN-. Gamma.homodimer; linker in bold; wild type IFN-. Gamma.monomer in italics)
SEQ ID NO. 47 (Single chain IFN-. Gamma.mutant A23V homodimer; linker bold; IFN-. Gamma.mutant monomer italic)
SEQ ID NO. 48 (Na Wu Liyou mab/Oudiewov anti-PD-1 Ab VH)
QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKGLEWVAVIWYDGSKRYYADSVKGRFTISRDNSKNTLFLQMNSLRAEDTAVYYCATNDDYWGQGTLVTVSSAS
SEQ ID NO. 49 (Na Wu Liyou mab/Oudiewov anti-PD-1 Ab VL)
EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQSSNWPRTFGQGTKVEI
SEQ ID NO. 50 (Na Wu Liyou mab/Oudiwa anti-PD-1 Ab LC; VL underlined)
EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGT DFTLTISSLEPEDFAVYYCQQSSNWPRTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
SEQ ID NO. 51 (anti-PD-1 Ab HC (IgG 1 Fc mutant 2); VH underlined; hinge is bold)
SEQ ID NO. 52 (wild type mature human IL-10 monomer)
SPGQGTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRN
SEQ ID NO. 53 (IL-10 mutant R24A monomer)
SEQ ID NO. 54 (IL-10 mutant D25A/L26A monomer)
SEQ ID NO. 55 (IL-10 mutant R27A monomer)
/>
SEQ ID NO. 56 (IL-10 mutant D28A/A29S monomer)
SEQ ID NO. 57 (IL-10 mutant F30A/S31A monomer)
SEQ ID NO. 58 (IL-10 mutant R32A monomer)
SEQ ID NO. 59 (Single chain "wild type" IL-10 homodimer; linker in bold; wild type IL-10 monomer in italic)
SEQ ID NO. 60 (Single chain IL-10 mutant R27A homodimer; linker in bold; IL-10 mutant monomer in italic)
SEQ ID NO. 61 (wild type mature human IL-12A (p 35) subunit)
RNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDHEDITKDKTSTVEACLPLELTKNESCLNSRETSFITNGSCLASRKTSFMMALCLSSIYEDLKMYQVEFKTMNAKLLMDPKRQIFLDQNMLAVIDELMQALNFNSETVPQKSSLEEPDFYKTKIKLCILLHAFRIRAVTIDRVMSYLNAS
SEQ ID NO. 62 (wild type mature human IL-12B (p 40) subunit)
IWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTLTIQVKEFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKNKTFLRCEAKNYSGRFTCWWLTTISTDLTFSVKSSRGSSDPQGVTCGAATLSAERVRGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFCVQVQGKSKREKKDRVFTDKTSATVICRKNASISVRAQDRYYSSSWSEWASVPCS
SEQ ID NO. 63 (IL-12B (p 40) mutant E59A/F60A subunit)
SEQ ID NO. 64 (IL-12B (p 40) mutant E59A subunit)
SEQ ID NO. 65 (IL-12B (p 40) mutant F60A subunit)
SEQ ID NO. 66 (IL-12B (p 40) mutant G64A subunit)
SEQ ID NO. 67 (Single chain "wild type" IL-12 heterodimer IL-12B (wt p 40) -linker-IL-12A (wt p 35); linker in bold)
SEQ ID NO. 68 (Single chain IL-12 mutant heterodimeric IL-12B (p 40E 59A/F60A) -linker-IL-12A (wt p 35); linker in bold, IL-12 subunit in italics)
SEQ ID NO. 69 (Single chain IL-12 mutant heterodimeric IL-12B (p 40E 59A) -linker-IL-12A (wt p 35), linker bold and underlined, IL-12 subunit italics
SEQ ID NO. 70 (Single chain IL-12 mutant heterodimeric IL-12B (p 40G 64A) -linker-IL-12A (wt p 35), linker bold and underlined, IL-12 subunit italics
SEQ ID NO. 71 (Single chain IL-12 mutant heterodimeric IL-12B (p 40F 60A) -linker-IL-12A (wt p 35), linker in bold, IL-12 subunit in italics
SEQ ID NO. 72 (mouse Single chain mutant heterodimer IL-12B (E59A/F60A) -linker-IL-12A (wt p 35), linker bold and underlined, mouse IL-12 subunit italics
SEQ ID NO. 73 (wild type mature human IL-23A (p 19) subunit)
RAVPGGSSPAWTQCQQLSQKLCTLAWSAHPLVGHMDLREEGDEETTNDVPHIQCGDGCDPQGLRD
NSQFCLQRIHQGLIFYEKLLGSDIFTGEPSLLPDSPVGQLHASLLGLSQLLQPEGHHWETQQIPSLSPS
QPWQRLLLRFKILRSLQAFVAVAARVFAHGAATLSP
SEQ ID NO. 74 (Single chain "wild type" IL-23 heterodimer IL-12B (wt p 40) -linker-IL-23A (wt p 19); linker in bold, IL-23 subunit in italics)
SEQ ID NO. 75 (Single chain IL-23 mutant heterodimeric IL-12B (p 40E 59A/F60A) -linker-IL-23A (wt p 19); linker in bold, IL-23 subunit in italics)
/>
SEQ ID NO. 76 (hinge)
EPKSCDKTHTCPPCPAPELLGGP
SEQ ID NO. 77 (hinge)
SEQ ID NO. 78 (hinge)
SEQ ID NO. 79 (hinge)
SEQ ID NO. 80 (hinge)
SEQ ID NO. 81 (hinge)
SEQ ID NO. 82 (hinge)
ERKCCVECPPCPAPPVAGP
SEQ ID NO. 83 (hinge)
ESKYGPPCPSCPAPEFLGGP
SEQ ID NO. 84 (hinge, e.g., hinge N' portion)
EPKSCDK
SEQ ID NO. 85 (hinge, e.g., hinge N' portion)
EPKSC
SEQ ID NO. 86 (hinge, e.g., hinge C' portion)
DKTHTCPPCPAPELLGGP
SEQ ID NO. 87 (hinge, e.g., hinge C' portion)
SEQ ID NO. 88 (hinge, e.g., hinge C' portion)
SEQ ID NO. 89 (hinge)
DKTHT
SEQ ID NO. 90 (hinge, e.g., hinge N' portion)
SEQ ID NO. 91 (hinge)
SEQ ID NO. 92 (hinge)
SEQ ID NO. 93 (hinge)
SEQ ID NO. 94 (hinge)
ESKYGPPCPPCPAPEFLGGP
SEQ ID NO. 95 (hinge)
SEQ ID NO. 96 (wild type human IgG1 Fc)
SVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
SEQ ID NO. 97 (IgG 1 Fc mutant 1T 350V/L351Y/S400E/F405A/Y407V)
SEQ ID NO. 98 (IgG 1 Fc mutant 2T 350V/T366L/N390R/K392M/T394W)
SEQ ID NO. 99 (wild type human IgG4 Fc)
SVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK
SEQ ID NO. 100 (IgG 1 Fc mutant)
SEQ ID NO. 101 (IgG 1 Fc mutant)
SEQ ID NO. 102 (IgG 1 Fc mutant)
SEQ ID NO. 103 (anti-PD-1 Ab HC (IgG 1 Fc mutant); VH underlined)
SEQ ID NO 104 (Na Wu Liyou mab/Oudeao anti-PD-1 Ab HC; VH underlined; hinge is bold)
SEQ ID NO. 105 (wild type human PD-L2; signal peptide in italics; extracellular domain underlined; cytoplasmic domain in bold)
SEQ ID NO. 106 (wild type human PD-L2 ectodomain)
LFTVTVPKELYIIEHGSNVTLECNFDTGSHVNLGAITASLQKVENDTSPHRERATLLEEQLPLGKASFHIPQVQVRDEGQYQCIIIYGVAWDYKYLTLKVKASYRKINTHILKVPETDEVELTCQATGYPLAEVSWPNVSVPANTSHSRTPEGLYQVTSVLRLKPPPGRNFSCVFWNTHVRELTLASIDLQSQMEPRTHPT
SEQ ID NO. 107 (human PD-L2 ectodomain mutant 1T 56V)
SEQ ID NO. 108 (human PD-L2 ectodomain mutant 2S 58V)
SEQ ID NO. 109 (human PD-L2 ectodomain mutant 3Q 60L)
SEQ ID NO. 110 (human PD-L2 ectodomain mutant 4T 56V/S58V/Q60L)
SEQ ID NO. 111 (wild type human PD-L2 ectodomain-hinge-IgG 1 Fc mutant; PD-L2 ectodomain underlined; hinge bold)
SEQ ID NO. 112 (wild type human PD-L2 ectodomain-hinge-IgG 1 Fc mutant 2; PD-L2 ectodomain underlined; hinge bold)
SEQ ID NO. 113 (wild type human PD-L2 ectodomain-hinge-IgG 1 Fc mutant 2; PD-L2 ectodomain underlined; linker bold and underlined; hinge bold)
SEQ ID NO. 114 (wild type human PD-L2 ectodomain-linker-hinge-IgG 1 Fc mutant 2; PD-L2 ectodomain underlined; linker bold and underlined; hinge bold)
SEQ ID NO. 115 (wild type human PD-L2 ectodomain-hinge-IgG 1 Fc mutant 2; PD-L2 ectodomain underlined; hinge bold; linker bold and underlined)
SEQ ID NO. 116 (human PD-L2 ectodomain mutant 2S58V ectodomain-linker-hinge-IgG 1 Fc mutant 1; PD-L2 ectodomain underlined; linker bold and underlined; hinge bold)
SEQ ID NO. 117 (human PD-L2 ectodomain mutant 4T56V/S58V/Q60L ectodomain-linker-hinge-IgG 1 Fc mutant 1; PD-L2 ectodomain underlined; linker bold and underlined; hinge bold)
/>
SEQ ID NO. 118 (human PD-L2 ectodomain mutant 2S58V ectodomain-linker-hinge-IgG 1 Fc mutant 2; PD-L2 ectodomain underlined; linker bold and underlined; hinge bold)
SEQ ID NO. 119 (human PD-L2 ectodomain mutant 4T56V/S58V/Q60L ectodomain-linker-hinge-IgG 1 Fc mutant 2; PD-L2 ectodomain underlined; linker bold and underlined; hinge bold)
SEQ ID NO. 120 (wild type human PD-L1; signal peptide in italics; extracellular domain underlined; cytoplasmic domain in bold)
SEQ ID NO. 121 (wild type human PD-L1 ectodomain)
FTVTVPKDLYVVEYGSNMTIECKFPVEKQLDLAALIVYWEMEDKNIIQFVHGEEDLKVQHSSYRQR
ARLLKDQLSLGNAALQITDVKLQDAGVYRCMISYGGADYKRITVKVNAPYNKINQRILVVDPVTSE
HELTCQAEGYPKAEVIWTSSDHQVLSGKTTTTNSKREEKLFNVTSTLRINTTTNEIFYCTFRRLDPEE
NHTAELVIPELPLAHPPNER
SEQ ID NO. 122 (human PD-L1 ectodomain mutant 1E 58M/R113T/M115L/S117A/G119K)
SEQ ID NO. 123 (human PD-L1 ectodomain mutant 2I 54Q/E58M/R113T/M115L/S117A/G119K)
SEQ ID NO. 124 (human PD-L1 ectodomain mutant 3I 54Q/R113T/M115L/S117A/G119K)
SEQ ID NO. 125 (human PD-L1 ectodomain mutant 4I 54Q/E58M/M115L/S117A/G119K)
SEQ ID NO. 126 (human PD-L1 ectodomain mutant 5I 54Q/E58M/R113T/S117A/G119K)
SEQ ID NO. 127 (human PD-L1 ectodomain mutant 6I 54Q/E58M/R113T/M115L/G119K)
SEQ ID NO. 128 (human PD-L1 ectodomain mutant 7I 54Q/E58M/R113T/M115L/S117A)
SEQ ID NO. 129 (human PD-L1 ectodomain mutant 8I 54Q/Y56F/E58M/R113T/M115L/S117A/G119K)
SEQ ID NO. 130 (wild type human PD-L1 ectodomain-linker-hinge-IgG 1 Fc mutant 1; PD-L1 ectodomain underlined; linker bold and underlined; hinge bold)
SEQ ID NO. 131 (wild type human PD-L1 ectodomain-linker-hinge-IgG 1 Fc mutant 2; PD-L1 ectodomain underlined; linker bold and underlined; hinge bold)
SEQ ID NO. 132 (human PD-L1 ectodomain mutant 2I54Q/E58M/R113T/M115L/S117A/G119K ectodomain-linker-hinge-IgG 1 Fc mutant 1; PD-L1 ectodomain underlined; linker bold and underlined; hinge bold)
SEQ ID NO. 133 (human PD-L1 ectodomain mutant 7I54Q/E58M/R113T/M115L/S117A ectodomain-linker-hinge-IgG 1 Fc mutant 1; PD-L1 ectodomain underlined; linker bold and underlined; hinge bold)
SEQ ID NO. 134 (human PD-L1 ectodomain mutant 2I54Q/E58M/R113T/M115L/S117A/G119K ectodomain-linker-hinge-IgG 1 Fc mutant 2, PD-L1 ectodomain underlined; linker bold and underlined; hinge bold)
SEQ ID NO. 135 (human PD-L1 ectodomain mutant 7I54Q/E58M/R113T/M115L/S117A ectodomain-linker-hinge-IgG 1Fc mutant 2; PD-L1 ectodomain underlined; linker bold and underlined; hinge bold)
SEQ ID NO. 136 (wild type human CD 155)
MARAMAAAWPLLLVALLVLSWPPPGTGDVVVQAPTQVPGFLGDSVTLPCYLQVPNMEVTHVSQLTWARHGESGSMAVFHQTQGPSYSESKRLEFVAARLGAELRNASLRMFGLRVEDEGNYTCLFVTFPQGSRSVDIWLRVLAKPQNTAEVQKVQLTGEPVPMARCVSTGGRPPAQITWHSDLGGMPNTSQVPGFLSGTVTVTSLWILVPSSQVDGKNVTCKVEHESFEKPQLLTVNLTVYYPPEVSISGYDNNWYLGQNEATLTCDARSNPEPTGYNWSTTMGPLPPFAVAQGAQLLIRPVDKPINTTLICNVTNALGARQAELTVQVKEGPPSEHSGISRNAIIFLVLGILVFLILLGIGIYFYWSKCSREVLWHCHLCPSSTEHASASANGHVSYSAVSRENSSSQDPQTEGTR
SEQ ID NO. 137 (wild type human CD155 ectodomain)
WPPPGTGDVVVQAPTQVPGFLGDSVTLPCYLQVPNMEVTHVSQLTWARHGESGSMAVFHQTQGPSYSESKRLEFVAARLGAELRNASLRMFGLRVEDEGNYTCLFVTFPQGSRSVDIWLRVLAKPQNTAEVQKVQLTGEPVPMARCVSTGGRPPAQITWHSDLGGMPNTSQVPGFLSGTVTVTSLWILVPSSQVDGKNVTCKVEHESFEKPQLLTVNLTVYYPPEVSISGYDNNWYLGQNEATLTCDARSNPEPTGYNWSTTMGPLPPFAVAQGAQLLIRPVDKPINTTLICNVTNALGARQAELTVQVKEGPPSEHSGISRN
SEQ ID NO. 138 (human CD155 ectodomain-linker-hinge-IgG 1Fc mutant 1; CD155 ectodomain underlined; linker bold and underlined; hinge bold)
SEQ ID NO. 139 (human CD155 ectodomain-linker-hinge-IgG 1Fc mutant 2; CD155 ectodomain underlined; linker bold and underlined; hinge bold)
SEQ ID NO. 140 (IL-12B (p 40) mutant F60D subunit)
IWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTLTIQVKEDGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKNKTFLRCEAKNYSGRFTCWWLTTISTDLTFSVKSSRGSSDPQGVTCGAATLSAERVRGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFCVQVQGKSKREKKDRVFTDKTSATVICRKNASISVRAQDRYYSSSWSEWASVPCS
SEQ ID NO. 141 (wild type human CTLA-4 extracellular domain)
KAMHVAQPAVVLASSRGIASFVCEYASPGKATEVRVTVLRQADSQVTEVCAATYMMGNELTFLDDSICTGTSSGNQVNLTIQGLRAMDTGLYICKVELMYPPPYYLGIGNGTQIYVIDPEPCPDSD
SEQ ID NO. 142 (human PD-L2 ectodomain hinge portion-linker-Single chain IL-12 mutant heterodimeric IL-12B (p 40F 60A) -linker IL-12A (wt p 35) -C' hinge portion-IgG 1Fc mutant 1; VH underlined; hinge is bold, linker is bold and underlined; IL-12 subunit is italic)
/>
SEQ ID NO 143 (human PD-L2 ectodomain-linker-hinge portion-IgG 1Fc mutant 1-linker-Single chain IL-12 mutant heterodimeric IL-12B (p 40F 60A) -linker IL-12A (wt p 35); VH underlined; hinge bold; linker bold and underlined; IL-12 subunit italics)
SEQ ID NO. 144 (anti-PD-1 antibody VH-CH1-N 'hinge part-IL-2 mutant R38D/K43E/E61R-C' hinge part-IgG 1Fc mutant 1; VH underlined; hinge is bold; IL-2 mutant is italic)
SEQ ID NO. 145 (anti-PD-1 antibody VH-CH1-N' hinge portion-linker-single chain IL-23 mutant heterodimeric IL-12B (p 40)
E59A/F60A) -linker-IL-23A (wt p 19) -C' hinge portion-IgG 1Fc mutant 1; VH bands underlined; the hinge is bold; the joints are bold and underlined; IL-23 subunit is italic
/>
SEQ ID NO. 146 (anti-PD-1 antibody VH-CH1-N 'hinge portion-linker-single chain IL-10 mutant R27A homodimer-C' hinge portion-IgG 1Fc mutant 1; VH underlined; hinge bold; linker bold and underlined; IL-10 mutant monomer italic)
SEQ ID NO. 147 (anti-PD-1 antibody VH-CH1-N 'hinge part-linker-single chain IFN-gamma mutant A23V homodimer-C' hinge part-IgG 1Fc mutant 1; VH underlined; hinge bold; linker bold and underlined; IFN-gamma mutant monomer italic)
SEQ ID NO. 148 (anti-PD-1 Ab VH-CH1-N 'hinge part-linker-IFN-. Alpha.2b mutant L30A-C' hinge part-IgG 1Fc mutant 1; VH underlined; hinge is bold, linker is bold and underlined; IFN-. Alpha.2b mutant is italic)
SEQ ID NO. 149 (anti-PD-1 Ab VH (D100N) -CH1-N 'hinge part-linker-Single chain IL-12 mutant heterodimeric IL-12B (p 40E 59A/F60A) -linker IL-12A (wt p 35) -C' hinge part-IgG 1 Fc mutant 1; VH underlined; hinge is bold; linker is bold and underlined; IL-12 subunit is italic)
SEQ ID NO. 150 (anti-PD-1 Ab VH (D100G) -CH1-N 'hinge part-linker-Single chain IL-12 mutant heterodimeric IL-12B (p 40E 59A/F60A) -linker IL-12A (wt p 35) -C' hinge part-IgG 1 Fc mutant 1; VH underlined; hinge is bold; linker is bold and underlined; IL-12 subunit is italic)
SEQ ID NO. 151 (anti-PD-1 Ab VH (D100R) -CH1-N 'hinge portion-linker-Single chain IL-12 mutant heterodimeric IL-12B (p 40E 59A/F60A) -linker IL-12A (wt p 35) -C' hinge portion-IgG 1 Fc mutant 1; VH underlined; hinge is bold; linker is bold and underlined; IL-12 subunit is italic)
SEQ ID NO. 152 (anti-PD-1 Ab VH (N99G) -CH1-N 'hinge portion-linker-Single chain IL-12 mutant heterodimeric IL-12B (p 40E 59A/F60A) -linker IL-12A (wt p 35) -C' hinge portion-IgG 1 Fc mutant 1; VH underlined; hinge is bold; linker is bold and underlined; IL-12 subunit is italic)
SEQ ID NO 153 (anti-PD-1 Ab VH (N99A) -CH1-N 'hinge part-linker-Single chain IL-12 mutant heterodimeric IL-12B (p 40E 59A/F60A) -linker IL-12A (wt p 35) -C' hinge part-IgG 1 Fc mutant 1; VH underlined; hinge is bold; linker is bold and underlined; IL-12 subunit is italic)
SEQ ID NO. 154 (anti-PD-1 Ab VH (N99M) -CH1-N 'hinge portion-linker-Single chain IL-12 mutant heterodimeric IL-12B (p 40E 59A/F60A) -linker IL-12A (wt p 35) -C' hinge portion-IgG 1 Fc mutant 1; VH underlined; hinge is bold; linker is bold and underlined; IL-12 subunit is italic)
SEQ ID NO. 155 (human PD-L1 ectodomain mutant 2I54Q/E58M/R113T/M115L/S117A/G119K ectodomain-linker-single chain IL-12 mutant heterodimeric IL-12B (p 40E 59A/F60A) -linker IL-12A (wt p 35) -hinge-IgG 1 Fc mutant 1; PD-L1 ectodomain underlined; linker bold and underlined; hinge bold; IL-12 subunit italics)
SEQ ID NO. 156 (human PD-L1 ectodomain mutant 7I54Q/E58M/R113T/M115L/S117A ectodomain-linker-Single chain IL-12 mutant heterodimeric IL-12B (p 40E 59A/F60A) -linker IL-12A (wt p 35) -hinge-IgG 1 Fc mutant 1; PD-L1 ectodomain underlined; linker bold and underlined; hinge bold; IL-12 subunit italics)
SEQ ID NO. 157 (human PD-L1 ectodomain mutant 2I54Q/E58M/R113T/M115L/S117A/G119K ectodomain-linker-hinge-IgG 1 Fc mutant 1-linker-single chain IL-12 mutant heterodimeric IL-12B (p 40E 59A/F60A) -linker IL-12A (wt p 35); PD-L1 ectodomain underlined; linker bold and underlined; hinge bold; IL-12 subunit italics)
/>
SEQ ID NO. 158 (human PD-L1 ectodomain mutant 7I54Q/E58M/R113T/M115L/S117A ectodomain-linker-hinge-IgG 1 Fc mutant 1-linker-single chain IL-12 mutant heterodimeric IL-12B (p 40E 59A/F60A) -linker IL-12A (wt p 35); PD-L1 ectodomain underlined; linker bold and underlined; hinge bold; IL-12 subunit italics)
SEQ ID NO. 159 (human PD-L1 ectodomain mutant 2I54Q/E58M/R113T/M115L/S117A/G119K ectodomain-linker-IL-2 mutant (L18R/Q22E/R38D/K43E/E61R) -linker-hinge-IgG 1 Fc mutant 2; PD-L1 ectodomain underlined; linker bold and underlined; hinge bold; IL-12 subunit italics)
SEQ ID NO. 160 (human PD-L1 ectodomain mutant 2I54Q/E58M/R113T/M115L/S117A/G119K ectodomain-linker-IL-2 mutant (R38D/K43E/E61R/Q126T) -linker-hinge-IgG 1 Fc mutant 2; PD-L1 ectodomain underlined; linker bold and underlined; hinge bold; IL-12 subunit italics)
SEQ ID NO. 161 (human PD-L1 ectodomain mutant 2I54Q/E58M/R113T/M115L/S117A/G119K ectodomain-linker-IL-2 mutant (L18R/Q22E/R38D/K43E/E61R/Q126T) -linker-hinge-IgG 1 Fc mutant 2; PD-L1 ectodomain underlined; linker bold and underlined; hinge bold; IL-12 subunit italics)
SEQ ID NO. 162 (human PD-L1 ectodomain mutant 2I54Q/E58M/R113T/M115L/S117A/G119K ectodomain-linker-IL-2 mutant (L18R/Q22E/R38D/K43E/E61R/Q126T/S130R) -linker-hinge-IgG 1 Fc mutant 2; PD-L1 ectodomain underlined; linker bold and underlined; hinge bold; IL-12 subunit italics)
SEQ ID NO. 163 (human PD-L1 ectodomain mutant 7I54Q/E58M/R113T/M115L/S117A/G119K ectodomain-linker-IL-2 mutant (L18R/Q22E/R38D/K43E/E61R) -linker-hinge-IgG 1 Fc mutant 2; PD-L1 ectodomain underlined; linker bold and underlined; hinge bold; IL-12 subunit italics)
SEQ ID NO. 164 (human PD-L1 ectodomain mutant 7I54Q/E58M/R113T/M115L/S117A ectodomain-linker-IL-2 mutant (R38D/K43E/E61R/Q126T) -linker-hinge-IgG 1 Fc mutant 2, PD-L1 ectodomain underlined; linker bold and underlined; hinge bold; IL-12 subunit italics)
SEQ ID NO. 165 (human PD-L1 ectodomain mutant 7I54Q/E58M/R113T/M115L/S117A ectodomain-linker-IL-2 mutant (L18R/Q22E/R38D/K43E/E61R/Q126T) -linker-hinge-IgG 1 Fc mutant 2; PD-L1 ectodomain underlined; linker bold and underlined; hinge bold; IL-12 subunit italics)
SEQ ID NO. 166 (human PD-L1 ectodomain mutant 7I54Q/E58M/R113T/M115L/S117A ectodomain-linker-IL-2 mutant (L18R/Q22E/R38D/K43E/E61R/Q126T/S130R) -linker-hinge-IgG 1 Fc mutant 2; PD-L1 ectodomain underlined; linker bold and underlined; hinge bold; IL-12 subunit italics)
167 (human PD-L2 ectodomain mutant 2S58V ectodomain-linker-Single chain IL-12 mutant heterodimeric IL-12B (p 40)
E59A/F60A) -linker IL-12A (wt p 35) -hinge-IgG 1 Fc mutant 1; the PD-L2 ectodomain is underlined; the joints are bold and underlined; the hinge is bold; IL-12 subunit is italic
SEQ ID NO. 168 (human PD-L2 ectodomain mutant 4T56V/S58V/Q60L ectodomain-linker-Single chain IL-12 mutant heterodimeric IL-12B (p 40E 59A/F60A) -linker IL-12A (wt p 35) -hinge-IgG 1 Fc mutant 1; PD-L2 ectodomain underlined; linker bold and underlined; hinge bold; IL-12 subunit italics)
SEQ ID NO. 169 (human PD-L2 ectodomain mutant 2S58V ectodomain-linker-hinge-IgG 1 Fc mutant 1-linker-single chain IL-12 mutant heterodimeric IL-12B (p 40E 59A/F60A) -linker IL-12A (wt p 35); PD-L2 ectodomain underlined; linker bold and underlined; hinge bold; IL-12 subunit italics)
SEQ ID NO. 170 (human PD-L2 ectodomain mutant 4T56V/S58V/Q60L ectodomain-linker-hinge-IgG 1 Fc mutant 1-linker-single chain IL-12 mutant heterodimeric IL-12B (p 40E 59A/F60A) -linker IL-12A (wt p 35); PD-L2 ectodomain underlined; linker bold and underlined; hinge bold; IL-12 subunit italics)
SEQ ID NO. 171 (human PD-L2 ectodomain mutant 2S58V ectodomain-linker-IL-2 mutant (L18R/Q22E/R38D/K43E/E61R) -linker-hinge-IgG 1 Fc mutant 2; PD-L2 ectodomain underlined; linker bold and underlined; hinge bold; IL-12 subunit italics)
SEQ ID NO. 172 (human PD-L2 ectodomain mutant 2S58V ectodomain-linker-IL-2 mutant (R38D/K43E/E61R/Q126T) -linker-hinge-IgG 1 Fc mutant 2; PD-L2 ectodomain underlined; linker bold and underlined; hinge bold; IL-12 subunit italics)
SEQ ID NO. 173 (human PD-L2 ectodomain mutant 2S58V ectodomain-linker-IL-2 mutant (L18R/Q22E/R38D/K43E/E61R/Q126T) -linker-hinge-IgG 1 Fc mutant 2; PD-L2 ectodomain underlined; linker bold and underlined; hinge bold; IL-12 subunit italics)
SEQ ID NO. 174 (human PD-L2 ectodomain mutant 2S58V ectodomain-linker-IL-2 mutant (R38D/K43E/E61R) (L18R/Q22E/Q126T/S130R) -linker-hinge-IgG 1 Fc mutant 2; PD-L2 ectodomain underlined; linker bold and underlined; hinge bold; IL-12 subunit italics)
SEQ ID NO. 175 (human PD-L2 ectodomain mutant 4T56V/S58V/Q60L ectodomain-linker-IL-2 mutant (L18R/Q22E/R38D/K43E/E61R) -linker-hinge-IgG 1 Fc mutant 2; PD-L2 ectodomain underlined; linker bold and underlined; hinge bold; IL-12 subunit italics)
SEQ ID NO. 176 (human PD-L2 ectodomain mutant 4T56V/S58V/Q60L ectodomain-linker-IL-2 mutant (R38D/K43E/E61R/Q126T) -linker-hinge-IgG 1 Fc mutant 2; PD-L2 ectodomain underlined; linker bold and underlined; hinge bold; IL-12 subunit italics)
SEQ ID NO. 177 (human PD-L2 mutant 4T56V/S58V/Q60L ectodomain-linker-IL-2 mutant (L18R/Q22E/R38D/K43E/E61R/Q126T) -linker-hinge-IgG 1 Fc mutant 2; PD-L2 ectodomain underlined; linker bold and underlined; hinge bold; IL-12 subunit italics)
SEQ ID NO. 178 (human PD-L2 mutant 4T56V/S58V/Q60L ectodomain-linker-IL-2 mutant (L18R/Q22E/R38D/K43E/E61R/Q126T/S130R) -linker-hinge-IgG 1 Fc mutant 2; PD-L2 ectodomain underlined; linker bold and underlined; hinge bold; IL-12 subunit italics)
SEQ ID NO. 179 (human PD-L1 mutant 2I54Q/E58M/R113T/M115L/S117A/G119K ectodomain-linker-IL-2 mutant (R38D/K43E/E61R) linker-hinge-IgG 1 Fc mutant 2; PD-L1 ectodomain underlined; linker bold and underlined; hinge bold; IL-12 subunit italics)
SEQ ID NO. 180 (human PD-L1 ectodomain mutant 2I54Q/E58M/R113T/M115L/S117A/G119K ectodomain-linker-Single chain mouse IL-12 mutant heterodimeric IL-12B (p 40E 59A/F60A) -and hunger' S figure IL-12A (wt p 35) -hinge-IgG 1 Fc mutant 1; PD-L1 ectodomain underlined; linker bold and underlined; hinge bold; mouse IL-12 subunit italics)
SEQ ID NO 181 (human PD-L1 ectodomain mutant 2I54Q/E58M/R113T/M115L/S117A/G119K ectodomain-linker-hinge-IgG 1 Fc mutant 1-linker-single chain mouse IL-12 mutant heterodimeric IL-12B (p 40E 59A/F60A) -linker IL-12A (wt p 35); PD-L1 ectodomain underlined; linker bold and underlined; hinge bold; mouse IL-12 subunit italics)
SEQ ID NO. 182 (human PD-L1 ectodomain mutant 2I54Q/E58M/R113T/M115L/S117A/G119K ectodomain-linker-single chain mutant homodimer IFN-. Gamma. (A23V/A23V) -linker hinge-IgG 1 Fc mutant 1; PD-L1 ectodomain underlined; linker bold and underlined; hinge bold; IFN-. Gamma. Italics)
SEQ ID NO. 183 (human PD-L1 ectodomain mutant 7I54Q/E58M/R113T/M115L/S117A ectodomain-linker-single chain mutant homodimer IFN-gamma (A23V/A23V) -linker hinge-IgG 1 Fc mutant 1; PD-L1 ectodomain underlined; linker bold and underlined; hinge bold; IFN-gamma italic)
SEQ ID NO. 184 (human PD-L1 ectodomain mutant 2I54Q/E58M/R113T/M115L/S117A/G119K ectodomain-linker-single chain mutant homodimer IFN-. Gamma. (A23V/A23V) -linker-hinge-IgG 1 Fc mutant 2; PD-L1 is an underlined ectodomain; linker is shaded; hinge is bold; IFN-. Gamma. Is italic)
SEQ ID NO. 185 (human PD-L1 ectodomain mutant 7I54Q/E58M/R113T/M115L/S117A/G119K ectodomain-linker-single chain mutant homodimer IFN-. Gamma. (A23V/A23V) -linker-hinge-IgG 1 Fc mutant 2; PD-L1 ectodomain underlined; linker bold and underlined; hinge bold; IFN-. Gamma. Italics)
SEQ ID NO. 186 (human PD-L2 ectodomain mutant 2S58V ectodomain-linker-single chain mutant homodimer IFN-gamma (A23V/A23V) -linker-hinge-IgG 1 Fc mutant 1; PD-L2 ectodomain underlined; linker bold and underlined; hinge bold; IFN-gamma italics)
SEQ ID NO. 187 (human PD-L2 ectodomain mutant 4T56V/S58V/Q60L ectodomain-linker-single chain mutant homodimer IFN-gamma (A23V/A23V) -linker-hinge-IgG 1 Fc mutant 1; PD-L2 ectodomain underlined; linker bold and underlined; hinge bold; IFN-gamma italic)
SEQ ID NO. 188 (human PD-L2 ectodomain mutant 2S58V ectodomain-linker-single chain mutant homodimer IFN-gamma (A23V/A23V) -linker-hinge-IgG 1 Fc mutant 2; PD-L2 ectodomain underlined; linker bold and underlined; hinge bold; IFN-gamma italic)
SEQ ID NO. 189 (human PD-L2 ectodomain mutant 4T56V/S58V/Q60L ectodomain-linker-single chain mutant homodimer IFN-gamma (A23V/A23V) -linker-hinge-IgG 1 Fc mutant 2; PD-L2 ectodomain underlined; linker bold and underlined; hinge bold; IFN-gamma italic)
SEQ ID NO. 190 (human CD155 ectodomain-linker-Single chain IL-12 mutant heterodimeric IL-12B (p 40E 59A/F60A) -linker IL-12A (wt p 35) -hinge-IgG 1 Fc mutant 1; CD155 ectodomain underlined; linker bold and underlined; hinge bold; IL-12 subunit italics)
SEQ ID NO. 191 (human CD155 ectodomain-linker-hinge-IgG 1 Fc mutant 1-linker-single chain IL-12 mutant heterodimeric IL-12B (p 40E 59A/F60A) -linker IL-12A (wt p 35); CD155 ectodomain underlined; linker bold and underlined; hinge bold; IL-12 subunit italics)
/>
SEQ ID NO. 192 (human CD155 ectodomain-linker-single chain mutant homodimer IFN-. Gamma. (A23V/A23V) -linker hinge-IgG 1)
Fc mutant 1; CD155 ectodomain underlined; the joints are bold and underlined; the hinge is bold; IFN-gamma is italic
SEQ ID NO 193 (human CD155 ectodomain-linker-Single chain mutant homodimer IFN-. Gamma. (A23V/A23V) -linker-hinge-IgG 1)
Fc mutant 2; CD155 ectodomain underlined; the joints are bold and underlined; the hinge is bold; IFN-gamma is italic
SEQ ID NO. 194 (Joint; n is an integer of at least 1)
(G) n
SEQ ID NO. 195 (Joint; n is an integer of at least 1)
(GS) n
SEQ ID NO 196 (linker; n is an integer of at least 1)
(GGS) n
SEQ ID NO 197 (Joint; n is an integer of at least 1)
(GGGS) n
SEQ ID NO. 198 (linker; n is an integer of at least 1)
(GGS) n (GGGS) n
SEQ ID NO 199 (linker; n is an integer of at least 1)
(GSGGS) n
SEQ ID NO. 200 (Joint; n is an integer of at least 1)
(GGSGS) n
SEQ ID NO. 201 (linker; n is an integer of at least 1)
(GGGGS) n
SEQ ID NO 202 (Joint)
GG
SEQ ID NO. 203 (Joint)
GSG
SEQ ID NO. 204 (Joint)
GGSG
SEQ ID NO. 205 (Joint)
GGSGG
SEQ ID NO. 206 (Joint)
GSGGGGG
SEQ ID NO. 207 (Joint)
GSGSG
SEQ ID NO. 208 (Joint)
GSGGG
SEQ ID NO. 209 (Joint)
GGGSG
SEQ ID NO. 210 (Joint)
GSSSG
SEQ ID NO. 211 (Joint)
GGSGGS
SEQ ID NO. 212 (Joint)
SGGGGS
SEQ ID NO. 213 (Joint)
GGGGS
SEQ ID NO. 214 (linker; n is an integer of at least 1)
(GA) n
SEQ ID NO. 215 (Joint)
GRAGGGGAGGGG
SEQ ID NO. 216 (Joint)
GRAGGG
SEQ ID NO 217 (Joint)
GSGGGSGGGGSGGGGS
SEQ ID NO. 218 (Joint)
GGGSGGGGSGGGGS
SEQ ID NO 219 (Joint)
GGGSGGSGGS
SEQ ID NO. 220 (Joint)
GGSGGSGGSGGSGGG
SEQ ID NO. 221 (Joint)
GGSGGSGGGGSGGGGS
SEQ ID NO. 222 (Joint)
GGSGGSGGSGGSGGSGGS
SEQ ID NO. 223 (Joint)
GGGGGGSGGGGSGGGGSA
SEQ ID NO. 224 (Joint)
GSGGGSGGGGSGGGGSGGGGS
SEQ ID NO 225 (Joint)
KTGGGSGGGS
SEQ ID NO. 226 (Joint)
GGPGGGGSGGGSGGGGS
SEQ ID NO 227 (Joint)
GGGSGGGGSGGGGSGGGGS
SEQ ID NO. 228 (Joint)
GGGGSGGGGSGGGGSGGGGSG
SEQ ID NO 229 (Joint)
GGGGSGGGGSGGGGS
SEQ ID NO. 230 (Joint)
ASTKGP
SEQ ID NO. 231 (Joint)
DKP
SEQ ID NO. 232 (Joint)
DKPGS
SEQ ID NO. 233 (Joint)
PGS
SEQ ID NO. 234 (Joint)
GS
SEQ ID NO. 235 (Joint)
DKPGSG
SEQ ID NO. 236 (Joint)
PGSG
SEQ ID NO. 237 (Joint)
DKPGSGS
SEQ ID NO. 238 (Joint)
PGSGS
SEQ ID NO 239 (Joint)
GSGS
SEQ ID NO. 240 (Joint)
DKPGSGGGGG
SEQ ID NO 241 (Joint)
PGSGGGGG
SEQ ID NO. 242 (Joint)
P
SEQ ID NO:243
GGGGSGGGSGGGG
SEQ ID NO:244
GGGGSGGG
SEQ ID NO:245
GGPGGGGSGGGSGGGG
SEQ ID NO:246
GGGGSGGGSGGGGS
SEQ ID NO. 247 (human CD155 ectodomain-linker-IL-2 mutant (L18R/Q22E/R38D/K43E/E61R) -linker-hinge-IgG 1 Fc mutant 2; CD155 ectodomain underlined; linker bold and underlined; hinge bold; IL-12 subunit italics)
SEQ ID NO. 248 (human CD155 ectodomain-linker-IL-2 mutant (R38D/K43E/E61R/Q126T) -linker-hinge-IgG 1 Fc mutant 2; CD155 ectodomain underlined; linker bold and underlined; hinge bold; IL-12 subunit italics)
SEQ ID NO. 249 (human CD155 ectodomain-linker-IL-2 mutant (L18R/Q22E/R38D/K43E/E61R/Q126T) -linker-hinge-IgG 1 Fc mutant 2; CD155 ectodomain underlined; linker bold and underlined; hinge bold; IL-12 subunit italics)
SEQ ID NO. 250 (human CD155 ectodomain-linker-IL-2 mutant (L18R/Q22E/R38D/K43E/E61 RQ 126T/S130R) -linker-hinge-IgG 1 Fc mutant 2; CD155 ectodomain underlined; linker bold and underlined; hinge bold; IL-12 subunit italics)
SEQ ID NO. 251 (Single-chain "wild type" IFN-. Gamma.homodimer; linker in bold; wild type IFN-. Gamma.monomer in italics)
SEQ ID NO. 252 (Single chain IFN-. Gamma.mutant A23V homodimer; linker bold; IFN-. Gamma.mutant monomer italic)
SEQ ID NO 253 (Single-chain "wild type" IL-12 heterodimer IL-12B (wt p 40) -linker-IL-12A (wt p 35); linker in bold)
SEQ ID NO. 254 (Single chain IL-12 mutant heterodimeric IL-12B (p 40E 59A/F60A) -linker-IL-12A (wt p 35); linker in bold, IL-12 subunit in italics)
/>
Sequence listing
<110> immune wakeup Co
<120> immunoregulatory molecules and uses thereof
<130> PG03302A
<140> not yet allocated
<141> at the same time
<150> PCT/US2021/073107
<151> 2021-12-23
<150> US 63/159,441
<151> 2021-03-10
<160> 254
<170> FastSEQ Windows version 4.0
<210> 1
<211> 356
<212> PRT
<213> Homo sapiens (Homo sapiens)
<400> 1
Lys Ala Met His Val Ala Gln Pro Ala Val Val Leu Ala Ser Ser Arg
1 5 10 15
Gly Ile Ala Ser Phe Val Cys Glu Tyr Ala Ser Pro Gly Lys Ala Thr
20 25 30
Glu Val Arg Val Thr Val Leu Arg Gln Ala Asp Ser Gln Val Thr Glu
35 40 45
Val Cys Ala Ala Thr Tyr Met Met Gly Asn Glu Leu Thr Phe Leu Asp
50 55 60
Asp Ser Ile Cys Thr Gly Thr Ser Ser Gly Asn Gln Val Asn Leu Thr
65 70 75 80
Ile Gln Gly Leu Arg Ala Met Asp Thr Gly Leu Tyr Ile Cys Lys Val
85 90 95
Glu Leu Met Tyr Pro Pro Pro Tyr Tyr Leu Gly Ile Gly Asn Gly Thr
100 105 110
Gln Ile Tyr Val Ile Asp Pro Glu Pro Cys Pro Asp Ser Asp Gly Ser
115 120 125
Gly Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Lys Lys
130 135 140
Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu
145 150 155 160
Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser
165 170 175
His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu
180 185 190
Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr
195 200 205
Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn
210 215 220
Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro
225 230 235 240
Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln
245 250 255
Val Tyr Val Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val
260 265 270
Ser Leu Leu Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val
275 280 285
Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Arg Tyr Met Thr Trp Pro
290 295 300
Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr
305 310 315 320
Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val
325 330 335
Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu
340 345 350
Ser Pro Gly Lys
355
<210> 2
<211> 895
<212> PRT
<213> Homo sapiens (Homo sapiens)
<400> 2
Lys Ala Met His Val Ala Gln Pro Ala Val Val Leu Ala Ser Ser Arg
1 5 10 15
Gly Ile Ala Ser Phe Val Cys Glu Tyr Ala Ser Pro Gly Lys Ala Thr
20 25 30
Glu Val Arg Val Thr Val Leu Arg Gln Ala Asp Ser Gln Val Thr Glu
35 40 45
Val Cys Ala Ala Thr Tyr Met Met Gly Asn Glu Leu Thr Phe Leu Asp
50 55 60
Asp Ser Ile Cys Thr Gly Thr Ser Ser Gly Asn Gln Val Asn Leu Thr
65 70 75 80
Ile Gln Gly Leu Arg Ala Met Asp Thr Gly Leu Tyr Ile Cys Lys Val
85 90 95
Glu Leu Met Tyr Pro Pro Pro Tyr Tyr Leu Gly Ile Gly Asn Gly Thr
100 105 110
Gln Ile Tyr Val Ile Asp Pro Glu Pro Cys Pro Asp Ser Asp Gly Ser
115 120 125
Gly Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Asp Glu
130 135 140
Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu
145 150 155 160
Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser
165 170 175
His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu
180 185 190
Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr
195 200 205
Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn
210 215 220
Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro
225 230 235 240
Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln
245 250 255
Val Tyr Val Tyr Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val
260 265 270
Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val
275 280 285
Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro
290 295 300
Pro Val Leu Asp Glu Asp Gly Ser Phe Ala Leu Val Ser Lys Leu Thr
305 310 315 320
Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val
325 330 335
Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu
340 345 350
Ser Pro Gly Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly
355 360 365
Gly Gly Ser Ile Trp Glu Leu Lys Lys Asp Val Tyr Val Val Glu Leu
370 375 380
Asp Trp Tyr Pro Asp Ala Pro Gly Glu Met Val Val Leu Thr Cys Asp
385 390 395 400
Thr Pro Glu Glu Asp Gly Ile Thr Trp Thr Leu Asp Gln Ser Ser Glu
405 410 415
Val Leu Gly Ser Gly Lys Thr Leu Thr Ile Gln Val Lys Ala Ala Gly
420 425 430
Asp Ala Gly Gln Tyr Thr Cys His Lys Gly Gly Glu Val Leu Ser His
435 440 445
Ser Leu Leu Leu Leu His Lys Lys Glu Asp Gly Ile Trp Ser Thr Asp
450 455 460
Ile Leu Lys Asp Gln Lys Glu Pro Lys Asn Lys Thr Phe Leu Arg Cys
465 470 475 480
Glu Ala Lys Asn Tyr Ser Gly Arg Phe Thr Cys Trp Trp Leu Thr Thr
485 490 495
Ile Ser Thr Asp Leu Thr Phe Ser Val Lys Ser Ser Arg Gly Ser Ser
500 505 510
Asp Pro Gln Gly Val Thr Cys Gly Ala Ala Thr Leu Ser Ala Glu Arg
515 520 525
Val Arg Gly Asp Asn Lys Glu Tyr Glu Tyr Ser Val Glu Cys Gln Glu
530 535 540
Asp Ser Ala Cys Pro Ala Ala Glu Glu Ser Leu Pro Ile Glu Val Met
545 550 555 560
Val Asp Ala Val His Lys Leu Lys Tyr Glu Asn Tyr Thr Ser Ser Phe
565 570 575
Phe Ile Arg Asp Ile Ile Lys Pro Asp Pro Pro Lys Asn Leu Gln Leu
580 585 590
Lys Pro Leu Lys Asn Ser Arg Gln Val Glu Val Ser Trp Glu Tyr Pro
595 600 605
Asp Thr Trp Ser Thr Pro His Ser Tyr Phe Ser Leu Thr Phe Cys Val
610 615 620
Gln Val Gln Gly Lys Ser Lys Arg Glu Lys Lys Asp Arg Val Phe Thr
625 630 635 640
Asp Lys Thr Ser Ala Thr Val Ile Cys Arg Lys Asn Ala Ser Ile Ser
645 650 655
Val Arg Ala Gln Asp Arg Tyr Tyr Ser Ser Ser Trp Ser Glu Trp Ala
660 665 670
Ser Val Pro Cys Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly
675 680 685
Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Arg Asn Leu Pro Val Ala
690 695 700
Thr Pro Asp Pro Gly Met Phe Pro Cys Leu His His Ser Gln Asn Leu
705 710 715 720
Leu Arg Ala Val Ser Asn Met Leu Gln Lys Ala Arg Gln Thr Leu Glu
725 730 735
Phe Tyr Pro Cys Thr Ser Glu Glu Ile Asp His Glu Asp Ile Thr Lys
740 745 750
Asp Lys Thr Ser Thr Val Glu Ala Cys Leu Pro Leu Glu Leu Thr Lys
755 760 765
Asn Glu Ser Cys Leu Asn Ser Arg Glu Thr Ser Phe Ile Thr Asn Gly
770 775 780
Ser Cys Leu Ala Ser Arg Lys Thr Ser Phe Met Met Ala Leu Cys Leu
785 790 795 800
Ser Ser Ile Tyr Glu Asp Leu Lys Met Tyr Gln Val Glu Phe Lys Thr
805 810 815
Met Asn Ala Lys Leu Leu Met Asp Pro Lys Arg Gln Ile Phe Leu Asp
820 825 830
Gln Asn Met Leu Ala Val Ile Asp Glu Leu Met Gln Ala Leu Asn Phe
835 840 845
Asn Ser Glu Thr Val Pro Gln Lys Ser Ser Leu Glu Glu Pro Asp Phe
850 855 860
Tyr Lys Thr Lys Ile Lys Leu Cys Ile Leu Leu His Ala Phe Arg Ile
865 870 875 880
Arg Ala Val Thr Ile Asp Arg Val Met Ser Tyr Leu Asn Ala Ser
885 890 895
<210> 3
<211> 895
<212> PRT
<213> Homo sapiens (Homo sapiens)
<400> 3
Lys Ala Met His Val Ala Gln Pro Ala Val Val Leu Ala Ser Ser Arg
1 5 10 15
Gly Ile Ala Ser Phe Val Cys Glu Tyr Ala Ser Pro Gly Lys Ala Thr
20 25 30
Glu Val Arg Val Thr Val Leu Arg Gln Ala Asp Ser Gln Val Thr Glu
35 40 45
Val Cys Ala Ala Thr Tyr Met Met Gly Asn Glu Leu Thr Phe Leu Asp
50 55 60
Asp Ser Ile Cys Thr Gly Thr Ser Ser Gly Asn Gln Val Asn Leu Thr
65 70 75 80
Ile Gln Gly Leu Arg Ala Met Asp Thr Gly Leu Tyr Ile Cys Lys Val
85 90 95
Glu Leu Met Tyr Pro Pro Pro Tyr Tyr Leu Gly Ile Gly Asn Gly Thr
100 105 110
Gln Ile Tyr Val Ile Asp Pro Glu Pro Cys Pro Asp Ser Asp Gly Ser
115 120 125
Gly Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Asp Glu
130 135 140
Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu
145 150 155 160
Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser
165 170 175
His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu
180 185 190
Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr
195 200 205
Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn
210 215 220
Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro
225 230 235 240
Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln
245 250 255
Val Tyr Val Tyr Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val
260 265 270
Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val
275 280 285
Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro
290 295 300
Pro Val Leu Asp Glu Asp Gly Ser Phe Ala Leu Val Ser Lys Leu Thr
305 310 315 320
Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val
325 330 335
Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu
340 345 350
Ser Pro Gly Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly
355 360 365
Gly Gly Ser Ile Trp Glu Leu Lys Lys Asp Val Tyr Val Val Glu Leu
370 375 380
Asp Trp Tyr Pro Asp Ala Pro Gly Glu Met Val Val Leu Thr Cys Asp
385 390 395 400
Thr Pro Glu Glu Asp Gly Ile Thr Trp Thr Leu Asp Gln Ser Ser Glu
405 410 415
Val Leu Gly Ser Gly Lys Thr Leu Thr Ile Gln Val Lys Glu Ala Gly
420 425 430
Asp Ala Gly Gln Tyr Thr Cys His Lys Gly Gly Glu Val Leu Ser His
435 440 445
Ser Leu Leu Leu Leu His Lys Lys Glu Asp Gly Ile Trp Ser Thr Asp
450 455 460
Ile Leu Lys Asp Gln Lys Glu Pro Lys Asn Lys Thr Phe Leu Arg Cys
465 470 475 480
Glu Ala Lys Asn Tyr Ser Gly Arg Phe Thr Cys Trp Trp Leu Thr Thr
485 490 495
Ile Ser Thr Asp Leu Thr Phe Ser Val Lys Ser Ser Arg Gly Ser Ser
500 505 510
Asp Pro Gln Gly Val Thr Cys Gly Ala Ala Thr Leu Ser Ala Glu Arg
515 520 525
Val Arg Gly Asp Asn Lys Glu Tyr Glu Tyr Ser Val Glu Cys Gln Glu
530 535 540
Asp Ser Ala Cys Pro Ala Ala Glu Glu Ser Leu Pro Ile Glu Val Met
545 550 555 560
Val Asp Ala Val His Lys Leu Lys Tyr Glu Asn Tyr Thr Ser Ser Phe
565 570 575
Phe Ile Arg Asp Ile Ile Lys Pro Asp Pro Pro Lys Asn Leu Gln Leu
580 585 590
Lys Pro Leu Lys Asn Ser Arg Gln Val Glu Val Ser Trp Glu Tyr Pro
595 600 605
Asp Thr Trp Ser Thr Pro His Ser Tyr Phe Ser Leu Thr Phe Cys Val
610 615 620
Gln Val Gln Gly Lys Ser Lys Arg Glu Lys Lys Asp Arg Val Phe Thr
625 630 635 640
Asp Lys Thr Ser Ala Thr Val Ile Cys Arg Lys Asn Ala Ser Ile Ser
645 650 655
Val Arg Ala Gln Asp Arg Tyr Tyr Ser Ser Ser Trp Ser Glu Trp Ala
660 665 670
Ser Val Pro Cys Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly
675 680 685
Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Arg Asn Leu Pro Val Ala
690 695 700
Thr Pro Asp Pro Gly Met Phe Pro Cys Leu His His Ser Gln Asn Leu
705 710 715 720
Leu Arg Ala Val Ser Asn Met Leu Gln Lys Ala Arg Gln Thr Leu Glu
725 730 735
Phe Tyr Pro Cys Thr Ser Glu Glu Ile Asp His Glu Asp Ile Thr Lys
740 745 750
Asp Lys Thr Ser Thr Val Glu Ala Cys Leu Pro Leu Glu Leu Thr Lys
755 760 765
Asn Glu Ser Cys Leu Asn Ser Arg Glu Thr Ser Phe Ile Thr Asn Gly
770 775 780
Ser Cys Leu Ala Ser Arg Lys Thr Ser Phe Met Met Ala Leu Cys Leu
785 790 795 800
Ser Ser Ile Tyr Glu Asp Leu Lys Met Tyr Gln Val Glu Phe Lys Thr
805 810 815
Met Asn Ala Lys Leu Leu Met Asp Pro Lys Arg Gln Ile Phe Leu Asp
820 825 830
Gln Asn Met Leu Ala Val Ile Asp Glu Leu Met Gln Ala Leu Asn Phe
835 840 845
Asn Ser Glu Thr Val Pro Gln Lys Ser Ser Leu Glu Glu Pro Asp Phe
850 855 860
Tyr Lys Thr Lys Ile Lys Leu Cys Ile Leu Leu His Ala Phe Arg Ile
865 870 875 880
Arg Ala Val Thr Ile Asp Arg Val Met Ser Tyr Leu Asn Ala Ser
885 890 895
<210> 4
<211> 880
<212> PRT
<213> Homo sapiens (Homo sapiens)
<400> 4
Lys Ala Met His Val Ala Gln Pro Ala Val Val Leu Ala Ser Ser Arg
1 5 10 15
Gly Ile Ala Ser Phe Val Cys Glu Tyr Ala Ser Pro Gly Lys Ala Thr
20 25 30
Glu Val Arg Val Thr Val Leu Arg Gln Ala Asp Ser Gln Val Thr Glu
35 40 45
Val Cys Ala Ala Thr Tyr Met Met Gly Asn Glu Leu Thr Phe Leu Asp
50 55 60
Asp Ser Ile Cys Thr Gly Thr Ser Ser Gly Asn Gln Val Asn Leu Thr
65 70 75 80
Ile Gln Gly Leu Arg Ala Met Asp Thr Gly Leu Tyr Ile Cys Lys Val
85 90 95
Glu Leu Met Tyr Pro Pro Pro Tyr Tyr Leu Gly Ile Gly Asn Gly Thr
100 105 110
Gln Ile Tyr Val Ile Asp Pro Glu Pro Cys Pro Asp Ser Asp Gly Ser
115 120 125
Gly Ile Trp Glu Leu Lys Lys Asp Val Tyr Val Val Glu Leu Asp Trp
130 135 140
Tyr Pro Asp Ala Pro Gly Glu Met Val Val Leu Thr Cys Asp Thr Pro
145 150 155 160
Glu Glu Asp Gly Ile Thr Trp Thr Leu Asp Gln Ser Ser Glu Val Leu
165 170 175
Gly Ser Gly Lys Thr Leu Thr Ile Gln Val Lys Ala Ala Gly Asp Ala
180 185 190
Gly Gln Tyr Thr Cys His Lys Gly Gly Glu Val Leu Ser His Ser Leu
195 200 205
Leu Leu Leu His Lys Lys Glu Asp Gly Ile Trp Ser Thr Asp Ile Leu
210 215 220
Lys Asp Gln Lys Glu Pro Lys Asn Lys Thr Phe Leu Arg Cys Glu Ala
225 230 235 240
Lys Asn Tyr Ser Gly Arg Phe Thr Cys Trp Trp Leu Thr Thr Ile Ser
245 250 255
Thr Asp Leu Thr Phe Ser Val Lys Ser Ser Arg Gly Ser Ser Asp Pro
260 265 270
Gln Gly Val Thr Cys Gly Ala Ala Thr Leu Ser Ala Glu Arg Val Arg
275 280 285
Gly Asp Asn Lys Glu Tyr Glu Tyr Ser Val Glu Cys Gln Glu Asp Ser
290 295 300
Ala Cys Pro Ala Ala Glu Glu Ser Leu Pro Ile Glu Val Met Val Asp
305 310 315 320
Ala Val His Lys Leu Lys Tyr Glu Asn Tyr Thr Ser Ser Phe Phe Ile
325 330 335
Arg Asp Ile Ile Lys Pro Asp Pro Pro Lys Asn Leu Gln Leu Lys Pro
340 345 350
Leu Lys Asn Ser Arg Gln Val Glu Val Ser Trp Glu Tyr Pro Asp Thr
355 360 365
Trp Ser Thr Pro His Ser Tyr Phe Ser Leu Thr Phe Cys Val Gln Val
370 375 380
Gln Gly Lys Ser Lys Arg Glu Lys Lys Asp Arg Val Phe Thr Asp Lys
385 390 395 400
Thr Ser Ala Thr Val Ile Cys Arg Lys Asn Ala Ser Ile Ser Val Arg
405 410 415
Ala Gln Asp Arg Tyr Tyr Ser Ser Ser Trp Ser Glu Trp Ala Ser Val
420 425 430
Pro Cys Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
435 440 445
Gly Ser Gly Gly Gly Gly Ser Gly Arg Asn Leu Pro Val Ala Thr Pro
450 455 460
Asp Pro Gly Met Phe Pro Cys Leu His His Ser Gln Asn Leu Leu Arg
465 470 475 480
Ala Val Ser Asn Met Leu Gln Lys Ala Arg Gln Thr Leu Glu Phe Tyr
485 490 495
Pro Cys Thr Ser Glu Glu Ile Asp His Glu Asp Ile Thr Lys Asp Lys
500 505 510
Thr Ser Thr Val Glu Ala Cys Leu Pro Leu Glu Leu Thr Lys Asn Glu
515 520 525
Ser Cys Leu Asn Ser Arg Glu Thr Ser Phe Ile Thr Asn Gly Ser Cys
530 535 540
Leu Ala Ser Arg Lys Thr Ser Phe Met Met Ala Leu Cys Leu Ser Ser
545 550 555 560
Ile Tyr Glu Asp Leu Lys Met Tyr Gln Val Glu Phe Lys Thr Met Asn
565 570 575
Ala Lys Leu Leu Met Asp Pro Lys Arg Gln Ile Phe Leu Asp Gln Asn
580 585 590
Met Leu Ala Val Ile Asp Glu Leu Met Gln Ala Leu Asn Phe Asn Ser
595 600 605
Glu Thr Val Pro Gln Lys Ser Ser Leu Glu Glu Pro Asp Phe Tyr Lys
610 615 620
Thr Lys Ile Lys Leu Cys Ile Leu Leu His Ala Phe Arg Ile Arg Ala
625 630 635 640
Val Thr Ile Asp Arg Val Met Ser Tyr Leu Asn Ala Ser Asp Lys Thr
645 650 655
His Thr Cys Pro Pro Cys Pro Ala Pro Glu Asp Glu Gly Gly Pro Ser
660 665 670
Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg
675 680 685
Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro
690 695 700
Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala
705 710 715 720
Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val
725 730 735
Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr
740 745 750
Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr
755 760 765
Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Val Tyr
770 775 780
Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys
785 790 795 800
Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser
805 810 815
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp
820 825 830
Glu Asp Gly Ser Phe Ala Leu Val Ser Lys Leu Thr Val Asp Lys Ser
835 840 845
Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala
850 855 860
Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
865 870 875 880
<210> 5
<211> 880
<212> PRT
<213> Homo sapiens (Homo sapiens)
<400> 5
Lys Ala Met His Val Ala Gln Pro Ala Val Val Leu Ala Ser Ser Arg
1 5 10 15
Gly Ile Ala Ser Phe Val Cys Glu Tyr Ala Ser Pro Gly Lys Ala Thr
20 25 30
Glu Val Arg Val Thr Val Leu Arg Gln Ala Asp Ser Gln Val Thr Glu
35 40 45
Val Cys Ala Ala Thr Tyr Met Met Gly Asn Glu Leu Thr Phe Leu Asp
50 55 60
Asp Ser Ile Cys Thr Gly Thr Ser Ser Gly Asn Gln Val Asn Leu Thr
65 70 75 80
Ile Gln Gly Leu Arg Ala Met Asp Thr Gly Leu Tyr Ile Cys Lys Val
85 90 95
Glu Leu Met Tyr Pro Pro Pro Tyr Tyr Leu Gly Ile Gly Asn Gly Thr
100 105 110
Gln Ile Tyr Val Ile Asp Pro Glu Pro Cys Pro Asp Ser Asp Gly Ser
115 120 125
Gly Ile Trp Glu Leu Lys Lys Asp Val Tyr Val Val Glu Leu Asp Trp
130 135 140
Tyr Pro Asp Ala Pro Gly Glu Met Val Val Leu Thr Cys Asp Thr Pro
145 150 155 160
Glu Glu Asp Gly Ile Thr Trp Thr Leu Asp Gln Ser Ser Glu Val Leu
165 170 175
Gly Ser Gly Lys Thr Leu Thr Ile Gln Val Lys Glu Ala Gly Asp Ala
180 185 190
Gly Gln Tyr Thr Cys His Lys Gly Gly Glu Val Leu Ser His Ser Leu
195 200 205
Leu Leu Leu His Lys Lys Glu Asp Gly Ile Trp Ser Thr Asp Ile Leu
210 215 220
Lys Asp Gln Lys Glu Pro Lys Asn Lys Thr Phe Leu Arg Cys Glu Ala
225 230 235 240
Lys Asn Tyr Ser Gly Arg Phe Thr Cys Trp Trp Leu Thr Thr Ile Ser
245 250 255
Thr Asp Leu Thr Phe Ser Val Lys Ser Ser Arg Gly Ser Ser Asp Pro
260 265 270
Gln Gly Val Thr Cys Gly Ala Ala Thr Leu Ser Ala Glu Arg Val Arg
275 280 285
Gly Asp Asn Lys Glu Tyr Glu Tyr Ser Val Glu Cys Gln Glu Asp Ser
290 295 300
Ala Cys Pro Ala Ala Glu Glu Ser Leu Pro Ile Glu Val Met Val Asp
305 310 315 320
Ala Val His Lys Leu Lys Tyr Glu Asn Tyr Thr Ser Ser Phe Phe Ile
325 330 335
Arg Asp Ile Ile Lys Pro Asp Pro Pro Lys Asn Leu Gln Leu Lys Pro
340 345 350
Leu Lys Asn Ser Arg Gln Val Glu Val Ser Trp Glu Tyr Pro Asp Thr
355 360 365
Trp Ser Thr Pro His Ser Tyr Phe Ser Leu Thr Phe Cys Val Gln Val
370 375 380
Gln Gly Lys Ser Lys Arg Glu Lys Lys Asp Arg Val Phe Thr Asp Lys
385 390 395 400
Thr Ser Ala Thr Val Ile Cys Arg Lys Asn Ala Ser Ile Ser Val Arg
405 410 415
Ala Gln Asp Arg Tyr Tyr Ser Ser Ser Trp Ser Glu Trp Ala Ser Val
420 425 430
Pro Cys Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
435 440 445
Gly Ser Gly Gly Gly Gly Ser Gly Arg Asn Leu Pro Val Ala Thr Pro
450 455 460
Asp Pro Gly Met Phe Pro Cys Leu His His Ser Gln Asn Leu Leu Arg
465 470 475 480
Ala Val Ser Asn Met Leu Gln Lys Ala Arg Gln Thr Leu Glu Phe Tyr
485 490 495
Pro Cys Thr Ser Glu Glu Ile Asp His Glu Asp Ile Thr Lys Asp Lys
500 505 510
Thr Ser Thr Val Glu Ala Cys Leu Pro Leu Glu Leu Thr Lys Asn Glu
515 520 525
Ser Cys Leu Asn Ser Arg Glu Thr Ser Phe Ile Thr Asn Gly Ser Cys
530 535 540
Leu Ala Ser Arg Lys Thr Ser Phe Met Met Ala Leu Cys Leu Ser Ser
545 550 555 560
Ile Tyr Glu Asp Leu Lys Met Tyr Gln Val Glu Phe Lys Thr Met Asn
565 570 575
Ala Lys Leu Leu Met Asp Pro Lys Arg Gln Ile Phe Leu Asp Gln Asn
580 585 590
Met Leu Ala Val Ile Asp Glu Leu Met Gln Ala Leu Asn Phe Asn Ser
595 600 605
Glu Thr Val Pro Gln Lys Ser Ser Leu Glu Glu Pro Asp Phe Tyr Lys
610 615 620
Thr Lys Ile Lys Leu Cys Ile Leu Leu His Ala Phe Arg Ile Arg Ala
625 630 635 640
Val Thr Ile Asp Arg Val Met Ser Tyr Leu Asn Ala Ser Asp Lys Thr
645 650 655
His Thr Cys Pro Pro Cys Pro Ala Pro Glu Asp Glu Gly Gly Pro Ser
660 665 670
Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg
675 680 685
Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro
690 695 700
Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala
705 710 715 720
Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val
725 730 735
Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr
740 745 750
Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr
755 760 765
Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Val Tyr
770 775 780
Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys
785 790 795 800
Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser
805 810 815
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp
820 825 830
Glu Asp Gly Ser Phe Ala Leu Val Ser Lys Leu Thr Val Asp Lys Ser
835 840 845
Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala
850 855 860
Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
865 870 875 880
<210> 6
<211> 450
<212> PRT
<213> Homo sapiens (Homo sapiens)
<400> 6
Phe Thr Val Thr Val Pro Lys Asp Leu Tyr Val Val Glu Tyr Gly Ser
1 5 10 15
Asn Met Thr Ile Glu Cys Lys Phe Pro Val Glu Lys Gln Leu Asp Leu
20 25 30
Ala Ala Leu Ile Val Tyr Trp Glu Met Glu Asp Lys Asn Ile Ile Gln
35 40 45
Phe Val His Gly Glu Glu Asp Leu Lys Val Gln His Ser Ser Tyr Arg
50 55 60
Gln Arg Ala Arg Leu Leu Lys Asp Gln Leu Ser Leu Gly Asn Ala Ala
65 70 75 80
Leu Gln Ile Thr Asp Val Lys Leu Gln Asp Ala Gly Val Tyr Arg Cys
85 90 95
Met Ile Ser Tyr Gly Gly Ala Asp Tyr Lys Arg Ile Thr Val Lys Val
100 105 110
Asn Ala Pro Tyr Asn Lys Ile Asn Gln Arg Ile Leu Val Val Asp Pro
115 120 125
Val Thr Ser Glu His Glu Leu Thr Cys Gln Ala Glu Gly Tyr Pro Lys
130 135 140
Ala Glu Val Ile Trp Thr Ser Ser Asp His Gln Val Leu Ser Gly Lys
145 150 155 160
Thr Thr Thr Thr Asn Ser Lys Arg Glu Glu Lys Leu Phe Asn Val Thr
165 170 175
Ser Thr Leu Arg Ile Asn Thr Thr Thr Asn Glu Ile Phe Tyr Cys Thr
180 185 190
Phe Arg Arg Leu Asp Pro Glu Glu Asn His Thr Ala Glu Leu Val Ile
195 200 205
Pro Glu Leu Pro Leu Ala His Pro Pro Asn Glu Arg Gly Ser Gly Asp
210 215 220
Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Lys Lys Gly Gly
225 230 235 240
Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile
245 250 255
Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu
260 265 270
Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His
275 280 285
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg
290 295 300
Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys
305 310 315 320
Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu
325 330 335
Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr
340 345 350
Val Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu
355 360 365
Leu Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp
370 375 380
Glu Ser Asn Gly Gln Pro Glu Asn Arg Tyr Met Thr Trp Pro Pro Val
385 390 395 400
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp
405 410 415
Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His
420 425 430
Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro
435 440 445
Gly Lys
450
<210> 7
<211> 989
<212> PRT
<213> Homo sapiens (Homo sapiens)
<400> 7
Phe Thr Val Thr Val Pro Lys Asp Leu Tyr Val Val Glu Tyr Gly Ser
1 5 10 15
Asn Met Thr Ile Glu Cys Lys Phe Pro Val Glu Lys Gln Leu Asp Leu
20 25 30
Ala Ala Leu Ile Val Tyr Trp Glu Met Glu Asp Lys Asn Ile Ile Gln
35 40 45
Phe Val His Gly Glu Glu Asp Leu Lys Val Gln His Ser Ser Tyr Arg
50 55 60
Gln Arg Ala Arg Leu Leu Lys Asp Gln Leu Ser Leu Gly Asn Ala Ala
65 70 75 80
Leu Gln Ile Thr Asp Val Lys Leu Gln Asp Ala Gly Val Tyr Arg Cys
85 90 95
Met Ile Ser Tyr Gly Gly Ala Asp Tyr Lys Arg Ile Thr Val Lys Val
100 105 110
Asn Ala Pro Tyr Asn Lys Ile Asn Gln Arg Ile Leu Val Val Asp Pro
115 120 125
Val Thr Ser Glu His Glu Leu Thr Cys Gln Ala Glu Gly Tyr Pro Lys
130 135 140
Ala Glu Val Ile Trp Thr Ser Ser Asp His Gln Val Leu Ser Gly Lys
145 150 155 160
Thr Thr Thr Thr Asn Ser Lys Arg Glu Glu Lys Leu Phe Asn Val Thr
165 170 175
Ser Thr Leu Arg Ile Asn Thr Thr Thr Asn Glu Ile Phe Tyr Cys Thr
180 185 190
Phe Arg Arg Leu Asp Pro Glu Glu Asn His Thr Ala Glu Leu Val Ile
195 200 205
Pro Glu Leu Pro Leu Ala His Pro Pro Asn Glu Arg Gly Ser Gly Asp
210 215 220
Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Asp Glu Gly Gly
225 230 235 240
Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile
245 250 255
Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu
260 265 270
Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His
275 280 285
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg
290 295 300
Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys
305 310 315 320
Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu
325 330 335
Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr
340 345 350
Val Tyr Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu
355 360 365
Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp
370 375 380
Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val
385 390 395 400
Leu Asp Glu Asp Gly Ser Phe Ala Leu Val Ser Lys Leu Thr Val Asp
405 410 415
Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His
420 425 430
Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro
435 440 445
Gly Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
450 455 460
Ser Ile Trp Glu Leu Lys Lys Asp Val Tyr Val Val Glu Leu Asp Trp
465 470 475 480
Tyr Pro Asp Ala Pro Gly Glu Met Val Val Leu Thr Cys Asp Thr Pro
485 490 495
Glu Glu Asp Gly Ile Thr Trp Thr Leu Asp Gln Ser Ser Glu Val Leu
500 505 510
Gly Ser Gly Lys Thr Leu Thr Ile Gln Val Lys Ala Ala Gly Asp Ala
515 520 525
Gly Gln Tyr Thr Cys His Lys Gly Gly Glu Val Leu Ser His Ser Leu
530 535 540
Leu Leu Leu His Lys Lys Glu Asp Gly Ile Trp Ser Thr Asp Ile Leu
545 550 555 560
Lys Asp Gln Lys Glu Pro Lys Asn Lys Thr Phe Leu Arg Cys Glu Ala
565 570 575
Lys Asn Tyr Ser Gly Arg Phe Thr Cys Trp Trp Leu Thr Thr Ile Ser
580 585 590
Thr Asp Leu Thr Phe Ser Val Lys Ser Ser Arg Gly Ser Ser Asp Pro
595 600 605
Gln Gly Val Thr Cys Gly Ala Ala Thr Leu Ser Ala Glu Arg Val Arg
610 615 620
Gly Asp Asn Lys Glu Tyr Glu Tyr Ser Val Glu Cys Gln Glu Asp Ser
625 630 635 640
Ala Cys Pro Ala Ala Glu Glu Ser Leu Pro Ile Glu Val Met Val Asp
645 650 655
Ala Val His Lys Leu Lys Tyr Glu Asn Tyr Thr Ser Ser Phe Phe Ile
660 665 670
Arg Asp Ile Ile Lys Pro Asp Pro Pro Lys Asn Leu Gln Leu Lys Pro
675 680 685
Leu Lys Asn Ser Arg Gln Val Glu Val Ser Trp Glu Tyr Pro Asp Thr
690 695 700
Trp Ser Thr Pro His Ser Tyr Phe Ser Leu Thr Phe Cys Val Gln Val
705 710 715 720
Gln Gly Lys Ser Lys Arg Glu Lys Lys Asp Arg Val Phe Thr Asp Lys
725 730 735
Thr Ser Ala Thr Val Ile Cys Arg Lys Asn Ala Ser Ile Ser Val Arg
740 745 750
Ala Gln Asp Arg Tyr Tyr Ser Ser Ser Trp Ser Glu Trp Ala Ser Val
755 760 765
Pro Cys Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
770 775 780
Gly Ser Gly Gly Gly Gly Ser Gly Arg Asn Leu Pro Val Ala Thr Pro
785 790 795 800
Asp Pro Gly Met Phe Pro Cys Leu His His Ser Gln Asn Leu Leu Arg
805 810 815
Ala Val Ser Asn Met Leu Gln Lys Ala Arg Gln Thr Leu Glu Phe Tyr
820 825 830
Pro Cys Thr Ser Glu Glu Ile Asp His Glu Asp Ile Thr Lys Asp Lys
835 840 845
Thr Ser Thr Val Glu Ala Cys Leu Pro Leu Glu Leu Thr Lys Asn Glu
850 855 860
Ser Cys Leu Asn Ser Arg Glu Thr Ser Phe Ile Thr Asn Gly Ser Cys
865 870 875 880
Leu Ala Ser Arg Lys Thr Ser Phe Met Met Ala Leu Cys Leu Ser Ser
885 890 895
Ile Tyr Glu Asp Leu Lys Met Tyr Gln Val Glu Phe Lys Thr Met Asn
900 905 910
Ala Lys Leu Leu Met Asp Pro Lys Arg Gln Ile Phe Leu Asp Gln Asn
915 920 925
Met Leu Ala Val Ile Asp Glu Leu Met Gln Ala Leu Asn Phe Asn Ser
930 935 940
Glu Thr Val Pro Gln Lys Ser Ser Leu Glu Glu Pro Asp Phe Tyr Lys
945 950 955 960
Thr Lys Ile Lys Leu Cys Ile Leu Leu His Ala Phe Arg Ile Arg Ala
965 970 975
Val Thr Ile Asp Arg Val Met Ser Tyr Leu Asn Ala Ser
980 985
<210> 8
<211> 989
<212> PRT
<213> Homo sapiens (Homo sapiens)
<400> 8
Phe Thr Val Thr Val Pro Lys Asp Leu Tyr Val Val Glu Tyr Gly Ser
1 5 10 15
Asn Met Thr Ile Glu Cys Lys Phe Pro Val Glu Lys Gln Leu Asp Leu
20 25 30
Ala Ala Leu Ile Val Tyr Trp Glu Met Glu Asp Lys Asn Ile Ile Gln
35 40 45
Phe Val His Gly Glu Glu Asp Leu Lys Val Gln His Ser Ser Tyr Arg
50 55 60
Gln Arg Ala Arg Leu Leu Lys Asp Gln Leu Ser Leu Gly Asn Ala Ala
65 70 75 80
Leu Gln Ile Thr Asp Val Lys Leu Gln Asp Ala Gly Val Tyr Arg Cys
85 90 95
Met Ile Ser Tyr Gly Gly Ala Asp Tyr Lys Arg Ile Thr Val Lys Val
100 105 110
Asn Ala Pro Tyr Asn Lys Ile Asn Gln Arg Ile Leu Val Val Asp Pro
115 120 125
Val Thr Ser Glu His Glu Leu Thr Cys Gln Ala Glu Gly Tyr Pro Lys
130 135 140
Ala Glu Val Ile Trp Thr Ser Ser Asp His Gln Val Leu Ser Gly Lys
145 150 155 160
Thr Thr Thr Thr Asn Ser Lys Arg Glu Glu Lys Leu Phe Asn Val Thr
165 170 175
Ser Thr Leu Arg Ile Asn Thr Thr Thr Asn Glu Ile Phe Tyr Cys Thr
180 185 190
Phe Arg Arg Leu Asp Pro Glu Glu Asn His Thr Ala Glu Leu Val Ile
195 200 205
Pro Glu Leu Pro Leu Ala His Pro Pro Asn Glu Arg Gly Ser Gly Asp
210 215 220
Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Asp Glu Gly Gly
225 230 235 240
Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile
245 250 255
Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu
260 265 270
Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His
275 280 285
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg
290 295 300
Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys
305 310 315 320
Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu
325 330 335
Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr
340 345 350
Val Tyr Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu
355 360 365
Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp
370 375 380
Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val
385 390 395 400
Leu Asp Glu Asp Gly Ser Phe Ala Leu Val Ser Lys Leu Thr Val Asp
405 410 415
Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His
420 425 430
Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro
435 440 445
Gly Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
450 455 460
Ser Ile Trp Glu Leu Lys Lys Asp Val Tyr Val Val Glu Leu Asp Trp
465 470 475 480
Tyr Pro Asp Ala Pro Gly Glu Met Val Val Leu Thr Cys Asp Thr Pro
485 490 495
Glu Glu Asp Gly Ile Thr Trp Thr Leu Asp Gln Ser Ser Glu Val Leu
500 505 510
Gly Ser Gly Lys Thr Leu Thr Ile Gln Val Lys Glu Ala Gly Asp Ala
515 520 525
Gly Gln Tyr Thr Cys His Lys Gly Gly Glu Val Leu Ser His Ser Leu
530 535 540
Leu Leu Leu His Lys Lys Glu Asp Gly Ile Trp Ser Thr Asp Ile Leu
545 550 555 560
Lys Asp Gln Lys Glu Pro Lys Asn Lys Thr Phe Leu Arg Cys Glu Ala
565 570 575
Lys Asn Tyr Ser Gly Arg Phe Thr Cys Trp Trp Leu Thr Thr Ile Ser
580 585 590
Thr Asp Leu Thr Phe Ser Val Lys Ser Ser Arg Gly Ser Ser Asp Pro
595 600 605
Gln Gly Val Thr Cys Gly Ala Ala Thr Leu Ser Ala Glu Arg Val Arg
610 615 620
Gly Asp Asn Lys Glu Tyr Glu Tyr Ser Val Glu Cys Gln Glu Asp Ser
625 630 635 640
Ala Cys Pro Ala Ala Glu Glu Ser Leu Pro Ile Glu Val Met Val Asp
645 650 655
Ala Val His Lys Leu Lys Tyr Glu Asn Tyr Thr Ser Ser Phe Phe Ile
660 665 670
Arg Asp Ile Ile Lys Pro Asp Pro Pro Lys Asn Leu Gln Leu Lys Pro
675 680 685
Leu Lys Asn Ser Arg Gln Val Glu Val Ser Trp Glu Tyr Pro Asp Thr
690 695 700
Trp Ser Thr Pro His Ser Tyr Phe Ser Leu Thr Phe Cys Val Gln Val
705 710 715 720
Gln Gly Lys Ser Lys Arg Glu Lys Lys Asp Arg Val Phe Thr Asp Lys
725 730 735
Thr Ser Ala Thr Val Ile Cys Arg Lys Asn Ala Ser Ile Ser Val Arg
740 745 750
Ala Gln Asp Arg Tyr Tyr Ser Ser Ser Trp Ser Glu Trp Ala Ser Val
755 760 765
Pro Cys Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
770 775 780
Gly Ser Gly Gly Gly Gly Ser Gly Arg Asn Leu Pro Val Ala Thr Pro
785 790 795 800
Asp Pro Gly Met Phe Pro Cys Leu His His Ser Gln Asn Leu Leu Arg
805 810 815
Ala Val Ser Asn Met Leu Gln Lys Ala Arg Gln Thr Leu Glu Phe Tyr
820 825 830
Pro Cys Thr Ser Glu Glu Ile Asp His Glu Asp Ile Thr Lys Asp Lys
835 840 845
Thr Ser Thr Val Glu Ala Cys Leu Pro Leu Glu Leu Thr Lys Asn Glu
850 855 860
Ser Cys Leu Asn Ser Arg Glu Thr Ser Phe Ile Thr Asn Gly Ser Cys
865 870 875 880
Leu Ala Ser Arg Lys Thr Ser Phe Met Met Ala Leu Cys Leu Ser Ser
885 890 895
Ile Tyr Glu Asp Leu Lys Met Tyr Gln Val Glu Phe Lys Thr Met Asn
900 905 910
Ala Lys Leu Leu Met Asp Pro Lys Arg Gln Ile Phe Leu Asp Gln Asn
915 920 925
Met Leu Ala Val Ile Asp Glu Leu Met Gln Ala Leu Asn Phe Asn Ser
930 935 940
Glu Thr Val Pro Gln Lys Ser Ser Leu Glu Glu Pro Asp Phe Tyr Lys
945 950 955 960
Thr Lys Ile Lys Leu Cys Ile Leu Leu His Ala Phe Arg Ile Arg Ala
965 970 975
Val Thr Ile Asp Arg Val Met Ser Tyr Leu Asn Ala Ser
980 985
<210> 9
<211> 974
<212> PRT
<213> Homo sapiens (Homo sapiens)
<400> 9
Phe Thr Val Thr Val Pro Lys Asp Leu Tyr Val Val Glu Tyr Gly Ser
1 5 10 15
Asn Met Thr Ile Glu Cys Lys Phe Pro Val Glu Lys Gln Leu Asp Leu
20 25 30
Ala Ala Leu Ile Val Tyr Trp Glu Met Glu Asp Lys Asn Ile Ile Gln
35 40 45
Phe Val His Gly Glu Glu Asp Leu Lys Val Gln His Ser Ser Tyr Arg
50 55 60
Gln Arg Ala Arg Leu Leu Lys Asp Gln Leu Ser Leu Gly Asn Ala Ala
65 70 75 80
Leu Gln Ile Thr Asp Val Lys Leu Gln Asp Ala Gly Val Tyr Arg Cys
85 90 95
Met Ile Ser Tyr Gly Gly Ala Asp Tyr Lys Arg Ile Thr Val Lys Val
100 105 110
Asn Ala Pro Tyr Asn Lys Ile Asn Gln Arg Ile Leu Val Val Asp Pro
115 120 125
Val Thr Ser Glu His Glu Leu Thr Cys Gln Ala Glu Gly Tyr Pro Lys
130 135 140
Ala Glu Val Ile Trp Thr Ser Ser Asp His Gln Val Leu Ser Gly Lys
145 150 155 160
Thr Thr Thr Thr Asn Ser Lys Arg Glu Glu Lys Leu Phe Asn Val Thr
165 170 175
Ser Thr Leu Arg Ile Asn Thr Thr Thr Asn Glu Ile Phe Tyr Cys Thr
180 185 190
Phe Arg Arg Leu Asp Pro Glu Glu Asn His Thr Ala Glu Leu Val Ile
195 200 205
Pro Glu Leu Pro Leu Ala His Pro Pro Asn Glu Arg Gly Ser Gly Ile
210 215 220
Trp Glu Leu Lys Lys Asp Val Tyr Val Val Glu Leu Asp Trp Tyr Pro
225 230 235 240
Asp Ala Pro Gly Glu Met Val Val Leu Thr Cys Asp Thr Pro Glu Glu
245 250 255
Asp Gly Ile Thr Trp Thr Leu Asp Gln Ser Ser Glu Val Leu Gly Ser
260 265 270
Gly Lys Thr Leu Thr Ile Gln Val Lys Ala Ala Gly Asp Ala Gly Gln
275 280 285
Tyr Thr Cys His Lys Gly Gly Glu Val Leu Ser His Ser Leu Leu Leu
290 295 300
Leu His Lys Lys Glu Asp Gly Ile Trp Ser Thr Asp Ile Leu Lys Asp
305 310 315 320
Gln Lys Glu Pro Lys Asn Lys Thr Phe Leu Arg Cys Glu Ala Lys Asn
325 330 335
Tyr Ser Gly Arg Phe Thr Cys Trp Trp Leu Thr Thr Ile Ser Thr Asp
340 345 350
Leu Thr Phe Ser Val Lys Ser Ser Arg Gly Ser Ser Asp Pro Gln Gly
355 360 365
Val Thr Cys Gly Ala Ala Thr Leu Ser Ala Glu Arg Val Arg Gly Asp
370 375 380
Asn Lys Glu Tyr Glu Tyr Ser Val Glu Cys Gln Glu Asp Ser Ala Cys
385 390 395 400
Pro Ala Ala Glu Glu Ser Leu Pro Ile Glu Val Met Val Asp Ala Val
405 410 415
His Lys Leu Lys Tyr Glu Asn Tyr Thr Ser Ser Phe Phe Ile Arg Asp
420 425 430
Ile Ile Lys Pro Asp Pro Pro Lys Asn Leu Gln Leu Lys Pro Leu Lys
435 440 445
Asn Ser Arg Gln Val Glu Val Ser Trp Glu Tyr Pro Asp Thr Trp Ser
450 455 460
Thr Pro His Ser Tyr Phe Ser Leu Thr Phe Cys Val Gln Val Gln Gly
465 470 475 480
Lys Ser Lys Arg Glu Lys Lys Asp Arg Val Phe Thr Asp Lys Thr Ser
485 490 495
Ala Thr Val Ile Cys Arg Lys Asn Ala Ser Ile Ser Val Arg Ala Gln
500 505 510
Asp Arg Tyr Tyr Ser Ser Ser Trp Ser Glu Trp Ala Ser Val Pro Cys
515 520 525
Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
530 535 540
Gly Gly Gly Gly Ser Gly Arg Asn Leu Pro Val Ala Thr Pro Asp Pro
545 550 555 560
Gly Met Phe Pro Cys Leu His His Ser Gln Asn Leu Leu Arg Ala Val
565 570 575
Ser Asn Met Leu Gln Lys Ala Arg Gln Thr Leu Glu Phe Tyr Pro Cys
580 585 590
Thr Ser Glu Glu Ile Asp His Glu Asp Ile Thr Lys Asp Lys Thr Ser
595 600 605
Thr Val Glu Ala Cys Leu Pro Leu Glu Leu Thr Lys Asn Glu Ser Cys
610 615 620
Leu Asn Ser Arg Glu Thr Ser Phe Ile Thr Asn Gly Ser Cys Leu Ala
625 630 635 640
Ser Arg Lys Thr Ser Phe Met Met Ala Leu Cys Leu Ser Ser Ile Tyr
645 650 655
Glu Asp Leu Lys Met Tyr Gln Val Glu Phe Lys Thr Met Asn Ala Lys
660 665 670
Leu Leu Met Asp Pro Lys Arg Gln Ile Phe Leu Asp Gln Asn Met Leu
675 680 685
Ala Val Ile Asp Glu Leu Met Gln Ala Leu Asn Phe Asn Ser Glu Thr
690 695 700
Val Pro Gln Lys Ser Ser Leu Glu Glu Pro Asp Phe Tyr Lys Thr Lys
705 710 715 720
Ile Lys Leu Cys Ile Leu Leu His Ala Phe Arg Ile Arg Ala Val Thr
725 730 735
Ile Asp Arg Val Met Ser Tyr Leu Asn Ala Ser Asp Lys Thr His Thr
740 745 750
Cys Pro Pro Cys Pro Ala Pro Glu Asp Glu Gly Gly Pro Ser Val Phe
755 760 765
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro
770 775 780
Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val
785 790 795 800
Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr
805 810 815
Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val
820 825 830
Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys
835 840 845
Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser
850 855 860
Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Val Tyr Pro Pro
865 870 875 880
Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val
885 890 895
Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly
900 905 910
Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Glu Asp
915 920 925
Gly Ser Phe Ala Leu Val Ser Lys Leu Thr Val Asp Lys Ser Arg Trp
930 935 940
Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His
945 950 955 960
Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
965 970
<210> 10
<211> 974
<212> PRT
<213> Homo sapiens (Homo sapiens)
<400> 10
Phe Thr Val Thr Val Pro Lys Asp Leu Tyr Val Val Glu Tyr Gly Ser
1 5 10 15
Asn Met Thr Ile Glu Cys Lys Phe Pro Val Glu Lys Gln Leu Asp Leu
20 25 30
Ala Ala Leu Ile Val Tyr Trp Glu Met Glu Asp Lys Asn Ile Ile Gln
35 40 45
Phe Val His Gly Glu Glu Asp Leu Lys Val Gln His Ser Ser Tyr Arg
50 55 60
Gln Arg Ala Arg Leu Leu Lys Asp Gln Leu Ser Leu Gly Asn Ala Ala
65 70 75 80
Leu Gln Ile Thr Asp Val Lys Leu Gln Asp Ala Gly Val Tyr Arg Cys
85 90 95
Met Ile Ser Tyr Gly Gly Ala Asp Tyr Lys Arg Ile Thr Val Lys Val
100 105 110
Asn Ala Pro Tyr Asn Lys Ile Asn Gln Arg Ile Leu Val Val Asp Pro
115 120 125
Val Thr Ser Glu His Glu Leu Thr Cys Gln Ala Glu Gly Tyr Pro Lys
130 135 140
Ala Glu Val Ile Trp Thr Ser Ser Asp His Gln Val Leu Ser Gly Lys
145 150 155 160
Thr Thr Thr Thr Asn Ser Lys Arg Glu Glu Lys Leu Phe Asn Val Thr
165 170 175
Ser Thr Leu Arg Ile Asn Thr Thr Thr Asn Glu Ile Phe Tyr Cys Thr
180 185 190
Phe Arg Arg Leu Asp Pro Glu Glu Asn His Thr Ala Glu Leu Val Ile
195 200 205
Pro Glu Leu Pro Leu Ala His Pro Pro Asn Glu Arg Gly Ser Gly Ile
210 215 220
Trp Glu Leu Lys Lys Asp Val Tyr Val Val Glu Leu Asp Trp Tyr Pro
225 230 235 240
Asp Ala Pro Gly Glu Met Val Val Leu Thr Cys Asp Thr Pro Glu Glu
245 250 255
Asp Gly Ile Thr Trp Thr Leu Asp Gln Ser Ser Glu Val Leu Gly Ser
260 265 270
Gly Lys Thr Leu Thr Ile Gln Val Lys Glu Ala Gly Asp Ala Gly Gln
275 280 285
Tyr Thr Cys His Lys Gly Gly Glu Val Leu Ser His Ser Leu Leu Leu
290 295 300
Leu His Lys Lys Glu Asp Gly Ile Trp Ser Thr Asp Ile Leu Lys Asp
305 310 315 320
Gln Lys Glu Pro Lys Asn Lys Thr Phe Leu Arg Cys Glu Ala Lys Asn
325 330 335
Tyr Ser Gly Arg Phe Thr Cys Trp Trp Leu Thr Thr Ile Ser Thr Asp
340 345 350
Leu Thr Phe Ser Val Lys Ser Ser Arg Gly Ser Ser Asp Pro Gln Gly
355 360 365
Val Thr Cys Gly Ala Ala Thr Leu Ser Ala Glu Arg Val Arg Gly Asp
370 375 380
Asn Lys Glu Tyr Glu Tyr Ser Val Glu Cys Gln Glu Asp Ser Ala Cys
385 390 395 400
Pro Ala Ala Glu Glu Ser Leu Pro Ile Glu Val Met Val Asp Ala Val
405 410 415
His Lys Leu Lys Tyr Glu Asn Tyr Thr Ser Ser Phe Phe Ile Arg Asp
420 425 430
Ile Ile Lys Pro Asp Pro Pro Lys Asn Leu Gln Leu Lys Pro Leu Lys
435 440 445
Asn Ser Arg Gln Val Glu Val Ser Trp Glu Tyr Pro Asp Thr Trp Ser
450 455 460
Thr Pro His Ser Tyr Phe Ser Leu Thr Phe Cys Val Gln Val Gln Gly
465 470 475 480
Lys Ser Lys Arg Glu Lys Lys Asp Arg Val Phe Thr Asp Lys Thr Ser
485 490 495
Ala Thr Val Ile Cys Arg Lys Asn Ala Ser Ile Ser Val Arg Ala Gln
500 505 510
Asp Arg Tyr Tyr Ser Ser Ser Trp Ser Glu Trp Ala Ser Val Pro Cys
515 520 525
Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
530 535 540
Gly Gly Gly Gly Ser Gly Arg Asn Leu Pro Val Ala Thr Pro Asp Pro
545 550 555 560
Gly Met Phe Pro Cys Leu His His Ser Gln Asn Leu Leu Arg Ala Val
565 570 575
Ser Asn Met Leu Gln Lys Ala Arg Gln Thr Leu Glu Phe Tyr Pro Cys
580 585 590
Thr Ser Glu Glu Ile Asp His Glu Asp Ile Thr Lys Asp Lys Thr Ser
595 600 605
Thr Val Glu Ala Cys Leu Pro Leu Glu Leu Thr Lys Asn Glu Ser Cys
610 615 620
Leu Asn Ser Arg Glu Thr Ser Phe Ile Thr Asn Gly Ser Cys Leu Ala
625 630 635 640
Ser Arg Lys Thr Ser Phe Met Met Ala Leu Cys Leu Ser Ser Ile Tyr
645 650 655
Glu Asp Leu Lys Met Tyr Gln Val Glu Phe Lys Thr Met Asn Ala Lys
660 665 670
Leu Leu Met Asp Pro Lys Arg Gln Ile Phe Leu Asp Gln Asn Met Leu
675 680 685
Ala Val Ile Asp Glu Leu Met Gln Ala Leu Asn Phe Asn Ser Glu Thr
690 695 700
Val Pro Gln Lys Ser Ser Leu Glu Glu Pro Asp Phe Tyr Lys Thr Lys
705 710 715 720
Ile Lys Leu Cys Ile Leu Leu His Ala Phe Arg Ile Arg Ala Val Thr
725 730 735
Ile Asp Arg Val Met Ser Tyr Leu Asn Ala Ser Asp Lys Thr His Thr
740 745 750
Cys Pro Pro Cys Pro Ala Pro Glu Asp Glu Gly Gly Pro Ser Val Phe
755 760 765
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro
770 775 780
Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val
785 790 795 800
Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr
805 810 815
Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val
820 825 830
Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys
835 840 845
Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser
850 855 860
Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Val Tyr Pro Pro
865 870 875 880
Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val
885 890 895
Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly
900 905 910
Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Glu Asp
915 920 925
Gly Ser Phe Ala Leu Val Ser Lys Leu Thr Val Asp Lys Ser Arg Trp
930 935 940
Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His
945 950 955 960
Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
965 970
<210> 11
<211> 450
<212> PRT
<213> Homo sapiens (Homo sapiens)
<400> 11
Phe Thr Val Thr Val Pro Lys Asp Leu Tyr Val Val Glu Tyr Gly Ser
1 5 10 15
Asn Met Thr Ile Glu Cys Lys Phe Pro Val Glu Lys Gln Leu Asp Leu
20 25 30
Ala Ala Leu Gln Val Phe Trp Met Met Glu Asp Lys Asn Ile Ile Gln
35 40 45
Phe Val His Gly Glu Glu Asp Leu Lys Val Gln His Ser Ser Tyr Arg
50 55 60
Gln Arg Ala Arg Leu Leu Lys Asp Gln Leu Ser Leu Gly Asn Ala Ala
65 70 75 80
Leu Gln Ile Thr Asp Val Lys Leu Gln Asp Ala Gly Val Tyr Thr Cys
85 90 95
Leu Ile Ala Tyr Lys Gly Ala Asp Tyr Lys Arg Ile Thr Val Lys Val
100 105 110
Asn Ala Pro Tyr Asn Lys Ile Asn Gln Arg Ile Leu Val Val Asp Pro
115 120 125
Val Thr Ser Glu His Glu Leu Thr Cys Gln Ala Glu Gly Tyr Pro Lys
130 135 140
Ala Glu Val Ile Trp Thr Ser Ser Asp His Gln Val Leu Ser Gly Lys
145 150 155 160
Thr Thr Thr Thr Asn Ser Lys Arg Glu Glu Lys Leu Phe Asn Val Thr
165 170 175
Ser Thr Leu Arg Ile Asn Thr Thr Thr Asn Glu Ile Phe Tyr Cys Thr
180 185 190
Phe Arg Arg Leu Asp Pro Glu Glu Asn His Thr Ala Glu Leu Val Ile
195 200 205
Pro Glu Leu Pro Leu Ala His Pro Pro Asn Glu Arg Gly Ser Gly Asp
210 215 220
Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Lys Lys Gly Gly
225 230 235 240
Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile
245 250 255
Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu
260 265 270
Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His
275 280 285
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg
290 295 300
Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys
305 310 315 320
Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu
325 330 335
Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr
340 345 350
Val Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu
355 360 365
Leu Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp
370 375 380
Glu Ser Asn Gly Gln Pro Glu Asn Arg Tyr Met Thr Trp Pro Pro Val
385 390 395 400
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp
405 410 415
Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His
420 425 430
Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro
435 440 445
Gly Lys
450
<210> 12
<211> 989
<212> PRT
<213> Homo sapiens (Homo sapiens)
<400> 12
Phe Thr Val Thr Val Pro Lys Asp Leu Tyr Val Val Glu Tyr Gly Ser
1 5 10 15
Asn Met Thr Ile Glu Cys Lys Phe Pro Val Glu Lys Gln Leu Asp Leu
20 25 30
Ala Ala Leu Gln Val Phe Trp Met Met Glu Asp Lys Asn Ile Ile Gln
35 40 45
Phe Val His Gly Glu Glu Asp Leu Lys Val Gln His Ser Ser Tyr Arg
50 55 60
Gln Arg Ala Arg Leu Leu Lys Asp Gln Leu Ser Leu Gly Asn Ala Ala
65 70 75 80
Leu Gln Ile Thr Asp Val Lys Leu Gln Asp Ala Gly Val Tyr Thr Cys
85 90 95
Leu Ile Ala Tyr Lys Gly Ala Asp Tyr Lys Arg Ile Thr Val Lys Val
100 105 110
Asn Ala Pro Tyr Asn Lys Ile Asn Gln Arg Ile Leu Val Val Asp Pro
115 120 125
Val Thr Ser Glu His Glu Leu Thr Cys Gln Ala Glu Gly Tyr Pro Lys
130 135 140
Ala Glu Val Ile Trp Thr Ser Ser Asp His Gln Val Leu Ser Gly Lys
145 150 155 160
Thr Thr Thr Thr Asn Ser Lys Arg Glu Glu Lys Leu Phe Asn Val Thr
165 170 175
Ser Thr Leu Arg Ile Asn Thr Thr Thr Asn Glu Ile Phe Tyr Cys Thr
180 185 190
Phe Arg Arg Leu Asp Pro Glu Glu Asn His Thr Ala Glu Leu Val Ile
195 200 205
Pro Glu Leu Pro Leu Ala His Pro Pro Asn Glu Arg Gly Ser Gly Asp
210 215 220
Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Asp Glu Gly Gly
225 230 235 240
Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile
245 250 255
Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu
260 265 270
Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His
275 280 285
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg
290 295 300
Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys
305 310 315 320
Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu
325 330 335
Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr
340 345 350
Val Tyr Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu
355 360 365
Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp
370 375 380
Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val
385 390 395 400
Leu Asp Glu Asp Gly Ser Phe Ala Leu Val Ser Lys Leu Thr Val Asp
405 410 415
Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His
420 425 430
Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro
435 440 445
Gly Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
450 455 460
Ser Ile Trp Glu Leu Lys Lys Asp Val Tyr Val Val Glu Leu Asp Trp
465 470 475 480
Tyr Pro Asp Ala Pro Gly Glu Met Val Val Leu Thr Cys Asp Thr Pro
485 490 495
Glu Glu Asp Gly Ile Thr Trp Thr Leu Asp Gln Ser Ser Glu Val Leu
500 505 510
Gly Ser Gly Lys Thr Leu Thr Ile Gln Val Lys Ala Ala Gly Asp Ala
515 520 525
Gly Gln Tyr Thr Cys His Lys Gly Gly Glu Val Leu Ser His Ser Leu
530 535 540
Leu Leu Leu His Lys Lys Glu Asp Gly Ile Trp Ser Thr Asp Ile Leu
545 550 555 560
Lys Asp Gln Lys Glu Pro Lys Asn Lys Thr Phe Leu Arg Cys Glu Ala
565 570 575
Lys Asn Tyr Ser Gly Arg Phe Thr Cys Trp Trp Leu Thr Thr Ile Ser
580 585 590
Thr Asp Leu Thr Phe Ser Val Lys Ser Ser Arg Gly Ser Ser Asp Pro
595 600 605
Gln Gly Val Thr Cys Gly Ala Ala Thr Leu Ser Ala Glu Arg Val Arg
610 615 620
Gly Asp Asn Lys Glu Tyr Glu Tyr Ser Val Glu Cys Gln Glu Asp Ser
625 630 635 640
Ala Cys Pro Ala Ala Glu Glu Ser Leu Pro Ile Glu Val Met Val Asp
645 650 655
Ala Val His Lys Leu Lys Tyr Glu Asn Tyr Thr Ser Ser Phe Phe Ile
660 665 670
Arg Asp Ile Ile Lys Pro Asp Pro Pro Lys Asn Leu Gln Leu Lys Pro
675 680 685
Leu Lys Asn Ser Arg Gln Val Glu Val Ser Trp Glu Tyr Pro Asp Thr
690 695 700
Trp Ser Thr Pro His Ser Tyr Phe Ser Leu Thr Phe Cys Val Gln Val
705 710 715 720
Gln Gly Lys Ser Lys Arg Glu Lys Lys Asp Arg Val Phe Thr Asp Lys
725 730 735
Thr Ser Ala Thr Val Ile Cys Arg Lys Asn Ala Ser Ile Ser Val Arg
740 745 750
Ala Gln Asp Arg Tyr Tyr Ser Ser Ser Trp Ser Glu Trp Ala Ser Val
755 760 765
Pro Cys Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
770 775 780
Gly Ser Gly Gly Gly Gly Ser Gly Arg Asn Leu Pro Val Ala Thr Pro
785 790 795 800
Asp Pro Gly Met Phe Pro Cys Leu His His Ser Gln Asn Leu Leu Arg
805 810 815
Ala Val Ser Asn Met Leu Gln Lys Ala Arg Gln Thr Leu Glu Phe Tyr
820 825 830
Pro Cys Thr Ser Glu Glu Ile Asp His Glu Asp Ile Thr Lys Asp Lys
835 840 845
Thr Ser Thr Val Glu Ala Cys Leu Pro Leu Glu Leu Thr Lys Asn Glu
850 855 860
Ser Cys Leu Asn Ser Arg Glu Thr Ser Phe Ile Thr Asn Gly Ser Cys
865 870 875 880
Leu Ala Ser Arg Lys Thr Ser Phe Met Met Ala Leu Cys Leu Ser Ser
885 890 895
Ile Tyr Glu Asp Leu Lys Met Tyr Gln Val Glu Phe Lys Thr Met Asn
900 905 910
Ala Lys Leu Leu Met Asp Pro Lys Arg Gln Ile Phe Leu Asp Gln Asn
915 920 925
Met Leu Ala Val Ile Asp Glu Leu Met Gln Ala Leu Asn Phe Asn Ser
930 935 940
Glu Thr Val Pro Gln Lys Ser Ser Leu Glu Glu Pro Asp Phe Tyr Lys
945 950 955 960
Thr Lys Ile Lys Leu Cys Ile Leu Leu His Ala Phe Arg Ile Arg Ala
965 970 975
Val Thr Ile Asp Arg Val Met Ser Tyr Leu Asn Ala Ser
980 985
<210> 13
<211> 989
<212> PRT
<213> Homo sapiens (Homo sapiens)
<400> 13
Phe Thr Val Thr Val Pro Lys Asp Leu Tyr Val Val Glu Tyr Gly Ser
1 5 10 15
Asn Met Thr Ile Glu Cys Lys Phe Pro Val Glu Lys Gln Leu Asp Leu
20 25 30
Ala Ala Leu Gln Val Phe Trp Met Met Glu Asp Lys Asn Ile Ile Gln
35 40 45
Phe Val His Gly Glu Glu Asp Leu Lys Val Gln His Ser Ser Tyr Arg
50 55 60
Gln Arg Ala Arg Leu Leu Lys Asp Gln Leu Ser Leu Gly Asn Ala Ala
65 70 75 80
Leu Gln Ile Thr Asp Val Lys Leu Gln Asp Ala Gly Val Tyr Thr Cys
85 90 95
Leu Ile Ala Tyr Lys Gly Ala Asp Tyr Lys Arg Ile Thr Val Lys Val
100 105 110
Asn Ala Pro Tyr Asn Lys Ile Asn Gln Arg Ile Leu Val Val Asp Pro
115 120 125
Val Thr Ser Glu His Glu Leu Thr Cys Gln Ala Glu Gly Tyr Pro Lys
130 135 140
Ala Glu Val Ile Trp Thr Ser Ser Asp His Gln Val Leu Ser Gly Lys
145 150 155 160
Thr Thr Thr Thr Asn Ser Lys Arg Glu Glu Lys Leu Phe Asn Val Thr
165 170 175
Ser Thr Leu Arg Ile Asn Thr Thr Thr Asn Glu Ile Phe Tyr Cys Thr
180 185 190
Phe Arg Arg Leu Asp Pro Glu Glu Asn His Thr Ala Glu Leu Val Ile
195 200 205
Pro Glu Leu Pro Leu Ala His Pro Pro Asn Glu Arg Gly Ser Gly Asp
210 215 220
Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Asp Glu Gly Gly
225 230 235 240
Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile
245 250 255
Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu
260 265 270
Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His
275 280 285
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg
290 295 300
Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys
305 310 315 320
Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu
325 330 335
Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr
340 345 350
Val Tyr Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu
355 360 365
Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp
370 375 380
Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val
385 390 395 400
Leu Asp Glu Asp Gly Ser Phe Ala Leu Val Ser Lys Leu Thr Val Asp
405 410 415
Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His
420 425 430
Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro
435 440 445
Gly Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
450 455 460
Ser Ile Trp Glu Leu Lys Lys Asp Val Tyr Val Val Glu Leu Asp Trp
465 470 475 480
Tyr Pro Asp Ala Pro Gly Glu Met Val Val Leu Thr Cys Asp Thr Pro
485 490 495
Glu Glu Asp Gly Ile Thr Trp Thr Leu Asp Gln Ser Ser Glu Val Leu
500 505 510
Gly Ser Gly Lys Thr Leu Thr Ile Gln Val Lys Glu Ala Gly Asp Ala
515 520 525
Gly Gln Tyr Thr Cys His Lys Gly Gly Glu Val Leu Ser His Ser Leu
530 535 540
Leu Leu Leu His Lys Lys Glu Asp Gly Ile Trp Ser Thr Asp Ile Leu
545 550 555 560
Lys Asp Gln Lys Glu Pro Lys Asn Lys Thr Phe Leu Arg Cys Glu Ala
565 570 575
Lys Asn Tyr Ser Gly Arg Phe Thr Cys Trp Trp Leu Thr Thr Ile Ser
580 585 590
Thr Asp Leu Thr Phe Ser Val Lys Ser Ser Arg Gly Ser Ser Asp Pro
595 600 605
Gln Gly Val Thr Cys Gly Ala Ala Thr Leu Ser Ala Glu Arg Val Arg
610 615 620
Gly Asp Asn Lys Glu Tyr Glu Tyr Ser Val Glu Cys Gln Glu Asp Ser
625 630 635 640
Ala Cys Pro Ala Ala Glu Glu Ser Leu Pro Ile Glu Val Met Val Asp
645 650 655
Ala Val His Lys Leu Lys Tyr Glu Asn Tyr Thr Ser Ser Phe Phe Ile
660 665 670
Arg Asp Ile Ile Lys Pro Asp Pro Pro Lys Asn Leu Gln Leu Lys Pro
675 680 685
Leu Lys Asn Ser Arg Gln Val Glu Val Ser Trp Glu Tyr Pro Asp Thr
690 695 700
Trp Ser Thr Pro His Ser Tyr Phe Ser Leu Thr Phe Cys Val Gln Val
705 710 715 720
Gln Gly Lys Ser Lys Arg Glu Lys Lys Asp Arg Val Phe Thr Asp Lys
725 730 735
Thr Ser Ala Thr Val Ile Cys Arg Lys Asn Ala Ser Ile Ser Val Arg
740 745 750
Ala Gln Asp Arg Tyr Tyr Ser Ser Ser Trp Ser Glu Trp Ala Ser Val
755 760 765
Pro Cys Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
770 775 780
Gly Ser Gly Gly Gly Gly Ser Gly Arg Asn Leu Pro Val Ala Thr Pro
785 790 795 800
Asp Pro Gly Met Phe Pro Cys Leu His His Ser Gln Asn Leu Leu Arg
805 810 815
Ala Val Ser Asn Met Leu Gln Lys Ala Arg Gln Thr Leu Glu Phe Tyr
820 825 830
Pro Cys Thr Ser Glu Glu Ile Asp His Glu Asp Ile Thr Lys Asp Lys
835 840 845
Thr Ser Thr Val Glu Ala Cys Leu Pro Leu Glu Leu Thr Lys Asn Glu
850 855 860
Ser Cys Leu Asn Ser Arg Glu Thr Ser Phe Ile Thr Asn Gly Ser Cys
865 870 875 880
Leu Ala Ser Arg Lys Thr Ser Phe Met Met Ala Leu Cys Leu Ser Ser
885 890 895
Ile Tyr Glu Asp Leu Lys Met Tyr Gln Val Glu Phe Lys Thr Met Asn
900 905 910
Ala Lys Leu Leu Met Asp Pro Lys Arg Gln Ile Phe Leu Asp Gln Asn
915 920 925
Met Leu Ala Val Ile Asp Glu Leu Met Gln Ala Leu Asn Phe Asn Ser
930 935 940
Glu Thr Val Pro Gln Lys Ser Ser Leu Glu Glu Pro Asp Phe Tyr Lys
945 950 955 960
Thr Lys Ile Lys Leu Cys Ile Leu Leu His Ala Phe Arg Ile Arg Ala
965 970 975
Val Thr Ile Asp Arg Val Met Ser Tyr Leu Asn Ala Ser
980 985
<210> 14
<211> 974
<212> PRT
<213> Homo sapiens (Homo sapiens)
<400> 14
Phe Thr Val Thr Val Pro Lys Asp Leu Tyr Val Val Glu Tyr Gly Ser
1 5 10 15
Asn Met Thr Ile Glu Cys Lys Phe Pro Val Glu Lys Gln Leu Asp Leu
20 25 30
Ala Ala Leu Gln Val Phe Trp Met Met Glu Asp Lys Asn Ile Ile Gln
35 40 45
Phe Val His Gly Glu Glu Asp Leu Lys Val Gln His Ser Ser Tyr Arg
50 55 60
Gln Arg Ala Arg Leu Leu Lys Asp Gln Leu Ser Leu Gly Asn Ala Ala
65 70 75 80
Leu Gln Ile Thr Asp Val Lys Leu Gln Asp Ala Gly Val Tyr Thr Cys
85 90 95
Leu Ile Ala Tyr Lys Gly Ala Asp Tyr Lys Arg Ile Thr Val Lys Val
100 105 110
Asn Ala Pro Tyr Asn Lys Ile Asn Gln Arg Ile Leu Val Val Asp Pro
115 120 125
Val Thr Ser Glu His Glu Leu Thr Cys Gln Ala Glu Gly Tyr Pro Lys
130 135 140
Ala Glu Val Ile Trp Thr Ser Ser Asp His Gln Val Leu Ser Gly Lys
145 150 155 160
Thr Thr Thr Thr Asn Ser Lys Arg Glu Glu Lys Leu Phe Asn Val Thr
165 170 175
Ser Thr Leu Arg Ile Asn Thr Thr Thr Asn Glu Ile Phe Tyr Cys Thr
180 185 190
Phe Arg Arg Leu Asp Pro Glu Glu Asn His Thr Ala Glu Leu Val Ile
195 200 205
Pro Glu Leu Pro Leu Ala His Pro Pro Asn Glu Arg Gly Ser Gly Ile
210 215 220
Trp Glu Leu Lys Lys Asp Val Tyr Val Val Glu Leu Asp Trp Tyr Pro
225 230 235 240
Asp Ala Pro Gly Glu Met Val Val Leu Thr Cys Asp Thr Pro Glu Glu
245 250 255
Asp Gly Ile Thr Trp Thr Leu Asp Gln Ser Ser Glu Val Leu Gly Ser
260 265 270
Gly Lys Thr Leu Thr Ile Gln Val Lys Ala Ala Gly Asp Ala Gly Gln
275 280 285
Tyr Thr Cys His Lys Gly Gly Glu Val Leu Ser His Ser Leu Leu Leu
290 295 300
Leu His Lys Lys Glu Asp Gly Ile Trp Ser Thr Asp Ile Leu Lys Asp
305 310 315 320
Gln Lys Glu Pro Lys Asn Lys Thr Phe Leu Arg Cys Glu Ala Lys Asn
325 330 335
Tyr Ser Gly Arg Phe Thr Cys Trp Trp Leu Thr Thr Ile Ser Thr Asp
340 345 350
Leu Thr Phe Ser Val Lys Ser Ser Arg Gly Ser Ser Asp Pro Gln Gly
355 360 365
Val Thr Cys Gly Ala Ala Thr Leu Ser Ala Glu Arg Val Arg Gly Asp
370 375 380
Asn Lys Glu Tyr Glu Tyr Ser Val Glu Cys Gln Glu Asp Ser Ala Cys
385 390 395 400
Pro Ala Ala Glu Glu Ser Leu Pro Ile Glu Val Met Val Asp Ala Val
405 410 415
His Lys Leu Lys Tyr Glu Asn Tyr Thr Ser Ser Phe Phe Ile Arg Asp
420 425 430
Ile Ile Lys Pro Asp Pro Pro Lys Asn Leu Gln Leu Lys Pro Leu Lys
435 440 445
Asn Ser Arg Gln Val Glu Val Ser Trp Glu Tyr Pro Asp Thr Trp Ser
450 455 460
Thr Pro His Ser Tyr Phe Ser Leu Thr Phe Cys Val Gln Val Gln Gly
465 470 475 480
Lys Ser Lys Arg Glu Lys Lys Asp Arg Val Phe Thr Asp Lys Thr Ser
485 490 495
Ala Thr Val Ile Cys Arg Lys Asn Ala Ser Ile Ser Val Arg Ala Gln
500 505 510
Asp Arg Tyr Tyr Ser Ser Ser Trp Ser Glu Trp Ala Ser Val Pro Cys
515 520 525
Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
530 535 540
Gly Gly Gly Gly Ser Gly Arg Asn Leu Pro Val Ala Thr Pro Asp Pro
545 550 555 560
Gly Met Phe Pro Cys Leu His His Ser Gln Asn Leu Leu Arg Ala Val
565 570 575
Ser Asn Met Leu Gln Lys Ala Arg Gln Thr Leu Glu Phe Tyr Pro Cys
580 585 590
Thr Ser Glu Glu Ile Asp His Glu Asp Ile Thr Lys Asp Lys Thr Ser
595 600 605
Thr Val Glu Ala Cys Leu Pro Leu Glu Leu Thr Lys Asn Glu Ser Cys
610 615 620
Leu Asn Ser Arg Glu Thr Ser Phe Ile Thr Asn Gly Ser Cys Leu Ala
625 630 635 640
Ser Arg Lys Thr Ser Phe Met Met Ala Leu Cys Leu Ser Ser Ile Tyr
645 650 655
Glu Asp Leu Lys Met Tyr Gln Val Glu Phe Lys Thr Met Asn Ala Lys
660 665 670
Leu Leu Met Asp Pro Lys Arg Gln Ile Phe Leu Asp Gln Asn Met Leu
675 680 685
Ala Val Ile Asp Glu Leu Met Gln Ala Leu Asn Phe Asn Ser Glu Thr
690 695 700
Val Pro Gln Lys Ser Ser Leu Glu Glu Pro Asp Phe Tyr Lys Thr Lys
705 710 715 720
Ile Lys Leu Cys Ile Leu Leu His Ala Phe Arg Ile Arg Ala Val Thr
725 730 735
Ile Asp Arg Val Met Ser Tyr Leu Asn Ala Ser Asp Lys Thr His Thr
740 745 750
Cys Pro Pro Cys Pro Ala Pro Glu Asp Glu Gly Gly Pro Ser Val Phe
755 760 765
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro
770 775 780
Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val
785 790 795 800
Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr
805 810 815
Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val
820 825 830
Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys
835 840 845
Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser
850 855 860
Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Val Tyr Pro Pro
865 870 875 880
Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val
885 890 895
Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly
900 905 910
Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Glu Asp
915 920 925
Gly Ser Phe Ala Leu Val Ser Lys Leu Thr Val Asp Lys Ser Arg Trp
930 935 940
Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His
945 950 955 960
Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
965 970
<210> 15
<211> 974
<212> PRT
<213> Homo sapiens (Homo sapiens)
<400> 15
Phe Thr Val Thr Val Pro Lys Asp Leu Tyr Val Val Glu Tyr Gly Ser
1 5 10 15
Asn Met Thr Ile Glu Cys Lys Phe Pro Val Glu Lys Gln Leu Asp Leu
20 25 30
Ala Ala Leu Gln Val Phe Trp Met Met Glu Asp Lys Asn Ile Ile Gln
35 40 45
Phe Val His Gly Glu Glu Asp Leu Lys Val Gln His Ser Ser Tyr Arg
50 55 60
Gln Arg Ala Arg Leu Leu Lys Asp Gln Leu Ser Leu Gly Asn Ala Ala
65 70 75 80
Leu Gln Ile Thr Asp Val Lys Leu Gln Asp Ala Gly Val Tyr Thr Cys
85 90 95
Leu Ile Ala Tyr Lys Gly Ala Asp Tyr Lys Arg Ile Thr Val Lys Val
100 105 110
Asn Ala Pro Tyr Asn Lys Ile Asn Gln Arg Ile Leu Val Val Asp Pro
115 120 125
Val Thr Ser Glu His Glu Leu Thr Cys Gln Ala Glu Gly Tyr Pro Lys
130 135 140
Ala Glu Val Ile Trp Thr Ser Ser Asp His Gln Val Leu Ser Gly Lys
145 150 155 160
Thr Thr Thr Thr Asn Ser Lys Arg Glu Glu Lys Leu Phe Asn Val Thr
165 170 175
Ser Thr Leu Arg Ile Asn Thr Thr Thr Asn Glu Ile Phe Tyr Cys Thr
180 185 190
Phe Arg Arg Leu Asp Pro Glu Glu Asn His Thr Ala Glu Leu Val Ile
195 200 205
Pro Glu Leu Pro Leu Ala His Pro Pro Asn Glu Arg Gly Ser Gly Ile
210 215 220
Trp Glu Leu Lys Lys Asp Val Tyr Val Val Glu Leu Asp Trp Tyr Pro
225 230 235 240
Asp Ala Pro Gly Glu Met Val Val Leu Thr Cys Asp Thr Pro Glu Glu
245 250 255
Asp Gly Ile Thr Trp Thr Leu Asp Gln Ser Ser Glu Val Leu Gly Ser
260 265 270
Gly Lys Thr Leu Thr Ile Gln Val Lys Glu Ala Gly Asp Ala Gly Gln
275 280 285
Tyr Thr Cys His Lys Gly Gly Glu Val Leu Ser His Ser Leu Leu Leu
290 295 300
Leu His Lys Lys Glu Asp Gly Ile Trp Ser Thr Asp Ile Leu Lys Asp
305 310 315 320
Gln Lys Glu Pro Lys Asn Lys Thr Phe Leu Arg Cys Glu Ala Lys Asn
325 330 335
Tyr Ser Gly Arg Phe Thr Cys Trp Trp Leu Thr Thr Ile Ser Thr Asp
340 345 350
Leu Thr Phe Ser Val Lys Ser Ser Arg Gly Ser Ser Asp Pro Gln Gly
355 360 365
Val Thr Cys Gly Ala Ala Thr Leu Ser Ala Glu Arg Val Arg Gly Asp
370 375 380
Asn Lys Glu Tyr Glu Tyr Ser Val Glu Cys Gln Glu Asp Ser Ala Cys
385 390 395 400
Pro Ala Ala Glu Glu Ser Leu Pro Ile Glu Val Met Val Asp Ala Val
405 410 415
His Lys Leu Lys Tyr Glu Asn Tyr Thr Ser Ser Phe Phe Ile Arg Asp
420 425 430
Ile Ile Lys Pro Asp Pro Pro Lys Asn Leu Gln Leu Lys Pro Leu Lys
435 440 445
Asn Ser Arg Gln Val Glu Val Ser Trp Glu Tyr Pro Asp Thr Trp Ser
450 455 460
Thr Pro His Ser Tyr Phe Ser Leu Thr Phe Cys Val Gln Val Gln Gly
465 470 475 480
Lys Ser Lys Arg Glu Lys Lys Asp Arg Val Phe Thr Asp Lys Thr Ser
485 490 495
Ala Thr Val Ile Cys Arg Lys Asn Ala Ser Ile Ser Val Arg Ala Gln
500 505 510
Asp Arg Tyr Tyr Ser Ser Ser Trp Ser Glu Trp Ala Ser Val Pro Cys
515 520 525
Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
530 535 540
Gly Gly Gly Gly Ser Gly Arg Asn Leu Pro Val Ala Thr Pro Asp Pro
545 550 555 560
Gly Met Phe Pro Cys Leu His His Ser Gln Asn Leu Leu Arg Ala Val
565 570 575
Ser Asn Met Leu Gln Lys Ala Arg Gln Thr Leu Glu Phe Tyr Pro Cys
580 585 590
Thr Ser Glu Glu Ile Asp His Glu Asp Ile Thr Lys Asp Lys Thr Ser
595 600 605
Thr Val Glu Ala Cys Leu Pro Leu Glu Leu Thr Lys Asn Glu Ser Cys
610 615 620
Leu Asn Ser Arg Glu Thr Ser Phe Ile Thr Asn Gly Ser Cys Leu Ala
625 630 635 640
Ser Arg Lys Thr Ser Phe Met Met Ala Leu Cys Leu Ser Ser Ile Tyr
645 650 655
Glu Asp Leu Lys Met Tyr Gln Val Glu Phe Lys Thr Met Asn Ala Lys
660 665 670
Leu Leu Met Asp Pro Lys Arg Gln Ile Phe Leu Asp Gln Asn Met Leu
675 680 685
Ala Val Ile Asp Glu Leu Met Gln Ala Leu Asn Phe Asn Ser Glu Thr
690 695 700
Val Pro Gln Lys Ser Ser Leu Glu Glu Pro Asp Phe Tyr Lys Thr Lys
705 710 715 720
Ile Lys Leu Cys Ile Leu Leu His Ala Phe Arg Ile Arg Ala Val Thr
725 730 735
Ile Asp Arg Val Met Ser Tyr Leu Asn Ala Ser Asp Lys Thr His Thr
740 745 750
Cys Pro Pro Cys Pro Ala Pro Glu Asp Glu Gly Gly Pro Ser Val Phe
755 760 765
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro
770 775 780
Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val
785 790 795 800
Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr
805 810 815
Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val
820 825 830
Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys
835 840 845
Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser
850 855 860
Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Val Tyr Pro Pro
865 870 875 880
Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val
885 890 895
Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly
900 905 910
Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Glu Asp
915 920 925
Gly Ser Phe Ala Leu Val Ser Lys Leu Thr Val Asp Lys Ser Arg Trp
930 935 940
Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His
945 950 955 960
Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
965 970
<210> 16
<211> 431
<212> PRT
<213> Homo sapiens (Homo sapiens)
<400> 16
Leu Phe Thr Val Thr Val Pro Lys Glu Leu Tyr Ile Ile Glu His Gly
1 5 10 15
Ser Asn Val Thr Leu Glu Cys Asn Phe Asp Thr Gly Ser His Val Asn
20 25 30
Leu Gly Ala Ile Thr Ala Ser Leu Gln Lys Val Glu Asn Asp Thr Ser
35 40 45
Pro His Arg Glu Arg Ala Thr Leu Leu Glu Glu Gln Leu Pro Leu Gly
50 55 60
Lys Ala Ser Phe His Ile Pro Gln Val Gln Val Arg Asp Glu Gly Gln
65 70 75 80
Tyr Gln Cys Ile Ile Ile Tyr Gly Val Ala Trp Asp Tyr Lys Tyr Leu
85 90 95
Thr Leu Lys Val Lys Ala Ser Tyr Arg Lys Ile Asn Thr His Ile Leu
100 105 110
Lys Val Pro Glu Thr Asp Glu Val Glu Leu Thr Cys Gln Ala Thr Gly
115 120 125
Tyr Pro Leu Ala Glu Val Ser Trp Pro Asn Val Ser Val Pro Ala Asn
130 135 140
Thr Ser His Ser Arg Thr Pro Glu Gly Leu Tyr Gln Val Thr Ser Val
145 150 155 160
Leu Arg Leu Lys Pro Pro Pro Gly Arg Asn Phe Ser Cys Val Phe Trp
165 170 175
Asn Thr His Val Arg Glu Leu Thr Leu Ala Ser Ile Asp Leu Gln Ser
180 185 190
Gln Met Glu Pro Arg Thr His Pro Thr Gly Ser Gly Asp Lys Thr His
195 200 205
Thr Cys Pro Pro Cys Pro Ala Pro Glu Lys Lys Gly Gly Pro Ser Val
210 215 220
Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr
225 230 235 240
Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu
245 250 255
Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys
260 265 270
Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser
275 280 285
Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys
290 295 300
Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile
305 310 315 320
Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Val Leu Pro
325 330 335
Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Leu Cys Leu
340 345 350
Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn
355 360 365
Gly Gln Pro Glu Asn Arg Tyr Met Thr Trp Pro Pro Val Leu Asp Ser
370 375 380
Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg
385 390 395 400
Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu
405 410 415
His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
420 425 430
<210> 17
<211> 955
<212> PRT
<213> Homo sapiens (Homo sapiens)
<400> 17
Leu Phe Thr Val Thr Val Pro Lys Glu Leu Tyr Ile Ile Glu His Gly
1 5 10 15
Ser Asn Val Thr Leu Glu Cys Asn Phe Asp Thr Gly Ser His Val Asn
20 25 30
Leu Gly Ala Ile Thr Ala Ser Leu Gln Lys Val Glu Asn Asp Thr Ser
35 40 45
Pro His Arg Glu Arg Ala Thr Leu Leu Glu Glu Gln Leu Pro Leu Gly
50 55 60
Lys Ala Ser Phe His Ile Pro Gln Val Gln Val Arg Asp Glu Gly Gln
65 70 75 80
Tyr Gln Cys Ile Ile Ile Tyr Gly Val Ala Trp Asp Tyr Lys Tyr Leu
85 90 95
Thr Leu Lys Val Lys Ala Ser Tyr Arg Lys Ile Asn Thr His Ile Leu
100 105 110
Lys Val Pro Glu Thr Asp Glu Val Glu Leu Thr Cys Gln Ala Thr Gly
115 120 125
Tyr Pro Leu Ala Glu Val Ser Trp Pro Asn Val Ser Val Pro Ala Asn
130 135 140
Thr Ser His Ser Arg Thr Pro Glu Gly Leu Tyr Gln Val Thr Ser Val
145 150 155 160
Leu Arg Leu Lys Pro Pro Pro Gly Arg Asn Phe Ser Cys Val Phe Trp
165 170 175
Asn Thr His Val Arg Glu Leu Thr Leu Ala Ser Ile Asp Leu Gln Ser
180 185 190
Gln Met Glu Pro Arg Thr His Pro Thr Gly Ser Gly Ile Trp Glu Leu
195 200 205
Lys Lys Asp Val Tyr Val Val Glu Leu Asp Trp Tyr Pro Asp Ala Pro
210 215 220
Gly Glu Met Val Val Leu Thr Cys Asp Thr Pro Glu Glu Asp Gly Ile
225 230 235 240
Thr Trp Thr Leu Asp Gln Ser Ser Glu Val Leu Gly Ser Gly Lys Thr
245 250 255
Leu Thr Ile Gln Val Lys Ala Ala Gly Asp Ala Gly Gln Tyr Thr Cys
260 265 270
His Lys Gly Gly Glu Val Leu Ser His Ser Leu Leu Leu Leu His Lys
275 280 285
Lys Glu Asp Gly Ile Trp Ser Thr Asp Ile Leu Lys Asp Gln Lys Glu
290 295 300
Pro Lys Asn Lys Thr Phe Leu Arg Cys Glu Ala Lys Asn Tyr Ser Gly
305 310 315 320
Arg Phe Thr Cys Trp Trp Leu Thr Thr Ile Ser Thr Asp Leu Thr Phe
325 330 335
Ser Val Lys Ser Ser Arg Gly Ser Ser Asp Pro Gln Gly Val Thr Cys
340 345 350
Gly Ala Ala Thr Leu Ser Ala Glu Arg Val Arg Gly Asp Asn Lys Glu
355 360 365
Tyr Glu Tyr Ser Val Glu Cys Gln Glu Asp Ser Ala Cys Pro Ala Ala
370 375 380
Glu Glu Ser Leu Pro Ile Glu Val Met Val Asp Ala Val His Lys Leu
385 390 395 400
Lys Tyr Glu Asn Tyr Thr Ser Ser Phe Phe Ile Arg Asp Ile Ile Lys
405 410 415
Pro Asp Pro Pro Lys Asn Leu Gln Leu Lys Pro Leu Lys Asn Ser Arg
420 425 430
Gln Val Glu Val Ser Trp Glu Tyr Pro Asp Thr Trp Ser Thr Pro His
435 440 445
Ser Tyr Phe Ser Leu Thr Phe Cys Val Gln Val Gln Gly Lys Ser Lys
450 455 460
Arg Glu Lys Lys Asp Arg Val Phe Thr Asp Lys Thr Ser Ala Thr Val
465 470 475 480
Ile Cys Arg Lys Asn Ala Ser Ile Ser Val Arg Ala Gln Asp Arg Tyr
485 490 495
Tyr Ser Ser Ser Trp Ser Glu Trp Ala Ser Val Pro Cys Ser Gly Gly
500 505 510
Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
515 520 525
Gly Ser Gly Arg Asn Leu Pro Val Ala Thr Pro Asp Pro Gly Met Phe
530 535 540
Pro Cys Leu His His Ser Gln Asn Leu Leu Arg Ala Val Ser Asn Met
545 550 555 560
Leu Gln Lys Ala Arg Gln Thr Leu Glu Phe Tyr Pro Cys Thr Ser Glu
565 570 575
Glu Ile Asp His Glu Asp Ile Thr Lys Asp Lys Thr Ser Thr Val Glu
580 585 590
Ala Cys Leu Pro Leu Glu Leu Thr Lys Asn Glu Ser Cys Leu Asn Ser
595 600 605
Arg Glu Thr Ser Phe Ile Thr Asn Gly Ser Cys Leu Ala Ser Arg Lys
610 615 620
Thr Ser Phe Met Met Ala Leu Cys Leu Ser Ser Ile Tyr Glu Asp Leu
625 630 635 640
Lys Met Tyr Gln Val Glu Phe Lys Thr Met Asn Ala Lys Leu Leu Met
645 650 655
Asp Pro Lys Arg Gln Ile Phe Leu Asp Gln Asn Met Leu Ala Val Ile
660 665 670
Asp Glu Leu Met Gln Ala Leu Asn Phe Asn Ser Glu Thr Val Pro Gln
675 680 685
Lys Ser Ser Leu Glu Glu Pro Asp Phe Tyr Lys Thr Lys Ile Lys Leu
690 695 700
Cys Ile Leu Leu His Ala Phe Arg Ile Arg Ala Val Thr Ile Asp Arg
705 710 715 720
Val Met Ser Tyr Leu Asn Ala Ser Asp Lys Thr His Thr Cys Pro Pro
725 730 735
Cys Pro Ala Pro Glu Asp Glu Gly Gly Pro Ser Val Phe Leu Phe Pro
740 745 750
Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr
755 760 765
Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn
770 775 780
Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg
785 790 795 800
Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val
805 810 815
Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser
820 825 830
Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys
835 840 845
Gly Gln Pro Arg Glu Pro Gln Val Tyr Val Tyr Pro Pro Ser Arg Glu
850 855 860
Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe
865 870 875 880
Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu
885 890 895
Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Glu Asp Gly Ser Phe
900 905 910
Ala Leu Val Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly
915 920 925
Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr
930 935 940
Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
945 950 955
<210> 18
<211> 955
<212> PRT
<213> Homo sapiens (Homo sapiens)
<400> 18
Leu Phe Thr Val Thr Val Pro Lys Glu Leu Tyr Ile Ile Glu His Gly
1 5 10 15
Ser Asn Val Thr Leu Glu Cys Asn Phe Asp Thr Gly Ser His Val Asn
20 25 30
Leu Gly Ala Ile Thr Ala Ser Leu Gln Lys Val Glu Asn Asp Thr Ser
35 40 45
Pro His Arg Glu Arg Ala Thr Leu Leu Glu Glu Gln Leu Pro Leu Gly
50 55 60
Lys Ala Ser Phe His Ile Pro Gln Val Gln Val Arg Asp Glu Gly Gln
65 70 75 80
Tyr Gln Cys Ile Ile Ile Tyr Gly Val Ala Trp Asp Tyr Lys Tyr Leu
85 90 95
Thr Leu Lys Val Lys Ala Ser Tyr Arg Lys Ile Asn Thr His Ile Leu
100 105 110
Lys Val Pro Glu Thr Asp Glu Val Glu Leu Thr Cys Gln Ala Thr Gly
115 120 125
Tyr Pro Leu Ala Glu Val Ser Trp Pro Asn Val Ser Val Pro Ala Asn
130 135 140
Thr Ser His Ser Arg Thr Pro Glu Gly Leu Tyr Gln Val Thr Ser Val
145 150 155 160
Leu Arg Leu Lys Pro Pro Pro Gly Arg Asn Phe Ser Cys Val Phe Trp
165 170 175
Asn Thr His Val Arg Glu Leu Thr Leu Ala Ser Ile Asp Leu Gln Ser
180 185 190
Gln Met Glu Pro Arg Thr His Pro Thr Gly Ser Gly Ile Trp Glu Leu
195 200 205
Lys Lys Asp Val Tyr Val Val Glu Leu Asp Trp Tyr Pro Asp Ala Pro
210 215 220
Gly Glu Met Val Val Leu Thr Cys Asp Thr Pro Glu Glu Asp Gly Ile
225 230 235 240
Thr Trp Thr Leu Asp Gln Ser Ser Glu Val Leu Gly Ser Gly Lys Thr
245 250 255
Leu Thr Ile Gln Val Lys Glu Ala Gly Asp Ala Gly Gln Tyr Thr Cys
260 265 270
His Lys Gly Gly Glu Val Leu Ser His Ser Leu Leu Leu Leu His Lys
275 280 285
Lys Glu Asp Gly Ile Trp Ser Thr Asp Ile Leu Lys Asp Gln Lys Glu
290 295 300
Pro Lys Asn Lys Thr Phe Leu Arg Cys Glu Ala Lys Asn Tyr Ser Gly
305 310 315 320
Arg Phe Thr Cys Trp Trp Leu Thr Thr Ile Ser Thr Asp Leu Thr Phe
325 330 335
Ser Val Lys Ser Ser Arg Gly Ser Ser Asp Pro Gln Gly Val Thr Cys
340 345 350
Gly Ala Ala Thr Leu Ser Ala Glu Arg Val Arg Gly Asp Asn Lys Glu
355 360 365
Tyr Glu Tyr Ser Val Glu Cys Gln Glu Asp Ser Ala Cys Pro Ala Ala
370 375 380
Glu Glu Ser Leu Pro Ile Glu Val Met Val Asp Ala Val His Lys Leu
385 390 395 400
Lys Tyr Glu Asn Tyr Thr Ser Ser Phe Phe Ile Arg Asp Ile Ile Lys
405 410 415
Pro Asp Pro Pro Lys Asn Leu Gln Leu Lys Pro Leu Lys Asn Ser Arg
420 425 430
Gln Val Glu Val Ser Trp Glu Tyr Pro Asp Thr Trp Ser Thr Pro His
435 440 445
Ser Tyr Phe Ser Leu Thr Phe Cys Val Gln Val Gln Gly Lys Ser Lys
450 455 460
Arg Glu Lys Lys Asp Arg Val Phe Thr Asp Lys Thr Ser Ala Thr Val
465 470 475 480
Ile Cys Arg Lys Asn Ala Ser Ile Ser Val Arg Ala Gln Asp Arg Tyr
485 490 495
Tyr Ser Ser Ser Trp Ser Glu Trp Ala Ser Val Pro Cys Ser Gly Gly
500 505 510
Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
515 520 525
Gly Ser Gly Arg Asn Leu Pro Val Ala Thr Pro Asp Pro Gly Met Phe
530 535 540
Pro Cys Leu His His Ser Gln Asn Leu Leu Arg Ala Val Ser Asn Met
545 550 555 560
Leu Gln Lys Ala Arg Gln Thr Leu Glu Phe Tyr Pro Cys Thr Ser Glu
565 570 575
Glu Ile Asp His Glu Asp Ile Thr Lys Asp Lys Thr Ser Thr Val Glu
580 585 590
Ala Cys Leu Pro Leu Glu Leu Thr Lys Asn Glu Ser Cys Leu Asn Ser
595 600 605
Arg Glu Thr Ser Phe Ile Thr Asn Gly Ser Cys Leu Ala Ser Arg Lys
610 615 620
Thr Ser Phe Met Met Ala Leu Cys Leu Ser Ser Ile Tyr Glu Asp Leu
625 630 635 640
Lys Met Tyr Gln Val Glu Phe Lys Thr Met Asn Ala Lys Leu Leu Met
645 650 655
Asp Pro Lys Arg Gln Ile Phe Leu Asp Gln Asn Met Leu Ala Val Ile
660 665 670
Asp Glu Leu Met Gln Ala Leu Asn Phe Asn Ser Glu Thr Val Pro Gln
675 680 685
Lys Ser Ser Leu Glu Glu Pro Asp Phe Tyr Lys Thr Lys Ile Lys Leu
690 695 700
Cys Ile Leu Leu His Ala Phe Arg Ile Arg Ala Val Thr Ile Asp Arg
705 710 715 720
Val Met Ser Tyr Leu Asn Ala Ser Asp Lys Thr His Thr Cys Pro Pro
725 730 735
Cys Pro Ala Pro Glu Asp Glu Gly Gly Pro Ser Val Phe Leu Phe Pro
740 745 750
Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr
755 760 765
Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn
770 775 780
Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg
785 790 795 800
Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val
805 810 815
Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser
820 825 830
Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys
835 840 845
Gly Gln Pro Arg Glu Pro Gln Val Tyr Val Tyr Pro Pro Ser Arg Glu
850 855 860
Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe
865 870 875 880
Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu
885 890 895
Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Glu Asp Gly Ser Phe
900 905 910
Ala Leu Val Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly
915 920 925
Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr
930 935 940
Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
945 950 955
<210> 19
<211> 970
<212> PRT
<213> Homo sapiens (Homo sapiens)
<400> 19
Leu Phe Thr Val Thr Val Pro Lys Glu Leu Tyr Ile Ile Glu His Gly
1 5 10 15
Ser Asn Val Thr Leu Glu Cys Asn Phe Asp Thr Gly Ser His Val Asn
20 25 30
Leu Gly Ala Ile Thr Ala Ser Leu Gln Lys Val Glu Asn Asp Thr Ser
35 40 45
Pro His Arg Glu Arg Ala Thr Leu Leu Glu Glu Gln Leu Pro Leu Gly
50 55 60
Lys Ala Ser Phe His Ile Pro Gln Val Gln Val Arg Asp Glu Gly Gln
65 70 75 80
Tyr Gln Cys Ile Ile Ile Tyr Gly Val Ala Trp Asp Tyr Lys Tyr Leu
85 90 95
Thr Leu Lys Val Lys Ala Ser Tyr Arg Lys Ile Asn Thr His Ile Leu
100 105 110
Lys Val Pro Glu Thr Asp Glu Val Glu Leu Thr Cys Gln Ala Thr Gly
115 120 125
Tyr Pro Leu Ala Glu Val Ser Trp Pro Asn Val Ser Val Pro Ala Asn
130 135 140
Thr Ser His Ser Arg Thr Pro Glu Gly Leu Tyr Gln Val Thr Ser Val
145 150 155 160
Leu Arg Leu Lys Pro Pro Pro Gly Arg Asn Phe Ser Cys Val Phe Trp
165 170 175
Asn Thr His Val Arg Glu Leu Thr Leu Ala Ser Ile Asp Leu Gln Ser
180 185 190
Gln Met Glu Pro Arg Thr His Pro Thr Gly Ser Gly Asp Lys Thr His
195 200 205
Thr Cys Pro Pro Cys Pro Ala Pro Glu Asp Glu Gly Gly Pro Ser Val
210 215 220
Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr
225 230 235 240
Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu
245 250 255
Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys
260 265 270
Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser
275 280 285
Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys
290 295 300
Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile
305 310 315 320
Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Val Tyr Pro
325 330 335
Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu
340 345 350
Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn
355 360 365
Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Glu
370 375 380
Asp Gly Ser Phe Ala Leu Val Ser Lys Leu Thr Val Asp Lys Ser Arg
385 390 395 400
Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu
405 410 415
His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Gly
420 425 430
Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ile Trp
435 440 445
Glu Leu Lys Lys Asp Val Tyr Val Val Glu Leu Asp Trp Tyr Pro Asp
450 455 460
Ala Pro Gly Glu Met Val Val Leu Thr Cys Asp Thr Pro Glu Glu Asp
465 470 475 480
Gly Ile Thr Trp Thr Leu Asp Gln Ser Ser Glu Val Leu Gly Ser Gly
485 490 495
Lys Thr Leu Thr Ile Gln Val Lys Ala Ala Gly Asp Ala Gly Gln Tyr
500 505 510
Thr Cys His Lys Gly Gly Glu Val Leu Ser His Ser Leu Leu Leu Leu
515 520 525
His Lys Lys Glu Asp Gly Ile Trp Ser Thr Asp Ile Leu Lys Asp Gln
530 535 540
Lys Glu Pro Lys Asn Lys Thr Phe Leu Arg Cys Glu Ala Lys Asn Tyr
545 550 555 560
Ser Gly Arg Phe Thr Cys Trp Trp Leu Thr Thr Ile Ser Thr Asp Leu
565 570 575
Thr Phe Ser Val Lys Ser Ser Arg Gly Ser Ser Asp Pro Gln Gly Val
580 585 590
Thr Cys Gly Ala Ala Thr Leu Ser Ala Glu Arg Val Arg Gly Asp Asn
595 600 605
Lys Glu Tyr Glu Tyr Ser Val Glu Cys Gln Glu Asp Ser Ala Cys Pro
610 615 620
Ala Ala Glu Glu Ser Leu Pro Ile Glu Val Met Val Asp Ala Val His
625 630 635 640
Lys Leu Lys Tyr Glu Asn Tyr Thr Ser Ser Phe Phe Ile Arg Asp Ile
645 650 655
Ile Lys Pro Asp Pro Pro Lys Asn Leu Gln Leu Lys Pro Leu Lys Asn
660 665 670
Ser Arg Gln Val Glu Val Ser Trp Glu Tyr Pro Asp Thr Trp Ser Thr
675 680 685
Pro His Ser Tyr Phe Ser Leu Thr Phe Cys Val Gln Val Gln Gly Lys
690 695 700
Ser Lys Arg Glu Lys Lys Asp Arg Val Phe Thr Asp Lys Thr Ser Ala
705 710 715 720
Thr Val Ile Cys Arg Lys Asn Ala Ser Ile Ser Val Arg Ala Gln Asp
725 730 735
Arg Tyr Tyr Ser Ser Ser Trp Ser Glu Trp Ala Ser Val Pro Cys Ser
740 745 750
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly
755 760 765
Gly Gly Gly Ser Gly Arg Asn Leu Pro Val Ala Thr Pro Asp Pro Gly
770 775 780
Met Phe Pro Cys Leu His His Ser Gln Asn Leu Leu Arg Ala Val Ser
785 790 795 800
Asn Met Leu Gln Lys Ala Arg Gln Thr Leu Glu Phe Tyr Pro Cys Thr
805 810 815
Ser Glu Glu Ile Asp His Glu Asp Ile Thr Lys Asp Lys Thr Ser Thr
820 825 830
Val Glu Ala Cys Leu Pro Leu Glu Leu Thr Lys Asn Glu Ser Cys Leu
835 840 845
Asn Ser Arg Glu Thr Ser Phe Ile Thr Asn Gly Ser Cys Leu Ala Ser
850 855 860
Arg Lys Thr Ser Phe Met Met Ala Leu Cys Leu Ser Ser Ile Tyr Glu
865 870 875 880
Asp Leu Lys Met Tyr Gln Val Glu Phe Lys Thr Met Asn Ala Lys Leu
885 890 895
Leu Met Asp Pro Lys Arg Gln Ile Phe Leu Asp Gln Asn Met Leu Ala
900 905 910
Val Ile Asp Glu Leu Met Gln Ala Leu Asn Phe Asn Ser Glu Thr Val
915 920 925
Pro Gln Lys Ser Ser Leu Glu Glu Pro Asp Phe Tyr Lys Thr Lys Ile
930 935 940
Lys Leu Cys Ile Leu Leu His Ala Phe Arg Ile Arg Ala Val Thr Ile
945 950 955 960
Asp Arg Val Met Ser Tyr Leu Asn Ala Ser
965 970
<210> 20
<211> 970
<212> PRT
<213> Homo sapiens (Homo sapiens)
<400> 20
Leu Phe Thr Val Thr Val Pro Lys Glu Leu Tyr Ile Ile Glu His Gly
1 5 10 15
Ser Asn Val Thr Leu Glu Cys Asn Phe Asp Thr Gly Ser His Val Asn
20 25 30
Leu Gly Ala Ile Thr Ala Ser Leu Gln Lys Val Glu Asn Asp Thr Ser
35 40 45
Pro His Arg Glu Arg Ala Thr Leu Leu Glu Glu Gln Leu Pro Leu Gly
50 55 60
Lys Ala Ser Phe His Ile Pro Gln Val Gln Val Arg Asp Glu Gly Gln
65 70 75 80
Tyr Gln Cys Ile Ile Ile Tyr Gly Val Ala Trp Asp Tyr Lys Tyr Leu
85 90 95
Thr Leu Lys Val Lys Ala Ser Tyr Arg Lys Ile Asn Thr His Ile Leu
100 105 110
Lys Val Pro Glu Thr Asp Glu Val Glu Leu Thr Cys Gln Ala Thr Gly
115 120 125
Tyr Pro Leu Ala Glu Val Ser Trp Pro Asn Val Ser Val Pro Ala Asn
130 135 140
Thr Ser His Ser Arg Thr Pro Glu Gly Leu Tyr Gln Val Thr Ser Val
145 150 155 160
Leu Arg Leu Lys Pro Pro Pro Gly Arg Asn Phe Ser Cys Val Phe Trp
165 170 175
Asn Thr His Val Arg Glu Leu Thr Leu Ala Ser Ile Asp Leu Gln Ser
180 185 190
Gln Met Glu Pro Arg Thr His Pro Thr Gly Ser Gly Asp Lys Thr His
195 200 205
Thr Cys Pro Pro Cys Pro Ala Pro Glu Asp Glu Gly Gly Pro Ser Val
210 215 220
Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr
225 230 235 240
Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu
245 250 255
Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys
260 265 270
Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser
275 280 285
Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys
290 295 300
Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile
305 310 315 320
Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Val Tyr Pro
325 330 335
Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu
340 345 350
Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn
355 360 365
Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Glu
370 375 380
Asp Gly Ser Phe Ala Leu Val Ser Lys Leu Thr Val Asp Lys Ser Arg
385 390 395 400
Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu
405 410 415
His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Gly
420 425 430
Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ile Trp
435 440 445
Glu Leu Lys Lys Asp Val Tyr Val Val Glu Leu Asp Trp Tyr Pro Asp
450 455 460
Ala Pro Gly Glu Met Val Val Leu Thr Cys Asp Thr Pro Glu Glu Asp
465 470 475 480
Gly Ile Thr Trp Thr Leu Asp Gln Ser Ser Glu Val Leu Gly Ser Gly
485 490 495
Lys Thr Leu Thr Ile Gln Val Lys Glu Ala Gly Asp Ala Gly Gln Tyr
500 505 510
Thr Cys His Lys Gly Gly Glu Val Leu Ser His Ser Leu Leu Leu Leu
515 520 525
His Lys Lys Glu Asp Gly Ile Trp Ser Thr Asp Ile Leu Lys Asp Gln
530 535 540
Lys Glu Pro Lys Asn Lys Thr Phe Leu Arg Cys Glu Ala Lys Asn Tyr
545 550 555 560
Ser Gly Arg Phe Thr Cys Trp Trp Leu Thr Thr Ile Ser Thr Asp Leu
565 570 575
Thr Phe Ser Val Lys Ser Ser Arg Gly Ser Ser Asp Pro Gln Gly Val
580 585 590
Thr Cys Gly Ala Ala Thr Leu Ser Ala Glu Arg Val Arg Gly Asp Asn
595 600 605
Lys Glu Tyr Glu Tyr Ser Val Glu Cys Gln Glu Asp Ser Ala Cys Pro
610 615 620
Ala Ala Glu Glu Ser Leu Pro Ile Glu Val Met Val Asp Ala Val His
625 630 635 640
Lys Leu Lys Tyr Glu Asn Tyr Thr Ser Ser Phe Phe Ile Arg Asp Ile
645 650 655
Ile Lys Pro Asp Pro Pro Lys Asn Leu Gln Leu Lys Pro Leu Lys Asn
660 665 670
Ser Arg Gln Val Glu Val Ser Trp Glu Tyr Pro Asp Thr Trp Ser Thr
675 680 685
Pro His Ser Tyr Phe Ser Leu Thr Phe Cys Val Gln Val Gln Gly Lys
690 695 700
Ser Lys Arg Glu Lys Lys Asp Arg Val Phe Thr Asp Lys Thr Ser Ala
705 710 715 720
Thr Val Ile Cys Arg Lys Asn Ala Ser Ile Ser Val Arg Ala Gln Asp
725 730 735
Arg Tyr Tyr Ser Ser Ser Trp Ser Glu Trp Ala Ser Val Pro Cys Ser
740 745 750
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly
755 760 765
Gly Gly Gly Ser Gly Arg Asn Leu Pro Val Ala Thr Pro Asp Pro Gly
770 775 780
Met Phe Pro Cys Leu His His Ser Gln Asn Leu Leu Arg Ala Val Ser
785 790 795 800
Asn Met Leu Gln Lys Ala Arg Gln Thr Leu Glu Phe Tyr Pro Cys Thr
805 810 815
Ser Glu Glu Ile Asp His Glu Asp Ile Thr Lys Asp Lys Thr Ser Thr
820 825 830
Val Glu Ala Cys Leu Pro Leu Glu Leu Thr Lys Asn Glu Ser Cys Leu
835 840 845
Asn Ser Arg Glu Thr Ser Phe Ile Thr Asn Gly Ser Cys Leu Ala Ser
850 855 860
Arg Lys Thr Ser Phe Met Met Ala Leu Cys Leu Ser Ser Ile Tyr Glu
865 870 875 880
Asp Leu Lys Met Tyr Gln Val Glu Phe Lys Thr Met Asn Ala Lys Leu
885 890 895
Leu Met Asp Pro Lys Arg Gln Ile Phe Leu Asp Gln Asn Met Leu Ala
900 905 910
Val Ile Asp Glu Leu Met Gln Ala Leu Asn Phe Asn Ser Glu Thr Val
915 920 925
Pro Gln Lys Ser Ser Leu Glu Glu Pro Asp Phe Tyr Lys Thr Lys Ile
930 935 940
Lys Leu Cys Ile Leu Leu His Ala Phe Arg Ile Arg Ala Val Thr Ile
945 950 955 960
Asp Arg Val Met Ser Tyr Leu Asn Ala Ser
965 970
<210> 21
<211> 449
<212> PRT
<213> artificial sequence
<220>
<223> synthetic construct
<400> 21
Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg
1 5 10 15
Ser Leu Arg Leu Asp Cys Lys Ala Ser Gly Ile Thr Phe Ser Asn Ser
20 25 30
Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Val Ile Trp Tyr Asp Gly Ser Lys Arg Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Phe
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Thr Asn Asp Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser
100 105 110
Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser
115 120 125
Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp
130 135 140
Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr
145 150 155 160
Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr
165 170 175
Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln
180 185 190
Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp
195 200 205
Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Pro Gly Ser Gly Asp Lys
210 215 220
Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Lys Lys Gly Gly Pro
225 230 235 240
Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser
245 250 255
Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp
260 265 270
Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn
275 280 285
Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val
290 295 300
Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu
305 310 315 320
Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys
325 330 335
Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Val
340 345 350
Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Leu
355 360 365
Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu
370 375 380
Ser Asn Gly Gln Pro Glu Asn Arg Tyr Met Thr Trp Pro Pro Val Leu
385 390 395 400
Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys
405 410 415
Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu
420 425 430
Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly
435 440 445
Lys
<210> 22
<211> 973
<212> PRT
<213> artificial sequence
<220>
<223> synthetic construct
<400> 22
Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg
1 5 10 15
Ser Leu Arg Leu Asp Cys Lys Ala Ser Gly Ile Thr Phe Ser Asn Ser
20 25 30
Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Val Ile Trp Tyr Asp Gly Ser Lys Arg Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Phe
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Thr Asn Asp Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser
100 105 110
Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser
115 120 125
Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp
130 135 140
Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr
145 150 155 160
Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr
165 170 175
Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln
180 185 190
Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp
195 200 205
Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Pro Gly Ser Gly Ile Trp
210 215 220
Glu Leu Lys Lys Asp Val Tyr Val Val Glu Leu Asp Trp Tyr Pro Asp
225 230 235 240
Ala Pro Gly Glu Met Val Val Leu Thr Cys Asp Thr Pro Glu Glu Asp
245 250 255
Gly Ile Thr Trp Thr Leu Asp Gln Ser Ser Glu Val Leu Gly Ser Gly
260 265 270
Lys Thr Leu Thr Ile Gln Val Lys Ala Ala Gly Asp Ala Gly Gln Tyr
275 280 285
Thr Cys His Lys Gly Gly Glu Val Leu Ser His Ser Leu Leu Leu Leu
290 295 300
His Lys Lys Glu Asp Gly Ile Trp Ser Thr Asp Ile Leu Lys Asp Gln
305 310 315 320
Lys Glu Pro Lys Asn Lys Thr Phe Leu Arg Cys Glu Ala Lys Asn Tyr
325 330 335
Ser Gly Arg Phe Thr Cys Trp Trp Leu Thr Thr Ile Ser Thr Asp Leu
340 345 350
Thr Phe Ser Val Lys Ser Ser Arg Gly Ser Ser Asp Pro Gln Gly Val
355 360 365
Thr Cys Gly Ala Ala Thr Leu Ser Ala Glu Arg Val Arg Gly Asp Asn
370 375 380
Lys Glu Tyr Glu Tyr Ser Val Glu Cys Gln Glu Asp Ser Ala Cys Pro
385 390 395 400
Ala Ala Glu Glu Ser Leu Pro Ile Glu Val Met Val Asp Ala Val His
405 410 415
Lys Leu Lys Tyr Glu Asn Tyr Thr Ser Ser Phe Phe Ile Arg Asp Ile
420 425 430
Ile Lys Pro Asp Pro Pro Lys Asn Leu Gln Leu Lys Pro Leu Lys Asn
435 440 445
Ser Arg Gln Val Glu Val Ser Trp Glu Tyr Pro Asp Thr Trp Ser Thr
450 455 460
Pro His Ser Tyr Phe Ser Leu Thr Phe Cys Val Gln Val Gln Gly Lys
465 470 475 480
Ser Lys Arg Glu Lys Lys Asp Arg Val Phe Thr Asp Lys Thr Ser Ala
485 490 495
Thr Val Ile Cys Arg Lys Asn Ala Ser Ile Ser Val Arg Ala Gln Asp
500 505 510
Arg Tyr Tyr Ser Ser Ser Trp Ser Glu Trp Ala Ser Val Pro Cys Ser
515 520 525
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly
530 535 540
Gly Gly Gly Ser Gly Arg Asn Leu Pro Val Ala Thr Pro Asp Pro Gly
545 550 555 560
Met Phe Pro Cys Leu His His Ser Gln Asn Leu Leu Arg Ala Val Ser
565 570 575
Asn Met Leu Gln Lys Ala Arg Gln Thr Leu Glu Phe Tyr Pro Cys Thr
580 585 590
Ser Glu Glu Ile Asp His Glu Asp Ile Thr Lys Asp Lys Thr Ser Thr
595 600 605
Val Glu Ala Cys Leu Pro Leu Glu Leu Thr Lys Asn Glu Ser Cys Leu
610 615 620
Asn Ser Arg Glu Thr Ser Phe Ile Thr Asn Gly Ser Cys Leu Ala Ser
625 630 635 640
Arg Lys Thr Ser Phe Met Met Ala Leu Cys Leu Ser Ser Ile Tyr Glu
645 650 655
Asp Leu Lys Met Tyr Gln Val Glu Phe Lys Thr Met Asn Ala Lys Leu
660 665 670
Leu Met Asp Pro Lys Arg Gln Ile Phe Leu Asp Gln Asn Met Leu Ala
675 680 685
Val Ile Asp Glu Leu Met Gln Ala Leu Asn Phe Asn Ser Glu Thr Val
690 695 700
Pro Gln Lys Ser Ser Leu Glu Glu Pro Asp Phe Tyr Lys Thr Lys Ile
705 710 715 720
Lys Leu Cys Ile Leu Leu His Ala Phe Arg Ile Arg Ala Val Thr Ile
725 730 735
Asp Arg Val Met Ser Tyr Leu Asn Ala Ser Asp Lys Thr His Thr Cys
740 745 750
Pro Pro Cys Pro Ala Pro Glu Asp Glu Gly Gly Pro Ser Val Phe Leu
755 760 765
Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu
770 775 780
Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys
785 790 795 800
Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys
805 810 815
Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu
820 825 830
Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys
835 840 845
Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys
850 855 860
Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Val Tyr Pro Pro Ser
865 870 875 880
Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys
885 890 895
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln
900 905 910
Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Glu Asp Gly
915 920 925
Ser Phe Ala Leu Val Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln
930 935 940
Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn
945 950 955 960
His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
965 970
<210> 23
<211> 973
<212> PRT
<213> artificial sequence
<220>
<223> synthetic construct
<400> 23
Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg
1 5 10 15
Ser Leu Arg Leu Asp Cys Lys Ala Ser Gly Ile Thr Phe Ser Asn Ser
20 25 30
Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Val Ile Trp Tyr Asp Gly Ser Lys Arg Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Phe
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Thr Asn Asp Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser
100 105 110
Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser
115 120 125
Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp
130 135 140
Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr
145 150 155 160
Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr
165 170 175
Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln
180 185 190
Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp
195 200 205
Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Pro Gly Ser Gly Ile Trp
210 215 220
Glu Leu Lys Lys Asp Val Tyr Val Val Glu Leu Asp Trp Tyr Pro Asp
225 230 235 240
Ala Pro Gly Glu Met Val Val Leu Thr Cys Asp Thr Pro Glu Glu Asp
245 250 255
Gly Ile Thr Trp Thr Leu Asp Gln Ser Ser Glu Val Leu Gly Ser Gly
260 265 270
Lys Thr Leu Thr Ile Gln Val Lys Glu Ala Gly Asp Ala Gly Gln Tyr
275 280 285
Thr Cys His Lys Gly Gly Glu Val Leu Ser His Ser Leu Leu Leu Leu
290 295 300
His Lys Lys Glu Asp Gly Ile Trp Ser Thr Asp Ile Leu Lys Asp Gln
305 310 315 320
Lys Glu Pro Lys Asn Lys Thr Phe Leu Arg Cys Glu Ala Lys Asn Tyr
325 330 335
Ser Gly Arg Phe Thr Cys Trp Trp Leu Thr Thr Ile Ser Thr Asp Leu
340 345 350
Thr Phe Ser Val Lys Ser Ser Arg Gly Ser Ser Asp Pro Gln Gly Val
355 360 365
Thr Cys Gly Ala Ala Thr Leu Ser Ala Glu Arg Val Arg Gly Asp Asn
370 375 380
Lys Glu Tyr Glu Tyr Ser Val Glu Cys Gln Glu Asp Ser Ala Cys Pro
385 390 395 400
Ala Ala Glu Glu Ser Leu Pro Ile Glu Val Met Val Asp Ala Val His
405 410 415
Lys Leu Lys Tyr Glu Asn Tyr Thr Ser Ser Phe Phe Ile Arg Asp Ile
420 425 430
Ile Lys Pro Asp Pro Pro Lys Asn Leu Gln Leu Lys Pro Leu Lys Asn
435 440 445
Ser Arg Gln Val Glu Val Ser Trp Glu Tyr Pro Asp Thr Trp Ser Thr
450 455 460
Pro His Ser Tyr Phe Ser Leu Thr Phe Cys Val Gln Val Gln Gly Lys
465 470 475 480
Ser Lys Arg Glu Lys Lys Asp Arg Val Phe Thr Asp Lys Thr Ser Ala
485 490 495
Thr Val Ile Cys Arg Lys Asn Ala Ser Ile Ser Val Arg Ala Gln Asp
500 505 510
Arg Tyr Tyr Ser Ser Ser Trp Ser Glu Trp Ala Ser Val Pro Cys Ser
515 520 525
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly
530 535 540
Gly Gly Gly Ser Gly Arg Asn Leu Pro Val Ala Thr Pro Asp Pro Gly
545 550 555 560
Met Phe Pro Cys Leu His His Ser Gln Asn Leu Leu Arg Ala Val Ser
565 570 575
Asn Met Leu Gln Lys Ala Arg Gln Thr Leu Glu Phe Tyr Pro Cys Thr
580 585 590
Ser Glu Glu Ile Asp His Glu Asp Ile Thr Lys Asp Lys Thr Ser Thr
595 600 605
Val Glu Ala Cys Leu Pro Leu Glu Leu Thr Lys Asn Glu Ser Cys Leu
610 615 620
Asn Ser Arg Glu Thr Ser Phe Ile Thr Asn Gly Ser Cys Leu Ala Ser
625 630 635 640
Arg Lys Thr Ser Phe Met Met Ala Leu Cys Leu Ser Ser Ile Tyr Glu
645 650 655
Asp Leu Lys Met Tyr Gln Val Glu Phe Lys Thr Met Asn Ala Lys Leu
660 665 670
Leu Met Asp Pro Lys Arg Gln Ile Phe Leu Asp Gln Asn Met Leu Ala
675 680 685
Val Ile Asp Glu Leu Met Gln Ala Leu Asn Phe Asn Ser Glu Thr Val
690 695 700
Pro Gln Lys Ser Ser Leu Glu Glu Pro Asp Phe Tyr Lys Thr Lys Ile
705 710 715 720
Lys Leu Cys Ile Leu Leu His Ala Phe Arg Ile Arg Ala Val Thr Ile
725 730 735
Asp Arg Val Met Ser Tyr Leu Asn Ala Ser Asp Lys Thr His Thr Cys
740 745 750
Pro Pro Cys Pro Ala Pro Glu Asp Glu Gly Gly Pro Ser Val Phe Leu
755 760 765
Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu
770 775 780
Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys
785 790 795 800
Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys
805 810 815
Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu
820 825 830
Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys
835 840 845
Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys
850 855 860
Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Val Tyr Pro Pro Ser
865 870 875 880
Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys
885 890 895
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln
900 905 910
Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Glu Asp Gly
915 920 925
Ser Phe Ala Leu Val Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln
930 935 940
Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn
945 950 955 960
His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
965 970
<210> 24
<211> 566
<212> PRT
<213> Homo sapiens (Homo sapiens)
<400> 24
Leu Phe Thr Val Thr Val Pro Lys Glu Leu Tyr Ile Ile Glu His Gly
1 5 10 15
Ser Asn Val Thr Leu Glu Cys Asn Phe Asp Thr Gly Ser His Val Asn
20 25 30
Leu Gly Ala Ile Thr Ala Ser Leu Gln Lys Val Glu Asn Asp Thr Ser
35 40 45
Pro His Arg Glu Arg Ala Thr Leu Leu Glu Glu Gln Leu Pro Leu Gly
50 55 60
Lys Ala Ser Phe His Ile Pro Gln Val Gln Val Arg Asp Glu Gly Gln
65 70 75 80
Tyr Gln Cys Ile Ile Ile Tyr Gly Val Ala Trp Asp Tyr Lys Tyr Leu
85 90 95
Thr Leu Lys Val Lys Ala Ser Tyr Arg Lys Ile Asn Thr His Ile Leu
100 105 110
Lys Val Pro Glu Thr Asp Glu Val Glu Leu Thr Cys Gln Ala Thr Gly
115 120 125
Tyr Pro Leu Ala Glu Val Ser Trp Pro Asn Val Ser Val Pro Ala Asn
130 135 140
Thr Ser His Ser Arg Thr Pro Glu Gly Leu Tyr Gln Val Thr Ser Val
145 150 155 160
Leu Arg Leu Lys Pro Pro Pro Gly Arg Asn Phe Ser Cys Val Phe Trp
165 170 175
Asn Thr His Val Arg Glu Leu Thr Leu Ala Ser Ile Asp Leu Gln Ser
180 185 190
Gln Met Glu Pro Arg Thr His Pro Thr Gly Gly Gly Gly Ser Ala Pro
195 200 205
Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu Leu
210 215 220
Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn Pro
225 230 235 240
Lys Leu Thr Asp Met Leu Thr Phe Glu Phe Tyr Met Pro Lys Lys Ala
245 250 255
Thr Glu Leu Lys His Leu Gln Cys Leu Glu Arg Glu Leu Lys Pro Leu
260 265 270
Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg Pro
275 280 285
Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys Gly
290 295 300
Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr Ile
305 310 315 320
Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile Ile Ser
325 330 335
Thr Leu Thr Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu
340 345 350
Asp Glu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp
355 360 365
Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp
370 375 380
Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly
385 390 395 400
Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn
405 410 415
Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp
420 425 430
Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro
435 440 445
Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu
450 455 460
Pro Gln Val Tyr Val Tyr Pro Pro Ser Arg Glu Glu Met Thr Lys Asn
465 470 475 480
Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile
485 490 495
Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr
500 505 510
Thr Pro Pro Val Leu Asp Glu Asp Gly Ser Phe Ala Leu Val Ser Lys
515 520 525
Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys
530 535 540
Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu
545 550 555 560
Ser Leu Ser Pro Gly Lys
565
<210> 25
<211> 133
<212> PRT
<213> Homo sapiens (Homo sapiens)
<400> 25
Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His
1 5 10 15
Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys
20 25 30
Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys
35 40 45
Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys
50 55 60
Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu
65 70 75 80
Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu
85 90 95
Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala
100 105 110
Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile
115 120 125
Ile Ser Thr Leu Thr
130
<210> 26
<211> 133
<212> PRT
<213> artificial sequence
<220>
<223> synthetic construct
<400> 26
Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His
1 5 10 15
Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys
20 25 30
Asn Pro Lys Leu Thr Asp Met Leu Thr Phe Glu Phe Tyr Met Pro Lys
35 40 45
Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Arg Glu Leu Lys
50 55 60
Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu
65 70 75 80
Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu
85 90 95
Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala
100 105 110
Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile
115 120 125
Ile Ser Thr Leu Thr
130
<210> 27
<211> 133
<212> PRT
<213> artificial sequence
<220>
<223> synthetic construct
<400> 27
Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His
1 5 10 15
Leu Arg Leu Asp Leu Glu Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys
20 25 30
Asn Pro Lys Leu Thr Asp Met Leu Thr Phe Glu Phe Tyr Met Pro Lys
35 40 45
Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Arg Glu Leu Lys
50 55 60
Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu
65 70 75 80
Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu
85 90 95
Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala
100 105 110
Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile
115 120 125
Ile Ser Thr Leu Thr
130
<210> 28
<211> 133
<212> PRT
<213> artificial sequence
<220>
<223> synthetic construct
<400> 28
Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His
1 5 10 15
Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys
20 25 30
Asn Pro Lys Leu Thr Asp Met Leu Thr Phe Glu Phe Tyr Met Pro Lys
35 40 45
Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Arg Glu Leu Lys
50 55 60
Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu
65 70 75 80
Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu
85 90 95
Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala
100 105 110
Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Thr Ser Ile
115 120 125
Ile Ser Thr Leu Thr
130
<210> 29
<211> 133
<212> PRT
<213> artificial sequence
<220>
<223> synthetic construct
<400> 29
Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His
1 5 10 15
Leu Arg Leu Asp Leu Glu Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys
20 25 30
Asn Pro Lys Leu Thr Asp Met Leu Thr Phe Glu Phe Tyr Met Pro Lys
35 40 45
Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Arg Glu Leu Lys
50 55 60
Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu
65 70 75 80
Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu
85 90 95
Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala
100 105 110
Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Thr Ser Ile
115 120 125
Ile Ser Thr Leu Thr
130
<210> 30
<211> 133
<212> PRT
<213> artificial sequence
<220>
<223> synthetic construct
<400> 30
Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His
1 5 10 15
Leu Arg Leu Asp Leu Glu Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys
20 25 30
Asn Pro Lys Leu Thr Asp Met Leu Thr Phe Glu Phe Tyr Met Pro Lys
35 40 45
Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Arg Glu Leu Lys
50 55 60
Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu
65 70 75 80
Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu
85 90 95
Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala
100 105 110
Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Thr Ser Ile
115 120 125
Ile Arg Thr Leu Thr
130
<210> 31
<211> 165
<212> PRT
<213> Homo sapiens (Homo sapiens)
<400> 31
Cys Asp Leu Pro Gln Thr His Ser Leu Gly Ser Arg Arg Thr Leu Met
1 5 10 15
Leu Leu Ala Gln Met Arg Lys Ile Ser Leu Phe Ser Cys Leu Lys Asp
20 25 30
Arg His Asp Phe Gly Phe Pro Gln Glu Glu Phe Gly Asn Gln Phe Gln
35 40 45
Lys Ala Glu Thr Ile Pro Val Leu His Glu Met Ile Gln Gln Ile Phe
50 55 60
Asn Leu Phe Ser Thr Lys Asp Ser Ser Ala Ala Trp Asp Glu Thr Leu
65 70 75 80
Leu Asp Lys Phe Tyr Thr Glu Leu Tyr Gln Gln Leu Asn Asp Leu Glu
85 90 95
Ala Cys Val Ile Gln Gly Val Gly Val Thr Glu Thr Pro Leu Met Lys
100 105 110
Glu Asp Ser Ile Leu Ala Val Arg Lys Tyr Phe Gln Arg Ile Thr Leu
115 120 125
Tyr Leu Lys Glu Lys Lys Tyr Ser Pro Cys Ala Trp Glu Val Val Arg
130 135 140
Ala Glu Ile Met Arg Ser Phe Ser Leu Ser Thr Asn Leu Gln Glu Ser
145 150 155 160
Leu Arg Ser Lys Glu
165
<210> 32
<211> 165
<212> PRT
<213> artificial sequence
<220>
<223> synthetic construct
<400> 32
Cys Asp Leu Pro Gln Thr His Ser Leu Gly Ser Arg Arg Thr Leu Met
1 5 10 15
Leu Leu Ala Gln Met Arg Lys Ile Ser Leu Phe Ser Cys Ala Lys Asp
20 25 30
Arg His Asp Phe Gly Phe Pro Gln Glu Glu Phe Gly Asn Gln Phe Gln
35 40 45
Lys Ala Glu Thr Ile Pro Val Leu His Glu Met Ile Gln Gln Ile Phe
50 55 60
Asn Leu Phe Ser Thr Lys Asp Ser Ser Ala Ala Trp Asp Glu Thr Leu
65 70 75 80
Leu Asp Lys Phe Tyr Thr Glu Leu Tyr Gln Gln Leu Asn Asp Leu Glu
85 90 95
Ala Cys Val Ile Gln Gly Val Gly Val Thr Glu Thr Pro Leu Met Lys
100 105 110
Glu Asp Ser Ile Leu Ala Val Arg Lys Tyr Phe Gln Arg Ile Thr Leu
115 120 125
Tyr Leu Lys Glu Lys Lys Tyr Ser Pro Cys Ala Trp Glu Val Val Arg
130 135 140
Ala Glu Ile Met Arg Ser Phe Ser Leu Ser Thr Asn Leu Gln Glu Ser
145 150 155 160
Leu Arg Ser Lys Glu
165
<210> 33
<211> 165
<212> PRT
<213> artificial sequence
<220>
<223> synthetic construct
<400> 33
Cys Asp Leu Pro Gln Thr His Ser Leu Gly Ser Arg Arg Thr Leu Met
1 5 10 15
Leu Leu Ala Gln Met Arg Lys Ile Ser Leu Phe Ser Cys Leu Ala Asp
20 25 30
Arg His Asp Phe Gly Phe Pro Gln Glu Glu Phe Gly Asn Gln Phe Gln
35 40 45
Lys Ala Glu Thr Ile Pro Val Leu His Glu Met Ile Gln Gln Ile Phe
50 55 60
Asn Leu Phe Ser Thr Lys Asp Ser Ser Ala Ala Trp Asp Glu Thr Leu
65 70 75 80
Leu Asp Lys Phe Tyr Thr Glu Leu Tyr Gln Gln Leu Asn Asp Leu Glu
85 90 95
Ala Cys Val Ile Gln Gly Val Gly Val Thr Glu Thr Pro Leu Met Lys
100 105 110
Glu Asp Ser Ile Leu Ala Val Arg Lys Tyr Phe Gln Arg Ile Thr Leu
115 120 125
Tyr Leu Lys Glu Lys Lys Tyr Ser Pro Cys Ala Trp Glu Val Val Arg
130 135 140
Ala Glu Ile Met Arg Ser Phe Ser Leu Ser Thr Asn Leu Gln Glu Ser
145 150 155 160
Leu Arg Ser Lys Glu
165
<210> 34
<211> 165
<212> PRT
<213> artificial sequence
<220>
<223> synthetic construct
<400> 34
Cys Asp Leu Pro Gln Thr His Ser Leu Gly Ser Arg Arg Thr Leu Met
1 5 10 15
Leu Leu Ala Gln Met Arg Lys Ile Ser Leu Phe Ser Cys Leu Lys Ala
20 25 30
Arg His Asp Phe Gly Phe Pro Gln Glu Glu Phe Gly Asn Gln Phe Gln
35 40 45
Lys Ala Glu Thr Ile Pro Val Leu His Glu Met Ile Gln Gln Ile Phe
50 55 60
Asn Leu Phe Ser Thr Lys Asp Ser Ser Ala Ala Trp Asp Glu Thr Leu
65 70 75 80
Leu Asp Lys Phe Tyr Thr Glu Leu Tyr Gln Gln Leu Asn Asp Leu Glu
85 90 95
Ala Cys Val Ile Gln Gly Val Gly Val Thr Glu Thr Pro Leu Met Lys
100 105 110
Glu Asp Ser Ile Leu Ala Val Arg Lys Tyr Phe Gln Arg Ile Thr Leu
115 120 125
Tyr Leu Lys Glu Lys Lys Tyr Ser Pro Cys Ala Trp Glu Val Val Arg
130 135 140
Ala Glu Ile Met Arg Ser Phe Ser Leu Ser Thr Asn Leu Gln Glu Ser
145 150 155 160
Leu Arg Ser Lys Glu
165
<210> 35
<211> 165
<212> PRT
<213> artificial sequence
<220>
<223> synthetic construct
<400> 35
Cys Asp Leu Pro Gln Thr His Ser Leu Gly Ser Arg Arg Thr Leu Met
1 5 10 15
Leu Leu Ala Gln Met Arg Lys Ile Ser Leu Phe Ser Cys Leu Lys Asp
20 25 30
Ala His Asp Phe Gly Phe Pro Gln Glu Glu Phe Gly Asn Gln Phe Gln
35 40 45
Lys Ala Glu Thr Ile Pro Val Leu His Glu Met Ile Gln Gln Ile Phe
50 55 60
Asn Leu Phe Ser Thr Lys Asp Ser Ser Ala Ala Trp Asp Glu Thr Leu
65 70 75 80
Leu Asp Lys Phe Tyr Thr Glu Leu Tyr Gln Gln Leu Asn Asp Leu Glu
85 90 95
Ala Cys Val Ile Gln Gly Val Gly Val Thr Glu Thr Pro Leu Met Lys
100 105 110
Glu Asp Ser Ile Leu Ala Val Arg Lys Tyr Phe Gln Arg Ile Thr Leu
115 120 125
Tyr Leu Lys Glu Lys Lys Tyr Ser Pro Cys Ala Trp Glu Val Val Arg
130 135 140
Ala Glu Ile Met Arg Ser Phe Ser Leu Ser Thr Asn Leu Gln Glu Ser
145 150 155 160
Leu Arg Ser Lys Glu
165
<210> 36
<211> 165
<212> PRT
<213> artificial sequence
<220>
<223> synthetic construct
<400> 36
Cys Asp Leu Pro Gln Thr His Ser Leu Gly Ser Arg Arg Thr Leu Met
1 5 10 15
Leu Leu Ala Gln Met Arg Lys Ile Ser Leu Phe Ser Cys Leu Lys Asp
20 25 30
Arg Ala Asp Phe Gly Phe Pro Gln Glu Glu Phe Gly Asn Gln Phe Gln
35 40 45
Lys Ala Glu Thr Ile Pro Val Leu His Glu Met Ile Gln Gln Ile Phe
50 55 60
Asn Leu Phe Ser Thr Lys Asp Ser Ser Ala Ala Trp Asp Glu Thr Leu
65 70 75 80
Leu Asp Lys Phe Tyr Thr Glu Leu Tyr Gln Gln Leu Asn Asp Leu Glu
85 90 95
Ala Cys Val Ile Gln Gly Val Gly Val Thr Glu Thr Pro Leu Met Lys
100 105 110
Glu Asp Ser Ile Leu Ala Val Arg Lys Tyr Phe Gln Arg Ile Thr Leu
115 120 125
Tyr Leu Lys Glu Lys Lys Tyr Ser Pro Cys Ala Trp Glu Val Val Arg
130 135 140
Ala Glu Ile Met Arg Ser Phe Ser Leu Ser Thr Asn Leu Gln Glu Ser
145 150 155 160
Leu Arg Ser Lys Glu
165
<210> 37
<211> 165
<212> PRT
<213> artificial sequence
<220>
<223> synthetic construct
<400> 37
Cys Asp Leu Pro Gln Thr His Ser Leu Gly Ser Arg Arg Thr Leu Met
1 5 10 15
Leu Leu Ala Gln Met Arg Lys Ile Ser Leu Phe Ser Cys Leu Lys Asp
20 25 30
Arg His Ala Phe Gly Phe Pro Gln Glu Glu Phe Gly Asn Gln Phe Gln
35 40 45
Lys Ala Glu Thr Ile Pro Val Leu His Glu Met Ile Gln Gln Ile Phe
50 55 60
Asn Leu Phe Ser Thr Lys Asp Ser Ser Ala Ala Trp Asp Glu Thr Leu
65 70 75 80
Leu Asp Lys Phe Tyr Thr Glu Leu Tyr Gln Gln Leu Asn Asp Leu Glu
85 90 95
Ala Cys Val Ile Gln Gly Val Gly Val Thr Glu Thr Pro Leu Met Lys
100 105 110
Glu Asp Ser Ile Leu Ala Val Arg Lys Tyr Phe Gln Arg Ile Thr Leu
115 120 125
Tyr Leu Lys Glu Lys Lys Tyr Ser Pro Cys Ala Trp Glu Val Val Arg
130 135 140
Ala Glu Ile Met Arg Ser Phe Ser Leu Ser Thr Asn Leu Gln Glu Ser
145 150 155 160
Leu Arg Ser Lys Glu
165
<210> 38
<211> 138
<212> PRT
<213> artificial sequence
<220>
<223> synthetic construct
<400> 38
Gln Asp Pro Tyr Val Lys Glu Ala Glu Asn Leu Lys Lys Tyr Phe Asn
1 5 10 15
Ala Gly His Ser Asp Val Ala Asp Asn Gly Thr Leu Phe Leu Gly Ile
20 25 30
Leu Lys Asn Trp Lys Glu Glu Ser Asp Arg Lys Ile Met Gln Ser Gln
35 40 45
Ile Val Ser Phe Tyr Phe Lys Leu Phe Lys Asn Phe Lys Asp Asp Gln
50 55 60
Ser Ile Gln Lys Ser Val Glu Thr Ile Lys Glu Asp Met Asn Val Lys
65 70 75 80
Phe Phe Asn Ser Asn Lys Lys Lys Arg Asp Asp Phe Glu Lys Leu Thr
85 90 95
Asn Tyr Ser Val Thr Asp Leu Asn Val Gln Arg Lys Ala Ile His Glu
100 105 110
Leu Ile Gln Val Met Ala Glu Leu Ser Pro Ala Ala Lys Thr Gly Lys
115 120 125
Arg Lys Arg Ser Gln Met Leu Phe Arg Gly
130 135
<210> 39
<211> 138
<212> PRT
<213> artificial sequence
<220>
<223> synthetic construct
<400> 39
Gln Asp Pro Tyr Val Lys Glu Ala Glu Asn Leu Lys Lys Tyr Phe Asn
1 5 10 15
Ala Gly His Ala Ala Val Ala Asp Asn Gly Thr Leu Phe Leu Gly Ile
20 25 30
Leu Lys Asn Trp Lys Glu Glu Ser Asp Arg Lys Ile Met Gln Ser Gln
35 40 45
Ile Val Ser Phe Tyr Phe Lys Leu Phe Lys Asn Phe Lys Asp Asp Gln
50 55 60
Ser Ile Gln Lys Ser Val Glu Thr Ile Lys Glu Asp Met Asn Val Lys
65 70 75 80
Phe Phe Asn Ser Asn Lys Lys Lys Arg Asp Asp Phe Glu Lys Leu Thr
85 90 95
Asn Tyr Ser Val Thr Asp Leu Asn Val Gln Arg Lys Ala Ile His Glu
100 105 110
Leu Ile Gln Val Met Ala Glu Leu Ser Pro Ala Ala Lys Thr Gly Lys
115 120 125
Arg Lys Arg Ser Gln Met Leu Phe Arg Gly
130 135
<210> 40
<211> 138
<212> PRT
<213> artificial sequence
<220>
<223> synthetic construct
<400> 40
Gln Asp Pro Tyr Val Lys Glu Ala Glu Asn Leu Lys Lys Tyr Phe Asn
1 5 10 15
Ala Gly His Ser Asp Ala Ser Asp Asn Gly Thr Leu Phe Leu Gly Ile
20 25 30
Leu Lys Asn Trp Lys Glu Glu Ser Asp Arg Lys Ile Met Gln Ser Gln
35 40 45
Ile Val Ser Phe Tyr Phe Lys Leu Phe Lys Asn Phe Lys Asp Asp Gln
50 55 60
Ser Ile Gln Lys Ser Val Glu Thr Ile Lys Glu Asp Met Asn Val Lys
65 70 75 80
Phe Phe Asn Ser Asn Lys Lys Lys Arg Asp Asp Phe Glu Lys Leu Thr
85 90 95
Asn Tyr Ser Val Thr Asp Leu Asn Val Gln Arg Lys Ala Ile His Glu
100 105 110
Leu Ile Gln Val Met Ala Glu Leu Ser Pro Ala Ala Lys Thr Gly Lys
115 120 125
Arg Lys Arg Ser Gln Met Leu Phe Arg Gly
130 135
<210> 41
<211> 138
<212> PRT
<213> artificial sequence
<220>
<223> synthetic construct
<400> 41
Gln Asp Pro Tyr Val Lys Glu Ala Glu Asn Leu Lys Lys Tyr Phe Asn
1 5 10 15
Ala Gly His Ser Asp Val Val Asp Asn Gly Thr Leu Phe Leu Gly Ile
20 25 30
Leu Lys Asn Trp Lys Glu Glu Ser Asp Arg Lys Ile Met Gln Ser Gln
35 40 45
Ile Val Ser Phe Tyr Phe Lys Leu Phe Lys Asn Phe Lys Asp Asp Gln
50 55 60
Ser Ile Gln Lys Ser Val Glu Thr Ile Lys Glu Asp Met Asn Val Lys
65 70 75 80
Phe Phe Asn Ser Asn Lys Lys Lys Arg Asp Asp Phe Glu Lys Leu Thr
85 90 95
Asn Tyr Ser Val Thr Asp Leu Asn Val Gln Arg Lys Ala Ile His Glu
100 105 110
Leu Ile Gln Val Met Ala Glu Leu Ser Pro Ala Ala Lys Thr Gly Lys
115 120 125
Arg Lys Arg Ser Gln Met Leu Phe Arg Gly
130 135
<210> 42
<211> 138
<212> PRT
<213> artificial sequence
<220>
<223> synthetic construct
<400> 42
Gln Asp Pro Tyr Val Lys Glu Ala Glu Asn Leu Lys Lys Tyr Phe Asn
1 5 10 15
Ala Gly His Ser Asp Val Ala Ala Ala Gly Thr Leu Phe Leu Gly Ile
20 25 30
Leu Lys Asn Trp Lys Glu Glu Ser Asp Arg Lys Ile Met Gln Ser Gln
35 40 45
Ile Val Ser Phe Tyr Phe Lys Leu Phe Lys Asn Phe Lys Asp Asp Gln
50 55 60
Ser Ile Gln Lys Ser Val Glu Thr Ile Lys Glu Asp Met Asn Val Lys
65 70 75 80
Phe Phe Asn Ser Asn Lys Lys Lys Arg Asp Asp Phe Glu Lys Leu Thr
85 90 95
Asn Tyr Ser Val Thr Asp Leu Asn Val Gln Arg Lys Ala Ile His Glu
100 105 110
Leu Ile Gln Val Met Ala Glu Leu Ser Pro Ala Ala Lys Thr Gly Lys
115 120 125
Arg Lys Arg Ser Gln Met Leu Phe Arg Gly
130 135
<210> 43
<211> 138
<212> PRT
<213> artificial sequence
<220>
<223> synthetic construct
<400> 43
Gln Asp Pro Tyr Val Lys Glu Ala Glu Asn Leu Lys Lys Tyr Phe Asn
1 5 10 15
Ala Gly His Ser Asp Val Glu Glu Lys Gly Thr Leu Phe Leu Gly Ile
20 25 30
Leu Lys Asn Trp Lys Glu Glu Ser Asp Arg Lys Ile Met Gln Ser Gln
35 40 45
Ile Val Ser Phe Tyr Phe Lys Leu Phe Lys Asn Phe Lys Asp Asp Gln
50 55 60
Ser Ile Gln Lys Ser Val Glu Thr Ile Lys Glu Asp Met Asn Val Lys
65 70 75 80
Phe Phe Asn Ser Asn Lys Lys Lys Arg Asp Asp Phe Glu Lys Leu Thr
85 90 95
Asn Tyr Ser Val Thr Asp Leu Asn Val Gln Arg Lys Ala Ile His Glu
100 105 110
Leu Ile Gln Val Met Ala Glu Leu Ser Pro Ala Ala Lys Thr Gly Lys
115 120 125
Arg Lys Arg Ser Gln Met Leu Phe Arg Gly
130 135
<210> 44
<211> 138
<212> PRT
<213> artificial sequence
<220>
<223> synthetic construct
<400> 44
Gln Asp Pro Tyr Val Lys Glu Ala Glu Asn Leu Lys Lys Tyr Phe Asn
1 5 10 15
Ala Gly His Ser Asp Val Gln Asp Asn Gly Thr Leu Phe Leu Gly Ile
20 25 30
Leu Lys Asn Trp Lys Glu Glu Ser Asp Arg Lys Ile Met Gln Ser Gln
35 40 45
Ile Val Ser Phe Tyr Phe Lys Leu Phe Lys Asn Phe Lys Asp Asp Gln
50 55 60
Ser Ile Gln Lys Ser Val Glu Thr Ile Lys Glu Asp Met Asn Val Lys
65 70 75 80
Phe Phe Asn Ser Asn Lys Lys Lys Arg Asp Asp Phe Glu Lys Leu Thr
85 90 95
Asn Tyr Ser Val Thr Asp Leu Asn Val Gln Arg Lys Ala Ile His Glu
100 105 110
Leu Ile Gln Val Met Ala Glu Leu Ser Pro Ala Ala Lys Thr Gly Lys
115 120 125
Arg Lys Arg Ser Gln Met Leu Phe Arg Gly
130 135
<210> 45
<211> 138
<212> PRT
<213> artificial sequence
<220>
<223> synthetic construct
<400> 45
Gln Asp Pro Tyr Val Lys Glu Ala Glu Asn Leu Lys Lys Tyr Phe Asn
1 5 10 15
Ala Gly His Ser Lys Val Ala Asp Asn Gly Thr Leu Phe Leu Gly Ile
20 25 30
Leu Lys Asn Trp Lys Glu Glu Ser Asp Arg Lys Ile Met Gln Ser Gln
35 40 45
Ile Val Ser Phe Tyr Phe Lys Leu Phe Lys Asn Phe Lys Asp Asp Gln
50 55 60
Ser Ile Gln Lys Ser Val Glu Thr Ile Lys Glu Asp Met Asn Val Lys
65 70 75 80
Phe Phe Asn Ser Asn Lys Lys Lys Arg Asp Asp Phe Glu Lys Leu Thr
85 90 95
Asn Tyr Ser Val Thr Asp Leu Asn Val Gln Arg Lys Ala Ile His Glu
100 105 110
Leu Ile Gln Val Met Ala Glu Leu Ser Pro Ala Ala Lys Thr Gly Lys
115 120 125
Arg Lys Arg Ser Gln Met Leu Phe Arg Gly
130 135
<210> 46
<211> 297
<212> PRT
<213> artificial sequence
<220>
<223> synthetic construct
<400> 46
Gln Asp Pro Tyr Val Lys Glu Ala Glu Asn Leu Lys Lys Tyr Phe Asn
1 5 10 15
Ala Gly His Ser Asp Val Ala Asp Asn Gly Thr Leu Phe Leu Gly Ile
20 25 30
Leu Lys Asn Trp Lys Glu Glu Ser Asp Arg Lys Ile Met Gln Ser Gln
35 40 45
Ile Val Ser Phe Tyr Phe Lys Leu Phe Lys Asn Phe Lys Asp Asp Gln
50 55 60
Ser Ile Gln Lys Ser Val Glu Thr Ile Lys Glu Asp Met Asn Val Lys
65 70 75 80
Phe Phe Asn Ser Asn Lys Lys Lys Arg Asp Asp Phe Glu Lys Leu Thr
85 90 95
Asn Tyr Ser Val Thr Asp Leu Asn Val Gln Arg Lys Ala Ile His Glu
100 105 110
Leu Ile Gln Val Met Ala Glu Leu Ser Pro Ala Ala Lys Thr Gly Lys
115 120 125
Arg Lys Arg Ser Gln Met Leu Phe Arg Gly Phe Glu Gly Gly Gly Ser
130 135 140
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln
145 150 155 160
Asp Pro Tyr Val Lys Glu Ala Glu Asn Leu Lys Lys Tyr Phe Asn Ala
165 170 175
Gly His Ser Asp Val Ala Asp Asn Gly Thr Leu Phe Leu Gly Ile Leu
180 185 190
Lys Asn Trp Lys Glu Glu Ser Asp Arg Lys Ile Met Gln Ser Gln Ile
195 200 205
Val Ser Phe Tyr Phe Lys Leu Phe Lys Asn Phe Lys Asp Asp Gln Ser
210 215 220
Ile Gln Lys Ser Val Glu Thr Ile Lys Glu Asp Met Asn Val Lys Phe
225 230 235 240
Phe Asn Ser Asn Lys Lys Lys Arg Asp Asp Phe Glu Lys Leu Thr Asn
245 250 255
Tyr Ser Val Thr Asp Leu Asn Val Gln Arg Lys Ala Ile His Glu Leu
260 265 270
Ile Gln Val Met Ala Glu Leu Ser Pro Ala Ala Lys Thr Gly Lys Arg
275 280 285
Lys Arg Ser Gln Met Leu Phe Arg Gly
290 295
<210> 47
<211> 297
<212> PRT
<213> artificial sequence
<220>
<223> synthetic construct
<400> 47
Gln Asp Pro Tyr Val Lys Glu Ala Glu Asn Leu Lys Lys Tyr Phe Asn
1 5 10 15
Ala Gly His Ser Asp Val Val Asp Asn Gly Thr Leu Phe Leu Gly Ile
20 25 30
Leu Lys Asn Trp Lys Glu Glu Ser Asp Arg Lys Ile Met Gln Ser Gln
35 40 45
Ile Val Ser Phe Tyr Phe Lys Leu Phe Lys Asn Phe Lys Asp Asp Gln
50 55 60
Ser Ile Gln Lys Ser Val Glu Thr Ile Lys Glu Asp Met Asn Val Lys
65 70 75 80
Phe Phe Asn Ser Asn Lys Lys Lys Arg Asp Asp Phe Glu Lys Leu Thr
85 90 95
Asn Tyr Ser Val Thr Asp Leu Asn Val Gln Arg Lys Ala Ile His Glu
100 105 110
Leu Ile Gln Val Met Ala Glu Leu Ser Pro Ala Ala Lys Thr Gly Lys
115 120 125
Arg Lys Arg Ser Gln Met Leu Phe Arg Gly Phe Glu Gly Gly Gly Ser
130 135 140
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln
145 150 155 160
Asp Pro Tyr Val Lys Glu Ala Glu Asn Leu Lys Lys Tyr Phe Asn Ala
165 170 175
Gly His Ser Asp Val Val Asp Asn Gly Thr Leu Phe Leu Gly Ile Leu
180 185 190
Lys Asn Trp Lys Glu Glu Ser Asp Arg Lys Ile Met Gln Ser Gln Ile
195 200 205
Val Ser Phe Tyr Phe Lys Leu Phe Lys Asn Phe Lys Asp Asp Gln Ser
210 215 220
Ile Gln Lys Ser Val Glu Thr Ile Lys Glu Asp Met Asn Val Lys Phe
225 230 235 240
Phe Asn Ser Asn Lys Lys Lys Arg Asp Asp Phe Glu Lys Leu Thr Asn
245 250 255
Tyr Ser Val Thr Asp Leu Asn Val Gln Arg Lys Ala Ile His Glu Leu
260 265 270
Ile Gln Val Met Ala Glu Leu Ser Pro Ala Ala Lys Thr Gly Lys Arg
275 280 285
Lys Arg Ser Gln Met Leu Phe Arg Gly
290 295
<210> 48
<211> 115
<212> PRT
<213> artificial sequence
<220>
<223> synthetic construct
<400> 48
Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg
1 5 10 15
Ser Leu Arg Leu Asp Cys Lys Ala Ser Gly Ile Thr Phe Ser Asn Ser
20 25 30
Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Val Ile Trp Tyr Asp Gly Ser Lys Arg Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Phe
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Thr Asn Asp Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser
100 105 110
Ser Ala Ser
115
<210> 49
<211> 106
<212> PRT
<213> artificial sequence
<220>
<223> synthetic construct
<400> 49
Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly
1 5 10 15
Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr
20 25 30
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile
35 40 45
Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro
65 70 75 80
Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Ser Ser Asn Trp Pro Arg
85 90 95
Thr Phe Gly Gln Gly Thr Lys Val Glu Ile
100 105
<210> 50
<211> 214
<212> PRT
<213> artificial sequence
<220>
<223> synthetic construct
<400> 50
Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly
1 5 10 15
Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr
20 25 30
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile
35 40 45
Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro
65 70 75 80
Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Ser Ser Asn Trp Pro Arg
85 90 95
Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala
100 105 110
Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly
115 120 125
Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala
130 135 140
Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln
145 150 155 160
Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser
165 170 175
Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr
180 185 190
Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser
195 200 205
Phe Asn Arg Gly Glu Cys
210
<210> 51
<211> 443
<212> PRT
<213> artificial sequence
<220>
<223> synthetic construct
<400> 51
Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg
1 5 10 15
Ser Leu Arg Leu Asp Cys Lys Ala Ser Gly Ile Thr Phe Ser Asn Ser
20 25 30
Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Val Ile Trp Tyr Asp Gly Ser Lys Arg Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Phe
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Thr Asn Asp Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser
100 105 110
Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser
115 120 125
Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp
130 135 140
Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr
145 150 155 160
Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr
165 170 175
Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln
180 185 190
Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp
195 200 205
Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro
210 215 220
Cys Pro Ala Pro Glu Lys Lys Gly Gly Pro Ser Val Phe Leu Phe Pro
225 230 235 240
Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr
245 250 255
Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn
260 265 270
Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg
275 280 285
Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val
290 295 300
Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser
305 310 315 320
Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys
325 330 335
Gly Gln Pro Arg Glu Pro Gln Val Tyr Val Leu Pro Pro Ser Arg Glu
340 345 350
Glu Met Thr Lys Asn Gln Val Ser Leu Leu Cys Leu Val Lys Gly Phe
355 360 365
Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu
370 375 380
Asn Arg Tyr Met Thr Trp Pro Pro Val Leu Asp Ser Asp Gly Ser Phe
385 390 395 400
Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly
405 410 415
Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr
420 425 430
Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
435 440
<210> 52
<211> 160
<212> PRT
<213> Homo sapiens (Homo sapiens)
<400> 52
Ser Pro Gly Gln Gly Thr Gln Ser Glu Asn Ser Cys Thr His Phe Pro
1 5 10 15
Gly Asn Leu Pro Asn Met Leu Arg Asp Leu Arg Asp Ala Phe Ser Arg
20 25 30
Val Lys Thr Phe Phe Gln Met Lys Asp Gln Leu Asp Asn Leu Leu Leu
35 40 45
Lys Glu Ser Leu Leu Glu Asp Phe Lys Gly Tyr Leu Gly Cys Gln Ala
50 55 60
Leu Ser Glu Met Ile Gln Phe Tyr Leu Glu Glu Val Met Pro Gln Ala
65 70 75 80
Glu Asn Gln Asp Pro Asp Ile Lys Ala His Val Asn Ser Leu Gly Glu
85 90 95
Asn Leu Lys Thr Leu Arg Leu Arg Leu Arg Arg Cys His Arg Phe Leu
100 105 110
Pro Cys Glu Asn Lys Ser Lys Ala Val Glu Gln Val Lys Asn Ala Phe
115 120 125
Asn Lys Leu Gln Glu Lys Gly Ile Tyr Lys Ala Met Ser Glu Phe Asp
130 135 140
Ile Phe Ile Asn Tyr Ile Glu Ala Tyr Met Thr Met Lys Ile Arg Asn
145 150 155 160
<210> 53
<211> 160
<212> PRT
<213> artificial sequence
<220>
<223> synthetic construct
<400> 53
Ser Pro Gly Gln Gly Thr Gln Ser Glu Asn Ser Cys Thr His Phe Pro
1 5 10 15
Gly Asn Leu Pro Asn Met Leu Ala Asp Leu Arg Asp Ala Phe Ser Arg
20 25 30
Val Lys Thr Phe Phe Gln Met Lys Asp Gln Leu Asp Asn Leu Leu Leu
35 40 45
Lys Glu Ser Leu Leu Glu Asp Phe Lys Gly Tyr Leu Gly Cys Gln Ala
50 55 60
Leu Ser Glu Met Ile Gln Phe Tyr Leu Glu Glu Val Met Pro Gln Ala
65 70 75 80
Glu Asn Gln Asp Pro Asp Ile Lys Ala His Val Asn Ser Leu Gly Glu
85 90 95
Asn Leu Lys Thr Leu Arg Leu Arg Leu Arg Arg Cys His Arg Phe Leu
100 105 110
Pro Cys Glu Asn Lys Ser Lys Ala Val Glu Gln Val Lys Asn Ala Phe
115 120 125
Asn Lys Leu Gln Glu Lys Gly Ile Tyr Lys Ala Met Ser Glu Phe Asp
130 135 140
Ile Phe Ile Asn Tyr Ile Glu Ala Tyr Met Thr Met Lys Ile Arg Asn
145 150 155 160
<210> 54
<211> 160
<212> PRT
<213> artificial sequence
<220>
<223> synthetic construct
<400> 54
Ser Pro Gly Gln Gly Thr Gln Ser Glu Asn Ser Cys Thr His Phe Pro
1 5 10 15
Gly Asn Leu Pro Asn Met Leu Arg Ala Ala Arg Asp Ala Phe Ser Arg
20 25 30
Val Lys Thr Phe Phe Gln Met Lys Asp Gln Leu Asp Asn Leu Leu Leu
35 40 45
Lys Glu Ser Leu Leu Glu Asp Phe Lys Gly Tyr Leu Gly Cys Gln Ala
50 55 60
Leu Ser Glu Met Ile Gln Phe Tyr Leu Glu Glu Val Met Pro Gln Ala
65 70 75 80
Glu Asn Gln Asp Pro Asp Ile Lys Ala His Val Asn Ser Leu Gly Glu
85 90 95
Asn Leu Lys Thr Leu Arg Leu Arg Leu Arg Arg Cys His Arg Phe Leu
100 105 110
Pro Cys Glu Asn Lys Ser Lys Ala Val Glu Gln Val Lys Asn Ala Phe
115 120 125
Asn Lys Leu Gln Glu Lys Gly Ile Tyr Lys Ala Met Ser Glu Phe Asp
130 135 140
Ile Phe Ile Asn Tyr Ile Glu Ala Tyr Met Thr Met Lys Ile Arg Asn
145 150 155 160
<210> 55
<211> 160
<212> PRT
<213> artificial sequence
<220>
<223> synthetic construct
<400> 55
Ser Pro Gly Gln Gly Thr Gln Ser Glu Asn Ser Cys Thr His Phe Pro
1 5 10 15
Gly Asn Leu Pro Asn Met Leu Arg Asp Leu Ala Asp Ala Phe Ser Arg
20 25 30
Val Lys Thr Phe Phe Gln Met Lys Asp Gln Leu Asp Asn Leu Leu Leu
35 40 45
Lys Glu Ser Leu Leu Glu Asp Phe Lys Gly Tyr Leu Gly Cys Gln Ala
50 55 60
Leu Ser Glu Met Ile Gln Phe Tyr Leu Glu Glu Val Met Pro Gln Ala
65 70 75 80
Glu Asn Gln Asp Pro Asp Ile Lys Ala His Val Asn Ser Leu Gly Glu
85 90 95
Asn Leu Lys Thr Leu Arg Leu Arg Leu Arg Arg Cys His Arg Phe Leu
100 105 110
Pro Cys Glu Asn Lys Ser Lys Ala Val Glu Gln Val Lys Asn Ala Phe
115 120 125
Asn Lys Leu Gln Glu Lys Gly Ile Tyr Lys Ala Met Ser Glu Phe Asp
130 135 140
Ile Phe Ile Asn Tyr Ile Glu Ala Tyr Met Thr Met Lys Ile Arg Asn
145 150 155 160
<210> 56
<211> 160
<212> PRT
<213> artificial sequence
<220>
<223> synthetic construct
<400> 56
Ser Pro Gly Gln Gly Thr Gln Ser Glu Asn Ser Cys Thr His Phe Pro
1 5 10 15
Gly Asn Leu Pro Asn Met Leu Arg Asp Leu Arg Ala Ser Phe Ser Arg
20 25 30
Val Lys Thr Phe Phe Gln Met Lys Asp Gln Leu Asp Asn Leu Leu Leu
35 40 45
Lys Glu Ser Leu Leu Glu Asp Phe Lys Gly Tyr Leu Gly Cys Gln Ala
50 55 60
Leu Ser Glu Met Ile Gln Phe Tyr Leu Glu Glu Val Met Pro Gln Ala
65 70 75 80
Glu Asn Gln Asp Pro Asp Ile Lys Ala His Val Asn Ser Leu Gly Glu
85 90 95
Asn Leu Lys Thr Leu Arg Leu Arg Leu Arg Arg Cys His Arg Phe Leu
100 105 110
Pro Cys Glu Asn Lys Ser Lys Ala Val Glu Gln Val Lys Asn Ala Phe
115 120 125
Asn Lys Leu Gln Glu Lys Gly Ile Tyr Lys Ala Met Ser Glu Phe Asp
130 135 140
Ile Phe Ile Asn Tyr Ile Glu Ala Tyr Met Thr Met Lys Ile Arg Asn
145 150 155 160
<210> 57
<211> 160
<212> PRT
<213> artificial sequence
<220>
<223> synthetic construct
<400> 57
Ser Pro Gly Gln Gly Thr Gln Ser Glu Asn Ser Cys Thr His Phe Pro
1 5 10 15
Gly Asn Leu Pro Asn Met Leu Arg Asp Leu Arg Asp Ala Ala Ala Arg
20 25 30
Val Lys Thr Phe Phe Gln Met Lys Asp Gln Leu Asp Asn Leu Leu Leu
35 40 45
Lys Glu Ser Leu Leu Glu Asp Phe Lys Gly Tyr Leu Gly Cys Gln Ala
50 55 60
Leu Ser Glu Met Ile Gln Phe Tyr Leu Glu Glu Val Met Pro Gln Ala
65 70 75 80
Glu Asn Gln Asp Pro Asp Ile Lys Ala His Val Asn Ser Leu Gly Glu
85 90 95
Asn Leu Lys Thr Leu Arg Leu Arg Leu Arg Arg Cys His Arg Phe Leu
100 105 110
Pro Cys Glu Asn Lys Ser Lys Ala Val Glu Gln Val Lys Asn Ala Phe
115 120 125
Asn Lys Leu Gln Glu Lys Gly Ile Tyr Lys Ala Met Ser Glu Phe Asp
130 135 140
Ile Phe Ile Asn Tyr Ile Glu Ala Tyr Met Thr Met Lys Ile Arg Asn
145 150 155 160
<210> 58
<211> 160
<212> PRT
<213> artificial sequence
<220>
<223> synthetic construct
<400> 58
Ser Pro Gly Gln Gly Thr Gln Ser Glu Asn Ser Cys Thr His Phe Pro
1 5 10 15
Gly Asn Leu Pro Asn Met Leu Arg Asp Leu Arg Asp Ala Phe Ser Ala
20 25 30
Val Lys Thr Phe Phe Gln Met Lys Asp Gln Leu Asp Asn Leu Leu Leu
35 40 45
Lys Glu Ser Leu Leu Glu Asp Phe Lys Gly Tyr Leu Gly Cys Gln Ala
50 55 60
Leu Ser Glu Met Ile Gln Phe Tyr Leu Glu Glu Val Met Pro Gln Ala
65 70 75 80
Glu Asn Gln Asp Pro Asp Ile Lys Ala His Val Asn Ser Leu Gly Glu
85 90 95
Asn Leu Lys Thr Leu Arg Leu Arg Leu Arg Arg Cys His Arg Phe Leu
100 105 110
Pro Cys Glu Asn Lys Ser Lys Ala Val Glu Gln Val Lys Asn Ala Phe
115 120 125
Asn Lys Leu Gln Glu Lys Gly Ile Tyr Lys Ala Met Ser Glu Phe Asp
130 135 140
Ile Phe Ile Asn Tyr Ile Glu Ala Tyr Met Thr Met Lys Ile Arg Asn
145 150 155 160
<210> 59
<211> 341
<212> PRT
<213> artificial sequence
<220>
<223> synthetic construct
<400> 59
Ser Pro Gly Gln Gly Thr Gln Ser Glu Asn Ser Cys Thr His Phe Pro
1 5 10 15
Gly Asn Leu Pro Asn Met Leu Arg Asp Leu Arg Asp Ala Phe Ser Arg
20 25 30
Val Lys Thr Phe Phe Gln Met Lys Asp Gln Leu Asp Asn Leu Leu Leu
35 40 45
Lys Glu Ser Leu Leu Glu Asp Phe Lys Gly Tyr Leu Gly Cys Gln Ala
50 55 60
Leu Ser Glu Met Ile Gln Phe Tyr Leu Glu Glu Val Met Pro Gln Ala
65 70 75 80
Glu Asn Gln Asp Pro Asp Ile Lys Ala His Val Asn Ser Leu Gly Glu
85 90 95
Asn Leu Lys Thr Leu Arg Leu Arg Leu Arg Arg Cys His Arg Phe Leu
100 105 110
Pro Cys Glu Asn Lys Ser Lys Ala Val Glu Gln Val Lys Asn Ala Phe
115 120 125
Asn Lys Leu Gln Glu Lys Gly Ile Tyr Lys Ala Met Ser Glu Phe Asp
130 135 140
Ile Phe Ile Asn Tyr Ile Glu Ala Tyr Met Thr Met Lys Ile Arg Asn
145 150 155 160
Phe Glu Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
165 170 175
Gly Gly Gly Gly Ser Ser Pro Gly Gln Gly Thr Gln Ser Glu Asn Ser
180 185 190
Cys Thr His Phe Pro Gly Asn Leu Pro Asn Met Leu Arg Asp Leu Arg
195 200 205
Asp Ala Phe Ser Arg Val Lys Thr Phe Phe Gln Met Lys Asp Gln Leu
210 215 220
Asp Asn Leu Leu Leu Lys Glu Ser Leu Leu Glu Asp Phe Lys Gly Tyr
225 230 235 240
Leu Gly Cys Gln Ala Leu Ser Glu Met Ile Gln Phe Tyr Leu Glu Glu
245 250 255
Val Met Pro Gln Ala Glu Asn Gln Asp Pro Asp Ile Lys Ala His Val
260 265 270
Asn Ser Leu Gly Glu Asn Leu Lys Thr Leu Arg Leu Arg Leu Arg Arg
275 280 285
Cys His Arg Phe Leu Pro Cys Glu Asn Lys Ser Lys Ala Val Glu Gln
290 295 300
Val Lys Asn Ala Phe Asn Lys Leu Gln Glu Lys Gly Ile Tyr Lys Ala
305 310 315 320
Met Ser Glu Phe Asp Ile Phe Ile Asn Tyr Ile Glu Ala Tyr Met Thr
325 330 335
Met Lys Ile Arg Asn
340
<210> 60
<211> 341
<212> PRT
<213> artificial sequence
<220>
<223> synthetic construct
<400> 60
Ser Pro Gly Gln Gly Thr Gln Ser Glu Asn Ser Cys Thr His Phe Pro
1 5 10 15
Gly Asn Leu Pro Asn Met Leu Arg Asp Leu Ala Asp Ala Phe Ser Arg
20 25 30
Val Lys Thr Phe Phe Gln Met Lys Asp Gln Leu Asp Asn Leu Leu Leu
35 40 45
Lys Glu Ser Leu Leu Glu Asp Phe Lys Gly Tyr Leu Gly Cys Gln Ala
50 55 60
Leu Ser Glu Met Ile Gln Phe Tyr Leu Glu Glu Val Met Pro Gln Ala
65 70 75 80
Glu Asn Gln Asp Pro Asp Ile Lys Ala His Val Asn Ser Leu Gly Glu
85 90 95
Asn Leu Lys Thr Leu Arg Leu Arg Leu Arg Arg Cys His Arg Phe Leu
100 105 110
Pro Cys Glu Asn Lys Ser Lys Ala Val Glu Gln Val Lys Asn Ala Phe
115 120 125
Asn Lys Leu Gln Glu Lys Gly Ile Tyr Lys Ala Met Ser Glu Phe Asp
130 135 140
Ile Phe Ile Asn Tyr Ile Glu Ala Tyr Met Thr Met Lys Ile Arg Asn
145 150 155 160
Phe Glu Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
165 170 175
Gly Gly Gly Gly Ser Ser Pro Gly Gln Gly Thr Gln Ser Glu Asn Ser
180 185 190
Cys Thr His Phe Pro Gly Asn Leu Pro Asn Met Leu Arg Asp Leu Ala
195 200 205
Asp Ala Phe Ser Arg Val Lys Thr Phe Phe Gln Met Lys Asp Gln Leu
210 215 220
Asp Asn Leu Leu Leu Lys Glu Ser Leu Leu Glu Asp Phe Lys Gly Tyr
225 230 235 240
Leu Gly Cys Gln Ala Leu Ser Glu Met Ile Gln Phe Tyr Leu Glu Glu
245 250 255
Val Met Pro Gln Ala Glu Asn Gln Asp Pro Asp Ile Lys Ala His Val
260 265 270
Asn Ser Leu Gly Glu Asn Leu Lys Thr Leu Arg Leu Arg Leu Arg Arg
275 280 285
Cys His Arg Phe Leu Pro Cys Glu Asn Lys Ser Lys Ala Val Glu Gln
290 295 300
Val Lys Asn Ala Phe Asn Lys Leu Gln Glu Lys Gly Ile Tyr Lys Ala
305 310 315 320
Met Ser Glu Phe Asp Ile Phe Ile Asn Tyr Ile Glu Ala Tyr Met Thr
325 330 335
Met Lys Ile Arg Asn
340
<210> 61
<211> 197
<212> PRT
<213> Homo sapiens (Homo sapiens)
<400> 61
Arg Asn Leu Pro Val Ala Thr Pro Asp Pro Gly Met Phe Pro Cys Leu
1 5 10 15
His His Ser Gln Asn Leu Leu Arg Ala Val Ser Asn Met Leu Gln Lys
20 25 30
Ala Arg Gln Thr Leu Glu Phe Tyr Pro Cys Thr Ser Glu Glu Ile Asp
35 40 45
His Glu Asp Ile Thr Lys Asp Lys Thr Ser Thr Val Glu Ala Cys Leu
50 55 60
Pro Leu Glu Leu Thr Lys Asn Glu Ser Cys Leu Asn Ser Arg Glu Thr
65 70 75 80
Ser Phe Ile Thr Asn Gly Ser Cys Leu Ala Ser Arg Lys Thr Ser Phe
85 90 95
Met Met Ala Leu Cys Leu Ser Ser Ile Tyr Glu Asp Leu Lys Met Tyr
100 105 110
Gln Val Glu Phe Lys Thr Met Asn Ala Lys Leu Leu Met Asp Pro Lys
115 120 125
Arg Gln Ile Phe Leu Asp Gln Asn Met Leu Ala Val Ile Asp Glu Leu
130 135 140
Met Gln Ala Leu Asn Phe Asn Ser Glu Thr Val Pro Gln Lys Ser Ser
145 150 155 160
Leu Glu Glu Pro Asp Phe Tyr Lys Thr Lys Ile Lys Leu Cys Ile Leu
165 170 175
Leu His Ala Phe Arg Ile Arg Ala Val Thr Ile Asp Arg Val Met Ser
180 185 190
Tyr Leu Asn Ala Ser
195
<210> 62
<211> 306
<212> PRT
<213> Homo sapiens (Homo sapiens)
<400> 62
Ile Trp Glu Leu Lys Lys Asp Val Tyr Val Val Glu Leu Asp Trp Tyr
1 5 10 15
Pro Asp Ala Pro Gly Glu Met Val Val Leu Thr Cys Asp Thr Pro Glu
20 25 30
Glu Asp Gly Ile Thr Trp Thr Leu Asp Gln Ser Ser Glu Val Leu Gly
35 40 45
Ser Gly Lys Thr Leu Thr Ile Gln Val Lys Glu Phe Gly Asp Ala Gly
50 55 60
Gln Tyr Thr Cys His Lys Gly Gly Glu Val Leu Ser His Ser Leu Leu
65 70 75 80
Leu Leu His Lys Lys Glu Asp Gly Ile Trp Ser Thr Asp Ile Leu Lys
85 90 95
Asp Gln Lys Glu Pro Lys Asn Lys Thr Phe Leu Arg Cys Glu Ala Lys
100 105 110
Asn Tyr Ser Gly Arg Phe Thr Cys Trp Trp Leu Thr Thr Ile Ser Thr
115 120 125
Asp Leu Thr Phe Ser Val Lys Ser Ser Arg Gly Ser Ser Asp Pro Gln
130 135 140
Gly Val Thr Cys Gly Ala Ala Thr Leu Ser Ala Glu Arg Val Arg Gly
145 150 155 160
Asp Asn Lys Glu Tyr Glu Tyr Ser Val Glu Cys Gln Glu Asp Ser Ala
165 170 175
Cys Pro Ala Ala Glu Glu Ser Leu Pro Ile Glu Val Met Val Asp Ala
180 185 190
Val His Lys Leu Lys Tyr Glu Asn Tyr Thr Ser Ser Phe Phe Ile Arg
195 200 205
Asp Ile Ile Lys Pro Asp Pro Pro Lys Asn Leu Gln Leu Lys Pro Leu
210 215 220
Lys Asn Ser Arg Gln Val Glu Val Ser Trp Glu Tyr Pro Asp Thr Trp
225 230 235 240
Ser Thr Pro His Ser Tyr Phe Ser Leu Thr Phe Cys Val Gln Val Gln
245 250 255
Gly Lys Ser Lys Arg Glu Lys Lys Asp Arg Val Phe Thr Asp Lys Thr
260 265 270
Ser Ala Thr Val Ile Cys Arg Lys Asn Ala Ser Ile Ser Val Arg Ala
275 280 285
Gln Asp Arg Tyr Tyr Ser Ser Ser Trp Ser Glu Trp Ala Ser Val Pro
290 295 300
Cys Ser
305
<210> 63
<211> 306
<212> PRT
<213> artificial sequence
<220>
<223> synthetic construct
<400> 63
Ile Trp Glu Leu Lys Lys Asp Val Tyr Val Val Glu Leu Asp Trp Tyr
1 5 10 15
Pro Asp Ala Pro Gly Glu Met Val Val Leu Thr Cys Asp Thr Pro Glu
20 25 30
Glu Asp Gly Ile Thr Trp Thr Leu Asp Gln Ser Ser Glu Val Leu Gly
35 40 45
Ser Gly Lys Thr Leu Thr Ile Gln Val Lys Ala Ala Gly Asp Ala Gly
50 55 60
Gln Tyr Thr Cys His Lys Gly Gly Glu Val Leu Ser His Ser Leu Leu
65 70 75 80
Leu Leu His Lys Lys Glu Asp Gly Ile Trp Ser Thr Asp Ile Leu Lys
85 90 95
Asp Gln Lys Glu Pro Lys Asn Lys Thr Phe Leu Arg Cys Glu Ala Lys
100 105 110
Asn Tyr Ser Gly Arg Phe Thr Cys Trp Trp Leu Thr Thr Ile Ser Thr
115 120 125
Asp Leu Thr Phe Ser Val Lys Ser Ser Arg Gly Ser Ser Asp Pro Gln
130 135 140
Gly Val Thr Cys Gly Ala Ala Thr Leu Ser Ala Glu Arg Val Arg Gly
145 150 155 160
Asp Asn Lys Glu Tyr Glu Tyr Ser Val Glu Cys Gln Glu Asp Ser Ala
165 170 175
Cys Pro Ala Ala Glu Glu Ser Leu Pro Ile Glu Val Met Val Asp Ala
180 185 190
Val His Lys Leu Lys Tyr Glu Asn Tyr Thr Ser Ser Phe Phe Ile Arg
195 200 205
Asp Ile Ile Lys Pro Asp Pro Pro Lys Asn Leu Gln Leu Lys Pro Leu
210 215 220
Lys Asn Ser Arg Gln Val Glu Val Ser Trp Glu Tyr Pro Asp Thr Trp
225 230 235 240
Ser Thr Pro His Ser Tyr Phe Ser Leu Thr Phe Cys Val Gln Val Gln
245 250 255
Gly Lys Ser Lys Arg Glu Lys Lys Asp Arg Val Phe Thr Asp Lys Thr
260 265 270
Ser Ala Thr Val Ile Cys Arg Lys Asn Ala Ser Ile Ser Val Arg Ala
275 280 285
Gln Asp Arg Tyr Tyr Ser Ser Ser Trp Ser Glu Trp Ala Ser Val Pro
290 295 300
Cys Ser
305
<210> 64
<211> 306
<212> PRT
<213> artificial sequence
<220>
<223> synthetic construct
<400> 64
Ile Trp Glu Leu Lys Lys Asp Val Tyr Val Val Glu Leu Asp Trp Tyr
1 5 10 15
Pro Asp Ala Pro Gly Glu Met Val Val Leu Thr Cys Asp Thr Pro Glu
20 25 30
Glu Asp Gly Ile Thr Trp Thr Leu Asp Gln Ser Ser Glu Val Leu Gly
35 40 45
Ser Gly Lys Thr Leu Thr Ile Gln Val Lys Ala Phe Gly Asp Ala Gly
50 55 60
Gln Tyr Thr Cys His Lys Gly Gly Glu Val Leu Ser His Ser Leu Leu
65 70 75 80
Leu Leu His Lys Lys Glu Asp Gly Ile Trp Ser Thr Asp Ile Leu Lys
85 90 95
Asp Gln Lys Glu Pro Lys Asn Lys Thr Phe Leu Arg Cys Glu Ala Lys
100 105 110
Asn Tyr Ser Gly Arg Phe Thr Cys Trp Trp Leu Thr Thr Ile Ser Thr
115 120 125
Asp Leu Thr Phe Ser Val Lys Ser Ser Arg Gly Ser Ser Asp Pro Gln
130 135 140
Gly Val Thr Cys Gly Ala Ala Thr Leu Ser Ala Glu Arg Val Arg Gly
145 150 155 160
Asp Asn Lys Glu Tyr Glu Tyr Ser Val Glu Cys Gln Glu Asp Ser Ala
165 170 175
Cys Pro Ala Ala Glu Glu Ser Leu Pro Ile Glu Val Met Val Asp Ala
180 185 190
Val His Lys Leu Lys Tyr Glu Asn Tyr Thr Ser Ser Phe Phe Ile Arg
195 200 205
Asp Ile Ile Lys Pro Asp Pro Pro Lys Asn Leu Gln Leu Lys Pro Leu
210 215 220
Lys Asn Ser Arg Gln Val Glu Val Ser Trp Glu Tyr Pro Asp Thr Trp
225 230 235 240
Ser Thr Pro His Ser Tyr Phe Ser Leu Thr Phe Cys Val Gln Val Gln
245 250 255
Gly Lys Ser Lys Arg Glu Lys Lys Asp Arg Val Phe Thr Asp Lys Thr
260 265 270
Ser Ala Thr Val Ile Cys Arg Lys Asn Ala Ser Ile Ser Val Arg Ala
275 280 285
Gln Asp Arg Tyr Tyr Ser Ser Ser Trp Ser Glu Trp Ala Ser Val Pro
290 295 300
Cys Ser
305
<210> 65
<211> 306
<212> PRT
<213> artificial sequence
<220>
<223> synthetic construct
<400> 65
Ile Trp Glu Leu Lys Lys Asp Val Tyr Val Val Glu Leu Asp Trp Tyr
1 5 10 15
Pro Asp Ala Pro Gly Glu Met Val Val Leu Thr Cys Asp Thr Pro Glu
20 25 30
Glu Asp Gly Ile Thr Trp Thr Leu Asp Gln Ser Ser Glu Val Leu Gly
35 40 45
Ser Gly Lys Thr Leu Thr Ile Gln Val Lys Glu Ala Gly Asp Ala Gly
50 55 60
Gln Tyr Thr Cys His Lys Gly Gly Glu Val Leu Ser His Ser Leu Leu
65 70 75 80
Leu Leu His Lys Lys Glu Asp Gly Ile Trp Ser Thr Asp Ile Leu Lys
85 90 95
Asp Gln Lys Glu Pro Lys Asn Lys Thr Phe Leu Arg Cys Glu Ala Lys
100 105 110
Asn Tyr Ser Gly Arg Phe Thr Cys Trp Trp Leu Thr Thr Ile Ser Thr
115 120 125
Asp Leu Thr Phe Ser Val Lys Ser Ser Arg Gly Ser Ser Asp Pro Gln
130 135 140
Gly Val Thr Cys Gly Ala Ala Thr Leu Ser Ala Glu Arg Val Arg Gly
145 150 155 160
Asp Asn Lys Glu Tyr Glu Tyr Ser Val Glu Cys Gln Glu Asp Ser Ala
165 170 175
Cys Pro Ala Ala Glu Glu Ser Leu Pro Ile Glu Val Met Val Asp Ala
180 185 190
Val His Lys Leu Lys Tyr Glu Asn Tyr Thr Ser Ser Phe Phe Ile Arg
195 200 205
Asp Ile Ile Lys Pro Asp Pro Pro Lys Asn Leu Gln Leu Lys Pro Leu
210 215 220
Lys Asn Ser Arg Gln Val Glu Val Ser Trp Glu Tyr Pro Asp Thr Trp
225 230 235 240
Ser Thr Pro His Ser Tyr Phe Ser Leu Thr Phe Cys Val Gln Val Gln
245 250 255
Gly Lys Ser Lys Arg Glu Lys Lys Asp Arg Val Phe Thr Asp Lys Thr
260 265 270
Ser Ala Thr Val Ile Cys Arg Lys Asn Ala Ser Ile Ser Val Arg Ala
275 280 285
Gln Asp Arg Tyr Tyr Ser Ser Ser Trp Ser Glu Trp Ala Ser Val Pro
290 295 300
Cys Ser
305
<210> 66
<211> 306
<212> PRT
<213> artificial sequence
<220>
<223> synthetic construct
<400> 66
Ile Trp Glu Leu Lys Lys Asp Val Tyr Val Val Glu Leu Asp Trp Tyr
1 5 10 15
Pro Asp Ala Pro Gly Glu Met Val Val Leu Thr Cys Asp Thr Pro Glu
20 25 30
Glu Asp Gly Ile Thr Trp Thr Leu Asp Gln Ser Ser Glu Val Leu Gly
35 40 45
Ser Gly Lys Thr Leu Thr Ile Gln Val Lys Glu Phe Gly Asp Ala Ala
50 55 60
Gln Tyr Thr Cys His Lys Gly Gly Glu Val Leu Ser His Ser Leu Leu
65 70 75 80
Leu Leu His Lys Lys Glu Asp Gly Ile Trp Ser Thr Asp Ile Leu Lys
85 90 95
Asp Gln Lys Glu Pro Lys Asn Lys Thr Phe Leu Arg Cys Glu Ala Lys
100 105 110
Asn Tyr Ser Gly Arg Phe Thr Cys Trp Trp Leu Thr Thr Ile Ser Thr
115 120 125
Asp Leu Thr Phe Ser Val Lys Ser Ser Arg Gly Ser Ser Asp Pro Gln
130 135 140
Gly Val Thr Cys Gly Ala Ala Thr Leu Ser Ala Glu Arg Val Arg Gly
145 150 155 160
Asp Asn Lys Glu Tyr Glu Tyr Ser Val Glu Cys Gln Glu Asp Ser Ala
165 170 175
Cys Pro Ala Ala Glu Glu Ser Leu Pro Ile Glu Val Met Val Asp Ala
180 185 190
Val His Lys Leu Lys Tyr Glu Asn Tyr Thr Ser Ser Phe Phe Ile Arg
195 200 205
Asp Ile Ile Lys Pro Asp Pro Pro Lys Asn Leu Gln Leu Lys Pro Leu
210 215 220
Lys Asn Ser Arg Gln Val Glu Val Ser Trp Glu Tyr Pro Asp Thr Trp
225 230 235 240
Ser Thr Pro His Ser Tyr Phe Ser Leu Thr Phe Cys Val Gln Val Gln
245 250 255
Gly Lys Ser Lys Arg Glu Lys Lys Asp Arg Val Phe Thr Asp Lys Thr
260 265 270
Ser Ala Thr Val Ile Cys Arg Lys Asn Ala Ser Ile Ser Val Arg Ala
275 280 285
Gln Asp Arg Tyr Tyr Ser Ser Ser Trp Ser Glu Trp Ala Ser Val Pro
290 295 300
Cys Ser
305
<210> 67
<211> 524
<212> PRT
<213> artificial sequence
<220>
<223> synthetic construct
<400> 67
Ile Trp Glu Leu Lys Lys Asp Val Tyr Val Val Glu Leu Asp Trp Tyr
1 5 10 15
Pro Asp Ala Pro Gly Glu Met Val Val Leu Thr Cys Asp Thr Pro Glu
20 25 30
Glu Asp Gly Ile Thr Trp Thr Leu Asp Gln Ser Ser Glu Val Leu Gly
35 40 45
Ser Gly Lys Thr Leu Thr Ile Gln Val Lys Glu Phe Gly Asp Ala Gly
50 55 60
Gln Tyr Thr Cys His Lys Gly Gly Glu Val Leu Ser His Ser Leu Leu
65 70 75 80
Leu Leu His Lys Lys Glu Asp Gly Ile Trp Ser Thr Asp Ile Leu Lys
85 90 95
Asp Gln Lys Glu Pro Lys Asn Lys Thr Phe Leu Arg Cys Glu Ala Lys
100 105 110
Asn Tyr Ser Gly Arg Phe Thr Cys Trp Trp Leu Thr Thr Ile Ser Thr
115 120 125
Asp Leu Thr Phe Ser Val Lys Ser Ser Arg Gly Ser Ser Asp Pro Gln
130 135 140
Gly Val Thr Cys Gly Ala Ala Thr Leu Ser Ala Glu Arg Val Arg Gly
145 150 155 160
Asp Asn Lys Glu Tyr Glu Tyr Ser Val Glu Cys Gln Glu Asp Ser Ala
165 170 175
Cys Pro Ala Ala Glu Glu Ser Leu Pro Ile Glu Val Met Val Asp Ala
180 185 190
Val His Lys Leu Lys Tyr Glu Asn Tyr Thr Ser Ser Phe Phe Ile Arg
195 200 205
Asp Ile Ile Lys Pro Asp Pro Pro Lys Asn Leu Gln Leu Lys Pro Leu
210 215 220
Lys Asn Ser Arg Gln Val Glu Val Ser Trp Glu Tyr Pro Asp Thr Trp
225 230 235 240
Ser Thr Pro His Ser Tyr Phe Ser Leu Thr Phe Cys Val Gln Val Gln
245 250 255
Gly Lys Ser Lys Arg Glu Lys Lys Asp Arg Val Phe Thr Asp Lys Thr
260 265 270
Ser Ala Thr Val Ile Cys Arg Lys Asn Ala Ser Ile Ser Val Arg Ala
275 280 285
Gln Asp Arg Tyr Tyr Ser Ser Ser Trp Ser Glu Trp Ala Ser Val Pro
290 295 300
Cys Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
305 310 315 320
Ser Gly Gly Gly Gly Ser Gly Arg Asn Leu Pro Val Ala Thr Pro Asp
325 330 335
Pro Gly Met Phe Pro Cys Leu His His Ser Gln Asn Leu Leu Arg Ala
340 345 350
Val Ser Asn Met Leu Gln Lys Ala Arg Gln Thr Leu Glu Phe Tyr Pro
355 360 365
Cys Thr Ser Glu Glu Ile Asp His Glu Asp Ile Thr Lys Asp Lys Thr
370 375 380
Ser Thr Val Glu Ala Cys Leu Pro Leu Glu Leu Thr Lys Asn Glu Ser
385 390 395 400
Cys Leu Asn Ser Arg Glu Thr Ser Phe Ile Thr Asn Gly Ser Cys Leu
405 410 415
Ala Ser Arg Lys Thr Ser Phe Met Met Ala Leu Cys Leu Ser Ser Ile
420 425 430
Tyr Glu Asp Leu Lys Met Tyr Gln Val Glu Phe Lys Thr Met Asn Ala
435 440 445
Lys Leu Leu Met Asp Pro Lys Arg Gln Ile Phe Leu Asp Gln Asn Met
450 455 460
Leu Ala Val Ile Asp Glu Leu Met Gln Ala Leu Asn Phe Asn Ser Glu
465 470 475 480
Thr Val Pro Gln Lys Ser Ser Leu Glu Glu Pro Asp Phe Tyr Lys Thr
485 490 495
Lys Ile Lys Leu Cys Ile Leu Leu His Ala Phe Arg Ile Arg Ala Val
500 505 510
Thr Ile Asp Arg Val Met Ser Tyr Leu Asn Ala Ser
515 520
<210> 68
<211> 524
<212> PRT
<213> artificial sequence
<220>
<223> synthetic construct
<400> 68
Ile Trp Glu Leu Lys Lys Asp Val Tyr Val Val Glu Leu Asp Trp Tyr
1 5 10 15
Pro Asp Ala Pro Gly Glu Met Val Val Leu Thr Cys Asp Thr Pro Glu
20 25 30
Glu Asp Gly Ile Thr Trp Thr Leu Asp Gln Ser Ser Glu Val Leu Gly
35 40 45
Ser Gly Lys Thr Leu Thr Ile Gln Val Lys Ala Ala Gly Asp Ala Gly
50 55 60
Gln Tyr Thr Cys His Lys Gly Gly Glu Val Leu Ser His Ser Leu Leu
65 70 75 80
Leu Leu His Lys Lys Glu Asp Gly Ile Trp Ser Thr Asp Ile Leu Lys
85 90 95
Asp Gln Lys Glu Pro Lys Asn Lys Thr Phe Leu Arg Cys Glu Ala Lys
100 105 110
Asn Tyr Ser Gly Arg Phe Thr Cys Trp Trp Leu Thr Thr Ile Ser Thr
115 120 125
Asp Leu Thr Phe Ser Val Lys Ser Ser Arg Gly Ser Ser Asp Pro Gln
130 135 140
Gly Val Thr Cys Gly Ala Ala Thr Leu Ser Ala Glu Arg Val Arg Gly
145 150 155 160
Asp Asn Lys Glu Tyr Glu Tyr Ser Val Glu Cys Gln Glu Asp Ser Ala
165 170 175
Cys Pro Ala Ala Glu Glu Ser Leu Pro Ile Glu Val Met Val Asp Ala
180 185 190
Val His Lys Leu Lys Tyr Glu Asn Tyr Thr Ser Ser Phe Phe Ile Arg
195 200 205
Asp Ile Ile Lys Pro Asp Pro Pro Lys Asn Leu Gln Leu Lys Pro Leu
210 215 220
Lys Asn Ser Arg Gln Val Glu Val Ser Trp Glu Tyr Pro Asp Thr Trp
225 230 235 240
Ser Thr Pro His Ser Tyr Phe Ser Leu Thr Phe Cys Val Gln Val Gln
245 250 255
Gly Lys Ser Lys Arg Glu Lys Lys Asp Arg Val Phe Thr Asp Lys Thr
260 265 270
Ser Ala Thr Val Ile Cys Arg Lys Asn Ala Ser Ile Ser Val Arg Ala
275 280 285
Gln Asp Arg Tyr Tyr Ser Ser Ser Trp Ser Glu Trp Ala Ser Val Pro
290 295 300
Cys Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
305 310 315 320
Ser Gly Gly Gly Gly Ser Gly Arg Asn Leu Pro Val Ala Thr Pro Asp
325 330 335
Pro Gly Met Phe Pro Cys Leu His His Ser Gln Asn Leu Leu Arg Ala
340 345 350
Val Ser Asn Met Leu Gln Lys Ala Arg Gln Thr Leu Glu Phe Tyr Pro
355 360 365
Cys Thr Ser Glu Glu Ile Asp His Glu Asp Ile Thr Lys Asp Lys Thr
370 375 380
Ser Thr Val Glu Ala Cys Leu Pro Leu Glu Leu Thr Lys Asn Glu Ser
385 390 395 400
Cys Leu Asn Ser Arg Glu Thr Ser Phe Ile Thr Asn Gly Ser Cys Leu
405 410 415
Ala Ser Arg Lys Thr Ser Phe Met Met Ala Leu Cys Leu Ser Ser Ile
420 425 430
Tyr Glu Asp Leu Lys Met Tyr Gln Val Glu Phe Lys Thr Met Asn Ala
435 440 445
Lys Leu Leu Met Asp Pro Lys Arg Gln Ile Phe Leu Asp Gln Asn Met
450 455 460
Leu Ala Val Ile Asp Glu Leu Met Gln Ala Leu Asn Phe Asn Ser Glu
465 470 475 480
Thr Val Pro Gln Lys Ser Ser Leu Glu Glu Pro Asp Phe Tyr Lys Thr
485 490 495
Lys Ile Lys Leu Cys Ile Leu Leu His Ala Phe Arg Ile Arg Ala Val
500 505 510
Thr Ile Asp Arg Val Met Ser Tyr Leu Asn Ala Ser
515 520
<210> 69
<211> 524
<212> PRT
<213> artificial sequence
<220>
<223> synthetic construct
<400> 69
Ile Trp Glu Leu Lys Lys Asp Val Tyr Val Val Glu Leu Asp Trp Tyr
1 5 10 15
Pro Asp Ala Pro Gly Glu Met Val Val Leu Thr Cys Asp Thr Pro Glu
20 25 30
Glu Asp Gly Ile Thr Trp Thr Leu Asp Gln Ser Ser Glu Val Leu Gly
35 40 45
Ser Gly Lys Thr Leu Thr Ile Gln Val Lys Ala Phe Gly Asp Ala Gly
50 55 60
Gln Tyr Thr Cys His Lys Gly Gly Glu Val Leu Ser His Ser Leu Leu
65 70 75 80
Leu Leu His Lys Lys Glu Asp Gly Ile Trp Ser Thr Asp Ile Leu Lys
85 90 95
Asp Gln Lys Glu Pro Lys Asn Lys Thr Phe Leu Arg Cys Glu Ala Lys
100 105 110
Asn Tyr Ser Gly Arg Phe Thr Cys Trp Trp Leu Thr Thr Ile Ser Thr
115 120 125
Asp Leu Thr Phe Ser Val Lys Ser Ser Arg Gly Ser Ser Asp Pro Gln
130 135 140
Gly Val Thr Cys Gly Ala Ala Thr Leu Ser Ala Glu Arg Val Arg Gly
145 150 155 160
Asp Asn Lys Glu Tyr Glu Tyr Ser Val Glu Cys Gln Glu Asp Ser Ala
165 170 175
Cys Pro Ala Ala Glu Glu Ser Leu Pro Ile Glu Val Met Val Asp Ala
180 185 190
Val His Lys Leu Lys Tyr Glu Asn Tyr Thr Ser Ser Phe Phe Ile Arg
195 200 205
Asp Ile Ile Lys Pro Asp Pro Pro Lys Asn Leu Gln Leu Lys Pro Leu
210 215 220
Lys Asn Ser Arg Gln Val Glu Val Ser Trp Glu Tyr Pro Asp Thr Trp
225 230 235 240
Ser Thr Pro His Ser Tyr Phe Ser Leu Thr Phe Cys Val Gln Val Gln
245 250 255
Gly Lys Ser Lys Arg Glu Lys Lys Asp Arg Val Phe Thr Asp Lys Thr
260 265 270
Ser Ala Thr Val Ile Cys Arg Lys Asn Ala Ser Ile Ser Val Arg Ala
275 280 285
Gln Asp Arg Tyr Tyr Ser Ser Ser Trp Ser Glu Trp Ala Ser Val Pro
290 295 300
Cys Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
305 310 315 320
Ser Gly Gly Gly Gly Ser Gly Arg Asn Leu Pro Val Ala Thr Pro Asp
325 330 335
Pro Gly Met Phe Pro Cys Leu His His Ser Gln Asn Leu Leu Arg Ala
340 345 350
Val Ser Asn Met Leu Gln Lys Ala Arg Gln Thr Leu Glu Phe Tyr Pro
355 360 365
Cys Thr Ser Glu Glu Ile Asp His Glu Asp Ile Thr Lys Asp Lys Thr
370 375 380
Ser Thr Val Glu Ala Cys Leu Pro Leu Glu Leu Thr Lys Asn Glu Ser
385 390 395 400
Cys Leu Asn Ser Arg Glu Thr Ser Phe Ile Thr Asn Gly Ser Cys Leu
405 410 415
Ala Ser Arg Lys Thr Ser Phe Met Met Ala Leu Cys Leu Ser Ser Ile
420 425 430
Tyr Glu Asp Leu Lys Met Tyr Gln Val Glu Phe Lys Thr Met Asn Ala
435 440 445
Lys Leu Leu Met Asp Pro Lys Arg Gln Ile Phe Leu Asp Gln Asn Met
450 455 460
Leu Ala Val Ile Asp Glu Leu Met Gln Ala Leu Asn Phe Asn Ser Glu
465 470 475 480
Thr Val Pro Gln Lys Ser Ser Leu Glu Glu Pro Asp Phe Tyr Lys Thr
485 490 495
Lys Ile Lys Leu Cys Ile Leu Leu His Ala Phe Arg Ile Arg Ala Val
500 505 510
Thr Ile Asp Arg Val Met Ser Tyr Leu Asn Ala Ser
515 520
<210> 70
<211> 524
<212> PRT
<213> artificial sequence
<220>
<223> synthetic construct
<400> 70
Ile Trp Glu Leu Lys Lys Asp Val Tyr Val Val Glu Leu Asp Trp Tyr
1 5 10 15
Pro Asp Ala Pro Gly Glu Met Val Val Leu Thr Cys Asp Thr Pro Glu
20 25 30
Glu Asp Gly Ile Thr Trp Thr Leu Asp Gln Ser Ser Glu Val Leu Gly
35 40 45
Ser Gly Lys Thr Leu Thr Ile Gln Val Lys Glu Phe Gly Asp Ala Ala
50 55 60
Gln Tyr Thr Cys His Lys Gly Gly Glu Val Leu Ser His Ser Leu Leu
65 70 75 80
Leu Leu His Lys Lys Glu Asp Gly Ile Trp Ser Thr Asp Ile Leu Lys
85 90 95
Asp Gln Lys Glu Pro Lys Asn Lys Thr Phe Leu Arg Cys Glu Ala Lys
100 105 110
Asn Tyr Ser Gly Arg Phe Thr Cys Trp Trp Leu Thr Thr Ile Ser Thr
115 120 125
Asp Leu Thr Phe Ser Val Lys Ser Ser Arg Gly Ser Ser Asp Pro Gln
130 135 140
Gly Val Thr Cys Gly Ala Ala Thr Leu Ser Ala Glu Arg Val Arg Gly
145 150 155 160
Asp Asn Lys Glu Tyr Glu Tyr Ser Val Glu Cys Gln Glu Asp Ser Ala
165 170 175
Cys Pro Ala Ala Glu Glu Ser Leu Pro Ile Glu Val Met Val Asp Ala
180 185 190
Val His Lys Leu Lys Tyr Glu Asn Tyr Thr Ser Ser Phe Phe Ile Arg
195 200 205
Asp Ile Ile Lys Pro Asp Pro Pro Lys Asn Leu Gln Leu Lys Pro Leu
210 215 220
Lys Asn Ser Arg Gln Val Glu Val Ser Trp Glu Tyr Pro Asp Thr Trp
225 230 235 240
Ser Thr Pro His Ser Tyr Phe Ser Leu Thr Phe Cys Val Gln Val Gln
245 250 255
Gly Lys Ser Lys Arg Glu Lys Lys Asp Arg Val Phe Thr Asp Lys Thr
260 265 270
Ser Ala Thr Val Ile Cys Arg Lys Asn Ala Ser Ile Ser Val Arg Ala
275 280 285
Gln Asp Arg Tyr Tyr Ser Ser Ser Trp Ser Glu Trp Ala Ser Val Pro
290 295 300
Cys Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
305 310 315 320
Ser Gly Gly Gly Gly Ser Gly Arg Asn Leu Pro Val Ala Thr Pro Asp
325 330 335
Pro Gly Met Phe Pro Cys Leu His His Ser Gln Asn Leu Leu Arg Ala
340 345 350
Val Ser Asn Met Leu Gln Lys Ala Arg Gln Thr Leu Glu Phe Tyr Pro
355 360 365
Cys Thr Ser Glu Glu Ile Asp His Glu Asp Ile Thr Lys Asp Lys Thr
370 375 380
Ser Thr Val Glu Ala Cys Leu Pro Leu Glu Leu Thr Lys Asn Glu Ser
385 390 395 400
Cys Leu Asn Ser Arg Glu Thr Ser Phe Ile Thr Asn Gly Ser Cys Leu
405 410 415
Ala Ser Arg Lys Thr Ser Phe Met Met Ala Leu Cys Leu Ser Ser Ile
420 425 430
Tyr Glu Asp Leu Lys Met Tyr Gln Val Glu Phe Lys Thr Met Asn Ala
435 440 445
Lys Leu Leu Met Asp Pro Lys Arg Gln Ile Phe Leu Asp Gln Asn Met
450 455 460
Leu Ala Val Ile Asp Glu Leu Met Gln Ala Leu Asn Phe Asn Ser Glu
465 470 475 480
Thr Val Pro Gln Lys Ser Ser Leu Glu Glu Pro Asp Phe Tyr Lys Thr
485 490 495
Lys Ile Lys Leu Cys Ile Leu Leu His Ala Phe Arg Ile Arg Ala Val
500 505 510
Thr Ile Asp Arg Val Met Ser Tyr Leu Asn Ala Ser
515 520
<210> 71
<211> 524
<212> PRT
<213> artificial sequence
<220>
<223> synthetic construct
<400> 71
Ile Trp Glu Leu Lys Lys Asp Val Tyr Val Val Glu Leu Asp Trp Tyr
1 5 10 15
Pro Asp Ala Pro Gly Glu Met Val Val Leu Thr Cys Asp Thr Pro Glu
20 25 30
Glu Asp Gly Ile Thr Trp Thr Leu Asp Gln Ser Ser Glu Val Leu Gly
35 40 45
Ser Gly Lys Thr Leu Thr Ile Gln Val Lys Glu Ala Gly Asp Ala Gly
50 55 60
Gln Tyr Thr Cys His Lys Gly Gly Glu Val Leu Ser His Ser Leu Leu
65 70 75 80
Leu Leu His Lys Lys Glu Asp Gly Ile Trp Ser Thr Asp Ile Leu Lys
85 90 95
Asp Gln Lys Glu Pro Lys Asn Lys Thr Phe Leu Arg Cys Glu Ala Lys
100 105 110
Asn Tyr Ser Gly Arg Phe Thr Cys Trp Trp Leu Thr Thr Ile Ser Thr
115 120 125
Asp Leu Thr Phe Ser Val Lys Ser Ser Arg Gly Ser Ser Asp Pro Gln
130 135 140
Gly Val Thr Cys Gly Ala Ala Thr Leu Ser Ala Glu Arg Val Arg Gly
145 150 155 160
Asp Asn Lys Glu Tyr Glu Tyr Ser Val Glu Cys Gln Glu Asp Ser Ala
165 170 175
Cys Pro Ala Ala Glu Glu Ser Leu Pro Ile Glu Val Met Val Asp Ala
180 185 190
Val His Lys Leu Lys Tyr Glu Asn Tyr Thr Ser Ser Phe Phe Ile Arg
195 200 205
Asp Ile Ile Lys Pro Asp Pro Pro Lys Asn Leu Gln Leu Lys Pro Leu
210 215 220
Lys Asn Ser Arg Gln Val Glu Val Ser Trp Glu Tyr Pro Asp Thr Trp
225 230 235 240
Ser Thr Pro His Ser Tyr Phe Ser Leu Thr Phe Cys Val Gln Val Gln
245 250 255
Gly Lys Ser Lys Arg Glu Lys Lys Asp Arg Val Phe Thr Asp Lys Thr
260 265 270
Ser Ala Thr Val Ile Cys Arg Lys Asn Ala Ser Ile Ser Val Arg Ala
275 280 285
Gln Asp Arg Tyr Tyr Ser Ser Ser Trp Ser Glu Trp Ala Ser Val Pro
290 295 300
Cys Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
305 310 315 320
Ser Gly Gly Gly Gly Ser Gly Arg Asn Leu Pro Val Ala Thr Pro Asp
325 330 335
Pro Gly Met Phe Pro Cys Leu His His Ser Gln Asn Leu Leu Arg Ala
340 345 350
Val Ser Asn Met Leu Gln Lys Ala Arg Gln Thr Leu Glu Phe Tyr Pro
355 360 365
Cys Thr Ser Glu Glu Ile Asp His Glu Asp Ile Thr Lys Asp Lys Thr
370 375 380
Ser Thr Val Glu Ala Cys Leu Pro Leu Glu Leu Thr Lys Asn Glu Ser
385 390 395 400
Cys Leu Asn Ser Arg Glu Thr Ser Phe Ile Thr Asn Gly Ser Cys Leu
405 410 415
Ala Ser Arg Lys Thr Ser Phe Met Met Ala Leu Cys Leu Ser Ser Ile
420 425 430
Tyr Glu Asp Leu Lys Met Tyr Gln Val Glu Phe Lys Thr Met Asn Ala
435 440 445
Lys Leu Leu Met Asp Pro Lys Arg Gln Ile Phe Leu Asp Gln Asn Met
450 455 460
Leu Ala Val Ile Asp Glu Leu Met Gln Ala Leu Asn Phe Asn Ser Glu
465 470 475 480
Thr Val Pro Gln Lys Ser Ser Leu Glu Glu Pro Asp Phe Tyr Lys Thr
485 490 495
Lys Ile Lys Leu Cys Ile Leu Leu His Ala Phe Arg Ile Arg Ala Val
500 505 510
Thr Ile Asp Arg Val Met Ser Tyr Leu Asn Ala Ser
515 520
<210> 72
<211> 524
<212> PRT
<213> artificial sequence
<220>
<223> synthetic construct
<400> 72
Met Trp Glu Leu Glu Lys Asp Val Tyr Val Val Glu Val Asp Trp Thr
1 5 10 15
Pro Asp Ala Pro Gly Glu Thr Val Asn Leu Thr Cys Asp Thr Pro Glu
20 25 30
Glu Asp Asp Ile Thr Trp Thr Ser Asp Gln Arg His Gly Val Ile Gly
35 40 45
Ser Gly Lys Thr Leu Thr Ile Thr Val Lys Ala Ala Leu Asp Ala Gly
50 55 60
Gln Tyr Thr Cys His Lys Gly Gly Glu Thr Leu Ser His Ser His Leu
65 70 75 80
Leu Leu His Lys Lys Glu Asn Gly Ile Trp Ser Thr Glu Ile Leu Lys
85 90 95
Asn Phe Lys Asn Lys Thr Phe Leu Lys Cys Glu Ala Pro Asn Tyr Ser
100 105 110
Gly Arg Phe Thr Cys Ser Trp Leu Val Gln Arg Asn Met Asp Leu Lys
115 120 125
Phe Asn Ile Lys Ser Ser Ser Ser Ser Pro Asp Ser Arg Ala Val Thr
130 135 140
Cys Gly Met Ala Ser Leu Ser Ala Glu Lys Val Thr Leu Asp Gln Arg
145 150 155 160
Asp Tyr Glu Lys Tyr Ser Val Ser Cys Gln Glu Asp Val Thr Cys Pro
165 170 175
Thr Ala Glu Glu Thr Leu Pro Ile Glu Leu Ala Leu Glu Ala Arg Gln
180 185 190
Gln Asn Lys Tyr Glu Asn Tyr Ser Thr Ser Phe Phe Ile Arg Asp Ile
195 200 205
Ile Lys Pro Asp Pro Pro Lys Asn Leu Gln Met Lys Pro Leu Lys Asn
210 215 220
Ser Gln Val Glu Val Ser Trp Glu Tyr Pro Asp Ser Trp Ser Thr Pro
225 230 235 240
His Ser Tyr Phe Ser Leu Lys Phe Phe Val Arg Ile Gln Arg Lys Lys
245 250 255
Glu Lys Met Lys Glu Thr Glu Glu Gly Cys Asn Gln Lys Gly Ala Phe
260 265 270
Leu Val Glu Lys Thr Ser Thr Glu Val Gln Cys Lys Gly Gly Asn Val
275 280 285
Cys Val Gln Ala Gln Asp Arg Tyr Tyr Asn Ser Ser Cys Ser Lys Trp
290 295 300
Ala Cys Val Pro Cys Arg Val Arg Ser Gly Gly Pro Gly Gly Gly Gly
305 310 315 320
Ser Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Arg Val Ile Pro Val
325 330 335
Ser Gly Pro Ala Arg Cys Leu Ser Gln Ser Arg Asn Leu Leu Lys Thr
340 345 350
Thr Asp Asp Met Val Lys Thr Ala Arg Glu Lys Leu Lys His Tyr Ser
355 360 365
Cys Thr Ala Glu Asp Ile Asp His Glu Asp Ile Thr Arg Asp Gln Thr
370 375 380
Ser Thr Leu Lys Thr Cys Leu Pro Leu Glu Leu His Lys Asn Glu Ser
385 390 395 400
Cys Leu Ala Thr Arg Glu Thr Ser Ser Thr Thr Arg Gly Ser Cys Leu
405 410 415
Pro Pro Gln Lys Thr Ser Leu Met Met Thr Leu Cys Leu Gly Ser Ile
420 425 430
Tyr Glu Asp Leu Lys Met Tyr Gln Thr Glu Phe Gln Ala Ile Asn Ala
435 440 445
Ala Leu Gln Asn His Asn His Gln Gln Ile Ile Leu Asp Lys Gly Met
450 455 460
Leu Val Ala Ile Asp Glu Leu Met Gln Ser Leu Asn His Asn Gly Glu
465 470 475 480
Thr Leu Arg Gln Lys Pro Pro Val Gly Glu Ala Asp Pro Tyr Arg Val
485 490 495
Lys Met Lys Leu Cys Ile Leu Leu His Ala Phe Ser Thr Arg Val Val
500 505 510
Thr Ile Asn Arg Val Met Gly Tyr Leu Ser Ser Ala
515 520
<210> 73
<211> 170
<212> PRT
<213> Homo sapiens (Homo sapiens)
<400> 73
Arg Ala Val Pro Gly Gly Ser Ser Pro Ala Trp Thr Gln Cys Gln Gln
1 5 10 15
Leu Ser Gln Lys Leu Cys Thr Leu Ala Trp Ser Ala His Pro Leu Val
20 25 30
Gly His Met Asp Leu Arg Glu Glu Gly Asp Glu Glu Thr Thr Asn Asp
35 40 45
Val Pro His Ile Gln Cys Gly Asp Gly Cys Asp Pro Gln Gly Leu Arg
50 55 60
Asp Asn Ser Gln Phe Cys Leu Gln Arg Ile His Gln Gly Leu Ile Phe
65 70 75 80
Tyr Glu Lys Leu Leu Gly Ser Asp Ile Phe Thr Gly Glu Pro Ser Leu
85 90 95
Leu Pro Asp Ser Pro Val Gly Gln Leu His Ala Ser Leu Leu Gly Leu
100 105 110
Ser Gln Leu Leu Gln Pro Glu Gly His His Trp Glu Thr Gln Gln Ile
115 120 125
Pro Ser Leu Ser Pro Ser Gln Pro Trp Gln Arg Leu Leu Leu Arg Phe
130 135 140
Lys Ile Leu Arg Ser Leu Gln Ala Phe Val Ala Val Ala Ala Arg Val
145 150 155 160
Phe Ala His Gly Ala Ala Thr Leu Ser Pro
165 170
<210> 74
<211> 497
<212> PRT
<213> artificial sequence
<220>
<223> synthetic construct
<400> 74
Ile Trp Glu Leu Lys Lys Asp Val Tyr Val Val Glu Leu Asp Trp Tyr
1 5 10 15
Pro Asp Ala Pro Gly Glu Met Val Val Leu Thr Cys Asp Thr Pro Glu
20 25 30
Glu Asp Gly Ile Thr Trp Thr Leu Asp Gln Ser Ser Glu Val Leu Gly
35 40 45
Ser Gly Lys Thr Leu Thr Ile Gln Val Lys Glu Phe Gly Asp Ala Gly
50 55 60
Gln Tyr Thr Cys His Lys Gly Gly Glu Val Leu Ser His Ser Leu Leu
65 70 75 80
Leu Leu His Lys Lys Glu Asp Gly Ile Trp Ser Thr Asp Ile Leu Lys
85 90 95
Asp Gln Lys Glu Pro Lys Asn Lys Thr Phe Leu Arg Cys Glu Ala Lys
100 105 110
Asn Tyr Ser Gly Arg Phe Thr Cys Trp Trp Leu Thr Thr Ile Ser Thr
115 120 125
Asp Leu Thr Phe Ser Val Lys Ser Ser Arg Gly Ser Ser Asp Pro Gln
130 135 140
Gly Val Thr Cys Gly Ala Ala Thr Leu Ser Ala Glu Arg Val Arg Gly
145 150 155 160
Asp Asn Lys Glu Tyr Glu Tyr Ser Val Glu Cys Gln Glu Asp Ser Ala
165 170 175
Cys Pro Ala Ala Glu Glu Ser Leu Pro Ile Glu Val Met Val Asp Ala
180 185 190
Val His Lys Leu Lys Tyr Glu Asn Tyr Thr Ser Ser Phe Phe Ile Arg
195 200 205
Asp Ile Ile Lys Pro Asp Pro Pro Lys Asn Leu Gln Leu Lys Pro Leu
210 215 220
Lys Asn Ser Arg Gln Val Glu Val Ser Trp Glu Tyr Pro Asp Thr Trp
225 230 235 240
Ser Thr Pro His Ser Tyr Phe Ser Leu Thr Phe Cys Val Gln Val Gln
245 250 255
Gly Lys Ser Lys Arg Glu Lys Lys Asp Arg Val Phe Thr Asp Lys Thr
260 265 270
Ser Ala Thr Val Ile Cys Arg Lys Asn Ala Ser Ile Ser Val Arg Ala
275 280 285
Gln Asp Arg Tyr Tyr Ser Ser Ser Trp Ser Glu Trp Ala Ser Val Pro
290 295 300
Cys Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
305 310 315 320
Ser Gly Gly Gly Gly Ser Gly Arg Ala Val Pro Gly Gly Ser Ser Pro
325 330 335
Ala Trp Thr Gln Cys Gln Gln Leu Ser Gln Lys Leu Cys Thr Leu Ala
340 345 350
Trp Ser Ala His Pro Leu Val Gly His Met Asp Leu Arg Glu Glu Gly
355 360 365
Asp Glu Glu Thr Thr Asn Asp Val Pro His Ile Gln Cys Gly Asp Gly
370 375 380
Cys Asp Pro Gln Gly Leu Arg Asp Asn Ser Gln Phe Cys Leu Gln Arg
385 390 395 400
Ile His Gln Gly Leu Ile Phe Tyr Glu Lys Leu Leu Gly Ser Asp Ile
405 410 415
Phe Thr Gly Glu Pro Ser Leu Leu Pro Asp Ser Pro Val Gly Gln Leu
420 425 430
His Ala Ser Leu Leu Gly Leu Ser Gln Leu Leu Gln Pro Glu Gly His
435 440 445
His Trp Glu Thr Gln Gln Ile Pro Ser Leu Ser Pro Ser Gln Pro Trp
450 455 460
Gln Arg Leu Leu Leu Arg Phe Lys Ile Leu Arg Ser Leu Gln Ala Phe
465 470 475 480
Val Ala Val Ala Ala Arg Val Phe Ala His Gly Ala Ala Thr Leu Ser
485 490 495
Pro
<210> 75
<211> 497
<212> PRT
<213> artificial sequence
<220>
<223> synthetic construct
<400> 75
Ile Trp Glu Leu Lys Lys Asp Val Tyr Val Val Glu Leu Asp Trp Tyr
1 5 10 15
Pro Asp Ala Pro Gly Glu Met Val Val Leu Thr Cys Asp Thr Pro Glu
20 25 30
Glu Asp Gly Ile Thr Trp Thr Leu Asp Gln Ser Ser Glu Val Leu Gly
35 40 45
Ser Gly Lys Thr Leu Thr Ile Gln Val Lys Ala Ala Gly Asp Ala Gly
50 55 60
Gln Tyr Thr Cys His Lys Gly Gly Glu Val Leu Ser His Ser Leu Leu
65 70 75 80
Leu Leu His Lys Lys Glu Asp Gly Ile Trp Ser Thr Asp Ile Leu Lys
85 90 95
Asp Gln Lys Glu Pro Lys Asn Lys Thr Phe Leu Arg Cys Glu Ala Lys
100 105 110
Asn Tyr Ser Gly Arg Phe Thr Cys Trp Trp Leu Thr Thr Ile Ser Thr
115 120 125
Asp Leu Thr Phe Ser Val Lys Ser Ser Arg Gly Ser Ser Asp Pro Gln
130 135 140
Gly Val Thr Cys Gly Ala Ala Thr Leu Ser Ala Glu Arg Val Arg Gly
145 150 155 160
Asp Asn Lys Glu Tyr Glu Tyr Ser Val Glu Cys Gln Glu Asp Ser Ala
165 170 175
Cys Pro Ala Ala Glu Glu Ser Leu Pro Ile Glu Val Met Val Asp Ala
180 185 190
Val His Lys Leu Lys Tyr Glu Asn Tyr Thr Ser Ser Phe Phe Ile Arg
195 200 205
Asp Ile Ile Lys Pro Asp Pro Pro Lys Asn Leu Gln Leu Lys Pro Leu
210 215 220
Lys Asn Ser Arg Gln Val Glu Val Ser Trp Glu Tyr Pro Asp Thr Trp
225 230 235 240
Ser Thr Pro His Ser Tyr Phe Ser Leu Thr Phe Cys Val Gln Val Gln
245 250 255
Gly Lys Ser Lys Arg Glu Lys Lys Asp Arg Val Phe Thr Asp Lys Thr
260 265 270
Ser Ala Thr Val Ile Cys Arg Lys Asn Ala Ser Ile Ser Val Arg Ala
275 280 285
Gln Asp Arg Tyr Tyr Ser Ser Ser Trp Ser Glu Trp Ala Ser Val Pro
290 295 300
Cys Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
305 310 315 320
Ser Gly Gly Gly Gly Ser Gly Arg Ala Val Pro Gly Gly Ser Ser Pro
325 330 335
Ala Trp Thr Gln Cys Gln Gln Leu Ser Gln Lys Leu Cys Thr Leu Ala
340 345 350
Trp Ser Ala His Pro Leu Val Gly His Met Asp Leu Arg Glu Glu Gly
355 360 365
Asp Glu Glu Thr Thr Asn Asp Val Pro His Ile Gln Cys Gly Asp Gly
370 375 380
Cys Asp Pro Gln Gly Leu Arg Asp Asn Ser Gln Phe Cys Leu Gln Arg
385 390 395 400
Ile His Gln Gly Leu Ile Phe Tyr Glu Lys Leu Leu Gly Ser Asp Ile
405 410 415
Phe Thr Gly Glu Pro Ser Leu Leu Pro Asp Ser Pro Val Gly Gln Leu
420 425 430
His Ala Ser Leu Leu Gly Leu Ser Gln Leu Leu Gln Pro Glu Gly His
435 440 445
His Trp Glu Thr Gln Gln Ile Pro Ser Leu Ser Pro Ser Gln Pro Trp
450 455 460
Gln Arg Leu Leu Leu Arg Phe Lys Ile Leu Arg Ser Leu Gln Ala Phe
465 470 475 480
Val Ala Val Ala Ala Arg Val Phe Ala His Gly Ala Ala Thr Leu Ser
485 490 495
Pro
<210> 76
<211> 23
<212> PRT
<213> artificial sequence
<220>
<223> synthetic construct
<400> 76
Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala
1 5 10 15
Pro Glu Leu Leu Gly Gly Pro
20
<210> 77
<211> 23
<212> PRT
<213> artificial sequence
<220>
<223> synthetic construct
<400> 77
Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala
1 5 10 15
Pro Glu Lys Lys Gly Gly Pro
20
<210> 78
<211> 23
<212> PRT
<213> artificial sequence
<220>
<223> synthetic construct
<400> 78
Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala
1 5 10 15
Pro Glu Asp Glu Gly Gly Pro
20
<210> 79
<211> 25
<212> PRT
<213> artificial sequence
<220>
<223> synthetic construct
<400> 79
Glu Pro Lys Ser Cys Asp Lys Asp Lys Thr His Thr Cys Pro Pro Cys
1 5 10 15
Pro Ala Pro Glu Leu Leu Gly Gly Pro
20 25
<210> 80
<211> 25
<212> PRT
<213> artificial sequence
<220>
<223> synthetic construct
<400> 80
Glu Pro Lys Ser Cys Asp Lys Asp Lys Thr His Thr Cys Pro Pro Cys
1 5 10 15
Pro Ala Pro Glu Lys Lys Gly Gly Pro
20 25
<210> 81
<211> 25
<212> PRT
<213> artificial sequence
<220>
<223> synthetic construct
<400> 81
Glu Pro Lys Ser Cys Asp Lys Asp Lys Thr His Thr Cys Pro Pro Cys
1 5 10 15
Pro Ala Pro Glu Asp Glu Gly Gly Pro
20 25
<210> 82
<211> 19
<212> PRT
<213> artificial sequence
<220>
<223> synthetic construct
<400> 82
Glu Arg Lys Cys Cys Val Glu Cys Pro Pro Cys Pro Ala Pro Pro Val
1 5 10 15
Ala Gly Pro
<210> 83
<211> 20
<212> PRT
<213> artificial sequence
<220>
<223> synthetic construct
<400> 83
Glu Ser Lys Tyr Gly Pro Pro Cys Pro Ser Cys Pro Ala Pro Glu Phe
1 5 10 15
Leu Gly Gly Pro
20
<210> 84
<211> 7
<212> PRT
<213> artificial sequence
<220>
<223> synthetic construct
<400> 84
Glu Pro Lys Ser Cys Asp Lys
1 5
<210> 85
<211> 5
<212> PRT
<213> artificial sequence
<220>
<223> synthetic construct
<400> 85
Glu Pro Lys Ser Cys
1 5
<210> 86
<211> 18
<212> PRT
<213> artificial sequence
<220>
<223> synthetic construct
<400> 86
Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly
1 5 10 15
Gly Pro
<210> 87
<211> 18
<212> PRT
<213> artificial sequence
<220>
<223> synthetic construct
<400> 87
Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Lys Lys Gly
1 5 10 15
Gly Pro
<210> 88
<211> 18
<212> PRT
<213> artificial sequence
<220>
<223> synthetic construct
<400> 88
Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Asp Glu Gly
1 5 10 15
Gly Pro
<210> 89
<211> 5
<212> PRT
<213> artificial sequence
<220>
<223> synthetic construct
<400> 89
Asp Lys Thr His Thr
1 5
<210> 90
<211> 8
<212> PRT
<213> artificial sequence
<220>
<223> synthetic construct
<400> 90
Glu Pro Lys Ser Cys Asp Lys Pro
1 5
<210> 91
<211> 26
<212> PRT
<213> artificial sequence
<220>
<223> synthetic construct
<400> 91
Glu Pro Lys Ser Cys Asp Lys Pro Asp Lys Thr His Thr Cys Pro Pro
1 5 10 15
Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro
20 25
<210> 92
<211> 26
<212> PRT
<213> artificial sequence
<220>
<223> synthetic construct
<400> 92
Glu Pro Lys Ser Cys Asp Lys Pro Asp Lys Thr His Thr Cys Pro Pro
1 5 10 15
Cys Pro Ala Pro Glu Lys Lys Gly Gly Pro
20 25
<210> 93
<211> 26
<212> PRT
<213> artificial sequence
<220>
<223> synthetic construct
<400> 93
Glu Pro Lys Ser Cys Asp Lys Pro Asp Lys Thr His Thr Cys Pro Pro
1 5 10 15
Cys Pro Ala Pro Glu Asp Glu Gly Gly Pro
20 25
<210> 94
<211> 20
<212> PRT
<213> artificial sequence
<220>
<223> synthetic construct
<400> 94
Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe
1 5 10 15
Leu Gly Gly Pro
20
<210> 95
<211> 24
<212> PRT
<213> artificial sequence
<220>
<223> synthetic construct
<400> 95
Glu Pro Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro
1 5 10 15
Ala Pro Glu Leu Leu Gly Gly Pro
20
<210> 96
<211> 209
<212> PRT
<213> Homo sapiens (Homo sapiens)
<400> 96
Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser
1 5 10 15
Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp
20 25 30
Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn
35 40 45
Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val
50 55 60
Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu
65 70 75 80
Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys
85 90 95
Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr
100 105 110
Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr
115 120 125
Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu
130 135 140
Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu
145 150 155 160
Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys
165 170 175
Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu
180 185 190
Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly
195 200 205
Lys
<210> 97
<211> 209
<212> PRT
<213> artificial sequence
<220>
<223> synthetic construct
<400> 97
Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser
1 5 10 15
Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp
20 25 30
Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn
35 40 45
Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val
50 55 60
Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu
65 70 75 80
Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys
85 90 95
Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Val
100 105 110
Tyr Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr
115 120 125
Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu
130 135 140
Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu
145 150 155 160
Asp Glu Asp Gly Ser Phe Ala Leu Val Ser Lys Leu Thr Val Asp Lys
165 170 175
Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu
180 185 190
Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly
195 200 205
Lys
<210> 98
<211> 209
<212> PRT
<213> artificial sequence
<220>
<223> synthetic construct
<400> 98
Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser
1 5 10 15
Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp
20 25 30
Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn
35 40 45
Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val
50 55 60
Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu
65 70 75 80
Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys
85 90 95
Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Val
100 105 110
Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Leu
115 120 125
Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu
130 135 140
Ser Asn Gly Gln Pro Glu Asn Arg Tyr Met Thr Trp Pro Pro Val Leu
145 150 155 160
Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys
165 170 175
Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu
180 185 190
Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly
195 200 205
Lys
<210> 99
<211> 209
<212> PRT
<213> Homo sapiens (Homo sapiens)
<400> 99
Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser
1 5 10 15
Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp
20 25 30
Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn
35 40 45
Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val
50 55 60
Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu
65 70 75 80
Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys
85 90 95
Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr
100 105 110
Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr
115 120 125
Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu
130 135 140
Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu
145 150 155 160
Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys
165 170 175
Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu
180 185 190
Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly
195 200 205
Lys
<210> 100
<211> 209
<212> PRT
<213> artificial sequence
<220>
<223> synthetic construct
<400> 100
Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser
1 5 10 15
Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp
20 25 30
Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn
35 40 45
Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Ala Ser Thr Tyr Arg Val
50 55 60
Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu
65 70 75 80
Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys
85 90 95
Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr
100 105 110
Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr
115 120 125
Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu
130 135 140
Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu
145 150 155 160
Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys
165 170 175
Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu
180 185 190
Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly
195 200 205
Lys
<210> 101
<211> 209
<212> PRT
<213> artificial sequence
<220>
<223> synthetic construct
<400> 101
Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser
1 5 10 15
Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp
20 25 30
Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn
35 40 45
Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Ala Ser Thr Tyr Arg Val
50 55 60
Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu
65 70 75 80
Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys
85 90 95
Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr
100 105 110
Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr
115 120 125
Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu
130 135 140
Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu
145 150 155 160
Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys
165 170 175
Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu
180 185 190
Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly
195 200 205
Lys
<210> 102
<211> 209
<212> PRT
<213> artificial sequence
<220>
<223> synthetic construct
<400> 102
Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser
1 5 10 15
Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp
20 25 30
Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn
35 40 45
Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val
50 55 60
Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu
65 70 75 80
Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys
85 90 95
Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr
100 105 110
Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr
115 120 125
Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu
130 135 140
Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu
145 150 155 160
Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys
165 170 175
Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu
180 185 190
Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly
195 200 205
Lys
<210> 103
<211> 443
<212> PRT
<213> artificial sequence
<220>
<223> synthetic construct
<400> 103
Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg
1 5 10 15
Ser Leu Arg Leu Asp Cys Lys Ala Ser Gly Ile Thr Phe Ser Asn Ser
20 25 30
Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Val Ile Trp Tyr Asp Gly Ser Lys Arg Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Phe
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Thr Asn Asp Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser
100 105 110
Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser
115 120 125
Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp
130 135 140
Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr
145 150 155 160
Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr
165 170 175
Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln
180 185 190
Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp
195 200 205
Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro
210 215 220
Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro
225 230 235 240
Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr
245 250 255
Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn
260 265 270
Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg
275 280 285
Glu Glu Gln Tyr Ala Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val
290 295 300
Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser
305 310 315 320
Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys
325 330 335
Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp
340 345 350
Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe
355 360 365
Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu
370 375 380
Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe
385 390 395 400
Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly
405 410 415
Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr
420 425 430
Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
435 440
<210> 104
<211> 440
<212> PRT
<213> artificial sequence
<220>
<223> synthetic construct
<400> 104
Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg
1 5 10 15
Ser Leu Arg Leu Asp Cys Lys Ala Ser Gly Ile Thr Phe Ser Asn Ser
20 25 30
Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Val Ile Trp Tyr Asp Gly Ser Lys Arg Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Phe
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Thr Asn Asp Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser
100 105 110
Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser
115 120 125
Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp
130 135 140
Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr
145 150 155 160
Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr
165 170 175
Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys
180 185 190
Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp
195 200 205
Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala
210 215 220
Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro
225 230 235 240
Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val
245 250 255
Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val
260 265 270
Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln
275 280 285
Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln
290 295 300
Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly
305 310 315 320
Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro
325 330 335
Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr
340 345 350
Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser
355 360 365
Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr
370 375 380
Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr
385 390 395 400
Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe
405 410 415
Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys
420 425 430
Ser Leu Ser Leu Ser Leu Gly Lys
435 440
<210> 105
<211> 273
<212> PRT
<213> Homo sapiens (Homo sapiens)
<400> 105
Met Ile Phe Leu Leu Leu Met Leu Ser Leu Glu Leu Gln Leu His Gln
1 5 10 15
Ile Ala Ala Leu Phe Thr Val Thr Val Pro Lys Glu Leu Tyr Ile Ile
20 25 30
Glu His Gly Ser Asn Val Thr Leu Glu Cys Asn Phe Asp Thr Gly Ser
35 40 45
His Val Asn Leu Gly Ala Ile Thr Ala Ser Leu Gln Lys Val Glu Asn
50 55 60
Asp Thr Ser Pro His Arg Glu Arg Ala Thr Leu Leu Glu Glu Gln Leu
65 70 75 80
Pro Leu Gly Lys Ala Ser Phe His Ile Pro Gln Val Gln Val Arg Asp
85 90 95
Glu Gly Gln Tyr Gln Cys Ile Ile Ile Tyr Gly Val Ala Trp Asp Tyr
100 105 110
Lys Tyr Leu Thr Leu Lys Val Lys Ala Ser Tyr Arg Lys Ile Asn Thr
115 120 125
His Ile Leu Lys Val Pro Glu Thr Asp Glu Val Glu Leu Thr Cys Gln
130 135 140
Ala Thr Gly Tyr Pro Leu Ala Glu Val Ser Trp Pro Asn Val Ser Val
145 150 155 160
Pro Ala Asn Thr Ser His Ser Arg Thr Pro Glu Gly Leu Tyr Gln Val
165 170 175
Thr Ser Val Leu Arg Leu Lys Pro Pro Pro Gly Arg Asn Phe Ser Cys
180 185 190
Val Phe Trp Asn Thr His Val Arg Glu Leu Thr Leu Ala Ser Ile Asp
195 200 205
Leu Gln Ser Gln Met Glu Pro Arg Thr His Pro Thr Trp Leu Leu His
210 215 220
Ile Phe Ile Pro Phe Cys Ile Ile Ala Phe Ile Phe Ile Ala Thr Val
225 230 235 240
Ile Ala Leu Arg Lys Gln Leu Cys Gln Lys Leu Tyr Ser Ser Lys Asp
245 250 255
Thr Thr Lys Arg Pro Val Thr Thr Thr Lys Arg Glu Val Asn Ser Ala
260 265 270
Ile
<210> 106
<211> 201
<212> PRT
<213> Homo sapiens (Homo sapiens)
<400> 106
Leu Phe Thr Val Thr Val Pro Lys Glu Leu Tyr Ile Ile Glu His Gly
1 5 10 15
Ser Asn Val Thr Leu Glu Cys Asn Phe Asp Thr Gly Ser His Val Asn
20 25 30
Leu Gly Ala Ile Thr Ala Ser Leu Gln Lys Val Glu Asn Asp Thr Ser
35 40 45
Pro His Arg Glu Arg Ala Thr Leu Leu Glu Glu Gln Leu Pro Leu Gly
50 55 60
Lys Ala Ser Phe His Ile Pro Gln Val Gln Val Arg Asp Glu Gly Gln
65 70 75 80
Tyr Gln Cys Ile Ile Ile Tyr Gly Val Ala Trp Asp Tyr Lys Tyr Leu
85 90 95
Thr Leu Lys Val Lys Ala Ser Tyr Arg Lys Ile Asn Thr His Ile Leu
100 105 110
Lys Val Pro Glu Thr Asp Glu Val Glu Leu Thr Cys Gln Ala Thr Gly
115 120 125
Tyr Pro Leu Ala Glu Val Ser Trp Pro Asn Val Ser Val Pro Ala Asn
130 135 140
Thr Ser His Ser Arg Thr Pro Glu Gly Leu Tyr Gln Val Thr Ser Val
145 150 155 160
Leu Arg Leu Lys Pro Pro Pro Gly Arg Asn Phe Ser Cys Val Phe Trp
165 170 175
Asn Thr His Val Arg Glu Leu Thr Leu Ala Ser Ile Asp Leu Gln Ser
180 185 190
Gln Met Glu Pro Arg Thr His Pro Thr
195 200
<210> 107
<211> 201
<212> PRT
<213> Homo sapiens (Homo sapiens)
<400> 107
Leu Phe Thr Val Thr Val Pro Lys Glu Leu Tyr Ile Ile Glu His Gly
1 5 10 15
Ser Asn Val Thr Leu Glu Cys Asn Phe Asp Thr Gly Ser His Val Asn
20 25 30
Leu Gly Ala Ile Val Ala Ser Leu Gln Lys Val Glu Asn Asp Thr Ser
35 40 45
Pro His Arg Glu Arg Ala Thr Leu Leu Glu Glu Gln Leu Pro Leu Gly
50 55 60
Lys Ala Ser Phe His Ile Pro Gln Val Gln Val Arg Asp Glu Gly Gln
65 70 75 80
Tyr Gln Cys Ile Ile Ile Tyr Gly Val Ala Trp Asp Tyr Lys Tyr Leu
85 90 95
Thr Leu Lys Val Lys Ala Ser Tyr Arg Lys Ile Asn Thr His Ile Leu
100 105 110
Lys Val Pro Glu Thr Asp Glu Val Glu Leu Thr Cys Gln Ala Thr Gly
115 120 125
Tyr Pro Leu Ala Glu Val Ser Trp Pro Asn Val Ser Val Pro Ala Asn
130 135 140
Thr Ser His Ser Arg Thr Pro Glu Gly Leu Tyr Gln Val Thr Ser Val
145 150 155 160
Leu Arg Leu Lys Pro Pro Pro Gly Arg Asn Phe Ser Cys Val Phe Trp
165 170 175
Asn Thr His Val Arg Glu Leu Thr Leu Ala Ser Ile Asp Leu Gln Ser
180 185 190
Gln Met Glu Pro Arg Thr His Pro Thr
195 200
<210> 108
<211> 201
<212> PRT
<213> Homo sapiens (Homo sapiens)
<400> 108
Leu Phe Thr Val Thr Val Pro Lys Glu Leu Tyr Ile Ile Glu His Gly
1 5 10 15
Ser Asn Val Thr Leu Glu Cys Asn Phe Asp Thr Gly Ser His Val Asn
20 25 30
Leu Gly Ala Ile Thr Ala Val Leu Gln Lys Val Glu Asn Asp Thr Ser
35 40 45
Pro His Arg Glu Arg Ala Thr Leu Leu Glu Glu Gln Leu Pro Leu Gly
50 55 60
Lys Ala Ser Phe His Ile Pro Gln Val Gln Val Arg Asp Glu Gly Gln
65 70 75 80
Tyr Gln Cys Ile Ile Ile Tyr Gly Val Ala Trp Asp Tyr Lys Tyr Leu
85 90 95
Thr Leu Lys Val Lys Ala Ser Tyr Arg Lys Ile Asn Thr His Ile Leu
100 105 110
Lys Val Pro Glu Thr Asp Glu Val Glu Leu Thr Cys Gln Ala Thr Gly
115 120 125
Tyr Pro Leu Ala Glu Val Ser Trp Pro Asn Val Ser Val Pro Ala Asn
130 135 140
Thr Ser His Ser Arg Thr Pro Glu Gly Leu Tyr Gln Val Thr Ser Val
145 150 155 160
Leu Arg Leu Lys Pro Pro Pro Gly Arg Asn Phe Ser Cys Val Phe Trp
165 170 175
Asn Thr His Val Arg Glu Leu Thr Leu Ala Ser Ile Asp Leu Gln Ser
180 185 190
Gln Met Glu Pro Arg Thr His Pro Thr
195 200
<210> 109
<211> 201
<212> PRT
<213> Homo sapiens (Homo sapiens)
<400> 109
Leu Phe Thr Val Thr Val Pro Lys Glu Leu Tyr Ile Ile Glu His Gly
1 5 10 15
Ser Asn Val Thr Leu Glu Cys Asn Phe Asp Thr Gly Ser His Val Asn
20 25 30
Leu Gly Ala Ile Thr Ala Ser Leu Leu Lys Val Glu Asn Asp Thr Ser
35 40 45
Pro His Arg Glu Arg Ala Thr Leu Leu Glu Glu Gln Leu Pro Leu Gly
50 55 60
Lys Ala Ser Phe His Ile Pro Gln Val Gln Val Arg Asp Glu Gly Gln
65 70 75 80
Tyr Gln Cys Ile Ile Ile Tyr Gly Val Ala Trp Asp Tyr Lys Tyr Leu
85 90 95
Thr Leu Lys Val Lys Ala Ser Tyr Arg Lys Ile Asn Thr His Ile Leu
100 105 110
Lys Val Pro Glu Thr Asp Glu Val Glu Leu Thr Cys Gln Ala Thr Gly
115 120 125
Tyr Pro Leu Ala Glu Val Ser Trp Pro Asn Val Ser Val Pro Ala Asn
130 135 140
Thr Ser His Ser Arg Thr Pro Glu Gly Leu Tyr Gln Val Thr Ser Val
145 150 155 160
Leu Arg Leu Lys Pro Pro Pro Gly Arg Asn Phe Ser Cys Val Phe Trp
165 170 175
Asn Thr His Val Arg Glu Leu Thr Leu Ala Ser Ile Asp Leu Gln Ser
180 185 190
Gln Met Glu Pro Arg Thr His Pro Thr
195 200
<210> 110
<211> 201
<212> PRT
<213> Homo sapiens (Homo sapiens)
<400> 110
Leu Phe Thr Val Thr Val Pro Lys Glu Leu Tyr Ile Ile Glu His Gly
1 5 10 15
Ser Asn Val Thr Leu Glu Cys Asn Phe Asp Thr Gly Ser His Val Asn
20 25 30
Leu Gly Ala Ile Val Ala Val Leu Leu Lys Val Glu Asn Asp Thr Ser
35 40 45
Pro His Arg Glu Arg Ala Thr Leu Leu Glu Glu Gln Leu Pro Leu Gly
50 55 60
Lys Ala Ser Phe His Ile Pro Gln Val Gln Val Arg Asp Glu Gly Gln
65 70 75 80
Tyr Gln Cys Ile Ile Ile Tyr Gly Val Ala Trp Asp Tyr Lys Tyr Leu
85 90 95
Thr Leu Lys Val Lys Ala Ser Tyr Arg Lys Ile Asn Thr His Ile Leu
100 105 110
Lys Val Pro Glu Thr Asp Glu Val Glu Leu Thr Cys Gln Ala Thr Gly
115 120 125
Tyr Pro Leu Ala Glu Val Ser Trp Pro Asn Val Ser Val Pro Ala Asn
130 135 140
Thr Ser His Ser Arg Thr Pro Glu Gly Leu Tyr Gln Val Thr Ser Val
145 150 155 160
Leu Arg Leu Lys Pro Pro Pro Gly Arg Asn Phe Ser Cys Val Phe Trp
165 170 175
Asn Thr His Val Arg Glu Leu Thr Leu Ala Ser Ile Asp Leu Gln Ser
180 185 190
Gln Met Glu Pro Arg Thr His Pro Thr
195 200
<210> 111
<211> 428
<212> PRT
<213> Homo sapiens (Homo sapiens)
<400> 111
Leu Phe Thr Val Thr Val Pro Lys Glu Leu Tyr Ile Ile Glu His Gly
1 5 10 15
Ser Asn Val Thr Leu Glu Cys Asn Phe Asp Thr Gly Ser His Val Asn
20 25 30
Leu Gly Ala Ile Thr Ala Ser Leu Gln Lys Val Glu Asn Asp Thr Ser
35 40 45
Pro His Arg Glu Arg Ala Thr Leu Leu Glu Glu Gln Leu Pro Leu Gly
50 55 60
Lys Ala Ser Phe His Ile Pro Gln Val Gln Val Arg Asp Glu Gly Gln
65 70 75 80
Tyr Gln Cys Ile Ile Ile Tyr Gly Val Ala Trp Asp Tyr Lys Tyr Leu
85 90 95
Thr Leu Lys Val Lys Ala Ser Tyr Arg Lys Ile Asn Thr His Ile Leu
100 105 110
Lys Val Pro Glu Thr Asp Glu Val Glu Leu Thr Cys Gln Ala Thr Gly
115 120 125
Tyr Pro Leu Ala Glu Val Ser Trp Pro Asn Val Ser Val Pro Ala Asn
130 135 140
Thr Ser His Ser Arg Thr Pro Glu Gly Leu Tyr Gln Val Thr Ser Val
145 150 155 160
Leu Arg Leu Lys Pro Pro Pro Gly Arg Asn Phe Ser Cys Val Phe Trp
165 170 175
Asn Thr His Val Arg Glu Leu Thr Leu Ala Ser Ile Asp Leu Gln Ser
180 185 190
Gln Met Glu Pro Arg Thr His Pro Thr Asp Lys Thr His Thr Cys Pro
195 200 205
Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe
210 215 220
Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val
225 230 235 240
Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe
245 250 255
Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro
260 265 270
Arg Glu Glu Gln Tyr Ala Ser Thr Tyr Arg Val Val Ser Val Leu Thr
275 280 285
Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val
290 295 300
Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala
305 310 315 320
Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg
325 330 335
Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly
340 345 350
Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro
355 360 365
Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser
370 375 380
Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln
385 390 395 400
Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His
405 410 415
Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
420 425
<210> 112
<211> 428
<212> PRT
<213> Homo sapiens (Homo sapiens)
<400> 112
Leu Phe Thr Val Thr Val Pro Lys Glu Leu Tyr Ile Ile Glu His Gly
1 5 10 15
Ser Asn Val Thr Leu Glu Cys Asn Phe Asp Thr Gly Ser His Val Asn
20 25 30
Leu Gly Ala Ile Thr Ala Ser Leu Gln Lys Val Glu Asn Asp Thr Ser
35 40 45
Pro His Arg Glu Arg Ala Thr Leu Leu Glu Glu Gln Leu Pro Leu Gly
50 55 60
Lys Ala Ser Phe His Ile Pro Gln Val Gln Val Arg Asp Glu Gly Gln
65 70 75 80
Tyr Gln Cys Ile Ile Ile Tyr Gly Val Ala Trp Asp Tyr Lys Tyr Leu
85 90 95
Thr Leu Lys Val Lys Ala Ser Tyr Arg Lys Ile Asn Thr His Ile Leu
100 105 110
Lys Val Pro Glu Thr Asp Glu Val Glu Leu Thr Cys Gln Ala Thr Gly
115 120 125
Tyr Pro Leu Ala Glu Val Ser Trp Pro Asn Val Ser Val Pro Ala Asn
130 135 140
Thr Ser His Ser Arg Thr Pro Glu Gly Leu Tyr Gln Val Thr Ser Val
145 150 155 160
Leu Arg Leu Lys Pro Pro Pro Gly Arg Asn Phe Ser Cys Val Phe Trp
165 170 175
Asn Thr His Val Arg Glu Leu Thr Leu Ala Ser Ile Asp Leu Gln Ser
180 185 190
Gln Met Glu Pro Arg Thr His Pro Thr Asp Lys Thr His Thr Cys Pro
195 200 205
Pro Cys Pro Ala Pro Glu Lys Lys Gly Gly Pro Ser Val Phe Leu Phe
210 215 220
Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val
225 230 235 240
Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe
245 250 255
Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro
260 265 270
Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr
275 280 285
Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val
290 295 300
Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala
305 310 315 320
Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Val Leu Pro Pro Ser Arg
325 330 335
Glu Glu Met Thr Lys Asn Gln Val Ser Leu Leu Cys Leu Val Lys Gly
340 345 350
Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro
355 360 365
Glu Asn Arg Tyr Met Thr Trp Pro Pro Val Leu Asp Ser Asp Gly Ser
370 375 380
Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln
385 390 395 400
Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His
405 410 415
Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
420 425
<210> 113
<211> 433
<212> PRT
<213> Homo sapiens (Homo sapiens)
<400> 113
Leu Phe Thr Val Thr Val Pro Lys Glu Leu Tyr Ile Ile Glu His Gly
1 5 10 15
Ser Asn Val Thr Leu Glu Cys Asn Phe Asp Thr Gly Ser His Val Asn
20 25 30
Leu Gly Ala Ile Thr Ala Ser Leu Gln Lys Val Glu Asn Asp Thr Ser
35 40 45
Pro His Arg Glu Arg Ala Thr Leu Leu Glu Glu Gln Leu Pro Leu Gly
50 55 60
Lys Ala Ser Phe His Ile Pro Gln Val Gln Val Arg Asp Glu Gly Gln
65 70 75 80
Tyr Gln Cys Ile Ile Ile Tyr Gly Val Ala Trp Asp Tyr Lys Tyr Leu
85 90 95
Thr Leu Lys Val Lys Ala Ser Tyr Arg Lys Ile Asn Thr His Ile Leu
100 105 110
Lys Val Pro Glu Thr Asp Glu Val Glu Leu Thr Cys Gln Ala Thr Gly
115 120 125
Tyr Pro Leu Ala Glu Val Ser Trp Pro Asn Val Ser Val Pro Ala Asn
130 135 140
Thr Ser His Ser Arg Thr Pro Glu Gly Leu Tyr Gln Val Thr Ser Val
145 150 155 160
Leu Arg Leu Lys Pro Pro Pro Gly Arg Asn Phe Ser Cys Val Phe Trp
165 170 175
Asn Thr His Val Arg Glu Leu Thr Leu Ala Ser Ile Asp Leu Gln Ser
180 185 190
Gln Met Glu Pro Arg Thr His Pro Thr Gly Gly Gly Gly Ser Asp Lys
195 200 205
Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Lys Lys Gly Gly Pro
210 215 220
Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser
225 230 235 240
Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp
245 250 255
Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn
260 265 270
Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val
275 280 285
Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu
290 295 300
Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys
305 310 315 320
Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Val
325 330 335
Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Leu
340 345 350
Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu
355 360 365
Ser Asn Gly Gln Pro Glu Asn Arg Tyr Met Thr Trp Pro Pro Val Leu
370 375 380
Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys
385 390 395 400
Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu
405 410 415
Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly
420 425 430
Lys
<210> 114
<211> 435
<212> PRT
<213> Homo sapiens (Homo sapiens)
<400> 114
Leu Phe Thr Val Thr Val Pro Lys Glu Leu Tyr Ile Ile Glu His Gly
1 5 10 15
Ser Asn Val Thr Leu Glu Cys Asn Phe Asp Thr Gly Ser His Val Asn
20 25 30
Leu Gly Ala Ile Thr Ala Ser Leu Gln Lys Val Glu Asn Asp Thr Ser
35 40 45
Pro His Arg Glu Arg Ala Thr Leu Leu Glu Glu Gln Leu Pro Leu Gly
50 55 60
Lys Ala Ser Phe His Ile Pro Gln Val Gln Val Arg Asp Glu Gly Gln
65 70 75 80
Tyr Gln Cys Ile Ile Ile Tyr Gly Val Ala Trp Asp Tyr Lys Tyr Leu
85 90 95
Thr Leu Lys Val Lys Ala Ser Tyr Arg Lys Ile Asn Thr His Ile Leu
100 105 110
Lys Val Pro Glu Thr Asp Glu Val Glu Leu Thr Cys Gln Ala Thr Gly
115 120 125
Tyr Pro Leu Ala Glu Val Ser Trp Pro Asn Val Ser Val Pro Ala Asn
130 135 140
Thr Ser His Ser Arg Thr Pro Glu Gly Leu Tyr Gln Val Thr Ser Val
145 150 155 160
Leu Arg Leu Lys Pro Pro Pro Gly Arg Asn Phe Ser Cys Val Phe Trp
165 170 175
Asn Thr His Val Arg Glu Leu Thr Leu Ala Ser Ile Asp Leu Gln Ser
180 185 190
Gln Met Glu Pro Arg Thr His Pro Thr Gly Ser Gly Gly Gly Gly Gly
195 200 205
Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Lys Lys Gly
210 215 220
Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met
225 230 235 240
Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His
245 250 255
Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val
260 265 270
His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr
275 280 285
Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly
290 295 300
Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile
305 310 315 320
Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val
325 330 335
Tyr Val Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser
340 345 350
Leu Leu Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu
355 360 365
Trp Glu Ser Asn Gly Gln Pro Glu Asn Arg Tyr Met Thr Trp Pro Pro
370 375 380
Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val
385 390 395 400
Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met
405 410 415
His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser
420 425 430
Pro Gly Lys
435
<210> 115
<211> 431
<212> PRT
<213> Homo sapiens (Homo sapiens)
<400> 115
Leu Phe Thr Val Thr Val Pro Lys Glu Leu Tyr Ile Ile Glu His Gly
1 5 10 15
Ser Asn Val Thr Leu Glu Cys Asn Phe Asp Thr Gly Ser His Val Asn
20 25 30
Leu Gly Ala Ile Thr Ala Ser Leu Gln Lys Val Glu Asn Asp Thr Ser
35 40 45
Pro His Arg Glu Arg Ala Thr Leu Leu Glu Glu Gln Leu Pro Leu Gly
50 55 60
Lys Ala Ser Phe His Ile Pro Gln Val Gln Val Arg Asp Glu Gly Gln
65 70 75 80
Tyr Gln Cys Ile Ile Ile Tyr Gly Val Ala Trp Asp Tyr Lys Tyr Leu
85 90 95
Thr Leu Lys Val Lys Ala Ser Tyr Arg Lys Ile Asn Thr His Ile Leu
100 105 110
Lys Val Pro Glu Thr Asp Glu Val Glu Leu Thr Cys Gln Ala Thr Gly
115 120 125
Tyr Pro Leu Ala Glu Val Ser Trp Pro Asn Val Ser Val Pro Ala Asn
130 135 140
Thr Ser His Ser Arg Thr Pro Glu Gly Leu Tyr Gln Val Thr Ser Val
145 150 155 160
Leu Arg Leu Lys Pro Pro Pro Gly Arg Asn Phe Ser Cys Val Phe Trp
165 170 175
Asn Thr His Val Arg Glu Leu Thr Leu Ala Ser Ile Asp Leu Gln Ser
180 185 190
Gln Met Glu Pro Arg Thr His Pro Thr Gly Ser Gly Asp Lys Thr His
195 200 205
Thr Cys Pro Pro Cys Pro Ala Pro Glu Lys Lys Gly Gly Pro Ser Val
210 215 220
Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr
225 230 235 240
Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu
245 250 255
Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys
260 265 270
Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser
275 280 285
Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys
290 295 300
Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile
305 310 315 320
Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Val Leu Pro
325 330 335
Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Leu Cys Leu
340 345 350
Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn
355 360 365
Gly Gln Pro Glu Asn Arg Tyr Met Thr Trp Pro Pro Val Leu Asp Ser
370 375 380
Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg
385 390 395 400
Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu
405 410 415
His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
420 425 430
<210> 116
<211> 436
<212> PRT
<213> Homo sapiens (Homo sapiens)
<400> 116
Leu Phe Thr Val Thr Val Pro Lys Glu Leu Tyr Ile Ile Glu His Gly
1 5 10 15
Ser Asn Val Thr Leu Glu Cys Asn Phe Asp Thr Gly Ser His Val Asn
20 25 30
Leu Gly Ala Ile Thr Ala Val Leu Gln Lys Val Glu Asn Asp Thr Ser
35 40 45
Pro His Arg Glu Arg Ala Thr Leu Leu Glu Glu Gln Leu Pro Leu Gly
50 55 60
Lys Ala Ser Phe His Ile Pro Gln Val Gln Val Arg Asp Glu Gly Gln
65 70 75 80
Tyr Gln Cys Ile Ile Ile Tyr Gly Val Ala Trp Asp Tyr Lys Tyr Leu
85 90 95
Thr Leu Lys Val Lys Ala Ser Tyr Arg Lys Ile Asn Thr His Ile Leu
100 105 110
Lys Val Pro Glu Thr Asp Glu Val Glu Leu Thr Cys Gln Ala Thr Gly
115 120 125
Tyr Pro Leu Ala Glu Val Ser Trp Pro Asn Val Ser Val Pro Ala Asn
130 135 140
Thr Ser His Ser Arg Thr Pro Glu Gly Leu Tyr Gln Val Thr Ser Val
145 150 155 160
Leu Arg Leu Lys Pro Pro Pro Gly Arg Asn Phe Ser Cys Val Phe Trp
165 170 175
Asn Thr His Val Arg Glu Leu Thr Leu Ala Ser Ile Asp Leu Gln Ser
180 185 190
Gln Met Glu Pro Arg Thr His Pro Thr Gly Gly Gly Gly Ser Gly Gly
195 200 205
Gly Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Asp Glu
210 215 220
Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu
225 230 235 240
Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser
245 250 255
His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu
260 265 270
Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr
275 280 285
Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn
290 295 300
Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro
305 310 315 320
Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln
325 330 335
Val Tyr Val Tyr Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val
340 345 350
Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val
355 360 365
Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro
370 375 380
Pro Val Leu Asp Glu Asp Gly Ser Phe Ala Leu Val Ser Lys Leu Thr
385 390 395 400
Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val
405 410 415
Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu
420 425 430
Ser Pro Gly Lys
435
<210> 117
<211> 436
<212> PRT
<213> Homo sapiens (Homo sapiens)
<400> 117
Leu Phe Thr Val Thr Val Pro Lys Glu Leu Tyr Ile Ile Glu His Gly
1 5 10 15
Ser Asn Val Thr Leu Glu Cys Asn Phe Asp Thr Gly Ser His Val Asn
20 25 30
Leu Gly Ala Ile Val Ala Val Leu Leu Lys Val Glu Asn Asp Thr Ser
35 40 45
Pro His Arg Glu Arg Ala Thr Leu Leu Glu Glu Gln Leu Pro Leu Gly
50 55 60
Lys Ala Ser Phe His Ile Pro Gln Val Gln Val Arg Asp Glu Gly Gln
65 70 75 80
Tyr Gln Cys Ile Ile Ile Tyr Gly Val Ala Trp Asp Tyr Lys Tyr Leu
85 90 95
Thr Leu Lys Val Lys Ala Ser Tyr Arg Lys Ile Asn Thr His Ile Leu
100 105 110
Lys Val Pro Glu Thr Asp Glu Val Glu Leu Thr Cys Gln Ala Thr Gly
115 120 125
Tyr Pro Leu Ala Glu Val Ser Trp Pro Asn Val Ser Val Pro Ala Asn
130 135 140
Thr Ser His Ser Arg Thr Pro Glu Gly Leu Tyr Gln Val Thr Ser Val
145 150 155 160
Leu Arg Leu Lys Pro Pro Pro Gly Arg Asn Phe Ser Cys Val Phe Trp
165 170 175
Asn Thr His Val Arg Glu Leu Thr Leu Ala Ser Ile Asp Leu Gln Ser
180 185 190
Gln Met Glu Pro Arg Thr His Pro Thr Gly Gly Gly Gly Ser Gly Gly
195 200 205
Gly Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Asp Glu
210 215 220
Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu
225 230 235 240
Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser
245 250 255
His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu
260 265 270
Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr
275 280 285
Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn
290 295 300
Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro
305 310 315 320
Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln
325 330 335
Val Tyr Val Tyr Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val
340 345 350
Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val
355 360 365
Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro
370 375 380
Pro Val Leu Asp Glu Asp Gly Ser Phe Ala Leu Val Ser Lys Leu Thr
385 390 395 400
Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val
405 410 415
Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu
420 425 430
Ser Pro Gly Lys
435
<210> 118
<211> 436
<212> PRT
<213> Homo sapiens (Homo sapiens)
<400> 118
Leu Phe Thr Val Thr Val Pro Lys Glu Leu Tyr Ile Ile Glu His Gly
1 5 10 15
Ser Asn Val Thr Leu Glu Cys Asn Phe Asp Thr Gly Ser His Val Asn
20 25 30
Leu Gly Ala Ile Thr Ala Val Leu Gln Lys Val Glu Asn Asp Thr Ser
35 40 45
Pro His Arg Glu Arg Ala Thr Leu Leu Glu Glu Gln Leu Pro Leu Gly
50 55 60
Lys Ala Ser Phe His Ile Pro Gln Val Gln Val Arg Asp Glu Gly Gln
65 70 75 80
Tyr Gln Cys Ile Ile Ile Tyr Gly Val Ala Trp Asp Tyr Lys Tyr Leu
85 90 95
Thr Leu Lys Val Lys Ala Ser Tyr Arg Lys Ile Asn Thr His Ile Leu
100 105 110
Lys Val Pro Glu Thr Asp Glu Val Glu Leu Thr Cys Gln Ala Thr Gly
115 120 125
Tyr Pro Leu Ala Glu Val Ser Trp Pro Asn Val Ser Val Pro Ala Asn
130 135 140
Thr Ser His Ser Arg Thr Pro Glu Gly Leu Tyr Gln Val Thr Ser Val
145 150 155 160
Leu Arg Leu Lys Pro Pro Pro Gly Arg Asn Phe Ser Cys Val Phe Trp
165 170 175
Asn Thr His Val Arg Glu Leu Thr Leu Ala Ser Ile Asp Leu Gln Ser
180 185 190
Gln Met Glu Pro Arg Thr His Pro Thr Gly Gly Gly Gly Ser Gly Gly
195 200 205
Gly Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Lys Lys
210 215 220
Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu
225 230 235 240
Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser
245 250 255
His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu
260 265 270
Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr
275 280 285
Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn
290 295 300
Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro
305 310 315 320
Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln
325 330 335
Val Tyr Val Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val
340 345 350
Ser Leu Leu Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val
355 360 365
Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Arg Tyr Met Thr Trp Pro
370 375 380
Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr
385 390 395 400
Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val
405 410 415
Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu
420 425 430
Ser Pro Gly Lys
435
<210> 119
<211> 436
<212> PRT
<213> Homo sapiens (Homo sapiens)
<400> 119
Leu Phe Thr Val Thr Val Pro Lys Glu Leu Tyr Ile Ile Glu His Gly
1 5 10 15
Ser Asn Val Thr Leu Glu Cys Asn Phe Asp Thr Gly Ser His Val Asn
20 25 30
Leu Gly Ala Ile Val Ala Val Leu Leu Lys Val Glu Asn Asp Thr Ser
35 40 45
Pro His Arg Glu Arg Ala Thr Leu Leu Glu Glu Gln Leu Pro Leu Gly
50 55 60
Lys Ala Ser Phe His Ile Pro Gln Val Gln Val Arg Asp Glu Gly Gln
65 70 75 80
Tyr Gln Cys Ile Ile Ile Tyr Gly Val Ala Trp Asp Tyr Lys Tyr Leu
85 90 95
Thr Leu Lys Val Lys Ala Ser Tyr Arg Lys Ile Asn Thr His Ile Leu
100 105 110
Lys Val Pro Glu Thr Asp Glu Val Glu Leu Thr Cys Gln Ala Thr Gly
115 120 125
Tyr Pro Leu Ala Glu Val Ser Trp Pro Asn Val Ser Val Pro Ala Asn
130 135 140
Thr Ser His Ser Arg Thr Pro Glu Gly Leu Tyr Gln Val Thr Ser Val
145 150 155 160
Leu Arg Leu Lys Pro Pro Pro Gly Arg Asn Phe Ser Cys Val Phe Trp
165 170 175
Asn Thr His Val Arg Glu Leu Thr Leu Ala Ser Ile Asp Leu Gln Ser
180 185 190
Gln Met Glu Pro Arg Thr His Pro Thr Gly Gly Gly Gly Ser Gly Gly
195 200 205
Gly Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Lys Lys
210 215 220
Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu
225 230 235 240
Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser
245 250 255
His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu
260 265 270
Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr
275 280 285
Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn
290 295 300
Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro
305 310 315 320
Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln
325 330 335
Val Tyr Val Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val
340 345 350
Ser Leu Leu Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val
355 360 365
Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Arg Tyr Met Thr Trp Pro
370 375 380
Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr
385 390 395 400
Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val
405 410 415
Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu
420 425 430
Ser Pro Gly Lys
435
<210> 120
<211> 290
<212> PRT
<213> Homo sapiens (Homo sapiens)
<400> 120
Met Arg Ile Phe Ala Val Phe Ile Phe Met Thr Tyr Trp His Leu Leu
1 5 10 15
Asn Ala Phe Thr Val Thr Val Pro Lys Asp Leu Tyr Val Val Glu Tyr
20 25 30
Gly Ser Asn Met Thr Ile Glu Cys Lys Phe Pro Val Glu Lys Gln Leu
35 40 45
Asp Leu Ala Ala Leu Ile Val Tyr Trp Glu Met Glu Asp Lys Asn Ile
50 55 60
Ile Gln Phe Val His Gly Glu Glu Asp Leu Lys Val Gln His Ser Ser
65 70 75 80
Tyr Arg Gln Arg Ala Arg Leu Leu Lys Asp Gln Leu Ser Leu Gly Asn
85 90 95
Ala Ala Leu Gln Ile Thr Asp Val Lys Leu Gln Asp Ala Gly Val Tyr
100 105 110
Arg Cys Met Ile Ser Tyr Gly Gly Ala Asp Tyr Lys Arg Ile Thr Val
115 120 125
Lys Val Asn Ala Pro Tyr Asn Lys Ile Asn Gln Arg Ile Leu Val Val
130 135 140
Asp Pro Val Thr Ser Glu His Glu Leu Thr Cys Gln Ala Glu Gly Tyr
145 150 155 160
Pro Lys Ala Glu Val Ile Trp Thr Ser Ser Asp His Gln Val Leu Ser
165 170 175
Gly Lys Thr Thr Thr Thr Asn Ser Lys Arg Glu Glu Lys Leu Phe Asn
180 185 190
Val Thr Ser Thr Leu Arg Ile Asn Thr Thr Thr Asn Glu Ile Phe Tyr
195 200 205
Cys Thr Phe Arg Arg Leu Asp Pro Glu Glu Asn His Thr Ala Glu Leu
210 215 220
Val Ile Pro Glu Leu Pro Leu Ala His Pro Pro Asn Glu Arg Thr His
225 230 235 240
Leu Val Ile Leu Gly Ala Ile Leu Leu Cys Leu Gly Val Ala Leu Thr
245 250 255
Phe Ile Phe Arg Leu Arg Lys Gly Arg Met Met Asp Val Lys Lys Cys
260 265 270
Gly Ile Gln Asp Thr Asn Ser Lys Lys Gln Ser Asp Thr His Leu Glu
275 280 285
Glu Thr
290
<210> 121
<211> 220
<212> PRT
<213> Homo sapiens (Homo sapiens)
<400> 121
Phe Thr Val Thr Val Pro Lys Asp Leu Tyr Val Val Glu Tyr Gly Ser
1 5 10 15
Asn Met Thr Ile Glu Cys Lys Phe Pro Val Glu Lys Gln Leu Asp Leu
20 25 30
Ala Ala Leu Ile Val Tyr Trp Glu Met Glu Asp Lys Asn Ile Ile Gln
35 40 45
Phe Val His Gly Glu Glu Asp Leu Lys Val Gln His Ser Ser Tyr Arg
50 55 60
Gln Arg Ala Arg Leu Leu Lys Asp Gln Leu Ser Leu Gly Asn Ala Ala
65 70 75 80
Leu Gln Ile Thr Asp Val Lys Leu Gln Asp Ala Gly Val Tyr Arg Cys
85 90 95
Met Ile Ser Tyr Gly Gly Ala Asp Tyr Lys Arg Ile Thr Val Lys Val
100 105 110
Asn Ala Pro Tyr Asn Lys Ile Asn Gln Arg Ile Leu Val Val Asp Pro
115 120 125
Val Thr Ser Glu His Glu Leu Thr Cys Gln Ala Glu Gly Tyr Pro Lys
130 135 140
Ala Glu Val Ile Trp Thr Ser Ser Asp His Gln Val Leu Ser Gly Lys
145 150 155 160
Thr Thr Thr Thr Asn Ser Lys Arg Glu Glu Lys Leu Phe Asn Val Thr
165 170 175
Ser Thr Leu Arg Ile Asn Thr Thr Thr Asn Glu Ile Phe Tyr Cys Thr
180 185 190
Phe Arg Arg Leu Asp Pro Glu Glu Asn His Thr Ala Glu Leu Val Ile
195 200 205
Pro Glu Leu Pro Leu Ala His Pro Pro Asn Glu Arg
210 215 220
<210> 122
<211> 220
<212> PRT
<213> Homo sapiens (Homo sapiens)
<400> 122
Phe Thr Val Thr Val Pro Lys Asp Leu Tyr Val Val Glu Tyr Gly Ser
1 5 10 15
Asn Met Thr Ile Glu Cys Lys Phe Pro Val Glu Lys Gln Leu Asp Leu
20 25 30
Ala Ala Leu Ile Val Tyr Trp Met Met Glu Asp Lys Asn Ile Ile Gln
35 40 45
Phe Val His Gly Glu Glu Asp Leu Lys Val Gln His Ser Ser Tyr Arg
50 55 60
Gln Arg Ala Arg Leu Leu Lys Asp Gln Leu Ser Leu Gly Asn Ala Ala
65 70 75 80
Leu Gln Ile Thr Asp Val Lys Leu Gln Asp Ala Gly Val Tyr Thr Cys
85 90 95
Leu Ile Ala Tyr Lys Gly Ala Asp Tyr Lys Arg Ile Thr Val Lys Val
100 105 110
Asn Ala Pro Tyr Asn Lys Ile Asn Gln Arg Ile Leu Val Val Asp Pro
115 120 125
Val Thr Ser Glu His Glu Leu Thr Cys Gln Ala Glu Gly Tyr Pro Lys
130 135 140
Ala Glu Val Ile Trp Thr Ser Ser Asp His Gln Val Leu Ser Gly Lys
145 150 155 160
Thr Thr Thr Thr Asn Ser Lys Arg Glu Glu Lys Leu Phe Asn Val Thr
165 170 175
Ser Thr Leu Arg Ile Asn Thr Thr Thr Asn Glu Ile Phe Tyr Cys Thr
180 185 190
Phe Arg Arg Leu Asp Pro Glu Glu Asn His Thr Ala Glu Leu Val Ile
195 200 205
Pro Glu Leu Pro Leu Ala His Pro Pro Asn Glu Arg
210 215 220
<210> 123
<211> 220
<212> PRT
<213> Homo sapiens (Homo sapiens)
<400> 123
Phe Thr Val Thr Val Pro Lys Asp Leu Tyr Val Val Glu Tyr Gly Ser
1 5 10 15
Asn Met Thr Ile Glu Cys Lys Phe Pro Val Glu Lys Gln Leu Asp Leu
20 25 30
Ala Ala Leu Gln Val Tyr Trp Met Met Glu Asp Lys Asn Ile Ile Gln
35 40 45
Phe Val His Gly Glu Glu Asp Leu Lys Val Gln His Ser Ser Tyr Arg
50 55 60
Gln Arg Ala Arg Leu Leu Lys Asp Gln Leu Ser Leu Gly Asn Ala Ala
65 70 75 80
Leu Gln Ile Thr Asp Val Lys Leu Gln Asp Ala Gly Val Tyr Thr Cys
85 90 95
Leu Ile Ala Tyr Lys Gly Ala Asp Tyr Lys Arg Ile Thr Val Lys Val
100 105 110
Asn Ala Pro Tyr Asn Lys Ile Asn Gln Arg Ile Leu Val Val Asp Pro
115 120 125
Val Thr Ser Glu His Glu Leu Thr Cys Gln Ala Glu Gly Tyr Pro Lys
130 135 140
Ala Glu Val Ile Trp Thr Ser Ser Asp His Gln Val Leu Ser Gly Lys
145 150 155 160
Thr Thr Thr Thr Asn Ser Lys Arg Glu Glu Lys Leu Phe Asn Val Thr
165 170 175
Ser Thr Leu Arg Ile Asn Thr Thr Thr Asn Glu Ile Phe Tyr Cys Thr
180 185 190
Phe Arg Arg Leu Asp Pro Glu Glu Asn His Thr Ala Glu Leu Val Ile
195 200 205
Pro Glu Leu Pro Leu Ala His Pro Pro Asn Glu Arg
210 215 220
<210> 124
<211> 220
<212> PRT
<213> Homo sapiens (Homo sapiens)
<400> 124
Phe Thr Val Thr Val Pro Lys Asp Leu Tyr Val Val Glu Tyr Gly Ser
1 5 10 15
Asn Met Thr Ile Glu Cys Lys Phe Pro Val Glu Lys Gln Leu Asp Leu
20 25 30
Ala Ala Leu Gln Val Tyr Trp Glu Met Glu Asp Lys Asn Ile Ile Gln
35 40 45
Phe Val His Gly Glu Glu Asp Leu Lys Val Gln His Ser Ser Tyr Arg
50 55 60
Gln Arg Ala Arg Leu Leu Lys Asp Gln Leu Ser Leu Gly Asn Ala Ala
65 70 75 80
Leu Gln Ile Thr Asp Val Lys Leu Gln Asp Ala Gly Val Tyr Thr Cys
85 90 95
Leu Ile Ala Tyr Lys Gly Ala Asp Tyr Lys Arg Ile Thr Val Lys Val
100 105 110
Asn Ala Pro Tyr Asn Lys Ile Asn Gln Arg Ile Leu Val Val Asp Pro
115 120 125
Val Thr Ser Glu His Glu Leu Thr Cys Gln Ala Glu Gly Tyr Pro Lys
130 135 140
Ala Glu Val Ile Trp Thr Ser Ser Asp His Gln Val Leu Ser Gly Lys
145 150 155 160
Thr Thr Thr Thr Asn Ser Lys Arg Glu Glu Lys Leu Phe Asn Val Thr
165 170 175
Ser Thr Leu Arg Ile Asn Thr Thr Thr Asn Glu Ile Phe Tyr Cys Thr
180 185 190
Phe Arg Arg Leu Asp Pro Glu Glu Asn His Thr Ala Glu Leu Val Ile
195 200 205
Pro Glu Leu Pro Leu Ala His Pro Pro Asn Glu Arg
210 215 220
<210> 125
<211> 220
<212> PRT
<213> Homo sapiens (Homo sapiens)
<400> 125
Phe Thr Val Thr Val Pro Lys Asp Leu Tyr Val Val Glu Tyr Gly Ser
1 5 10 15
Asn Met Thr Ile Glu Cys Lys Phe Pro Val Glu Lys Gln Leu Asp Leu
20 25 30
Ala Ala Leu Gln Val Tyr Trp Met Met Glu Asp Lys Asn Ile Ile Gln
35 40 45
Phe Val His Gly Glu Glu Asp Leu Lys Val Gln His Ser Ser Tyr Arg
50 55 60
Gln Arg Ala Arg Leu Leu Lys Asp Gln Leu Ser Leu Gly Asn Ala Ala
65 70 75 80
Leu Gln Ile Thr Asp Val Lys Leu Gln Asp Ala Gly Val Tyr Arg Cys
85 90 95
Leu Ile Ala Tyr Lys Gly Ala Asp Tyr Lys Arg Ile Thr Val Lys Val
100 105 110
Asn Ala Pro Tyr Asn Lys Ile Asn Gln Arg Ile Leu Val Val Asp Pro
115 120 125
Val Thr Ser Glu His Glu Leu Thr Cys Gln Ala Glu Gly Tyr Pro Lys
130 135 140
Ala Glu Val Ile Trp Thr Ser Ser Asp His Gln Val Leu Ser Gly Lys
145 150 155 160
Thr Thr Thr Thr Asn Ser Lys Arg Glu Glu Lys Leu Phe Asn Val Thr
165 170 175
Ser Thr Leu Arg Ile Asn Thr Thr Thr Asn Glu Ile Phe Tyr Cys Thr
180 185 190
Phe Arg Arg Leu Asp Pro Glu Glu Asn His Thr Ala Glu Leu Val Ile
195 200 205
Pro Glu Leu Pro Leu Ala His Pro Pro Asn Glu Arg
210 215 220
<210> 126
<211> 220
<212> PRT
<213> Homo sapiens (Homo sapiens)
<400> 126
Phe Thr Val Thr Val Pro Lys Asp Leu Tyr Val Val Glu Tyr Gly Ser
1 5 10 15
Asn Met Thr Ile Glu Cys Lys Phe Pro Val Glu Lys Gln Leu Asp Leu
20 25 30
Ala Ala Leu Gln Val Tyr Trp Met Met Glu Asp Lys Asn Ile Ile Gln
35 40 45
Phe Val His Gly Glu Glu Asp Leu Lys Val Gln His Ser Ser Tyr Arg
50 55 60
Gln Arg Ala Arg Leu Leu Lys Asp Gln Leu Ser Leu Gly Asn Ala Ala
65 70 75 80
Leu Gln Ile Thr Asp Val Lys Leu Gln Asp Ala Gly Val Tyr Thr Cys
85 90 95
Met Ile Ala Tyr Lys Gly Ala Asp Tyr Lys Arg Ile Thr Val Lys Val
100 105 110
Asn Ala Pro Tyr Asn Lys Ile Asn Gln Arg Ile Leu Val Val Asp Pro
115 120 125
Val Thr Ser Glu His Glu Leu Thr Cys Gln Ala Glu Gly Tyr Pro Lys
130 135 140
Ala Glu Val Ile Trp Thr Ser Ser Asp His Gln Val Leu Ser Gly Lys
145 150 155 160
Thr Thr Thr Thr Asn Ser Lys Arg Glu Glu Lys Leu Phe Asn Val Thr
165 170 175
Ser Thr Leu Arg Ile Asn Thr Thr Thr Asn Glu Ile Phe Tyr Cys Thr
180 185 190
Phe Arg Arg Leu Asp Pro Glu Glu Asn His Thr Ala Glu Leu Val Ile
195 200 205
Pro Glu Leu Pro Leu Ala His Pro Pro Asn Glu Arg
210 215 220
<210> 127
<211> 220
<212> PRT
<213> Homo sapiens (Homo sapiens)
<400> 127
Phe Thr Val Thr Val Pro Lys Asp Leu Tyr Val Val Glu Tyr Gly Ser
1 5 10 15
Asn Met Thr Ile Glu Cys Lys Phe Pro Val Glu Lys Gln Leu Asp Leu
20 25 30
Ala Ala Leu Gln Val Tyr Trp Met Met Glu Asp Lys Asn Ile Ile Gln
35 40 45
Phe Val His Gly Glu Glu Asp Leu Lys Val Gln His Ser Ser Tyr Arg
50 55 60
Gln Arg Ala Arg Leu Leu Lys Asp Gln Leu Ser Leu Gly Asn Ala Ala
65 70 75 80
Leu Gln Ile Thr Asp Val Lys Leu Gln Asp Ala Gly Val Tyr Thr Cys
85 90 95
Leu Ile Ser Tyr Lys Gly Ala Asp Tyr Lys Arg Ile Thr Val Lys Val
100 105 110
Asn Ala Pro Tyr Asn Lys Ile Asn Gln Arg Ile Leu Val Val Asp Pro
115 120 125
Val Thr Ser Glu His Glu Leu Thr Cys Gln Ala Glu Gly Tyr Pro Lys
130 135 140
Ala Glu Val Ile Trp Thr Ser Ser Asp His Gln Val Leu Ser Gly Lys
145 150 155 160
Thr Thr Thr Thr Asn Ser Lys Arg Glu Glu Lys Leu Phe Asn Val Thr
165 170 175
Ser Thr Leu Arg Ile Asn Thr Thr Thr Asn Glu Ile Phe Tyr Cys Thr
180 185 190
Phe Arg Arg Leu Asp Pro Glu Glu Asn His Thr Ala Glu Leu Val Ile
195 200 205
Pro Glu Leu Pro Leu Ala His Pro Pro Asn Glu Arg
210 215 220
<210> 128
<211> 220
<212> PRT
<213> Homo sapiens (Homo sapiens)
<400> 128
Phe Thr Val Thr Val Pro Lys Asp Leu Tyr Val Val Glu Tyr Gly Ser
1 5 10 15
Asn Met Thr Ile Glu Cys Lys Phe Pro Val Glu Lys Gln Leu Asp Leu
20 25 30
Ala Ala Leu Gln Val Tyr Trp Met Met Glu Asp Lys Asn Ile Ile Gln
35 40 45
Phe Val His Gly Glu Glu Asp Leu Lys Val Gln His Ser Ser Tyr Arg
50 55 60
Gln Arg Ala Arg Leu Leu Lys Asp Gln Leu Ser Leu Gly Asn Ala Ala
65 70 75 80
Leu Gln Ile Thr Asp Val Lys Leu Gln Asp Ala Gly Val Tyr Thr Cys
85 90 95
Leu Ile Ala Tyr Gly Gly Ala Asp Tyr Lys Arg Ile Thr Val Lys Val
100 105 110
Asn Ala Pro Tyr Asn Lys Ile Asn Gln Arg Ile Leu Val Val Asp Pro
115 120 125
Val Thr Ser Glu His Glu Leu Thr Cys Gln Ala Glu Gly Tyr Pro Lys
130 135 140
Ala Glu Val Ile Trp Thr Ser Ser Asp His Gln Val Leu Ser Gly Lys
145 150 155 160
Thr Thr Thr Thr Asn Ser Lys Arg Glu Glu Lys Leu Phe Asn Val Thr
165 170 175
Ser Thr Leu Arg Ile Asn Thr Thr Thr Asn Glu Ile Phe Tyr Cys Thr
180 185 190
Phe Arg Arg Leu Asp Pro Glu Glu Asn His Thr Ala Glu Leu Val Ile
195 200 205
Pro Glu Leu Pro Leu Ala His Pro Pro Asn Glu Arg
210 215 220
<210> 129
<211> 220
<212> PRT
<213> Homo sapiens (Homo sapiens)
<400> 129
Phe Thr Val Thr Val Pro Lys Asp Leu Tyr Val Val Glu Tyr Gly Ser
1 5 10 15
Asn Met Thr Ile Glu Cys Lys Phe Pro Val Glu Lys Gln Leu Asp Leu
20 25 30
Ala Ala Leu Gln Val Phe Trp Met Met Glu Asp Lys Asn Ile Ile Gln
35 40 45
Phe Val His Gly Glu Glu Asp Leu Lys Val Gln His Ser Ser Tyr Arg
50 55 60
Gln Arg Ala Arg Leu Leu Lys Asp Gln Leu Ser Leu Gly Asn Ala Ala
65 70 75 80
Leu Gln Ile Thr Asp Val Lys Leu Gln Asp Ala Gly Val Tyr Thr Cys
85 90 95
Leu Ile Ala Tyr Lys Gly Ala Asp Tyr Lys Arg Ile Thr Val Lys Val
100 105 110
Asn Ala Pro Tyr Asn Lys Ile Asn Gln Arg Ile Leu Val Val Asp Pro
115 120 125
Val Thr Ser Glu His Glu Leu Thr Cys Gln Ala Glu Gly Tyr Pro Lys
130 135 140
Ala Glu Val Ile Trp Thr Ser Ser Asp His Gln Val Leu Ser Gly Lys
145 150 155 160
Thr Thr Thr Thr Asn Ser Lys Arg Glu Glu Lys Leu Phe Asn Val Thr
165 170 175
Ser Thr Leu Arg Ile Asn Thr Thr Thr Asn Glu Ile Phe Tyr Cys Thr
180 185 190
Phe Arg Arg Leu Asp Pro Glu Glu Asn His Thr Ala Glu Leu Val Ile
195 200 205
Pro Glu Leu Pro Leu Ala His Pro Pro Asn Glu Arg
210 215 220
<210> 130
<211> 455
<212> PRT
<213> Homo sapiens (Homo sapiens)
<400> 130
Phe Thr Val Thr Val Pro Lys Asp Leu Tyr Val Val Glu Tyr Gly Ser
1 5 10 15
Asn Met Thr Ile Glu Cys Lys Phe Pro Val Glu Lys Gln Leu Asp Leu
20 25 30
Ala Ala Leu Ile Val Tyr Trp Glu Met Glu Asp Lys Asn Ile Ile Gln
35 40 45
Phe Val His Gly Glu Glu Asp Leu Lys Val Gln His Ser Ser Tyr Arg
50 55 60
Gln Arg Ala Arg Leu Leu Lys Asp Gln Leu Ser Leu Gly Asn Ala Ala
65 70 75 80
Leu Gln Ile Thr Asp Val Lys Leu Gln Asp Ala Gly Val Tyr Arg Cys
85 90 95
Met Ile Ser Tyr Gly Gly Ala Asp Tyr Lys Arg Ile Thr Val Lys Val
100 105 110
Asn Ala Pro Tyr Asn Lys Ile Asn Gln Arg Ile Leu Val Val Asp Pro
115 120 125
Val Thr Ser Glu His Glu Leu Thr Cys Gln Ala Glu Gly Tyr Pro Lys
130 135 140
Ala Glu Val Ile Trp Thr Ser Ser Asp His Gln Val Leu Ser Gly Lys
145 150 155 160
Thr Thr Thr Thr Asn Ser Lys Arg Glu Glu Lys Leu Phe Asn Val Thr
165 170 175
Ser Thr Leu Arg Ile Asn Thr Thr Thr Asn Glu Ile Phe Tyr Cys Thr
180 185 190
Phe Arg Arg Leu Asp Pro Glu Glu Asn His Thr Ala Glu Leu Val Ile
195 200 205
Pro Glu Leu Pro Leu Ala His Pro Pro Asn Glu Arg Gly Gly Gly Gly
210 215 220
Ser Gly Gly Gly Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro
225 230 235 240
Glu Asp Glu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys
245 250 255
Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val
260 265 270
Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp
275 280 285
Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr
290 295 300
Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp
305 310 315 320
Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu
325 330 335
Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg
340 345 350
Glu Pro Gln Val Tyr Val Tyr Pro Pro Ser Arg Glu Glu Met Thr Lys
355 360 365
Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp
370 375 380
Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
385 390 395 400
Thr Thr Pro Pro Val Leu Asp Glu Asp Gly Ser Phe Ala Leu Val Ser
405 410 415
Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser
420 425 430
Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser
435 440 445
Leu Ser Leu Ser Pro Gly Lys
450 455
<210> 131
<211> 455
<212> PRT
<213> Homo sapiens (Homo sapiens)
<400> 131
Phe Thr Val Thr Val Pro Lys Asp Leu Tyr Val Val Glu Tyr Gly Ser
1 5 10 15
Asn Met Thr Ile Glu Cys Lys Phe Pro Val Glu Lys Gln Leu Asp Leu
20 25 30
Ala Ala Leu Ile Val Tyr Trp Glu Met Glu Asp Lys Asn Ile Ile Gln
35 40 45
Phe Val His Gly Glu Glu Asp Leu Lys Val Gln His Ser Ser Tyr Arg
50 55 60
Gln Arg Ala Arg Leu Leu Lys Asp Gln Leu Ser Leu Gly Asn Ala Ala
65 70 75 80
Leu Gln Ile Thr Asp Val Lys Leu Gln Asp Ala Gly Val Tyr Arg Cys
85 90 95
Met Ile Ser Tyr Gly Gly Ala Asp Tyr Lys Arg Ile Thr Val Lys Val
100 105 110
Asn Ala Pro Tyr Asn Lys Ile Asn Gln Arg Ile Leu Val Val Asp Pro
115 120 125
Val Thr Ser Glu His Glu Leu Thr Cys Gln Ala Glu Gly Tyr Pro Lys
130 135 140
Ala Glu Val Ile Trp Thr Ser Ser Asp His Gln Val Leu Ser Gly Lys
145 150 155 160
Thr Thr Thr Thr Asn Ser Lys Arg Glu Glu Lys Leu Phe Asn Val Thr
165 170 175
Ser Thr Leu Arg Ile Asn Thr Thr Thr Asn Glu Ile Phe Tyr Cys Thr
180 185 190
Phe Arg Arg Leu Asp Pro Glu Glu Asn His Thr Ala Glu Leu Val Ile
195 200 205
Pro Glu Leu Pro Leu Ala His Pro Pro Asn Glu Arg Gly Gly Gly Gly
210 215 220
Ser Gly Gly Gly Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro
225 230 235 240
Glu Lys Lys Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys
245 250 255
Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val
260 265 270
Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp
275 280 285
Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr
290 295 300
Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp
305 310 315 320
Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu
325 330 335
Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg
340 345 350
Glu Pro Gln Val Tyr Val Leu Pro Pro Ser Arg Glu Glu Met Thr Lys
355 360 365
Asn Gln Val Ser Leu Leu Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp
370 375 380
Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Arg Tyr Met
385 390 395 400
Thr Trp Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
405 410 415
Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser
420 425 430
Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser
435 440 445
Leu Ser Leu Ser Pro Gly Lys
450 455
<210> 132
<211> 455
<212> PRT
<213> Homo sapiens (Homo sapiens)
<400> 132
Phe Thr Val Thr Val Pro Lys Asp Leu Tyr Val Val Glu Tyr Gly Ser
1 5 10 15
Asn Met Thr Ile Glu Cys Lys Phe Pro Val Glu Lys Gln Leu Asp Leu
20 25 30
Ala Ala Leu Gln Val Tyr Trp Met Met Glu Asp Lys Asn Ile Ile Gln
35 40 45
Phe Val His Gly Glu Glu Asp Leu Lys Val Gln His Ser Ser Tyr Arg
50 55 60
Gln Arg Ala Arg Leu Leu Lys Asp Gln Leu Ser Leu Gly Asn Ala Ala
65 70 75 80
Leu Gln Ile Thr Asp Val Lys Leu Gln Asp Ala Gly Val Tyr Thr Cys
85 90 95
Leu Ile Ala Tyr Lys Gly Ala Asp Tyr Lys Arg Ile Thr Val Lys Val
100 105 110
Asn Ala Pro Tyr Asn Lys Ile Asn Gln Arg Ile Leu Val Val Asp Pro
115 120 125
Val Thr Ser Glu His Glu Leu Thr Cys Gln Ala Glu Gly Tyr Pro Lys
130 135 140
Ala Glu Val Ile Trp Thr Ser Ser Asp His Gln Val Leu Ser Gly Lys
145 150 155 160
Thr Thr Thr Thr Asn Ser Lys Arg Glu Glu Lys Leu Phe Asn Val Thr
165 170 175
Ser Thr Leu Arg Ile Asn Thr Thr Thr Asn Glu Ile Phe Tyr Cys Thr
180 185 190
Phe Arg Arg Leu Asp Pro Glu Glu Asn His Thr Ala Glu Leu Val Ile
195 200 205
Pro Glu Leu Pro Leu Ala His Pro Pro Asn Glu Arg Gly Gly Gly Gly
210 215 220
Ser Gly Gly Gly Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro
225 230 235 240
Glu Asp Glu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys
245 250 255
Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val
260 265 270
Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp
275 280 285
Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr
290 295 300
Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp
305 310 315 320
Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu
325 330 335
Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg
340 345 350
Glu Pro Gln Val Tyr Val Tyr Pro Pro Ser Arg Glu Glu Met Thr Lys
355 360 365
Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp
370 375 380
Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
385 390 395 400
Thr Thr Pro Pro Val Leu Asp Glu Asp Gly Ser Phe Ala Leu Val Ser
405 410 415
Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser
420 425 430
Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser
435 440 445
Leu Ser Leu Ser Pro Gly Lys
450 455
<210> 133
<211> 455
<212> PRT
<213> Homo sapiens (Homo sapiens)
<400> 133
Phe Thr Val Thr Val Pro Lys Asp Leu Tyr Val Val Glu Tyr Gly Ser
1 5 10 15
Asn Met Thr Ile Glu Cys Lys Phe Pro Val Glu Lys Gln Leu Asp Leu
20 25 30
Ala Ala Leu Gln Val Tyr Trp Met Met Glu Asp Lys Asn Ile Ile Gln
35 40 45
Phe Val His Gly Glu Glu Asp Leu Lys Val Gln His Ser Ser Tyr Arg
50 55 60
Gln Arg Ala Arg Leu Leu Lys Asp Gln Leu Ser Leu Gly Asn Ala Ala
65 70 75 80
Leu Gln Ile Thr Asp Val Lys Leu Gln Asp Ala Gly Val Tyr Thr Cys
85 90 95
Leu Ile Ala Tyr Gly Gly Ala Asp Tyr Lys Arg Ile Thr Val Lys Val
100 105 110
Asn Ala Pro Tyr Asn Lys Ile Asn Gln Arg Ile Leu Val Val Asp Pro
115 120 125
Val Thr Ser Glu His Glu Leu Thr Cys Gln Ala Glu Gly Tyr Pro Lys
130 135 140
Ala Glu Val Ile Trp Thr Ser Ser Asp His Gln Val Leu Ser Gly Lys
145 150 155 160
Thr Thr Thr Thr Asn Ser Lys Arg Glu Glu Lys Leu Phe Asn Val Thr
165 170 175
Ser Thr Leu Arg Ile Asn Thr Thr Thr Asn Glu Ile Phe Tyr Cys Thr
180 185 190
Phe Arg Arg Leu Asp Pro Glu Glu Asn His Thr Ala Glu Leu Val Ile
195 200 205
Pro Glu Leu Pro Leu Ala His Pro Pro Asn Glu Arg Gly Gly Gly Gly
210 215 220
Ser Gly Gly Gly Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro
225 230 235 240
Glu Asp Glu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys
245 250 255
Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val
260 265 270
Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp
275 280 285
Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr
290 295 300
Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp
305 310 315 320
Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu
325 330 335
Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg
340 345 350
Glu Pro Gln Val Tyr Val Tyr Pro Pro Ser Arg Glu Glu Met Thr Lys
355 360 365
Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp
370 375 380
Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
385 390 395 400
Thr Thr Pro Pro Val Leu Asp Glu Asp Gly Ser Phe Ala Leu Val Ser
405 410 415
Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser
420 425 430
Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser
435 440 445
Leu Ser Leu Ser Pro Gly Lys
450 455
<210> 134
<211> 455
<212> PRT
<213> Homo sapiens (Homo sapiens)
<400> 134
Phe Thr Val Thr Val Pro Lys Asp Leu Tyr Val Val Glu Tyr Gly Ser
1 5 10 15
Asn Met Thr Ile Glu Cys Lys Phe Pro Val Glu Lys Gln Leu Asp Leu
20 25 30
Ala Ala Leu Gln Val Tyr Trp Met Met Glu Asp Lys Asn Ile Ile Gln
35 40 45
Phe Val His Gly Glu Glu Asp Leu Lys Val Gln His Ser Ser Tyr Arg
50 55 60
Gln Arg Ala Arg Leu Leu Lys Asp Gln Leu Ser Leu Gly Asn Ala Ala
65 70 75 80
Leu Gln Ile Thr Asp Val Lys Leu Gln Asp Ala Gly Val Tyr Thr Cys
85 90 95
Leu Ile Ala Tyr Lys Gly Ala Asp Tyr Lys Arg Ile Thr Val Lys Val
100 105 110
Asn Ala Pro Tyr Asn Lys Ile Asn Gln Arg Ile Leu Val Val Asp Pro
115 120 125
Val Thr Ser Glu His Glu Leu Thr Cys Gln Ala Glu Gly Tyr Pro Lys
130 135 140
Ala Glu Val Ile Trp Thr Ser Ser Asp His Gln Val Leu Ser Gly Lys
145 150 155 160
Thr Thr Thr Thr Asn Ser Lys Arg Glu Glu Lys Leu Phe Asn Val Thr
165 170 175
Ser Thr Leu Arg Ile Asn Thr Thr Thr Asn Glu Ile Phe Tyr Cys Thr
180 185 190
Phe Arg Arg Leu Asp Pro Glu Glu Asn His Thr Ala Glu Leu Val Ile
195 200 205
Pro Glu Leu Pro Leu Ala His Pro Pro Asn Glu Arg Gly Gly Gly Gly
210 215 220
Ser Gly Gly Gly Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro
225 230 235 240
Glu Lys Lys Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys
245 250 255
Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val
260 265 270
Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp
275 280 285
Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr
290 295 300
Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp
305 310 315 320
Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu
325 330 335
Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg
340 345 350
Glu Pro Gln Val Tyr Val Leu Pro Pro Ser Arg Glu Glu Met Thr Lys
355 360 365
Asn Gln Val Ser Leu Leu Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp
370 375 380
Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Arg Tyr Met
385 390 395 400
Thr Trp Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
405 410 415
Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser
420 425 430
Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser
435 440 445
Leu Ser Leu Ser Pro Gly Lys
450 455
<210> 135
<211> 455
<212> PRT
<213> Homo sapiens (Homo sapiens)
<400> 135
Phe Thr Val Thr Val Pro Lys Asp Leu Tyr Val Val Glu Tyr Gly Ser
1 5 10 15
Asn Met Thr Ile Glu Cys Lys Phe Pro Val Glu Lys Gln Leu Asp Leu
20 25 30
Ala Ala Leu Gln Val Tyr Trp Met Met Glu Asp Lys Asn Ile Ile Gln
35 40 45
Phe Val His Gly Glu Glu Asp Leu Lys Val Gln His Ser Ser Tyr Arg
50 55 60
Gln Arg Ala Arg Leu Leu Lys Asp Gln Leu Ser Leu Gly Asn Ala Ala
65 70 75 80
Leu Gln Ile Thr Asp Val Lys Leu Gln Asp Ala Gly Val Tyr Thr Cys
85 90 95
Leu Ile Ala Tyr Gly Gly Ala Asp Tyr Lys Arg Ile Thr Val Lys Val
100 105 110
Asn Ala Pro Tyr Asn Lys Ile Asn Gln Arg Ile Leu Val Val Asp Pro
115 120 125
Val Thr Ser Glu His Glu Leu Thr Cys Gln Ala Glu Gly Tyr Pro Lys
130 135 140
Ala Glu Val Ile Trp Thr Ser Ser Asp His Gln Val Leu Ser Gly Lys
145 150 155 160
Thr Thr Thr Thr Asn Ser Lys Arg Glu Glu Lys Leu Phe Asn Val Thr
165 170 175
Ser Thr Leu Arg Ile Asn Thr Thr Thr Asn Glu Ile Phe Tyr Cys Thr
180 185 190
Phe Arg Arg Leu Asp Pro Glu Glu Asn His Thr Ala Glu Leu Val Ile
195 200 205
Pro Glu Leu Pro Leu Ala His Pro Pro Asn Glu Arg Gly Gly Gly Gly
210 215 220
Ser Gly Gly Gly Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro
225 230 235 240
Glu Lys Lys Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys
245 250 255
Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val
260 265 270
Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp
275 280 285
Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr
290 295 300
Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp
305 310 315 320
Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu
325 330 335
Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg
340 345 350
Glu Pro Gln Val Tyr Val Leu Pro Pro Ser Arg Glu Glu Met Thr Lys
355 360 365
Asn Gln Val Ser Leu Leu Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp
370 375 380
Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Arg Tyr Met
385 390 395 400
Thr Trp Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
405 410 415
Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser
420 425 430
Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser
435 440 445
Leu Ser Leu Ser Pro Gly Lys
450 455
<210> 136
<211> 417
<212> PRT
<213> Homo sapiens (Homo sapiens)
<400> 136
Met Ala Arg Ala Met Ala Ala Ala Trp Pro Leu Leu Leu Val Ala Leu
1 5 10 15
Leu Val Leu Ser Trp Pro Pro Pro Gly Thr Gly Asp Val Val Val Gln
20 25 30
Ala Pro Thr Gln Val Pro Gly Phe Leu Gly Asp Ser Val Thr Leu Pro
35 40 45
Cys Tyr Leu Gln Val Pro Asn Met Glu Val Thr His Val Ser Gln Leu
50 55 60
Thr Trp Ala Arg His Gly Glu Ser Gly Ser Met Ala Val Phe His Gln
65 70 75 80
Thr Gln Gly Pro Ser Tyr Ser Glu Ser Lys Arg Leu Glu Phe Val Ala
85 90 95
Ala Arg Leu Gly Ala Glu Leu Arg Asn Ala Ser Leu Arg Met Phe Gly
100 105 110
Leu Arg Val Glu Asp Glu Gly Asn Tyr Thr Cys Leu Phe Val Thr Phe
115 120 125
Pro Gln Gly Ser Arg Ser Val Asp Ile Trp Leu Arg Val Leu Ala Lys
130 135 140
Pro Gln Asn Thr Ala Glu Val Gln Lys Val Gln Leu Thr Gly Glu Pro
145 150 155 160
Val Pro Met Ala Arg Cys Val Ser Thr Gly Gly Arg Pro Pro Ala Gln
165 170 175
Ile Thr Trp His Ser Asp Leu Gly Gly Met Pro Asn Thr Ser Gln Val
180 185 190
Pro Gly Phe Leu Ser Gly Thr Val Thr Val Thr Ser Leu Trp Ile Leu
195 200 205
Val Pro Ser Ser Gln Val Asp Gly Lys Asn Val Thr Cys Lys Val Glu
210 215 220
His Glu Ser Phe Glu Lys Pro Gln Leu Leu Thr Val Asn Leu Thr Val
225 230 235 240
Tyr Tyr Pro Pro Glu Val Ser Ile Ser Gly Tyr Asp Asn Asn Trp Tyr
245 250 255
Leu Gly Gln Asn Glu Ala Thr Leu Thr Cys Asp Ala Arg Ser Asn Pro
260 265 270
Glu Pro Thr Gly Tyr Asn Trp Ser Thr Thr Met Gly Pro Leu Pro Pro
275 280 285
Phe Ala Val Ala Gln Gly Ala Gln Leu Leu Ile Arg Pro Val Asp Lys
290 295 300
Pro Ile Asn Thr Thr Leu Ile Cys Asn Val Thr Asn Ala Leu Gly Ala
305 310 315 320
Arg Gln Ala Glu Leu Thr Val Gln Val Lys Glu Gly Pro Pro Ser Glu
325 330 335
His Ser Gly Ile Ser Arg Asn Ala Ile Ile Phe Leu Val Leu Gly Ile
340 345 350
Leu Val Phe Leu Ile Leu Leu Gly Ile Gly Ile Tyr Phe Tyr Trp Ser
355 360 365
Lys Cys Ser Arg Glu Val Leu Trp His Cys His Leu Cys Pro Ser Ser
370 375 380
Thr Glu His Ala Ser Ala Ser Ala Asn Gly His Val Ser Tyr Ser Ala
385 390 395 400
Val Ser Arg Glu Asn Ser Ser Ser Gln Asp Pro Gln Thr Glu Gly Thr
405 410 415
Arg
<210> 137
<211> 323
<212> PRT
<213> Homo sapiens (Homo sapiens)
<400> 137
Trp Pro Pro Pro Gly Thr Gly Asp Val Val Val Gln Ala Pro Thr Gln
1 5 10 15
Val Pro Gly Phe Leu Gly Asp Ser Val Thr Leu Pro Cys Tyr Leu Gln
20 25 30
Val Pro Asn Met Glu Val Thr His Val Ser Gln Leu Thr Trp Ala Arg
35 40 45
His Gly Glu Ser Gly Ser Met Ala Val Phe His Gln Thr Gln Gly Pro
50 55 60
Ser Tyr Ser Glu Ser Lys Arg Leu Glu Phe Val Ala Ala Arg Leu Gly
65 70 75 80
Ala Glu Leu Arg Asn Ala Ser Leu Arg Met Phe Gly Leu Arg Val Glu
85 90 95
Asp Glu Gly Asn Tyr Thr Cys Leu Phe Val Thr Phe Pro Gln Gly Ser
100 105 110
Arg Ser Val Asp Ile Trp Leu Arg Val Leu Ala Lys Pro Gln Asn Thr
115 120 125
Ala Glu Val Gln Lys Val Gln Leu Thr Gly Glu Pro Val Pro Met Ala
130 135 140
Arg Cys Val Ser Thr Gly Gly Arg Pro Pro Ala Gln Ile Thr Trp His
145 150 155 160
Ser Asp Leu Gly Gly Met Pro Asn Thr Ser Gln Val Pro Gly Phe Leu
165 170 175
Ser Gly Thr Val Thr Val Thr Ser Leu Trp Ile Leu Val Pro Ser Ser
180 185 190
Gln Val Asp Gly Lys Asn Val Thr Cys Lys Val Glu His Glu Ser Phe
195 200 205
Glu Lys Pro Gln Leu Leu Thr Val Asn Leu Thr Val Tyr Tyr Pro Pro
210 215 220
Glu Val Ser Ile Ser Gly Tyr Asp Asn Asn Trp Tyr Leu Gly Gln Asn
225 230 235 240
Glu Ala Thr Leu Thr Cys Asp Ala Arg Ser Asn Pro Glu Pro Thr Gly
245 250 255
Tyr Asn Trp Ser Thr Thr Met Gly Pro Leu Pro Pro Phe Ala Val Ala
260 265 270
Gln Gly Ala Gln Leu Leu Ile Arg Pro Val Asp Lys Pro Ile Asn Thr
275 280 285
Thr Leu Ile Cys Asn Val Thr Asn Ala Leu Gly Ala Arg Gln Ala Glu
290 295 300
Leu Thr Val Gln Val Lys Glu Gly Pro Pro Ser Glu His Ser Gly Ile
305 310 315 320
Ser Arg Asn
<210> 138
<211> 558
<212> PRT
<213> Homo sapiens (Homo sapiens)
<400> 138
Trp Pro Pro Pro Gly Thr Gly Asp Val Val Val Gln Ala Pro Thr Gln
1 5 10 15
Val Pro Gly Phe Leu Gly Asp Ser Val Thr Leu Pro Cys Tyr Leu Gln
20 25 30
Val Pro Asn Met Glu Val Thr His Val Ser Gln Leu Thr Trp Ala Arg
35 40 45
His Gly Glu Ser Gly Ser Met Ala Val Phe His Gln Thr Gln Gly Pro
50 55 60
Ser Tyr Ser Glu Ser Lys Arg Leu Glu Phe Val Ala Ala Arg Leu Gly
65 70 75 80
Ala Glu Leu Arg Asn Ala Ser Leu Arg Met Phe Gly Leu Arg Val Glu
85 90 95
Asp Glu Gly Asn Tyr Thr Cys Leu Phe Val Thr Phe Pro Gln Gly Ser
100 105 110
Arg Ser Val Asp Ile Trp Leu Arg Val Leu Ala Lys Pro Gln Asn Thr
115 120 125
Ala Glu Val Gln Lys Val Gln Leu Thr Gly Glu Pro Val Pro Met Ala
130 135 140
Arg Cys Val Ser Thr Gly Gly Arg Pro Pro Ala Gln Ile Thr Trp His
145 150 155 160
Ser Asp Leu Gly Gly Met Pro Asn Thr Ser Gln Val Pro Gly Phe Leu
165 170 175
Ser Gly Thr Val Thr Val Thr Ser Leu Trp Ile Leu Val Pro Ser Ser
180 185 190
Gln Val Asp Gly Lys Asn Val Thr Cys Lys Val Glu His Glu Ser Phe
195 200 205
Glu Lys Pro Gln Leu Leu Thr Val Asn Leu Thr Val Tyr Tyr Pro Pro
210 215 220
Glu Val Ser Ile Ser Gly Tyr Asp Asn Asn Trp Tyr Leu Gly Gln Asn
225 230 235 240
Glu Ala Thr Leu Thr Cys Asp Ala Arg Ser Asn Pro Glu Pro Thr Gly
245 250 255
Tyr Asn Trp Ser Thr Thr Met Gly Pro Leu Pro Pro Phe Ala Val Ala
260 265 270
Gln Gly Ala Gln Leu Leu Ile Arg Pro Val Asp Lys Pro Ile Asn Thr
275 280 285
Thr Leu Ile Cys Asn Val Thr Asn Ala Leu Gly Ala Arg Gln Ala Glu
290 295 300
Leu Thr Val Gln Val Lys Glu Gly Pro Pro Ser Glu His Ser Gly Ile
305 310 315 320
Ser Arg Asn Gly Gly Gly Gly Ser Gly Gly Gly Asp Lys Thr His Thr
325 330 335
Cys Pro Pro Cys Pro Ala Pro Glu Asp Glu Gly Gly Pro Ser Val Phe
340 345 350
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro
355 360 365
Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val
370 375 380
Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr
385 390 395 400
Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val
405 410 415
Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys
420 425 430
Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser
435 440 445
Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Val Tyr Pro Pro
450 455 460
Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val
465 470 475 480
Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly
485 490 495
Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Glu Asp
500 505 510
Gly Ser Phe Ala Leu Val Ser Lys Leu Thr Val Asp Lys Ser Arg Trp
515 520 525
Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His
530 535 540
Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
545 550 555
<210> 139
<211> 558
<212> PRT
<213> Homo sapiens (Homo sapiens)
<400> 139
Trp Pro Pro Pro Gly Thr Gly Asp Val Val Val Gln Ala Pro Thr Gln
1 5 10 15
Val Pro Gly Phe Leu Gly Asp Ser Val Thr Leu Pro Cys Tyr Leu Gln
20 25 30
Val Pro Asn Met Glu Val Thr His Val Ser Gln Leu Thr Trp Ala Arg
35 40 45
His Gly Glu Ser Gly Ser Met Ala Val Phe His Gln Thr Gln Gly Pro
50 55 60
Ser Tyr Ser Glu Ser Lys Arg Leu Glu Phe Val Ala Ala Arg Leu Gly
65 70 75 80
Ala Glu Leu Arg Asn Ala Ser Leu Arg Met Phe Gly Leu Arg Val Glu
85 90 95
Asp Glu Gly Asn Tyr Thr Cys Leu Phe Val Thr Phe Pro Gln Gly Ser
100 105 110
Arg Ser Val Asp Ile Trp Leu Arg Val Leu Ala Lys Pro Gln Asn Thr
115 120 125
Ala Glu Val Gln Lys Val Gln Leu Thr Gly Glu Pro Val Pro Met Ala
130 135 140
Arg Cys Val Ser Thr Gly Gly Arg Pro Pro Ala Gln Ile Thr Trp His
145 150 155 160
Ser Asp Leu Gly Gly Met Pro Asn Thr Ser Gln Val Pro Gly Phe Leu
165 170 175
Ser Gly Thr Val Thr Val Thr Ser Leu Trp Ile Leu Val Pro Ser Ser
180 185 190
Gln Val Asp Gly Lys Asn Val Thr Cys Lys Val Glu His Glu Ser Phe
195 200 205
Glu Lys Pro Gln Leu Leu Thr Val Asn Leu Thr Val Tyr Tyr Pro Pro
210 215 220
Glu Val Ser Ile Ser Gly Tyr Asp Asn Asn Trp Tyr Leu Gly Gln Asn
225 230 235 240
Glu Ala Thr Leu Thr Cys Asp Ala Arg Ser Asn Pro Glu Pro Thr Gly
245 250 255
Tyr Asn Trp Ser Thr Thr Met Gly Pro Leu Pro Pro Phe Ala Val Ala
260 265 270
Gln Gly Ala Gln Leu Leu Ile Arg Pro Val Asp Lys Pro Ile Asn Thr
275 280 285
Thr Leu Ile Cys Asn Val Thr Asn Ala Leu Gly Ala Arg Gln Ala Glu
290 295 300
Leu Thr Val Gln Val Lys Glu Gly Pro Pro Ser Glu His Ser Gly Ile
305 310 315 320
Ser Arg Asn Gly Gly Gly Gly Ser Gly Gly Gly Asp Lys Thr His Thr
325 330 335
Cys Pro Pro Cys Pro Ala Pro Glu Lys Lys Gly Gly Pro Ser Val Phe
340 345 350
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro
355 360 365
Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val
370 375 380
Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr
385 390 395 400
Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val
405 410 415
Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys
420 425 430
Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser
435 440 445
Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Val Leu Pro Pro
450 455 460
Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Leu Cys Leu Val
465 470 475 480
Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly
485 490 495
Gln Pro Glu Asn Arg Tyr Met Thr Trp Pro Pro Val Leu Asp Ser Asp
500 505 510
Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp
515 520 525
Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His
530 535 540
Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
545 550 555
<210> 140
<211> 306
<212> PRT
<213> artificial sequence
<220>
<223> synthetic construct
<400> 140
Ile Trp Glu Leu Lys Lys Asp Val Tyr Val Val Glu Leu Asp Trp Tyr
1 5 10 15
Pro Asp Ala Pro Gly Glu Met Val Val Leu Thr Cys Asp Thr Pro Glu
20 25 30
Glu Asp Gly Ile Thr Trp Thr Leu Asp Gln Ser Ser Glu Val Leu Gly
35 40 45
Ser Gly Lys Thr Leu Thr Ile Gln Val Lys Glu Asp Gly Asp Ala Gly
50 55 60
Gln Tyr Thr Cys His Lys Gly Gly Glu Val Leu Ser His Ser Leu Leu
65 70 75 80
Leu Leu His Lys Lys Glu Asp Gly Ile Trp Ser Thr Asp Ile Leu Lys
85 90 95
Asp Gln Lys Glu Pro Lys Asn Lys Thr Phe Leu Arg Cys Glu Ala Lys
100 105 110
Asn Tyr Ser Gly Arg Phe Thr Cys Trp Trp Leu Thr Thr Ile Ser Thr
115 120 125
Asp Leu Thr Phe Ser Val Lys Ser Ser Arg Gly Ser Ser Asp Pro Gln
130 135 140
Gly Val Thr Cys Gly Ala Ala Thr Leu Ser Ala Glu Arg Val Arg Gly
145 150 155 160
Asp Asn Lys Glu Tyr Glu Tyr Ser Val Glu Cys Gln Glu Asp Ser Ala
165 170 175
Cys Pro Ala Ala Glu Glu Ser Leu Pro Ile Glu Val Met Val Asp Ala
180 185 190
Val His Lys Leu Lys Tyr Glu Asn Tyr Thr Ser Ser Phe Phe Ile Arg
195 200 205
Asp Ile Ile Lys Pro Asp Pro Pro Lys Asn Leu Gln Leu Lys Pro Leu
210 215 220
Lys Asn Ser Arg Gln Val Glu Val Ser Trp Glu Tyr Pro Asp Thr Trp
225 230 235 240
Ser Thr Pro His Ser Tyr Phe Ser Leu Thr Phe Cys Val Gln Val Gln
245 250 255
Gly Lys Ser Lys Arg Glu Lys Lys Asp Arg Val Phe Thr Asp Lys Thr
260 265 270
Ser Ala Thr Val Ile Cys Arg Lys Asn Ala Ser Ile Ser Val Arg Ala
275 280 285
Gln Asp Arg Tyr Tyr Ser Ser Ser Trp Ser Glu Trp Ala Ser Val Pro
290 295 300
Cys Ser
305
<210> 141
<211> 126
<212> PRT
<213> Homo sapiens (Homo sapiens)
<400> 141
Lys Ala Met His Val Ala Gln Pro Ala Val Val Leu Ala Ser Ser Arg
1 5 10 15
Gly Ile Ala Ser Phe Val Cys Glu Tyr Ala Ser Pro Gly Lys Ala Thr
20 25 30
Glu Val Arg Val Thr Val Leu Arg Gln Ala Asp Ser Gln Val Thr Glu
35 40 45
Val Cys Ala Ala Thr Tyr Met Met Gly Asn Glu Leu Thr Phe Leu Asp
50 55 60
Asp Ser Ile Cys Thr Gly Thr Ser Ser Gly Asn Gln Val Asn Leu Thr
65 70 75 80
Ile Gln Gly Leu Arg Ala Met Asp Thr Gly Leu Tyr Ile Cys Lys Val
85 90 95
Glu Leu Met Tyr Pro Pro Pro Tyr Tyr Leu Gly Ile Gly Asn Gly Thr
100 105 110
Gln Ile Tyr Val Ile Asp Pro Glu Pro Cys Pro Asp Ser Asp
115 120 125
<210> 142
<211> 955
<212> PRT
<213> Homo sapiens (Homo sapiens)
<400> 142
Leu Phe Thr Val Thr Val Pro Lys Glu Leu Tyr Ile Ile Glu His Gly
1 5 10 15
Ser Asn Val Thr Leu Glu Cys Asn Phe Asp Thr Gly Ser His Val Asn
20 25 30
Leu Gly Ala Ile Thr Ala Ser Leu Gln Lys Val Glu Asn Asp Thr Ser
35 40 45
Pro His Arg Glu Arg Ala Thr Leu Leu Glu Glu Gln Leu Pro Leu Gly
50 55 60
Lys Ala Ser Phe His Ile Pro Gln Val Gln Val Arg Asp Glu Gly Gln
65 70 75 80
Tyr Gln Cys Ile Ile Ile Tyr Gly Val Ala Trp Asp Tyr Lys Tyr Leu
85 90 95
Thr Leu Lys Val Lys Ala Ser Tyr Arg Lys Ile Asn Thr His Ile Leu
100 105 110
Lys Val Pro Glu Thr Asp Glu Val Glu Leu Thr Cys Gln Ala Thr Gly
115 120 125
Tyr Pro Leu Ala Glu Val Ser Trp Pro Asn Val Ser Val Pro Ala Asn
130 135 140
Thr Ser His Ser Arg Thr Pro Glu Gly Leu Tyr Gln Val Thr Ser Val
145 150 155 160
Leu Arg Leu Lys Pro Pro Pro Gly Arg Asn Phe Ser Cys Val Phe Trp
165 170 175
Asn Thr His Val Arg Glu Leu Thr Leu Ala Ser Ile Asp Leu Gln Ser
180 185 190
Gln Met Glu Pro Arg Thr His Pro Thr Gly Ser Gly Ile Trp Glu Leu
195 200 205
Lys Lys Asp Val Tyr Val Val Glu Leu Asp Trp Tyr Pro Asp Ala Pro
210 215 220
Gly Glu Met Val Val Leu Thr Cys Asp Thr Pro Glu Glu Asp Gly Ile
225 230 235 240
Thr Trp Thr Leu Asp Gln Ser Ser Glu Val Leu Gly Ser Gly Lys Thr
245 250 255
Leu Thr Ile Gln Val Lys Glu Ala Gly Asp Ala Gly Gln Tyr Thr Cys
260 265 270
His Lys Gly Gly Glu Val Leu Ser His Ser Leu Leu Leu Leu His Lys
275 280 285
Lys Glu Asp Gly Ile Trp Ser Thr Asp Ile Leu Lys Asp Gln Lys Glu
290 295 300
Pro Lys Asn Lys Thr Phe Leu Arg Cys Glu Ala Lys Asn Tyr Ser Gly
305 310 315 320
Arg Phe Thr Cys Trp Trp Leu Thr Thr Ile Ser Thr Asp Leu Thr Phe
325 330 335
Ser Val Lys Ser Ser Arg Gly Ser Ser Asp Pro Gln Gly Val Thr Cys
340 345 350
Gly Ala Ala Thr Leu Ser Ala Glu Arg Val Arg Gly Asp Asn Lys Glu
355 360 365
Tyr Glu Tyr Ser Val Glu Cys Gln Glu Asp Ser Ala Cys Pro Ala Ala
370 375 380
Glu Glu Ser Leu Pro Ile Glu Val Met Val Asp Ala Val His Lys Leu
385 390 395 400
Lys Tyr Glu Asn Tyr Thr Ser Ser Phe Phe Ile Arg Asp Ile Ile Lys
405 410 415
Pro Asp Pro Pro Lys Asn Leu Gln Leu Lys Pro Leu Lys Asn Ser Arg
420 425 430
Gln Val Glu Val Ser Trp Glu Tyr Pro Asp Thr Trp Ser Thr Pro His
435 440 445
Ser Tyr Phe Ser Leu Thr Phe Cys Val Gln Val Gln Gly Lys Ser Lys
450 455 460
Arg Glu Lys Lys Asp Arg Val Phe Thr Asp Lys Thr Ser Ala Thr Val
465 470 475 480
Ile Cys Arg Lys Asn Ala Ser Ile Ser Val Arg Ala Gln Asp Arg Tyr
485 490 495
Tyr Ser Ser Ser Trp Ser Glu Trp Ala Ser Val Pro Cys Ser Gly Gly
500 505 510
Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
515 520 525
Gly Ser Gly Arg Asn Leu Pro Val Ala Thr Pro Asp Pro Gly Met Phe
530 535 540
Pro Cys Leu His His Ser Gln Asn Leu Leu Arg Ala Val Ser Asn Met
545 550 555 560
Leu Gln Lys Ala Arg Gln Thr Leu Glu Phe Tyr Pro Cys Thr Ser Glu
565 570 575
Glu Ile Asp His Glu Asp Ile Thr Lys Asp Lys Thr Ser Thr Val Glu
580 585 590
Ala Cys Leu Pro Leu Glu Leu Thr Lys Asn Glu Ser Cys Leu Asn Ser
595 600 605
Arg Glu Thr Ser Phe Ile Thr Asn Gly Ser Cys Leu Ala Ser Arg Lys
610 615 620
Thr Ser Phe Met Met Ala Leu Cys Leu Ser Ser Ile Tyr Glu Asp Leu
625 630 635 640
Lys Met Tyr Gln Val Glu Phe Lys Thr Met Asn Ala Lys Leu Leu Met
645 650 655
Asp Pro Lys Arg Gln Ile Phe Leu Asp Gln Asn Met Leu Ala Val Ile
660 665 670
Asp Glu Leu Met Gln Ala Leu Asn Phe Asn Ser Glu Thr Val Pro Gln
675 680 685
Lys Ser Ser Leu Glu Glu Pro Asp Phe Tyr Lys Thr Lys Ile Lys Leu
690 695 700
Cys Ile Leu Leu His Ala Phe Arg Ile Arg Ala Val Thr Ile Asp Arg
705 710 715 720
Val Met Ser Tyr Leu Asn Ala Ser Asp Lys Thr His Thr Cys Pro Pro
725 730 735
Cys Pro Ala Pro Glu Asp Glu Gly Gly Pro Ser Val Phe Leu Phe Pro
740 745 750
Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr
755 760 765
Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn
770 775 780
Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg
785 790 795 800
Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val
805 810 815
Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser
820 825 830
Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys
835 840 845
Gly Gln Pro Arg Glu Pro Gln Val Tyr Val Tyr Pro Pro Ser Arg Glu
850 855 860
Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe
865 870 875 880
Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu
885 890 895
Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Glu Asp Gly Ser Phe
900 905 910
Ala Leu Val Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly
915 920 925
Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr
930 935 940
Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
945 950 955
<210> 143
<211> 970
<212> PRT
<213> Homo sapiens (Homo sapiens)
<400> 143
Leu Phe Thr Val Thr Val Pro Lys Glu Leu Tyr Ile Ile Glu His Gly
1 5 10 15
Ser Asn Val Thr Leu Glu Cys Asn Phe Asp Thr Gly Ser His Val Asn
20 25 30
Leu Gly Ala Ile Thr Ala Ser Leu Gln Lys Val Glu Asn Asp Thr Ser
35 40 45
Pro His Arg Glu Arg Ala Thr Leu Leu Glu Glu Gln Leu Pro Leu Gly
50 55 60
Lys Ala Ser Phe His Ile Pro Gln Val Gln Val Arg Asp Glu Gly Gln
65 70 75 80
Tyr Gln Cys Ile Ile Ile Tyr Gly Val Ala Trp Asp Tyr Lys Tyr Leu
85 90 95
Thr Leu Lys Val Lys Ala Ser Tyr Arg Lys Ile Asn Thr His Ile Leu
100 105 110
Lys Val Pro Glu Thr Asp Glu Val Glu Leu Thr Cys Gln Ala Thr Gly
115 120 125
Tyr Pro Leu Ala Glu Val Ser Trp Pro Asn Val Ser Val Pro Ala Asn
130 135 140
Thr Ser His Ser Arg Thr Pro Glu Gly Leu Tyr Gln Val Thr Ser Val
145 150 155 160
Leu Arg Leu Lys Pro Pro Pro Gly Arg Asn Phe Ser Cys Val Phe Trp
165 170 175
Asn Thr His Val Arg Glu Leu Thr Leu Ala Ser Ile Asp Leu Gln Ser
180 185 190
Gln Met Glu Pro Arg Thr His Pro Thr Gly Ser Gly Asp Lys Thr His
195 200 205
Thr Cys Pro Pro Cys Pro Ala Pro Glu Asp Glu Gly Gly Pro Ser Val
210 215 220
Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr
225 230 235 240
Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu
245 250 255
Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys
260 265 270
Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser
275 280 285
Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys
290 295 300
Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile
305 310 315 320
Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Val Tyr Pro
325 330 335
Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu
340 345 350
Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn
355 360 365
Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Glu
370 375 380
Asp Gly Ser Phe Ala Leu Val Ser Lys Leu Thr Val Asp Lys Ser Arg
385 390 395 400
Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu
405 410 415
His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Gly
420 425 430
Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ile Trp
435 440 445
Glu Leu Lys Lys Asp Val Tyr Val Val Glu Leu Asp Trp Tyr Pro Asp
450 455 460
Ala Pro Gly Glu Met Val Val Leu Thr Cys Asp Thr Pro Glu Glu Asp
465 470 475 480
Gly Ile Thr Trp Thr Leu Asp Gln Ser Ser Glu Val Leu Gly Ser Gly
485 490 495
Lys Thr Leu Thr Ile Gln Val Lys Glu Ala Gly Asp Ala Gly Gln Tyr
500 505 510
Thr Cys His Lys Gly Gly Glu Val Leu Ser His Ser Leu Leu Leu Leu
515 520 525
His Lys Lys Glu Asp Gly Ile Trp Ser Thr Asp Ile Leu Lys Asp Gln
530 535 540
Lys Glu Pro Lys Asn Lys Thr Phe Leu Arg Cys Glu Ala Lys Asn Tyr
545 550 555 560
Ser Gly Arg Phe Thr Cys Trp Trp Leu Thr Thr Ile Ser Thr Asp Leu
565 570 575
Thr Phe Ser Val Lys Ser Ser Arg Gly Ser Ser Asp Pro Gln Gly Val
580 585 590
Thr Cys Gly Ala Ala Thr Leu Ser Ala Glu Arg Val Arg Gly Asp Asn
595 600 605
Lys Glu Tyr Glu Tyr Ser Val Glu Cys Gln Glu Asp Ser Ala Cys Pro
610 615 620
Ala Ala Glu Glu Ser Leu Pro Ile Glu Val Met Val Asp Ala Val His
625 630 635 640
Lys Leu Lys Tyr Glu Asn Tyr Thr Ser Ser Phe Phe Ile Arg Asp Ile
645 650 655
Ile Lys Pro Asp Pro Pro Lys Asn Leu Gln Leu Lys Pro Leu Lys Asn
660 665 670
Ser Arg Gln Val Glu Val Ser Trp Glu Tyr Pro Asp Thr Trp Ser Thr
675 680 685
Pro His Ser Tyr Phe Ser Leu Thr Phe Cys Val Gln Val Gln Gly Lys
690 695 700
Ser Lys Arg Glu Lys Lys Asp Arg Val Phe Thr Asp Lys Thr Ser Ala
705 710 715 720
Thr Val Ile Cys Arg Lys Asn Ala Ser Ile Ser Val Arg Ala Gln Asp
725 730 735
Arg Tyr Tyr Ser Ser Ser Trp Ser Glu Trp Ala Ser Val Pro Cys Ser
740 745 750
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly
755 760 765
Gly Gly Gly Ser Gly Arg Asn Leu Pro Val Ala Thr Pro Asp Pro Gly
770 775 780
Met Phe Pro Cys Leu His His Ser Gln Asn Leu Leu Arg Ala Val Ser
785 790 795 800
Asn Met Leu Gln Lys Ala Arg Gln Thr Leu Glu Phe Tyr Pro Cys Thr
805 810 815
Ser Glu Glu Ile Asp His Glu Asp Ile Thr Lys Asp Lys Thr Ser Thr
820 825 830
Val Glu Ala Cys Leu Pro Leu Glu Leu Thr Lys Asn Glu Ser Cys Leu
835 840 845
Asn Ser Arg Glu Thr Ser Phe Ile Thr Asn Gly Ser Cys Leu Ala Ser
850 855 860
Arg Lys Thr Ser Phe Met Met Ala Leu Cys Leu Ser Ser Ile Tyr Glu
865 870 875 880
Asp Leu Lys Met Tyr Gln Val Glu Phe Lys Thr Met Asn Ala Lys Leu
885 890 895
Leu Met Asp Pro Lys Arg Gln Ile Phe Leu Asp Gln Asn Met Leu Ala
900 905 910
Val Ile Asp Glu Leu Met Gln Ala Leu Asn Phe Asn Ser Glu Thr Val
915 920 925
Pro Gln Lys Ser Ser Leu Glu Glu Pro Asp Phe Tyr Lys Thr Lys Ile
930 935 940
Lys Leu Cys Ile Leu Leu His Ala Phe Arg Ile Arg Ala Val Thr Ile
945 950 955 960
Asp Arg Val Met Ser Tyr Leu Asn Ala Ser
965 970
<210> 144
<211> 579
<212> PRT
<213> artificial sequence
<220>
<223> synthetic construct
<400> 144
Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg
1 5 10 15
Ser Leu Arg Leu Asp Cys Lys Ala Ser Gly Ile Thr Phe Ser Asn Ser
20 25 30
Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Val Ile Trp Tyr Asp Gly Ser Lys Arg Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Phe
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Thr Asn Asp Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser
100 105 110
Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser
115 120 125
Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp
130 135 140
Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr
145 150 155 160
Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr
165 170 175
Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln
180 185 190
Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp
195 200 205
Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Pro Ala Pro Thr Ser Ser
210 215 220
Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu Leu Leu Asp Leu
225 230 235 240
Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn Pro Lys Leu Thr
245 250 255
Asp Met Leu Thr Phe Glu Phe Tyr Met Pro Lys Lys Ala Thr Glu Leu
260 265 270
Lys His Leu Gln Cys Leu Glu Arg Glu Leu Lys Pro Leu Glu Glu Val
275 280 285
Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg Pro Arg Asp Leu
290 295 300
Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys Gly Ser Glu Thr
305 310 315 320
Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr Ile Val Glu Phe
325 330 335
Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile Ile Ser Thr Leu Thr
340 345 350
Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Asp Glu Gly
355 360 365
Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met
370 375 380
Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His
385 390 395 400
Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val
405 410 415
His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr
420 425 430
Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly
435 440 445
Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile
450 455 460
Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val
465 470 475 480
Tyr Val Tyr Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser
485 490 495
Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu
500 505 510
Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro
515 520 525
Val Leu Asp Glu Asp Gly Ser Phe Ala Leu Val Ser Lys Leu Thr Val
530 535 540
Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met
545 550 555 560
His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser
565 570 575
Pro Gly Lys
<210> 145
<211> 946
<212> PRT
<213> artificial sequence
<220>
<223> synthetic construct
<400> 145
Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg
1 5 10 15
Ser Leu Arg Leu Asp Cys Lys Ala Ser Gly Ile Thr Phe Ser Asn Ser
20 25 30
Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Val Ile Trp Tyr Asp Gly Ser Lys Arg Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Phe
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Thr Asn Asp Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser
100 105 110
Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser
115 120 125
Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp
130 135 140
Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr
145 150 155 160
Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr
165 170 175
Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln
180 185 190
Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp
195 200 205
Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Pro Gly Ser Gly Ile Trp
210 215 220
Glu Leu Lys Lys Asp Val Tyr Val Val Glu Leu Asp Trp Tyr Pro Asp
225 230 235 240
Ala Pro Gly Glu Met Val Val Leu Thr Cys Asp Thr Pro Glu Glu Asp
245 250 255
Gly Ile Thr Trp Thr Leu Asp Gln Ser Ser Glu Val Leu Gly Ser Gly
260 265 270
Lys Thr Leu Thr Ile Gln Val Lys Ala Ala Gly Asp Ala Gly Gln Tyr
275 280 285
Thr Cys His Lys Gly Gly Glu Val Leu Ser His Ser Leu Leu Leu Leu
290 295 300
His Lys Lys Glu Asp Gly Ile Trp Ser Thr Asp Ile Leu Lys Asp Gln
305 310 315 320
Lys Glu Pro Lys Asn Lys Thr Phe Leu Arg Cys Glu Ala Lys Asn Tyr
325 330 335
Ser Gly Arg Phe Thr Cys Trp Trp Leu Thr Thr Ile Ser Thr Asp Leu
340 345 350
Thr Phe Ser Val Lys Ser Ser Arg Gly Ser Ser Asp Pro Gln Gly Val
355 360 365
Thr Cys Gly Ala Ala Thr Leu Ser Ala Glu Arg Val Arg Gly Asp Asn
370 375 380
Lys Glu Tyr Glu Tyr Ser Val Glu Cys Gln Glu Asp Ser Ala Cys Pro
385 390 395 400
Ala Ala Glu Glu Ser Leu Pro Ile Glu Val Met Val Asp Ala Val His
405 410 415
Lys Leu Lys Tyr Glu Asn Tyr Thr Ser Ser Phe Phe Ile Arg Asp Ile
420 425 430
Ile Lys Pro Asp Pro Pro Lys Asn Leu Gln Leu Lys Pro Leu Lys Asn
435 440 445
Ser Arg Gln Val Glu Val Ser Trp Glu Tyr Pro Asp Thr Trp Ser Thr
450 455 460
Pro His Ser Tyr Phe Ser Leu Thr Phe Cys Val Gln Val Gln Gly Lys
465 470 475 480
Ser Lys Arg Glu Lys Lys Asp Arg Val Phe Thr Asp Lys Thr Ser Ala
485 490 495
Thr Val Ile Cys Arg Lys Asn Ala Ser Ile Ser Val Arg Ala Gln Asp
500 505 510
Arg Tyr Tyr Ser Ser Ser Trp Ser Glu Trp Ala Ser Val Pro Cys Ser
515 520 525
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly
530 535 540
Gly Gly Gly Ser Gly Arg Ala Val Pro Gly Gly Ser Ser Pro Ala Trp
545 550 555 560
Thr Gln Cys Gln Gln Leu Ser Gln Lys Leu Cys Thr Leu Ala Trp Ser
565 570 575
Ala His Pro Leu Val Gly His Met Asp Leu Arg Glu Glu Gly Asp Glu
580 585 590
Glu Thr Thr Asn Asp Val Pro His Ile Gln Cys Gly Asp Gly Cys Asp
595 600 605
Pro Gln Gly Leu Arg Asp Asn Ser Gln Phe Cys Leu Gln Arg Ile His
610 615 620
Gln Gly Leu Ile Phe Tyr Glu Lys Leu Leu Gly Ser Asp Ile Phe Thr
625 630 635 640
Gly Glu Pro Ser Leu Leu Pro Asp Ser Pro Val Gly Gln Leu His Ala
645 650 655
Ser Leu Leu Gly Leu Ser Gln Leu Leu Gln Pro Glu Gly His His Trp
660 665 670
Glu Thr Gln Gln Ile Pro Ser Leu Ser Pro Ser Gln Pro Trp Gln Arg
675 680 685
Leu Leu Leu Arg Phe Lys Ile Leu Arg Ser Leu Gln Ala Phe Val Ala
690 695 700
Val Ala Ala Arg Val Phe Ala His Gly Ala Ala Thr Leu Ser Pro Asp
705 710 715 720
Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Asp Glu Gly Gly
725 730 735
Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile
740 745 750
Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu
755 760 765
Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His
770 775 780
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg
785 790 795 800
Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys
805 810 815
Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu
820 825 830
Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr
835 840 845
Val Tyr Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu
850 855 860
Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp
865 870 875 880
Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val
885 890 895
Leu Asp Glu Asp Gly Ser Phe Ala Leu Val Ser Lys Leu Thr Val Asp
900 905 910
Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His
915 920 925
Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro
930 935 940
Gly Lys
945
<210> 146
<211> 790
<212> PRT
<213> artificial sequence
<220>
<223> synthetic construct
<400> 146
Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg
1 5 10 15
Ser Leu Arg Leu Asp Cys Lys Ala Ser Gly Ile Thr Phe Ser Asn Ser
20 25 30
Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Val Ile Trp Tyr Asp Gly Ser Lys Arg Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Phe
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Thr Asn Asp Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser
100 105 110
Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser
115 120 125
Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp
130 135 140
Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr
145 150 155 160
Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr
165 170 175
Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln
180 185 190
Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp
195 200 205
Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Pro Gly Ser Gly Ser Pro
210 215 220
Gly Gln Gly Thr Gln Ser Glu Asn Ser Cys Thr His Phe Pro Gly Asn
225 230 235 240
Leu Pro Asn Met Leu Arg Asp Leu Ala Asp Ala Phe Ser Arg Val Lys
245 250 255
Thr Phe Phe Gln Met Lys Asp Gln Leu Asp Asn Leu Leu Leu Lys Glu
260 265 270
Ser Leu Leu Glu Asp Phe Lys Gly Tyr Leu Gly Cys Gln Ala Leu Ser
275 280 285
Glu Met Ile Gln Phe Tyr Leu Glu Glu Val Met Pro Gln Ala Glu Asn
290 295 300
Gln Asp Pro Asp Ile Lys Ala His Val Asn Ser Leu Gly Glu Asn Leu
305 310 315 320
Lys Thr Leu Arg Leu Arg Leu Arg Arg Cys His Arg Phe Leu Pro Cys
325 330 335
Glu Asn Lys Ser Lys Ala Val Glu Gln Val Lys Asn Ala Phe Asn Lys
340 345 350
Leu Gln Glu Lys Gly Ile Tyr Lys Ala Met Ser Glu Phe Asp Ile Phe
355 360 365
Ile Asn Tyr Ile Glu Ala Tyr Met Thr Met Lys Ile Arg Asn Phe Glu
370 375 380
Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly
385 390 395 400
Gly Gly Ser Ser Pro Gly Gln Gly Thr Gln Ser Glu Asn Ser Cys Thr
405 410 415
His Phe Pro Gly Asn Leu Pro Asn Met Leu Arg Asp Leu Ala Asp Ala
420 425 430
Phe Ser Arg Val Lys Thr Phe Phe Gln Met Lys Asp Gln Leu Asp Asn
435 440 445
Leu Leu Leu Lys Glu Ser Leu Leu Glu Asp Phe Lys Gly Tyr Leu Gly
450 455 460
Cys Gln Ala Leu Ser Glu Met Ile Gln Phe Tyr Leu Glu Glu Val Met
465 470 475 480
Pro Gln Ala Glu Asn Gln Asp Pro Asp Ile Lys Ala His Val Asn Ser
485 490 495
Leu Gly Glu Asn Leu Lys Thr Leu Arg Leu Arg Leu Arg Arg Cys His
500 505 510
Arg Phe Leu Pro Cys Glu Asn Lys Ser Lys Ala Val Glu Gln Val Lys
515 520 525
Asn Ala Phe Asn Lys Leu Gln Glu Lys Gly Ile Tyr Lys Ala Met Ser
530 535 540
Glu Phe Asp Ile Phe Ile Asn Tyr Ile Glu Ala Tyr Met Thr Met Lys
545 550 555 560
Ile Arg Asn Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu
565 570 575
Asp Glu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp
580 585 590
Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp
595 600 605
Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly
610 615 620
Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn
625 630 635 640
Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp
645 650 655
Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro
660 665 670
Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu
675 680 685
Pro Gln Val Tyr Val Tyr Pro Pro Ser Arg Glu Glu Met Thr Lys Asn
690 695 700
Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile
705 710 715 720
Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr
725 730 735
Thr Pro Pro Val Leu Asp Glu Asp Gly Ser Phe Ala Leu Val Ser Lys
740 745 750
Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys
755 760 765
Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu
770 775 780
Ser Leu Ser Pro Gly Lys
785 790
<210> 147
<211> 746
<212> PRT
<213> artificial sequence
<220>
<223> synthetic construct
<400> 147
Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg
1 5 10 15
Ser Leu Arg Leu Asp Cys Lys Ala Ser Gly Ile Thr Phe Ser Asn Ser
20 25 30
Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Val Ile Trp Tyr Asp Gly Ser Lys Arg Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Phe
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Thr Asn Asp Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser
100 105 110
Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser
115 120 125
Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp
130 135 140
Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr
145 150 155 160
Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr
165 170 175
Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln
180 185 190
Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp
195 200 205
Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Pro Gly Ser Gly Gln Asp
210 215 220
Pro Tyr Val Lys Glu Ala Glu Asn Leu Lys Lys Tyr Phe Asn Ala Gly
225 230 235 240
His Ser Asp Val Val Asp Asn Gly Thr Leu Phe Leu Gly Ile Leu Lys
245 250 255
Asn Trp Lys Glu Glu Ser Asp Arg Lys Ile Met Gln Ser Gln Ile Val
260 265 270
Ser Phe Tyr Phe Lys Leu Phe Lys Asn Phe Lys Asp Asp Gln Ser Ile
275 280 285
Gln Lys Ser Val Glu Thr Ile Lys Glu Asp Met Asn Val Lys Phe Phe
290 295 300
Asn Ser Asn Lys Lys Lys Arg Asp Asp Phe Glu Lys Leu Thr Asn Tyr
305 310 315 320
Ser Val Thr Asp Leu Asn Val Gln Arg Lys Ala Ile His Glu Leu Ile
325 330 335
Gln Val Met Ala Glu Leu Ser Pro Ala Ala Lys Thr Gly Lys Arg Lys
340 345 350
Arg Ser Gln Met Leu Phe Arg Gly Phe Glu Gly Gly Gly Ser Gly Gly
355 360 365
Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Asp Pro
370 375 380
Tyr Val Lys Glu Ala Glu Asn Leu Lys Lys Tyr Phe Asn Ala Gly His
385 390 395 400
Ser Asp Val Val Asp Asn Gly Thr Leu Phe Leu Gly Ile Leu Lys Asn
405 410 415
Trp Lys Glu Glu Ser Asp Arg Lys Ile Met Gln Ser Gln Ile Val Ser
420 425 430
Phe Tyr Phe Lys Leu Phe Lys Asn Phe Lys Asp Asp Gln Ser Ile Gln
435 440 445
Lys Ser Val Glu Thr Ile Lys Glu Asp Met Asn Val Lys Phe Phe Asn
450 455 460
Ser Asn Lys Lys Lys Arg Asp Asp Phe Glu Lys Leu Thr Asn Tyr Ser
465 470 475 480
Val Thr Asp Leu Asn Val Gln Arg Lys Ala Ile His Glu Leu Ile Gln
485 490 495
Val Met Ala Glu Leu Ser Pro Ala Ala Lys Thr Gly Lys Arg Lys Arg
500 505 510
Ser Gln Met Leu Phe Arg Gly Asp Lys Thr His Thr Cys Pro Pro Cys
515 520 525
Pro Ala Pro Glu Asp Glu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro
530 535 540
Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys
545 550 555 560
Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp
565 570 575
Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu
580 585 590
Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu
595 600 605
His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn
610 615 620
Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly
625 630 635 640
Gln Pro Arg Glu Pro Gln Val Tyr Val Tyr Pro Pro Ser Arg Glu Glu
645 650 655
Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr
660 665 670
Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn
675 680 685
Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Glu Asp Gly Ser Phe Ala
690 695 700
Leu Val Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn
705 710 715 720
Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr
725 730 735
Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
740 745
<210> 148
<211> 618
<212> PRT
<213> artificial sequence
<220>
<223> synthetic construct
<400> 148
Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg
1 5 10 15
Ser Leu Arg Leu Asp Cys Lys Ala Ser Gly Ile Thr Phe Ser Asn Ser
20 25 30
Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Val Ile Trp Tyr Asp Gly Ser Lys Arg Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Phe
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Thr Asn Asp Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser
100 105 110
Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser
115 120 125
Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp
130 135 140
Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr
145 150 155 160
Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr
165 170 175
Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln
180 185 190
Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp
195 200 205
Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Pro Gly Ser Gly Gly Gly
210 215 220
Gly Gly Cys Asp Leu Pro Gln Thr His Ser Leu Gly Ser Arg Arg Thr
225 230 235 240
Leu Met Leu Leu Ala Gln Met Arg Lys Ile Ser Leu Phe Ser Cys Ala
245 250 255
Lys Asp Arg His Asp Phe Gly Phe Pro Gln Glu Glu Phe Gly Asn Gln
260 265 270
Phe Gln Lys Ala Glu Thr Ile Pro Val Leu His Glu Met Ile Gln Gln
275 280 285
Ile Phe Asn Leu Phe Ser Thr Lys Asp Ser Ser Ala Ala Trp Asp Glu
290 295 300
Thr Leu Leu Asp Lys Phe Tyr Thr Glu Leu Tyr Gln Gln Leu Asn Asp
305 310 315 320
Leu Glu Ala Cys Val Ile Gln Gly Val Gly Val Thr Glu Thr Pro Leu
325 330 335
Met Lys Glu Asp Ser Ile Leu Ala Val Arg Lys Tyr Phe Gln Arg Ile
340 345 350
Thr Leu Tyr Leu Lys Glu Lys Lys Tyr Ser Pro Cys Ala Trp Glu Val
355 360 365
Val Arg Ala Glu Ile Met Arg Ser Phe Ser Leu Ser Thr Asn Leu Gln
370 375 380
Glu Ser Leu Arg Ser Lys Glu Asp Lys Thr His Thr Cys Pro Pro Cys
385 390 395 400
Pro Ala Pro Glu Asp Glu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro
405 410 415
Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys
420 425 430
Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp
435 440 445
Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu
450 455 460
Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu
465 470 475 480
His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn
485 490 495
Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly
500 505 510
Gln Pro Arg Glu Pro Gln Val Tyr Val Tyr Pro Pro Ser Arg Glu Glu
515 520 525
Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr
530 535 540
Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn
545 550 555 560
Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Glu Asp Gly Ser Phe Ala
565 570 575
Leu Val Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn
580 585 590
Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr
595 600 605
Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
610 615
<210> 149
<211> 973
<212> PRT
<213> artificial sequence
<220>
<223> synthetic construct
<400> 149
Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg
1 5 10 15
Ser Leu Arg Leu Asp Cys Lys Ala Ser Gly Ile Thr Phe Ser Asn Ser
20 25 30
Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Val Ile Trp Tyr Asp Gly Ser Lys Arg Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Phe
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Thr Asn Asn Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser
100 105 110
Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser
115 120 125
Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp
130 135 140
Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr
145 150 155 160
Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr
165 170 175
Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln
180 185 190
Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp
195 200 205
Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Pro Gly Ser Gly Ile Trp
210 215 220
Glu Leu Lys Lys Asp Val Tyr Val Val Glu Leu Asp Trp Tyr Pro Asp
225 230 235 240
Ala Pro Gly Glu Met Val Val Leu Thr Cys Asp Thr Pro Glu Glu Asp
245 250 255
Gly Ile Thr Trp Thr Leu Asp Gln Ser Ser Glu Val Leu Gly Ser Gly
260 265 270
Lys Thr Leu Thr Ile Gln Val Lys Ala Ala Gly Asp Ala Gly Gln Tyr
275 280 285
Thr Cys His Lys Gly Gly Glu Val Leu Ser His Ser Leu Leu Leu Leu
290 295 300
His Lys Lys Glu Asp Gly Ile Trp Ser Thr Asp Ile Leu Lys Asp Gln
305 310 315 320
Lys Glu Pro Lys Asn Lys Thr Phe Leu Arg Cys Glu Ala Lys Asn Tyr
325 330 335
Ser Gly Arg Phe Thr Cys Trp Trp Leu Thr Thr Ile Ser Thr Asp Leu
340 345 350
Thr Phe Ser Val Lys Ser Ser Arg Gly Ser Ser Asp Pro Gln Gly Val
355 360 365
Thr Cys Gly Ala Ala Thr Leu Ser Ala Glu Arg Val Arg Gly Asp Asn
370 375 380
Lys Glu Tyr Glu Tyr Ser Val Glu Cys Gln Glu Asp Ser Ala Cys Pro
385 390 395 400
Ala Ala Glu Glu Ser Leu Pro Ile Glu Val Met Val Asp Ala Val His
405 410 415
Lys Leu Lys Tyr Glu Asn Tyr Thr Ser Ser Phe Phe Ile Arg Asp Ile
420 425 430
Ile Lys Pro Asp Pro Pro Lys Asn Leu Gln Leu Lys Pro Leu Lys Asn
435 440 445
Ser Arg Gln Val Glu Val Ser Trp Glu Tyr Pro Asp Thr Trp Ser Thr
450 455 460
Pro His Ser Tyr Phe Ser Leu Thr Phe Cys Val Gln Val Gln Gly Lys
465 470 475 480
Ser Lys Arg Glu Lys Lys Asp Arg Val Phe Thr Asp Lys Thr Ser Ala
485 490 495
Thr Val Ile Cys Arg Lys Asn Ala Ser Ile Ser Val Arg Ala Gln Asp
500 505 510
Arg Tyr Tyr Ser Ser Ser Trp Ser Glu Trp Ala Ser Val Pro Cys Ser
515 520 525
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly
530 535 540
Gly Gly Gly Ser Gly Arg Asn Leu Pro Val Ala Thr Pro Asp Pro Gly
545 550 555 560
Met Phe Pro Cys Leu His His Ser Gln Asn Leu Leu Arg Ala Val Ser
565 570 575
Asn Met Leu Gln Lys Ala Arg Gln Thr Leu Glu Phe Tyr Pro Cys Thr
580 585 590
Ser Glu Glu Ile Asp His Glu Asp Ile Thr Lys Asp Lys Thr Ser Thr
595 600 605
Val Glu Ala Cys Leu Pro Leu Glu Leu Thr Lys Asn Glu Ser Cys Leu
610 615 620
Asn Ser Arg Glu Thr Ser Phe Ile Thr Asn Gly Ser Cys Leu Ala Ser
625 630 635 640
Arg Lys Thr Ser Phe Met Met Ala Leu Cys Leu Ser Ser Ile Tyr Glu
645 650 655
Asp Leu Lys Met Tyr Gln Val Glu Phe Lys Thr Met Asn Ala Lys Leu
660 665 670
Leu Met Asp Pro Lys Arg Gln Ile Phe Leu Asp Gln Asn Met Leu Ala
675 680 685
Val Ile Asp Glu Leu Met Gln Ala Leu Asn Phe Asn Ser Glu Thr Val
690 695 700
Pro Gln Lys Ser Ser Leu Glu Glu Pro Asp Phe Tyr Lys Thr Lys Ile
705 710 715 720
Lys Leu Cys Ile Leu Leu His Ala Phe Arg Ile Arg Ala Val Thr Ile
725 730 735
Asp Arg Val Met Ser Tyr Leu Asn Ala Ser Asp Lys Thr His Thr Cys
740 745 750
Pro Pro Cys Pro Ala Pro Glu Asp Glu Gly Gly Pro Ser Val Phe Leu
755 760 765
Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu
770 775 780
Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys
785 790 795 800
Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys
805 810 815
Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu
820 825 830
Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys
835 840 845
Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys
850 855 860
Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Val Tyr Pro Pro Ser
865 870 875 880
Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys
885 890 895
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln
900 905 910
Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Glu Asp Gly
915 920 925
Ser Phe Ala Leu Val Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln
930 935 940
Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn
945 950 955 960
His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
965 970
<210> 150
<211> 973
<212> PRT
<213> artificial sequence
<220>
<223> synthetic construct
<400> 150
Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg
1 5 10 15
Ser Leu Arg Leu Asp Cys Lys Ala Ser Gly Ile Thr Phe Ser Asn Ser
20 25 30
Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Val Ile Trp Tyr Asp Gly Ser Lys Arg Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Phe
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Thr Asn Gly Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser
100 105 110
Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser
115 120 125
Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp
130 135 140
Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr
145 150 155 160
Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr
165 170 175
Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln
180 185 190
Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp
195 200 205
Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Pro Gly Ser Gly Ile Trp
210 215 220
Glu Leu Lys Lys Asp Val Tyr Val Val Glu Leu Asp Trp Tyr Pro Asp
225 230 235 240
Ala Pro Gly Glu Met Val Val Leu Thr Cys Asp Thr Pro Glu Glu Asp
245 250 255
Gly Ile Thr Trp Thr Leu Asp Gln Ser Ser Glu Val Leu Gly Ser Gly
260 265 270
Lys Thr Leu Thr Ile Gln Val Lys Ala Ala Gly Asp Ala Gly Gln Tyr
275 280 285
Thr Cys His Lys Gly Gly Glu Val Leu Ser His Ser Leu Leu Leu Leu
290 295 300
His Lys Lys Glu Asp Gly Ile Trp Ser Thr Asp Ile Leu Lys Asp Gln
305 310 315 320
Lys Glu Pro Lys Asn Lys Thr Phe Leu Arg Cys Glu Ala Lys Asn Tyr
325 330 335
Ser Gly Arg Phe Thr Cys Trp Trp Leu Thr Thr Ile Ser Thr Asp Leu
340 345 350
Thr Phe Ser Val Lys Ser Ser Arg Gly Ser Ser Asp Pro Gln Gly Val
355 360 365
Thr Cys Gly Ala Ala Thr Leu Ser Ala Glu Arg Val Arg Gly Asp Asn
370 375 380
Lys Glu Tyr Glu Tyr Ser Val Glu Cys Gln Glu Asp Ser Ala Cys Pro
385 390 395 400
Ala Ala Glu Glu Ser Leu Pro Ile Glu Val Met Val Asp Ala Val His
405 410 415
Lys Leu Lys Tyr Glu Asn Tyr Thr Ser Ser Phe Phe Ile Arg Asp Ile
420 425 430
Ile Lys Pro Asp Pro Pro Lys Asn Leu Gln Leu Lys Pro Leu Lys Asn
435 440 445
Ser Arg Gln Val Glu Val Ser Trp Glu Tyr Pro Asp Thr Trp Ser Thr
450 455 460
Pro His Ser Tyr Phe Ser Leu Thr Phe Cys Val Gln Val Gln Gly Lys
465 470 475 480
Ser Lys Arg Glu Lys Lys Asp Arg Val Phe Thr Asp Lys Thr Ser Ala
485 490 495
Thr Val Ile Cys Arg Lys Asn Ala Ser Ile Ser Val Arg Ala Gln Asp
500 505 510
Arg Tyr Tyr Ser Ser Ser Trp Ser Glu Trp Ala Ser Val Pro Cys Ser
515 520 525
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly
530 535 540
Gly Gly Gly Ser Gly Arg Asn Leu Pro Val Ala Thr Pro Asp Pro Gly
545 550 555 560
Met Phe Pro Cys Leu His His Ser Gln Asn Leu Leu Arg Ala Val Ser
565 570 575
Asn Met Leu Gln Lys Ala Arg Gln Thr Leu Glu Phe Tyr Pro Cys Thr
580 585 590
Ser Glu Glu Ile Asp His Glu Asp Ile Thr Lys Asp Lys Thr Ser Thr
595 600 605
Val Glu Ala Cys Leu Pro Leu Glu Leu Thr Lys Asn Glu Ser Cys Leu
610 615 620
Asn Ser Arg Glu Thr Ser Phe Ile Thr Asn Gly Ser Cys Leu Ala Ser
625 630 635 640
Arg Lys Thr Ser Phe Met Met Ala Leu Cys Leu Ser Ser Ile Tyr Glu
645 650 655
Asp Leu Lys Met Tyr Gln Val Glu Phe Lys Thr Met Asn Ala Lys Leu
660 665 670
Leu Met Asp Pro Lys Arg Gln Ile Phe Leu Asp Gln Asn Met Leu Ala
675 680 685
Val Ile Asp Glu Leu Met Gln Ala Leu Asn Phe Asn Ser Glu Thr Val
690 695 700
Pro Gln Lys Ser Ser Leu Glu Glu Pro Asp Phe Tyr Lys Thr Lys Ile
705 710 715 720
Lys Leu Cys Ile Leu Leu His Ala Phe Arg Ile Arg Ala Val Thr Ile
725 730 735
Asp Arg Val Met Ser Tyr Leu Asn Ala Ser Asp Lys Thr His Thr Cys
740 745 750
Pro Pro Cys Pro Ala Pro Glu Asp Glu Gly Gly Pro Ser Val Phe Leu
755 760 765
Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu
770 775 780
Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys
785 790 795 800
Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys
805 810 815
Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu
820 825 830
Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys
835 840 845
Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys
850 855 860
Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Val Tyr Pro Pro Ser
865 870 875 880
Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys
885 890 895
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln
900 905 910
Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Glu Asp Gly
915 920 925
Ser Phe Ala Leu Val Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln
930 935 940
Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn
945 950 955 960
His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
965 970
<210> 151
<211> 973
<212> PRT
<213> artificial sequence
<220>
<223> synthetic construct
<400> 151
Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg
1 5 10 15
Ser Leu Arg Leu Asp Cys Lys Ala Ser Gly Ile Thr Phe Ser Asn Ser
20 25 30
Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Val Ile Trp Tyr Asp Gly Ser Lys Arg Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Phe
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Thr Asn Arg Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser
100 105 110
Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser
115 120 125
Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp
130 135 140
Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr
145 150 155 160
Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr
165 170 175
Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln
180 185 190
Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp
195 200 205
Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Pro Gly Ser Gly Ile Trp
210 215 220
Glu Leu Lys Lys Asp Val Tyr Val Val Glu Leu Asp Trp Tyr Pro Asp
225 230 235 240
Ala Pro Gly Glu Met Val Val Leu Thr Cys Asp Thr Pro Glu Glu Asp
245 250 255
Gly Ile Thr Trp Thr Leu Asp Gln Ser Ser Glu Val Leu Gly Ser Gly
260 265 270
Lys Thr Leu Thr Ile Gln Val Lys Ala Ala Gly Asp Ala Gly Gln Tyr
275 280 285
Thr Cys His Lys Gly Gly Glu Val Leu Ser His Ser Leu Leu Leu Leu
290 295 300
His Lys Lys Glu Asp Gly Ile Trp Ser Thr Asp Ile Leu Lys Asp Gln
305 310 315 320
Lys Glu Pro Lys Asn Lys Thr Phe Leu Arg Cys Glu Ala Lys Asn Tyr
325 330 335
Ser Gly Arg Phe Thr Cys Trp Trp Leu Thr Thr Ile Ser Thr Asp Leu
340 345 350
Thr Phe Ser Val Lys Ser Ser Arg Gly Ser Ser Asp Pro Gln Gly Val
355 360 365
Thr Cys Gly Ala Ala Thr Leu Ser Ala Glu Arg Val Arg Gly Asp Asn
370 375 380
Lys Glu Tyr Glu Tyr Ser Val Glu Cys Gln Glu Asp Ser Ala Cys Pro
385 390 395 400
Ala Ala Glu Glu Ser Leu Pro Ile Glu Val Met Val Asp Ala Val His
405 410 415
Lys Leu Lys Tyr Glu Asn Tyr Thr Ser Ser Phe Phe Ile Arg Asp Ile
420 425 430
Ile Lys Pro Asp Pro Pro Lys Asn Leu Gln Leu Lys Pro Leu Lys Asn
435 440 445
Ser Arg Gln Val Glu Val Ser Trp Glu Tyr Pro Asp Thr Trp Ser Thr
450 455 460
Pro His Ser Tyr Phe Ser Leu Thr Phe Cys Val Gln Val Gln Gly Lys
465 470 475 480
Ser Lys Arg Glu Lys Lys Asp Arg Val Phe Thr Asp Lys Thr Ser Ala
485 490 495
Thr Val Ile Cys Arg Lys Asn Ala Ser Ile Ser Val Arg Ala Gln Asp
500 505 510
Arg Tyr Tyr Ser Ser Ser Trp Ser Glu Trp Ala Ser Val Pro Cys Ser
515 520 525
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly
530 535 540
Gly Gly Gly Ser Gly Arg Asn Leu Pro Val Ala Thr Pro Asp Pro Gly
545 550 555 560
Met Phe Pro Cys Leu His His Ser Gln Asn Leu Leu Arg Ala Val Ser
565 570 575
Asn Met Leu Gln Lys Ala Arg Gln Thr Leu Glu Phe Tyr Pro Cys Thr
580 585 590
Ser Glu Glu Ile Asp His Glu Asp Ile Thr Lys Asp Lys Thr Ser Thr
595 600 605
Val Glu Ala Cys Leu Pro Leu Glu Leu Thr Lys Asn Glu Ser Cys Leu
610 615 620
Asn Ser Arg Glu Thr Ser Phe Ile Thr Asn Gly Ser Cys Leu Ala Ser
625 630 635 640
Arg Lys Thr Ser Phe Met Met Ala Leu Cys Leu Ser Ser Ile Tyr Glu
645 650 655
Asp Leu Lys Met Tyr Gln Val Glu Phe Lys Thr Met Asn Ala Lys Leu
660 665 670
Leu Met Asp Pro Lys Arg Gln Ile Phe Leu Asp Gln Asn Met Leu Ala
675 680 685
Val Ile Asp Glu Leu Met Gln Ala Leu Asn Phe Asn Ser Glu Thr Val
690 695 700
Pro Gln Lys Ser Ser Leu Glu Glu Pro Asp Phe Tyr Lys Thr Lys Ile
705 710 715 720
Lys Leu Cys Ile Leu Leu His Ala Phe Arg Ile Arg Ala Val Thr Ile
725 730 735
Asp Arg Val Met Ser Tyr Leu Asn Ala Ser Asp Lys Thr His Thr Cys
740 745 750
Pro Pro Cys Pro Ala Pro Glu Asp Glu Gly Gly Pro Ser Val Phe Leu
755 760 765
Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu
770 775 780
Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys
785 790 795 800
Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys
805 810 815
Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu
820 825 830
Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys
835 840 845
Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys
850 855 860
Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Val Tyr Pro Pro Ser
865 870 875 880
Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys
885 890 895
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln
900 905 910
Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Glu Asp Gly
915 920 925
Ser Phe Ala Leu Val Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln
930 935 940
Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn
945 950 955 960
His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
965 970
<210> 152
<211> 973
<212> PRT
<213> artificial sequence
<220>
<223> synthetic construct
<400> 152
Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg
1 5 10 15
Ser Leu Arg Leu Asp Cys Lys Ala Ser Gly Ile Thr Phe Ser Asn Ser
20 25 30
Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Val Ile Trp Tyr Asp Gly Ser Lys Arg Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Phe
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Thr Gly Asp Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser
100 105 110
Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser
115 120 125
Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp
130 135 140
Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr
145 150 155 160
Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr
165 170 175
Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln
180 185 190
Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp
195 200 205
Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Pro Gly Ser Gly Ile Trp
210 215 220
Glu Leu Lys Lys Asp Val Tyr Val Val Glu Leu Asp Trp Tyr Pro Asp
225 230 235 240
Ala Pro Gly Glu Met Val Val Leu Thr Cys Asp Thr Pro Glu Glu Asp
245 250 255
Gly Ile Thr Trp Thr Leu Asp Gln Ser Ser Glu Val Leu Gly Ser Gly
260 265 270
Lys Thr Leu Thr Ile Gln Val Lys Ala Ala Gly Asp Ala Gly Gln Tyr
275 280 285
Thr Cys His Lys Gly Gly Glu Val Leu Ser His Ser Leu Leu Leu Leu
290 295 300
His Lys Lys Glu Asp Gly Ile Trp Ser Thr Asp Ile Leu Lys Asp Gln
305 310 315 320
Lys Glu Pro Lys Asn Lys Thr Phe Leu Arg Cys Glu Ala Lys Asn Tyr
325 330 335
Ser Gly Arg Phe Thr Cys Trp Trp Leu Thr Thr Ile Ser Thr Asp Leu
340 345 350
Thr Phe Ser Val Lys Ser Ser Arg Gly Ser Ser Asp Pro Gln Gly Val
355 360 365
Thr Cys Gly Ala Ala Thr Leu Ser Ala Glu Arg Val Arg Gly Asp Asn
370 375 380
Lys Glu Tyr Glu Tyr Ser Val Glu Cys Gln Glu Asp Ser Ala Cys Pro
385 390 395 400
Ala Ala Glu Glu Ser Leu Pro Ile Glu Val Met Val Asp Ala Val His
405 410 415
Lys Leu Lys Tyr Glu Asn Tyr Thr Ser Ser Phe Phe Ile Arg Asp Ile
420 425 430
Ile Lys Pro Asp Pro Pro Lys Asn Leu Gln Leu Lys Pro Leu Lys Asn
435 440 445
Ser Arg Gln Val Glu Val Ser Trp Glu Tyr Pro Asp Thr Trp Ser Thr
450 455 460
Pro His Ser Tyr Phe Ser Leu Thr Phe Cys Val Gln Val Gln Gly Lys
465 470 475 480
Ser Lys Arg Glu Lys Lys Asp Arg Val Phe Thr Asp Lys Thr Ser Ala
485 490 495
Thr Val Ile Cys Arg Lys Asn Ala Ser Ile Ser Val Arg Ala Gln Asp
500 505 510
Arg Tyr Tyr Ser Ser Ser Trp Ser Glu Trp Ala Ser Val Pro Cys Ser
515 520 525
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly
530 535 540
Gly Gly Gly Ser Gly Arg Asn Leu Pro Val Ala Thr Pro Asp Pro Gly
545 550 555 560
Met Phe Pro Cys Leu His His Ser Gln Asn Leu Leu Arg Ala Val Ser
565 570 575
Asn Met Leu Gln Lys Ala Arg Gln Thr Leu Glu Phe Tyr Pro Cys Thr
580 585 590
Ser Glu Glu Ile Asp His Glu Asp Ile Thr Lys Asp Lys Thr Ser Thr
595 600 605
Val Glu Ala Cys Leu Pro Leu Glu Leu Thr Lys Asn Glu Ser Cys Leu
610 615 620
Asn Ser Arg Glu Thr Ser Phe Ile Thr Asn Gly Ser Cys Leu Ala Ser
625 630 635 640
Arg Lys Thr Ser Phe Met Met Ala Leu Cys Leu Ser Ser Ile Tyr Glu
645 650 655
Asp Leu Lys Met Tyr Gln Val Glu Phe Lys Thr Met Asn Ala Lys Leu
660 665 670
Leu Met Asp Pro Lys Arg Gln Ile Phe Leu Asp Gln Asn Met Leu Ala
675 680 685
Val Ile Asp Glu Leu Met Gln Ala Leu Asn Phe Asn Ser Glu Thr Val
690 695 700
Pro Gln Lys Ser Ser Leu Glu Glu Pro Asp Phe Tyr Lys Thr Lys Ile
705 710 715 720
Lys Leu Cys Ile Leu Leu His Ala Phe Arg Ile Arg Ala Val Thr Ile
725 730 735
Asp Arg Val Met Ser Tyr Leu Asn Ala Ser Asp Lys Thr His Thr Cys
740 745 750
Pro Pro Cys Pro Ala Pro Glu Asp Glu Gly Gly Pro Ser Val Phe Leu
755 760 765
Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu
770 775 780
Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys
785 790 795 800
Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys
805 810 815
Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu
820 825 830
Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys
835 840 845
Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys
850 855 860
Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Val Tyr Pro Pro Ser
865 870 875 880
Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys
885 890 895
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln
900 905 910
Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Glu Asp Gly
915 920 925
Ser Phe Ala Leu Val Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln
930 935 940
Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn
945 950 955 960
His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
965 970
<210> 153
<211> 973
<212> PRT
<213> artificial sequence
<220>
<223> synthetic construct
<400> 153
Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg
1 5 10 15
Ser Leu Arg Leu Asp Cys Lys Ala Ser Gly Ile Thr Phe Ser Asn Ser
20 25 30
Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Val Ile Trp Tyr Asp Gly Ser Lys Arg Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Phe
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Thr Ala Asp Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser
100 105 110
Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser
115 120 125
Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp
130 135 140
Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr
145 150 155 160
Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr
165 170 175
Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln
180 185 190
Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp
195 200 205
Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Pro Gly Ser Gly Ile Trp
210 215 220
Glu Leu Lys Lys Asp Val Tyr Val Val Glu Leu Asp Trp Tyr Pro Asp
225 230 235 240
Ala Pro Gly Glu Met Val Val Leu Thr Cys Asp Thr Pro Glu Glu Asp
245 250 255
Gly Ile Thr Trp Thr Leu Asp Gln Ser Ser Glu Val Leu Gly Ser Gly
260 265 270
Lys Thr Leu Thr Ile Gln Val Lys Ala Ala Gly Asp Ala Gly Gln Tyr
275 280 285
Thr Cys His Lys Gly Gly Glu Val Leu Ser His Ser Leu Leu Leu Leu
290 295 300
His Lys Lys Glu Asp Gly Ile Trp Ser Thr Asp Ile Leu Lys Asp Gln
305 310 315 320
Lys Glu Pro Lys Asn Lys Thr Phe Leu Arg Cys Glu Ala Lys Asn Tyr
325 330 335
Ser Gly Arg Phe Thr Cys Trp Trp Leu Thr Thr Ile Ser Thr Asp Leu
340 345 350
Thr Phe Ser Val Lys Ser Ser Arg Gly Ser Ser Asp Pro Gln Gly Val
355 360 365
Thr Cys Gly Ala Ala Thr Leu Ser Ala Glu Arg Val Arg Gly Asp Asn
370 375 380
Lys Glu Tyr Glu Tyr Ser Val Glu Cys Gln Glu Asp Ser Ala Cys Pro
385 390 395 400
Ala Ala Glu Glu Ser Leu Pro Ile Glu Val Met Val Asp Ala Val His
405 410 415
Lys Leu Lys Tyr Glu Asn Tyr Thr Ser Ser Phe Phe Ile Arg Asp Ile
420 425 430
Ile Lys Pro Asp Pro Pro Lys Asn Leu Gln Leu Lys Pro Leu Lys Asn
435 440 445
Ser Arg Gln Val Glu Val Ser Trp Glu Tyr Pro Asp Thr Trp Ser Thr
450 455 460
Pro His Ser Tyr Phe Ser Leu Thr Phe Cys Val Gln Val Gln Gly Lys
465 470 475 480
Ser Lys Arg Glu Lys Lys Asp Arg Val Phe Thr Asp Lys Thr Ser Ala
485 490 495
Thr Val Ile Cys Arg Lys Asn Ala Ser Ile Ser Val Arg Ala Gln Asp
500 505 510
Arg Tyr Tyr Ser Ser Ser Trp Ser Glu Trp Ala Ser Val Pro Cys Ser
515 520 525
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly
530 535 540
Gly Gly Gly Ser Gly Arg Asn Leu Pro Val Ala Thr Pro Asp Pro Gly
545 550 555 560
Met Phe Pro Cys Leu His His Ser Gln Asn Leu Leu Arg Ala Val Ser
565 570 575
Asn Met Leu Gln Lys Ala Arg Gln Thr Leu Glu Phe Tyr Pro Cys Thr
580 585 590
Ser Glu Glu Ile Asp His Glu Asp Ile Thr Lys Asp Lys Thr Ser Thr
595 600 605
Val Glu Ala Cys Leu Pro Leu Glu Leu Thr Lys Asn Glu Ser Cys Leu
610 615 620
Asn Ser Arg Glu Thr Ser Phe Ile Thr Asn Gly Ser Cys Leu Ala Ser
625 630 635 640
Arg Lys Thr Ser Phe Met Met Ala Leu Cys Leu Ser Ser Ile Tyr Glu
645 650 655
Asp Leu Lys Met Tyr Gln Val Glu Phe Lys Thr Met Asn Ala Lys Leu
660 665 670
Leu Met Asp Pro Lys Arg Gln Ile Phe Leu Asp Gln Asn Met Leu Ala
675 680 685
Val Ile Asp Glu Leu Met Gln Ala Leu Asn Phe Asn Ser Glu Thr Val
690 695 700
Pro Gln Lys Ser Ser Leu Glu Glu Pro Asp Phe Tyr Lys Thr Lys Ile
705 710 715 720
Lys Leu Cys Ile Leu Leu His Ala Phe Arg Ile Arg Ala Val Thr Ile
725 730 735
Asp Arg Val Met Ser Tyr Leu Asn Ala Ser Asp Lys Thr His Thr Cys
740 745 750
Pro Pro Cys Pro Ala Pro Glu Asp Glu Gly Gly Pro Ser Val Phe Leu
755 760 765
Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu
770 775 780
Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys
785 790 795 800
Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys
805 810 815
Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu
820 825 830
Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys
835 840 845
Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys
850 855 860
Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Val Tyr Pro Pro Ser
865 870 875 880
Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys
885 890 895
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln
900 905 910
Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Glu Asp Gly
915 920 925
Ser Phe Ala Leu Val Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln
930 935 940
Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn
945 950 955 960
His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
965 970
<210> 154
<211> 973
<212> PRT
<213> artificial sequence
<220>
<223> synthetic construct
<400> 154
Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg
1 5 10 15
Ser Leu Arg Leu Asp Cys Lys Ala Ser Gly Ile Thr Phe Ser Asn Ser
20 25 30
Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Val Ile Trp Tyr Asp Gly Ser Lys Arg Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Phe
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Thr Met Asp Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser
100 105 110
Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser
115 120 125
Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp
130 135 140
Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr
145 150 155 160
Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr
165 170 175
Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln
180 185 190
Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp
195 200 205
Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Pro Gly Ser Gly Ile Trp
210 215 220
Glu Leu Lys Lys Asp Val Tyr Val Val Glu Leu Asp Trp Tyr Pro Asp
225 230 235 240
Ala Pro Gly Glu Met Val Val Leu Thr Cys Asp Thr Pro Glu Glu Asp
245 250 255
Gly Ile Thr Trp Thr Leu Asp Gln Ser Ser Glu Val Leu Gly Ser Gly
260 265 270
Lys Thr Leu Thr Ile Gln Val Lys Ala Ala Gly Asp Ala Gly Gln Tyr
275 280 285
Thr Cys His Lys Gly Gly Glu Val Leu Ser His Ser Leu Leu Leu Leu
290 295 300
His Lys Lys Glu Asp Gly Ile Trp Ser Thr Asp Ile Leu Lys Asp Gln
305 310 315 320
Lys Glu Pro Lys Asn Lys Thr Phe Leu Arg Cys Glu Ala Lys Asn Tyr
325 330 335
Ser Gly Arg Phe Thr Cys Trp Trp Leu Thr Thr Ile Ser Thr Asp Leu
340 345 350
Thr Phe Ser Val Lys Ser Ser Arg Gly Ser Ser Asp Pro Gln Gly Val
355 360 365
Thr Cys Gly Ala Ala Thr Leu Ser Ala Glu Arg Val Arg Gly Asp Asn
370 375 380
Lys Glu Tyr Glu Tyr Ser Val Glu Cys Gln Glu Asp Ser Ala Cys Pro
385 390 395 400
Ala Ala Glu Glu Ser Leu Pro Ile Glu Val Met Val Asp Ala Val His
405 410 415
Lys Leu Lys Tyr Glu Asn Tyr Thr Ser Ser Phe Phe Ile Arg Asp Ile
420 425 430
Ile Lys Pro Asp Pro Pro Lys Asn Leu Gln Leu Lys Pro Leu Lys Asn
435 440 445
Ser Arg Gln Val Glu Val Ser Trp Glu Tyr Pro Asp Thr Trp Ser Thr
450 455 460
Pro His Ser Tyr Phe Ser Leu Thr Phe Cys Val Gln Val Gln Gly Lys
465 470 475 480
Ser Lys Arg Glu Lys Lys Asp Arg Val Phe Thr Asp Lys Thr Ser Ala
485 490 495
Thr Val Ile Cys Arg Lys Asn Ala Ser Ile Ser Val Arg Ala Gln Asp
500 505 510
Arg Tyr Tyr Ser Ser Ser Trp Ser Glu Trp Ala Ser Val Pro Cys Ser
515 520 525
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly
530 535 540
Gly Gly Gly Ser Gly Arg Asn Leu Pro Val Ala Thr Pro Asp Pro Gly
545 550 555 560
Met Phe Pro Cys Leu His His Ser Gln Asn Leu Leu Arg Ala Val Ser
565 570 575
Asn Met Leu Gln Lys Ala Arg Gln Thr Leu Glu Phe Tyr Pro Cys Thr
580 585 590
Ser Glu Glu Ile Asp His Glu Asp Ile Thr Lys Asp Lys Thr Ser Thr
595 600 605
Val Glu Ala Cys Leu Pro Leu Glu Leu Thr Lys Asn Glu Ser Cys Leu
610 615 620
Asn Ser Arg Glu Thr Ser Phe Ile Thr Asn Gly Ser Cys Leu Ala Ser
625 630 635 640
Arg Lys Thr Ser Phe Met Met Ala Leu Cys Leu Ser Ser Ile Tyr Glu
645 650 655
Asp Leu Lys Met Tyr Gln Val Glu Phe Lys Thr Met Asn Ala Lys Leu
660 665 670
Leu Met Asp Pro Lys Arg Gln Ile Phe Leu Asp Gln Asn Met Leu Ala
675 680 685
Val Ile Asp Glu Leu Met Gln Ala Leu Asn Phe Asn Ser Glu Thr Val
690 695 700
Pro Gln Lys Ser Ser Leu Glu Glu Pro Asp Phe Tyr Lys Thr Lys Ile
705 710 715 720
Lys Leu Cys Ile Leu Leu His Ala Phe Arg Ile Arg Ala Val Thr Ile
725 730 735
Asp Arg Val Met Ser Tyr Leu Asn Ala Ser Asp Lys Thr His Thr Cys
740 745 750
Pro Pro Cys Pro Ala Pro Glu Asp Glu Gly Gly Pro Ser Val Phe Leu
755 760 765
Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu
770 775 780
Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys
785 790 795 800
Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys
805 810 815
Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu
820 825 830
Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys
835 840 845
Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys
850 855 860
Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Val Tyr Pro Pro Ser
865 870 875 880
Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys
885 890 895
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln
900 905 910
Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Glu Asp Gly
915 920 925
Ser Phe Ala Leu Val Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln
930 935 940
Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn
945 950 955 960
His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
965 970
<210> 155
<211> 980
<212> PRT
<213> Homo sapiens (Homo sapiens)
<400> 155
Phe Thr Val Thr Val Pro Lys Asp Leu Tyr Val Val Glu Tyr Gly Ser
1 5 10 15
Asn Met Thr Ile Glu Cys Lys Phe Pro Val Glu Lys Gln Leu Asp Leu
20 25 30
Ala Ala Leu Gln Val Tyr Trp Met Met Glu Asp Lys Asn Ile Ile Gln
35 40 45
Phe Val His Gly Glu Glu Asp Leu Lys Val Gln His Ser Ser Tyr Arg
50 55 60
Gln Arg Ala Arg Leu Leu Lys Asp Gln Leu Ser Leu Gly Asn Ala Ala
65 70 75 80
Leu Gln Ile Thr Asp Val Lys Leu Gln Asp Ala Gly Val Tyr Thr Cys
85 90 95
Leu Ile Ala Tyr Lys Gly Ala Asp Tyr Lys Arg Ile Thr Val Lys Val
100 105 110
Asn Ala Pro Tyr Asn Lys Ile Asn Gln Arg Ile Leu Val Val Asp Pro
115 120 125
Val Thr Ser Glu His Glu Leu Thr Cys Gln Ala Glu Gly Tyr Pro Lys
130 135 140
Ala Glu Val Ile Trp Thr Ser Ser Asp His Gln Val Leu Ser Gly Lys
145 150 155 160
Thr Thr Thr Thr Asn Ser Lys Arg Glu Glu Lys Leu Phe Asn Val Thr
165 170 175
Ser Thr Leu Arg Ile Asn Thr Thr Thr Asn Glu Ile Phe Tyr Cys Thr
180 185 190
Phe Arg Arg Leu Asp Pro Glu Glu Asn His Thr Ala Glu Leu Val Ile
195 200 205
Pro Glu Leu Pro Leu Ala His Pro Pro Asn Glu Arg Gly Gly Gly Gly
210 215 220
Ser Gly Gly Gly Ile Trp Glu Leu Lys Lys Asp Val Tyr Val Val Glu
225 230 235 240
Leu Asp Trp Tyr Pro Asp Ala Pro Gly Glu Met Val Val Leu Thr Cys
245 250 255
Asp Thr Pro Glu Glu Asp Gly Ile Thr Trp Thr Leu Asp Gln Ser Ser
260 265 270
Glu Val Leu Gly Ser Gly Lys Thr Leu Thr Ile Gln Val Lys Ala Ala
275 280 285
Gly Asp Ala Gly Gln Tyr Thr Cys His Lys Gly Gly Glu Val Leu Ser
290 295 300
His Ser Leu Leu Leu Leu His Lys Lys Glu Asp Gly Ile Trp Ser Thr
305 310 315 320
Asp Ile Leu Lys Asp Gln Lys Glu Pro Lys Asn Lys Thr Phe Leu Arg
325 330 335
Cys Glu Ala Lys Asn Tyr Ser Gly Arg Phe Thr Cys Trp Trp Leu Thr
340 345 350
Thr Ile Ser Thr Asp Leu Thr Phe Ser Val Lys Ser Ser Arg Gly Ser
355 360 365
Ser Asp Pro Gln Gly Val Thr Cys Gly Ala Ala Thr Leu Ser Ala Glu
370 375 380
Arg Val Arg Gly Asp Asn Lys Glu Tyr Glu Tyr Ser Val Glu Cys Gln
385 390 395 400
Glu Asp Ser Ala Cys Pro Ala Ala Glu Glu Ser Leu Pro Ile Glu Val
405 410 415
Met Val Asp Ala Val His Lys Leu Lys Tyr Glu Asn Tyr Thr Ser Ser
420 425 430
Phe Phe Ile Arg Asp Ile Ile Lys Pro Asp Pro Pro Lys Asn Leu Gln
435 440 445
Leu Lys Pro Leu Lys Asn Ser Arg Gln Val Glu Val Ser Trp Glu Tyr
450 455 460
Pro Asp Thr Trp Ser Thr Pro His Ser Tyr Phe Ser Leu Thr Phe Cys
465 470 475 480
Val Gln Val Gln Gly Lys Ser Lys Arg Glu Lys Lys Asp Arg Val Phe
485 490 495
Thr Asp Lys Thr Ser Ala Thr Val Ile Cys Arg Lys Asn Ala Ser Ile
500 505 510
Ser Val Arg Ala Gln Asp Arg Tyr Tyr Ser Ser Ser Trp Ser Glu Trp
515 520 525
Ala Ser Val Pro Cys Ser Gly Gly Gly Gly Ser Gly Gly Gly Ser Gly
530 535 540
Gly Gly Gly Ser Arg Asn Leu Pro Val Ala Thr Pro Asp Pro Gly Met
545 550 555 560
Phe Pro Cys Leu His His Ser Gln Asn Leu Leu Arg Ala Val Ser Asn
565 570 575
Met Leu Gln Lys Ala Arg Gln Thr Leu Glu Phe Tyr Pro Cys Thr Ser
580 585 590
Glu Glu Ile Asp His Glu Asp Ile Thr Lys Asp Lys Thr Ser Thr Val
595 600 605
Glu Ala Cys Leu Pro Leu Glu Leu Thr Lys Asn Glu Ser Cys Leu Asn
610 615 620
Ser Arg Glu Thr Ser Phe Ile Thr Asn Gly Ser Cys Leu Ala Ser Arg
625 630 635 640
Lys Thr Ser Phe Met Met Ala Leu Cys Leu Ser Ser Ile Tyr Glu Asp
645 650 655
Leu Lys Met Tyr Gln Val Glu Phe Lys Thr Met Asn Ala Lys Leu Leu
660 665 670
Met Asp Pro Lys Arg Gln Ile Phe Leu Asp Gln Asn Met Leu Ala Val
675 680 685
Ile Asp Glu Leu Met Gln Ala Leu Asn Phe Asn Ser Glu Thr Val Pro
690 695 700
Gln Lys Ser Ser Leu Glu Glu Pro Asp Phe Tyr Lys Thr Lys Ile Lys
705 710 715 720
Leu Cys Ile Leu Leu His Ala Phe Arg Ile Arg Ala Val Thr Ile Asp
725 730 735
Arg Val Met Ser Tyr Leu Asn Ala Ser Gly Gly Gly Gly Ser Gly Gly
740 745 750
Gly Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Asp Glu
755 760 765
Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu
770 775 780
Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser
785 790 795 800
His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu
805 810 815
Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr
820 825 830
Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn
835 840 845
Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro
850 855 860
Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln
865 870 875 880
Val Tyr Val Tyr Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val
885 890 895
Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val
900 905 910
Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro
915 920 925
Pro Val Leu Asp Glu Asp Gly Ser Phe Ala Leu Val Ser Lys Leu Thr
930 935 940
Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val
945 950 955 960
Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu
965 970 975
Ser Pro Gly Lys
980
<210> 156
<211> 980
<212> PRT
<213> Homo sapiens (Homo sapiens)
<400> 156
Phe Thr Val Thr Val Pro Lys Asp Leu Tyr Val Val Glu Tyr Gly Ser
1 5 10 15
Asn Met Thr Ile Glu Cys Lys Phe Pro Val Glu Lys Gln Leu Asp Leu
20 25 30
Ala Ala Leu Gln Val Tyr Trp Met Met Glu Asp Lys Asn Ile Ile Gln
35 40 45
Phe Val His Gly Glu Glu Asp Leu Lys Val Gln His Ser Ser Tyr Arg
50 55 60
Gln Arg Ala Arg Leu Leu Lys Asp Gln Leu Ser Leu Gly Asn Ala Ala
65 70 75 80
Leu Gln Ile Thr Asp Val Lys Leu Gln Asp Ala Gly Val Tyr Thr Cys
85 90 95
Leu Ile Ala Tyr Gly Gly Ala Asp Tyr Lys Arg Ile Thr Val Lys Val
100 105 110
Asn Ala Pro Tyr Asn Lys Ile Asn Gln Arg Ile Leu Val Val Asp Pro
115 120 125
Val Thr Ser Glu His Glu Leu Thr Cys Gln Ala Glu Gly Tyr Pro Lys
130 135 140
Ala Glu Val Ile Trp Thr Ser Ser Asp His Gln Val Leu Ser Gly Lys
145 150 155 160
Thr Thr Thr Thr Asn Ser Lys Arg Glu Glu Lys Leu Phe Asn Val Thr
165 170 175
Ser Thr Leu Arg Ile Asn Thr Thr Thr Asn Glu Ile Phe Tyr Cys Thr
180 185 190
Phe Arg Arg Leu Asp Pro Glu Glu Asn His Thr Ala Glu Leu Val Ile
195 200 205
Pro Glu Leu Pro Leu Ala His Pro Pro Asn Glu Arg Gly Gly Gly Gly
210 215 220
Ser Gly Gly Gly Ile Trp Glu Leu Lys Lys Asp Val Tyr Val Val Glu
225 230 235 240
Leu Asp Trp Tyr Pro Asp Ala Pro Gly Glu Met Val Val Leu Thr Cys
245 250 255
Asp Thr Pro Glu Glu Asp Gly Ile Thr Trp Thr Leu Asp Gln Ser Ser
260 265 270
Glu Val Leu Gly Ser Gly Lys Thr Leu Thr Ile Gln Val Lys Ala Ala
275 280 285
Gly Asp Ala Gly Gln Tyr Thr Cys His Lys Gly Gly Glu Val Leu Ser
290 295 300
His Ser Leu Leu Leu Leu His Lys Lys Glu Asp Gly Ile Trp Ser Thr
305 310 315 320
Asp Ile Leu Lys Asp Gln Lys Glu Pro Lys Asn Lys Thr Phe Leu Arg
325 330 335
Cys Glu Ala Lys Asn Tyr Ser Gly Arg Phe Thr Cys Trp Trp Leu Thr
340 345 350
Thr Ile Ser Thr Asp Leu Thr Phe Ser Val Lys Ser Ser Arg Gly Ser
355 360 365
Ser Asp Pro Gln Gly Val Thr Cys Gly Ala Ala Thr Leu Ser Ala Glu
370 375 380
Arg Val Arg Gly Asp Asn Lys Glu Tyr Glu Tyr Ser Val Glu Cys Gln
385 390 395 400
Glu Asp Ser Ala Cys Pro Ala Ala Glu Glu Ser Leu Pro Ile Glu Val
405 410 415
Met Val Asp Ala Val His Lys Leu Lys Tyr Glu Asn Tyr Thr Ser Ser
420 425 430
Phe Phe Ile Arg Asp Ile Ile Lys Pro Asp Pro Pro Lys Asn Leu Gln
435 440 445
Leu Lys Pro Leu Lys Asn Ser Arg Gln Val Glu Val Ser Trp Glu Tyr
450 455 460
Pro Asp Thr Trp Ser Thr Pro His Ser Tyr Phe Ser Leu Thr Phe Cys
465 470 475 480
Val Gln Val Gln Gly Lys Ser Lys Arg Glu Lys Lys Asp Arg Val Phe
485 490 495
Thr Asp Lys Thr Ser Ala Thr Val Ile Cys Arg Lys Asn Ala Ser Ile
500 505 510
Ser Val Arg Ala Gln Asp Arg Tyr Tyr Ser Ser Ser Trp Ser Glu Trp
515 520 525
Ala Ser Val Pro Cys Ser Gly Gly Gly Gly Ser Gly Gly Gly Ser Gly
530 535 540
Gly Gly Gly Ser Arg Asn Leu Pro Val Ala Thr Pro Asp Pro Gly Met
545 550 555 560
Phe Pro Cys Leu His His Ser Gln Asn Leu Leu Arg Ala Val Ser Asn
565 570 575
Met Leu Gln Lys Ala Arg Gln Thr Leu Glu Phe Tyr Pro Cys Thr Ser
580 585 590
Glu Glu Ile Asp His Glu Asp Ile Thr Lys Asp Lys Thr Ser Thr Val
595 600 605
Glu Ala Cys Leu Pro Leu Glu Leu Thr Lys Asn Glu Ser Cys Leu Asn
610 615 620
Ser Arg Glu Thr Ser Phe Ile Thr Asn Gly Ser Cys Leu Ala Ser Arg
625 630 635 640
Lys Thr Ser Phe Met Met Ala Leu Cys Leu Ser Ser Ile Tyr Glu Asp
645 650 655
Leu Lys Met Tyr Gln Val Glu Phe Lys Thr Met Asn Ala Lys Leu Leu
660 665 670
Met Asp Pro Lys Arg Gln Ile Phe Leu Asp Gln Asn Met Leu Ala Val
675 680 685
Ile Asp Glu Leu Met Gln Ala Leu Asn Phe Asn Ser Glu Thr Val Pro
690 695 700
Gln Lys Ser Ser Leu Glu Glu Pro Asp Phe Tyr Lys Thr Lys Ile Lys
705 710 715 720
Leu Cys Ile Leu Leu His Ala Phe Arg Ile Arg Ala Val Thr Ile Asp
725 730 735
Arg Val Met Ser Tyr Leu Asn Ala Ser Gly Gly Gly Gly Ser Gly Gly
740 745 750
Gly Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Asp Glu
755 760 765
Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu
770 775 780
Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser
785 790 795 800
His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu
805 810 815
Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr
820 825 830
Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn
835 840 845
Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro
850 855 860
Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln
865 870 875 880
Val Tyr Val Tyr Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val
885 890 895
Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val
900 905 910
Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro
915 920 925
Pro Val Leu Asp Glu Asp Gly Ser Phe Ala Leu Val Ser Lys Leu Thr
930 935 940
Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val
945 950 955 960
Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu
965 970 975
Ser Pro Gly Lys
980
<210> 157
<211> 980
<212> PRT
<213> Homo sapiens (Homo sapiens)
<400> 157
Phe Thr Val Thr Val Pro Lys Asp Leu Tyr Val Val Glu Tyr Gly Ser
1 5 10 15
Asn Met Thr Ile Glu Cys Lys Phe Pro Val Glu Lys Gln Leu Asp Leu
20 25 30
Ala Ala Leu Gln Val Tyr Trp Met Met Glu Asp Lys Asn Ile Ile Gln
35 40 45
Phe Val His Gly Glu Glu Asp Leu Lys Val Gln His Ser Ser Tyr Arg
50 55 60
Gln Arg Ala Arg Leu Leu Lys Asp Gln Leu Ser Leu Gly Asn Ala Ala
65 70 75 80
Leu Gln Ile Thr Asp Val Lys Leu Gln Asp Ala Gly Val Tyr Thr Cys
85 90 95
Leu Ile Ala Tyr Lys Gly Ala Asp Tyr Lys Arg Ile Thr Val Lys Val
100 105 110
Asn Ala Pro Tyr Asn Lys Ile Asn Gln Arg Ile Leu Val Val Asp Pro
115 120 125
Val Thr Ser Glu His Glu Leu Thr Cys Gln Ala Glu Gly Tyr Pro Lys
130 135 140
Ala Glu Val Ile Trp Thr Ser Ser Asp His Gln Val Leu Ser Gly Lys
145 150 155 160
Thr Thr Thr Thr Asn Ser Lys Arg Glu Glu Lys Leu Phe Asn Val Thr
165 170 175
Ser Thr Leu Arg Ile Asn Thr Thr Thr Asn Glu Ile Phe Tyr Cys Thr
180 185 190
Phe Arg Arg Leu Asp Pro Glu Glu Asn His Thr Ala Glu Leu Val Ile
195 200 205
Pro Glu Leu Pro Leu Ala His Pro Pro Asn Glu Arg Gly Gly Gly Gly
210 215 220
Ser Gly Gly Gly Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro
225 230 235 240
Glu Asp Glu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys
245 250 255
Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val
260 265 270
Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp
275 280 285
Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr
290 295 300
Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp
305 310 315 320
Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu
325 330 335
Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg
340 345 350
Glu Pro Gln Val Tyr Val Tyr Pro Pro Ser Arg Glu Glu Met Thr Lys
355 360 365
Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp
370 375 380
Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
385 390 395 400
Thr Thr Pro Pro Val Leu Asp Glu Asp Gly Ser Phe Ala Leu Val Ser
405 410 415
Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser
420 425 430
Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser
435 440 445
Leu Ser Leu Ser Pro Gly Lys Gly Gly Gly Gly Ser Gly Gly Gly Ile
450 455 460
Trp Glu Leu Lys Lys Asp Val Tyr Val Val Glu Leu Asp Trp Tyr Pro
465 470 475 480
Asp Ala Pro Gly Glu Met Val Val Leu Thr Cys Asp Thr Pro Glu Glu
485 490 495
Asp Gly Ile Thr Trp Thr Leu Asp Gln Ser Ser Glu Val Leu Gly Ser
500 505 510
Gly Lys Thr Leu Thr Ile Gln Val Lys Ala Ala Gly Asp Ala Gly Gln
515 520 525
Tyr Thr Cys His Lys Gly Gly Glu Val Leu Ser His Ser Leu Leu Leu
530 535 540
Leu His Lys Lys Glu Asp Gly Ile Trp Ser Thr Asp Ile Leu Lys Asp
545 550 555 560
Gln Lys Glu Pro Lys Asn Lys Thr Phe Leu Arg Cys Glu Ala Lys Asn
565 570 575
Tyr Ser Gly Arg Phe Thr Cys Trp Trp Leu Thr Thr Ile Ser Thr Asp
580 585 590
Leu Thr Phe Ser Val Lys Ser Ser Arg Gly Ser Ser Asp Pro Gln Gly
595 600 605
Val Thr Cys Gly Ala Ala Thr Leu Ser Ala Glu Arg Val Arg Gly Asp
610 615 620
Asn Lys Glu Tyr Glu Tyr Ser Val Glu Cys Gln Glu Asp Ser Ala Cys
625 630 635 640
Pro Ala Ala Glu Glu Ser Leu Pro Ile Glu Val Met Val Asp Ala Val
645 650 655
His Lys Leu Lys Tyr Glu Asn Tyr Thr Ser Ser Phe Phe Ile Arg Asp
660 665 670
Ile Ile Lys Pro Asp Pro Pro Lys Asn Leu Gln Leu Lys Pro Leu Lys
675 680 685
Asn Ser Arg Gln Val Glu Val Ser Trp Glu Tyr Pro Asp Thr Trp Ser
690 695 700
Thr Pro His Ser Tyr Phe Ser Leu Thr Phe Cys Val Gln Val Gln Gly
705 710 715 720
Lys Ser Lys Arg Glu Lys Lys Asp Arg Val Phe Thr Asp Lys Thr Ser
725 730 735
Ala Thr Val Ile Cys Arg Lys Asn Ala Ser Ile Ser Val Arg Ala Gln
740 745 750
Asp Arg Tyr Tyr Ser Ser Ser Trp Ser Glu Trp Ala Ser Val Pro Cys
755 760 765
Ser Gly Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Gly Ser Arg
770 775 780
Asn Leu Pro Val Ala Thr Pro Asp Pro Gly Met Phe Pro Cys Leu His
785 790 795 800
His Ser Gln Asn Leu Leu Arg Ala Val Ser Asn Met Leu Gln Lys Ala
805 810 815
Arg Gln Thr Leu Glu Phe Tyr Pro Cys Thr Ser Glu Glu Ile Asp His
820 825 830
Glu Asp Ile Thr Lys Asp Lys Thr Ser Thr Val Glu Ala Cys Leu Pro
835 840 845
Leu Glu Leu Thr Lys Asn Glu Ser Cys Leu Asn Ser Arg Glu Thr Ser
850 855 860
Phe Ile Thr Asn Gly Ser Cys Leu Ala Ser Arg Lys Thr Ser Phe Met
865 870 875 880
Met Ala Leu Cys Leu Ser Ser Ile Tyr Glu Asp Leu Lys Met Tyr Gln
885 890 895
Val Glu Phe Lys Thr Met Asn Ala Lys Leu Leu Met Asp Pro Lys Arg
900 905 910
Gln Ile Phe Leu Asp Gln Asn Met Leu Ala Val Ile Asp Glu Leu Met
915 920 925
Gln Ala Leu Asn Phe Asn Ser Glu Thr Val Pro Gln Lys Ser Ser Leu
930 935 940
Glu Glu Pro Asp Phe Tyr Lys Thr Lys Ile Lys Leu Cys Ile Leu Leu
945 950 955 960
His Ala Phe Arg Ile Arg Ala Val Thr Ile Asp Arg Val Met Ser Tyr
965 970 975
Leu Asn Ala Ser
980
<210> 158
<211> 980
<212> PRT
<213> Homo sapiens (Homo sapiens)
<400> 158
Phe Thr Val Thr Val Pro Lys Asp Leu Tyr Val Val Glu Tyr Gly Ser
1 5 10 15
Asn Met Thr Ile Glu Cys Lys Phe Pro Val Glu Lys Gln Leu Asp Leu
20 25 30
Ala Ala Leu Gln Val Tyr Trp Met Met Glu Asp Lys Asn Ile Ile Gln
35 40 45
Phe Val His Gly Glu Glu Asp Leu Lys Val Gln His Ser Ser Tyr Arg
50 55 60
Gln Arg Ala Arg Leu Leu Lys Asp Gln Leu Ser Leu Gly Asn Ala Ala
65 70 75 80
Leu Gln Ile Thr Asp Val Lys Leu Gln Asp Ala Gly Val Tyr Thr Cys
85 90 95
Leu Ile Ala Tyr Gly Gly Ala Asp Tyr Lys Arg Ile Thr Val Lys Val
100 105 110
Asn Ala Pro Tyr Asn Lys Ile Asn Gln Arg Ile Leu Val Val Asp Pro
115 120 125
Val Thr Ser Glu His Glu Leu Thr Cys Gln Ala Glu Gly Tyr Pro Lys
130 135 140
Ala Glu Val Ile Trp Thr Ser Ser Asp His Gln Val Leu Ser Gly Lys
145 150 155 160
Thr Thr Thr Thr Asn Ser Lys Arg Glu Glu Lys Leu Phe Asn Val Thr
165 170 175
Ser Thr Leu Arg Ile Asn Thr Thr Thr Asn Glu Ile Phe Tyr Cys Thr
180 185 190
Phe Arg Arg Leu Asp Pro Glu Glu Asn His Thr Ala Glu Leu Val Ile
195 200 205
Pro Glu Leu Pro Leu Ala His Pro Pro Asn Glu Arg Gly Gly Gly Gly
210 215 220
Ser Gly Gly Gly Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro
225 230 235 240
Glu Asp Glu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys
245 250 255
Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val
260 265 270
Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp
275 280 285
Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr
290 295 300
Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp
305 310 315 320
Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu
325 330 335
Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg
340 345 350
Glu Pro Gln Val Tyr Val Tyr Pro Pro Ser Arg Glu Glu Met Thr Lys
355 360 365
Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp
370 375 380
Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
385 390 395 400
Thr Thr Pro Pro Val Leu Asp Glu Asp Gly Ser Phe Ala Leu Val Ser
405 410 415
Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser
420 425 430
Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser
435 440 445
Leu Ser Leu Ser Pro Gly Lys Gly Gly Gly Gly Ser Gly Gly Gly Ile
450 455 460
Trp Glu Leu Lys Lys Asp Val Tyr Val Val Glu Leu Asp Trp Tyr Pro
465 470 475 480
Asp Ala Pro Gly Glu Met Val Val Leu Thr Cys Asp Thr Pro Glu Glu
485 490 495
Asp Gly Ile Thr Trp Thr Leu Asp Gln Ser Ser Glu Val Leu Gly Ser
500 505 510
Gly Lys Thr Leu Thr Ile Gln Val Lys Ala Ala Gly Asp Ala Gly Gln
515 520 525
Tyr Thr Cys His Lys Gly Gly Glu Val Leu Ser His Ser Leu Leu Leu
530 535 540
Leu His Lys Lys Glu Asp Gly Ile Trp Ser Thr Asp Ile Leu Lys Asp
545 550 555 560
Gln Lys Glu Pro Lys Asn Lys Thr Phe Leu Arg Cys Glu Ala Lys Asn
565 570 575
Tyr Ser Gly Arg Phe Thr Cys Trp Trp Leu Thr Thr Ile Ser Thr Asp
580 585 590
Leu Thr Phe Ser Val Lys Ser Ser Arg Gly Ser Ser Asp Pro Gln Gly
595 600 605
Val Thr Cys Gly Ala Ala Thr Leu Ser Ala Glu Arg Val Arg Gly Asp
610 615 620
Asn Lys Glu Tyr Glu Tyr Ser Val Glu Cys Gln Glu Asp Ser Ala Cys
625 630 635 640
Pro Ala Ala Glu Glu Ser Leu Pro Ile Glu Val Met Val Asp Ala Val
645 650 655
His Lys Leu Lys Tyr Glu Asn Tyr Thr Ser Ser Phe Phe Ile Arg Asp
660 665 670
Ile Ile Lys Pro Asp Pro Pro Lys Asn Leu Gln Leu Lys Pro Leu Lys
675 680 685
Asn Ser Arg Gln Val Glu Val Ser Trp Glu Tyr Pro Asp Thr Trp Ser
690 695 700
Thr Pro His Ser Tyr Phe Ser Leu Thr Phe Cys Val Gln Val Gln Gly
705 710 715 720
Lys Ser Lys Arg Glu Lys Lys Asp Arg Val Phe Thr Asp Lys Thr Ser
725 730 735
Ala Thr Val Ile Cys Arg Lys Asn Ala Ser Ile Ser Val Arg Ala Gln
740 745 750
Asp Arg Tyr Tyr Ser Ser Ser Trp Ser Glu Trp Ala Ser Val Pro Cys
755 760 765
Ser Gly Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Gly Ser Arg
770 775 780
Asn Leu Pro Val Ala Thr Pro Asp Pro Gly Met Phe Pro Cys Leu His
785 790 795 800
His Ser Gln Asn Leu Leu Arg Ala Val Ser Asn Met Leu Gln Lys Ala
805 810 815
Arg Gln Thr Leu Glu Phe Tyr Pro Cys Thr Ser Glu Glu Ile Asp His
820 825 830
Glu Asp Ile Thr Lys Asp Lys Thr Ser Thr Val Glu Ala Cys Leu Pro
835 840 845
Leu Glu Leu Thr Lys Asn Glu Ser Cys Leu Asn Ser Arg Glu Thr Ser
850 855 860
Phe Ile Thr Asn Gly Ser Cys Leu Ala Ser Arg Lys Thr Ser Phe Met
865 870 875 880
Met Ala Leu Cys Leu Ser Ser Ile Tyr Glu Asp Leu Lys Met Tyr Gln
885 890 895
Val Glu Phe Lys Thr Met Asn Ala Lys Leu Leu Met Asp Pro Lys Arg
900 905 910
Gln Ile Phe Leu Asp Gln Asn Met Leu Ala Val Ile Asp Glu Leu Met
915 920 925
Gln Ala Leu Asn Phe Asn Ser Glu Thr Val Pro Gln Lys Ser Ser Leu
930 935 940
Glu Glu Pro Asp Phe Tyr Lys Thr Lys Ile Lys Leu Cys Ile Leu Leu
945 950 955 960
His Ala Phe Arg Ile Arg Ala Val Thr Ile Asp Arg Val Met Ser Tyr
965 970 975
Leu Asn Ala Ser
980
<210> 159
<211> 593
<212> PRT
<213> Homo sapiens (Homo sapiens)
<400> 159
Phe Thr Val Thr Val Pro Lys Asp Leu Tyr Val Val Glu Tyr Gly Ser
1 5 10 15
Asn Met Thr Ile Glu Cys Lys Phe Pro Val Glu Lys Gln Leu Asp Leu
20 25 30
Ala Ala Leu Gln Val Tyr Trp Met Met Glu Asp Lys Asn Ile Ile Gln
35 40 45
Phe Val His Gly Glu Glu Asp Leu Lys Val Gln His Ser Ser Tyr Arg
50 55 60
Gln Arg Ala Arg Leu Leu Lys Asp Gln Leu Ser Leu Gly Asn Ala Ala
65 70 75 80
Leu Gln Ile Thr Asp Val Lys Leu Gln Asp Ala Gly Val Tyr Thr Cys
85 90 95
Leu Ile Ala Tyr Lys Gly Ala Asp Tyr Lys Arg Ile Thr Val Lys Val
100 105 110
Asn Ala Pro Tyr Asn Lys Ile Asn Gln Arg Ile Leu Val Val Asp Pro
115 120 125
Val Thr Ser Glu His Glu Leu Thr Cys Gln Ala Glu Gly Tyr Pro Lys
130 135 140
Ala Glu Val Ile Trp Thr Ser Ser Asp His Gln Val Leu Ser Gly Lys
145 150 155 160
Thr Thr Thr Thr Asn Ser Lys Arg Glu Glu Lys Leu Phe Asn Val Thr
165 170 175
Ser Thr Leu Arg Ile Asn Thr Thr Thr Asn Glu Ile Phe Tyr Cys Thr
180 185 190
Phe Arg Arg Leu Asp Pro Glu Glu Asn His Thr Ala Glu Leu Val Ile
195 200 205
Pro Glu Leu Pro Leu Ala His Pro Pro Asn Glu Arg Gly Gly Gly Ser
210 215 220
Gly Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu
225 230 235 240
His Leu Arg Leu Asp Leu Glu Met Ile Leu Asn Gly Ile Asn Asn Tyr
245 250 255
Lys Asn Pro Lys Leu Thr Asp Met Leu Thr Phe Glu Phe Tyr Met Pro
260 265 270
Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Arg Glu Leu
275 280 285
Lys Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His
290 295 300
Leu Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu
305 310 315 320
Leu Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr
325 330 335
Ala Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser
340 345 350
Ile Ile Ser Thr Leu Thr Gly Gly Gly Gly Ser Gly Gly Gly Asp Lys
355 360 365
Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Lys Lys Gly Gly Pro
370 375 380
Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser
385 390 395 400
Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp
405 410 415
Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn
420 425 430
Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val
435 440 445
Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu
450 455 460
Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys
465 470 475 480
Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Val
485 490 495
Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Leu
500 505 510
Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu
515 520 525
Ser Asn Gly Gln Pro Glu Asn Arg Tyr Met Thr Trp Pro Pro Val Leu
530 535 540
Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys
545 550 555 560
Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu
565 570 575
Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly
580 585 590
Lys
<210> 160
<211> 593
<212> PRT
<213> Homo sapiens (Homo sapiens)
<400> 160
Phe Thr Val Thr Val Pro Lys Asp Leu Tyr Val Val Glu Tyr Gly Ser
1 5 10 15
Asn Met Thr Ile Glu Cys Lys Phe Pro Val Glu Lys Gln Leu Asp Leu
20 25 30
Ala Ala Leu Gln Val Tyr Trp Met Met Glu Asp Lys Asn Ile Ile Gln
35 40 45
Phe Val His Gly Glu Glu Asp Leu Lys Val Gln His Ser Ser Tyr Arg
50 55 60
Gln Arg Ala Arg Leu Leu Lys Asp Gln Leu Ser Leu Gly Asn Ala Ala
65 70 75 80
Leu Gln Ile Thr Asp Val Lys Leu Gln Asp Ala Gly Val Tyr Thr Cys
85 90 95
Leu Ile Ala Tyr Lys Gly Ala Asp Tyr Lys Arg Ile Thr Val Lys Val
100 105 110
Asn Ala Pro Tyr Asn Lys Ile Asn Gln Arg Ile Leu Val Val Asp Pro
115 120 125
Val Thr Ser Glu His Glu Leu Thr Cys Gln Ala Glu Gly Tyr Pro Lys
130 135 140
Ala Glu Val Ile Trp Thr Ser Ser Asp His Gln Val Leu Ser Gly Lys
145 150 155 160
Thr Thr Thr Thr Asn Ser Lys Arg Glu Glu Lys Leu Phe Asn Val Thr
165 170 175
Ser Thr Leu Arg Ile Asn Thr Thr Thr Asn Glu Ile Phe Tyr Cys Thr
180 185 190
Phe Arg Arg Leu Asp Pro Glu Glu Asn His Thr Ala Glu Leu Val Ile
195 200 205
Pro Glu Leu Pro Leu Ala His Pro Pro Asn Glu Arg Gly Gly Gly Ser
210 215 220
Gly Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu
225 230 235 240
His Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr
245 250 255
Lys Asn Pro Lys Leu Thr Asp Met Leu Thr Phe Glu Phe Tyr Met Pro
260 265 270
Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Arg Glu Leu
275 280 285
Lys Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His
290 295 300
Leu Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu
305 310 315 320
Leu Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr
325 330 335
Ala Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Thr Ser
340 345 350
Ile Ile Ser Thr Leu Thr Gly Gly Gly Gly Ser Gly Gly Gly Asp Lys
355 360 365
Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Lys Lys Gly Gly Pro
370 375 380
Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser
385 390 395 400
Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp
405 410 415
Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn
420 425 430
Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val
435 440 445
Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu
450 455 460
Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys
465 470 475 480
Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Val
485 490 495
Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Leu
500 505 510
Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu
515 520 525
Ser Asn Gly Gln Pro Glu Asn Arg Tyr Met Thr Trp Pro Pro Val Leu
530 535 540
Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys
545 550 555 560
Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu
565 570 575
Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly
580 585 590
Lys
<210> 161
<211> 593
<212> PRT
<213> Homo sapiens (Homo sapiens)
<400> 161
Phe Thr Val Thr Val Pro Lys Asp Leu Tyr Val Val Glu Tyr Gly Ser
1 5 10 15
Asn Met Thr Ile Glu Cys Lys Phe Pro Val Glu Lys Gln Leu Asp Leu
20 25 30
Ala Ala Leu Gln Val Tyr Trp Met Met Glu Asp Lys Asn Ile Ile Gln
35 40 45
Phe Val His Gly Glu Glu Asp Leu Lys Val Gln His Ser Ser Tyr Arg
50 55 60
Gln Arg Ala Arg Leu Leu Lys Asp Gln Leu Ser Leu Gly Asn Ala Ala
65 70 75 80
Leu Gln Ile Thr Asp Val Lys Leu Gln Asp Ala Gly Val Tyr Thr Cys
85 90 95
Leu Ile Ala Tyr Lys Gly Ala Asp Tyr Lys Arg Ile Thr Val Lys Val
100 105 110
Asn Ala Pro Tyr Asn Lys Ile Asn Gln Arg Ile Leu Val Val Asp Pro
115 120 125
Val Thr Ser Glu His Glu Leu Thr Cys Gln Ala Glu Gly Tyr Pro Lys
130 135 140
Ala Glu Val Ile Trp Thr Ser Ser Asp His Gln Val Leu Ser Gly Lys
145 150 155 160
Thr Thr Thr Thr Asn Ser Lys Arg Glu Glu Lys Leu Phe Asn Val Thr
165 170 175
Ser Thr Leu Arg Ile Asn Thr Thr Thr Asn Glu Ile Phe Tyr Cys Thr
180 185 190
Phe Arg Arg Leu Asp Pro Glu Glu Asn His Thr Ala Glu Leu Val Ile
195 200 205
Pro Glu Leu Pro Leu Ala His Pro Pro Asn Glu Arg Gly Gly Gly Ser
210 215 220
Gly Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu
225 230 235 240
His Leu Arg Leu Asp Leu Glu Met Ile Leu Asn Gly Ile Asn Asn Tyr
245 250 255
Lys Asn Pro Lys Leu Thr Asp Met Leu Thr Phe Glu Phe Tyr Met Pro
260 265 270
Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Arg Glu Leu
275 280 285
Lys Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His
290 295 300
Leu Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu
305 310 315 320
Leu Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr
325 330 335
Ala Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Thr Ser
340 345 350
Ile Ile Ser Thr Leu Thr Gly Gly Gly Gly Ser Gly Gly Gly Asp Lys
355 360 365
Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Lys Lys Gly Gly Pro
370 375 380
Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser
385 390 395 400
Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp
405 410 415
Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn
420 425 430
Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val
435 440 445
Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu
450 455 460
Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys
465 470 475 480
Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Val
485 490 495
Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Leu
500 505 510
Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu
515 520 525
Ser Asn Gly Gln Pro Glu Asn Arg Tyr Met Thr Trp Pro Pro Val Leu
530 535 540
Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys
545 550 555 560
Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu
565 570 575
Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly
580 585 590
Lys
<210> 162
<211> 593
<212> PRT
<213> Homo sapiens (Homo sapiens)
<400> 162
Phe Thr Val Thr Val Pro Lys Asp Leu Tyr Val Val Glu Tyr Gly Ser
1 5 10 15
Asn Met Thr Ile Glu Cys Lys Phe Pro Val Glu Lys Gln Leu Asp Leu
20 25 30
Ala Ala Leu Gln Val Tyr Trp Met Met Glu Asp Lys Asn Ile Ile Gln
35 40 45
Phe Val His Gly Glu Glu Asp Leu Lys Val Gln His Ser Ser Tyr Arg
50 55 60
Gln Arg Ala Arg Leu Leu Lys Asp Gln Leu Ser Leu Gly Asn Ala Ala
65 70 75 80
Leu Gln Ile Thr Asp Val Lys Leu Gln Asp Ala Gly Val Tyr Thr Cys
85 90 95
Leu Ile Ala Tyr Lys Gly Ala Asp Tyr Lys Arg Ile Thr Val Lys Val
100 105 110
Asn Ala Pro Tyr Asn Lys Ile Asn Gln Arg Ile Leu Val Val Asp Pro
115 120 125
Val Thr Ser Glu His Glu Leu Thr Cys Gln Ala Glu Gly Tyr Pro Lys
130 135 140
Ala Glu Val Ile Trp Thr Ser Ser Asp His Gln Val Leu Ser Gly Lys
145 150 155 160
Thr Thr Thr Thr Asn Ser Lys Arg Glu Glu Lys Leu Phe Asn Val Thr
165 170 175
Ser Thr Leu Arg Ile Asn Thr Thr Thr Asn Glu Ile Phe Tyr Cys Thr
180 185 190
Phe Arg Arg Leu Asp Pro Glu Glu Asn His Thr Ala Glu Leu Val Ile
195 200 205
Pro Glu Leu Pro Leu Ala His Pro Pro Asn Glu Arg Gly Gly Gly Ser
210 215 220
Gly Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu
225 230 235 240
His Leu Arg Leu Asp Leu Glu Met Ile Leu Asn Gly Ile Asn Asn Tyr
245 250 255
Lys Asn Pro Lys Leu Thr Asp Met Leu Thr Phe Glu Phe Tyr Met Pro
260 265 270
Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Arg Glu Leu
275 280 285
Lys Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His
290 295 300
Leu Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu
305 310 315 320
Leu Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr
325 330 335
Ala Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Thr Ser
340 345 350
Ile Ile Arg Thr Leu Thr Gly Gly Gly Gly Ser Gly Gly Gly Asp Lys
355 360 365
Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Lys Lys Gly Gly Pro
370 375 380
Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser
385 390 395 400
Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp
405 410 415
Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn
420 425 430
Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val
435 440 445
Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu
450 455 460
Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys
465 470 475 480
Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Val
485 490 495
Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Leu
500 505 510
Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu
515 520 525
Ser Asn Gly Gln Pro Glu Asn Arg Tyr Met Thr Trp Pro Pro Val Leu
530 535 540
Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys
545 550 555 560
Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu
565 570 575
Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly
580 585 590
Lys
<210> 163
<211> 593
<212> PRT
<213> Homo sapiens (Homo sapiens)
<400> 163
Phe Thr Val Thr Val Pro Lys Asp Leu Tyr Val Val Glu Tyr Gly Ser
1 5 10 15
Asn Met Thr Ile Glu Cys Lys Phe Pro Val Glu Lys Gln Leu Asp Leu
20 25 30
Ala Ala Leu Gln Val Tyr Trp Met Met Glu Asp Lys Asn Ile Ile Gln
35 40 45
Phe Val His Gly Glu Glu Asp Leu Lys Val Gln His Ser Ser Tyr Arg
50 55 60
Gln Arg Ala Arg Leu Leu Lys Asp Gln Leu Ser Leu Gly Asn Ala Ala
65 70 75 80
Leu Gln Ile Thr Asp Val Lys Leu Gln Asp Ala Gly Val Tyr Thr Cys
85 90 95
Leu Ile Ala Tyr Gly Gly Ala Asp Tyr Lys Arg Ile Thr Val Lys Val
100 105 110
Asn Ala Pro Tyr Asn Lys Ile Asn Gln Arg Ile Leu Val Val Asp Pro
115 120 125
Val Thr Ser Glu His Glu Leu Thr Cys Gln Ala Glu Gly Tyr Pro Lys
130 135 140
Ala Glu Val Ile Trp Thr Ser Ser Asp His Gln Val Leu Ser Gly Lys
145 150 155 160
Thr Thr Thr Thr Asn Ser Lys Arg Glu Glu Lys Leu Phe Asn Val Thr
165 170 175
Ser Thr Leu Arg Ile Asn Thr Thr Thr Asn Glu Ile Phe Tyr Cys Thr
180 185 190
Phe Arg Arg Leu Asp Pro Glu Glu Asn His Thr Ala Glu Leu Val Ile
195 200 205
Pro Glu Leu Pro Leu Ala His Pro Pro Asn Glu Arg Gly Gly Gly Ser
210 215 220
Gly Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu
225 230 235 240
His Leu Arg Leu Asp Leu Glu Met Ile Leu Asn Gly Ile Asn Asn Tyr
245 250 255
Lys Asn Pro Lys Leu Thr Asp Met Leu Thr Phe Glu Phe Tyr Met Pro
260 265 270
Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Arg Glu Leu
275 280 285
Lys Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His
290 295 300
Leu Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu
305 310 315 320
Leu Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr
325 330 335
Ala Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser
340 345 350
Ile Ile Ser Thr Leu Thr Gly Gly Gly Gly Ser Gly Gly Gly Asp Lys
355 360 365
Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Lys Lys Gly Gly Pro
370 375 380
Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser
385 390 395 400
Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp
405 410 415
Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn
420 425 430
Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val
435 440 445
Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu
450 455 460
Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys
465 470 475 480
Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Val
485 490 495
Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Leu
500 505 510
Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu
515 520 525
Ser Asn Gly Gln Pro Glu Asn Arg Tyr Met Thr Trp Pro Pro Val Leu
530 535 540
Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys
545 550 555 560
Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu
565 570 575
Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly
580 585 590
Lys
<210> 164
<211> 593
<212> PRT
<213> Homo sapiens (Homo sapiens)
<400> 164
Phe Thr Val Thr Val Pro Lys Asp Leu Tyr Val Val Glu Tyr Gly Ser
1 5 10 15
Asn Met Thr Ile Glu Cys Lys Phe Pro Val Glu Lys Gln Leu Asp Leu
20 25 30
Ala Ala Leu Gln Val Tyr Trp Met Met Glu Asp Lys Asn Ile Ile Gln
35 40 45
Phe Val His Gly Glu Glu Asp Leu Lys Val Gln His Ser Ser Tyr Arg
50 55 60
Gln Arg Ala Arg Leu Leu Lys Asp Gln Leu Ser Leu Gly Asn Ala Ala
65 70 75 80
Leu Gln Ile Thr Asp Val Lys Leu Gln Asp Ala Gly Val Tyr Thr Cys
85 90 95
Leu Ile Ala Tyr Gly Gly Ala Asp Tyr Lys Arg Ile Thr Val Lys Val
100 105 110
Asn Ala Pro Tyr Asn Lys Ile Asn Gln Arg Ile Leu Val Val Asp Pro
115 120 125
Val Thr Ser Glu His Glu Leu Thr Cys Gln Ala Glu Gly Tyr Pro Lys
130 135 140
Ala Glu Val Ile Trp Thr Ser Ser Asp His Gln Val Leu Ser Gly Lys
145 150 155 160
Thr Thr Thr Thr Asn Ser Lys Arg Glu Glu Lys Leu Phe Asn Val Thr
165 170 175
Ser Thr Leu Arg Ile Asn Thr Thr Thr Asn Glu Ile Phe Tyr Cys Thr
180 185 190
Phe Arg Arg Leu Asp Pro Glu Glu Asn His Thr Ala Glu Leu Val Ile
195 200 205
Pro Glu Leu Pro Leu Ala His Pro Pro Asn Glu Arg Gly Gly Gly Ser
210 215 220
Gly Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu
225 230 235 240
His Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr
245 250 255
Lys Asn Pro Lys Leu Thr Asp Met Leu Thr Phe Glu Phe Tyr Met Pro
260 265 270
Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Arg Glu Leu
275 280 285
Lys Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His
290 295 300
Leu Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu
305 310 315 320
Leu Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr
325 330 335
Ala Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Thr Ser
340 345 350
Ile Ile Ser Thr Leu Thr Gly Gly Gly Gly Ser Gly Gly Gly Asp Lys
355 360 365
Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Lys Lys Gly Gly Pro
370 375 380
Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser
385 390 395 400
Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp
405 410 415
Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn
420 425 430
Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val
435 440 445
Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu
450 455 460
Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys
465 470 475 480
Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Val
485 490 495
Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Leu
500 505 510
Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu
515 520 525
Ser Asn Gly Gln Pro Glu Asn Arg Tyr Met Thr Trp Pro Pro Val Leu
530 535 540
Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys
545 550 555 560
Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu
565 570 575
Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly
580 585 590
Lys
<210> 165
<211> 593
<212> PRT
<213> Homo sapiens (Homo sapiens)
<400> 165
Phe Thr Val Thr Val Pro Lys Asp Leu Tyr Val Val Glu Tyr Gly Ser
1 5 10 15
Asn Met Thr Ile Glu Cys Lys Phe Pro Val Glu Lys Gln Leu Asp Leu
20 25 30
Ala Ala Leu Gln Val Tyr Trp Met Met Glu Asp Lys Asn Ile Ile Gln
35 40 45
Phe Val His Gly Glu Glu Asp Leu Lys Val Gln His Ser Ser Tyr Arg
50 55 60
Gln Arg Ala Arg Leu Leu Lys Asp Gln Leu Ser Leu Gly Asn Ala Ala
65 70 75 80
Leu Gln Ile Thr Asp Val Lys Leu Gln Asp Ala Gly Val Tyr Thr Cys
85 90 95
Leu Ile Ala Tyr Gly Gly Ala Asp Tyr Lys Arg Ile Thr Val Lys Val
100 105 110
Asn Ala Pro Tyr Asn Lys Ile Asn Gln Arg Ile Leu Val Val Asp Pro
115 120 125
Val Thr Ser Glu His Glu Leu Thr Cys Gln Ala Glu Gly Tyr Pro Lys
130 135 140
Ala Glu Val Ile Trp Thr Ser Ser Asp His Gln Val Leu Ser Gly Lys
145 150 155 160
Thr Thr Thr Thr Asn Ser Lys Arg Glu Glu Lys Leu Phe Asn Val Thr
165 170 175
Ser Thr Leu Arg Ile Asn Thr Thr Thr Asn Glu Ile Phe Tyr Cys Thr
180 185 190
Phe Arg Arg Leu Asp Pro Glu Glu Asn His Thr Ala Glu Leu Val Ile
195 200 205
Pro Glu Leu Pro Leu Ala His Pro Pro Asn Glu Arg Gly Gly Gly Ser
210 215 220
Gly Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu
225 230 235 240
His Leu Arg Leu Asp Leu Glu Met Ile Leu Asn Gly Ile Asn Asn Tyr
245 250 255
Lys Asn Pro Lys Leu Thr Asp Met Leu Thr Phe Glu Phe Tyr Met Pro
260 265 270
Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Arg Glu Leu
275 280 285
Lys Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His
290 295 300
Leu Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu
305 310 315 320
Leu Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr
325 330 335
Ala Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Thr Ser
340 345 350
Ile Ile Ser Thr Leu Thr Gly Gly Gly Gly Ser Gly Gly Gly Asp Lys
355 360 365
Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Lys Lys Gly Gly Pro
370 375 380
Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser
385 390 395 400
Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp
405 410 415
Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn
420 425 430
Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val
435 440 445
Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu
450 455 460
Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys
465 470 475 480
Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Val
485 490 495
Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Leu
500 505 510
Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu
515 520 525
Ser Asn Gly Gln Pro Glu Asn Arg Tyr Met Thr Trp Pro Pro Val Leu
530 535 540
Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys
545 550 555 560
Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu
565 570 575
Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly
580 585 590
Lys
<210> 166
<211> 593
<212> PRT
<213> Homo sapiens (Homo sapiens)
<400> 166
Phe Thr Val Thr Val Pro Lys Asp Leu Tyr Val Val Glu Tyr Gly Ser
1 5 10 15
Asn Met Thr Ile Glu Cys Lys Phe Pro Val Glu Lys Gln Leu Asp Leu
20 25 30
Ala Ala Leu Gln Val Tyr Trp Met Met Glu Asp Lys Asn Ile Ile Gln
35 40 45
Phe Val His Gly Glu Glu Asp Leu Lys Val Gln His Ser Ser Tyr Arg
50 55 60
Gln Arg Ala Arg Leu Leu Lys Asp Gln Leu Ser Leu Gly Asn Ala Ala
65 70 75 80
Leu Gln Ile Thr Asp Val Lys Leu Gln Asp Ala Gly Val Tyr Thr Cys
85 90 95
Leu Ile Ala Tyr Gly Gly Ala Asp Tyr Lys Arg Ile Thr Val Lys Val
100 105 110
Asn Ala Pro Tyr Asn Lys Ile Asn Gln Arg Ile Leu Val Val Asp Pro
115 120 125
Val Thr Ser Glu His Glu Leu Thr Cys Gln Ala Glu Gly Tyr Pro Lys
130 135 140
Ala Glu Val Ile Trp Thr Ser Ser Asp His Gln Val Leu Ser Gly Lys
145 150 155 160
Thr Thr Thr Thr Asn Ser Lys Arg Glu Glu Lys Leu Phe Asn Val Thr
165 170 175
Ser Thr Leu Arg Ile Asn Thr Thr Thr Asn Glu Ile Phe Tyr Cys Thr
180 185 190
Phe Arg Arg Leu Asp Pro Glu Glu Asn His Thr Ala Glu Leu Val Ile
195 200 205
Pro Glu Leu Pro Leu Ala His Pro Pro Asn Glu Arg Gly Gly Gly Ser
210 215 220
Gly Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu
225 230 235 240
His Leu Arg Leu Asp Leu Glu Met Ile Leu Asn Gly Ile Asn Asn Tyr
245 250 255
Lys Asn Pro Lys Leu Thr Asp Met Leu Thr Phe Glu Phe Tyr Met Pro
260 265 270
Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Arg Glu Leu
275 280 285
Lys Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His
290 295 300
Leu Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu
305 310 315 320
Leu Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr
325 330 335
Ala Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Thr Ser
340 345 350
Ile Ile Arg Thr Leu Thr Gly Gly Gly Gly Ser Gly Gly Gly Asp Lys
355 360 365
Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Lys Lys Gly Gly Pro
370 375 380
Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser
385 390 395 400
Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp
405 410 415
Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn
420 425 430
Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val
435 440 445
Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu
450 455 460
Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys
465 470 475 480
Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Val
485 490 495
Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Leu
500 505 510
Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu
515 520 525
Ser Asn Gly Gln Pro Glu Asn Arg Tyr Met Thr Trp Pro Pro Val Leu
530 535 540
Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys
545 550 555 560
Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu
565 570 575
Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly
580 585 590
Lys
<210> 167
<211> 961
<212> PRT
<213> Homo sapiens (Homo sapiens)
<400> 167
Leu Phe Thr Val Thr Val Pro Lys Glu Leu Tyr Ile Ile Glu His Gly
1 5 10 15
Ser Asn Val Thr Leu Glu Cys Asn Phe Asp Thr Gly Ser His Val Asn
20 25 30
Leu Gly Ala Ile Thr Ala Val Leu Gln Lys Val Glu Asn Asp Thr Ser
35 40 45
Pro His Arg Glu Arg Ala Thr Leu Leu Glu Glu Gln Leu Pro Leu Gly
50 55 60
Lys Ala Ser Phe His Ile Pro Gln Val Gln Val Arg Asp Glu Gly Gln
65 70 75 80
Tyr Gln Cys Ile Ile Ile Tyr Gly Val Ala Trp Asp Tyr Lys Tyr Leu
85 90 95
Thr Leu Lys Val Lys Ala Ser Tyr Arg Lys Ile Asn Thr His Ile Leu
100 105 110
Lys Val Pro Glu Thr Asp Glu Val Glu Leu Thr Cys Gln Ala Thr Gly
115 120 125
Tyr Pro Leu Ala Glu Val Ser Trp Pro Asn Val Ser Val Pro Ala Asn
130 135 140
Thr Ser His Ser Arg Thr Pro Glu Gly Leu Tyr Gln Val Thr Ser Val
145 150 155 160
Leu Arg Leu Lys Pro Pro Pro Gly Arg Asn Phe Ser Cys Val Phe Trp
165 170 175
Asn Thr His Val Arg Glu Leu Thr Leu Ala Ser Ile Asp Leu Gln Ser
180 185 190
Gln Met Glu Pro Arg Thr His Pro Thr Gly Gly Gly Gly Ser Gly Gly
195 200 205
Gly Ile Trp Glu Leu Lys Lys Asp Val Tyr Val Val Glu Leu Asp Trp
210 215 220
Tyr Pro Asp Ala Pro Gly Glu Met Val Val Leu Thr Cys Asp Thr Pro
225 230 235 240
Glu Glu Asp Gly Ile Thr Trp Thr Leu Asp Gln Ser Ser Glu Val Leu
245 250 255
Gly Ser Gly Lys Thr Leu Thr Ile Gln Val Lys Ala Ala Gly Asp Ala
260 265 270
Gly Gln Tyr Thr Cys His Lys Gly Gly Glu Val Leu Ser His Ser Leu
275 280 285
Leu Leu Leu His Lys Lys Glu Asp Gly Ile Trp Ser Thr Asp Ile Leu
290 295 300
Lys Asp Gln Lys Glu Pro Lys Asn Lys Thr Phe Leu Arg Cys Glu Ala
305 310 315 320
Lys Asn Tyr Ser Gly Arg Phe Thr Cys Trp Trp Leu Thr Thr Ile Ser
325 330 335
Thr Asp Leu Thr Phe Ser Val Lys Ser Ser Arg Gly Ser Ser Asp Pro
340 345 350
Gln Gly Val Thr Cys Gly Ala Ala Thr Leu Ser Ala Glu Arg Val Arg
355 360 365
Gly Asp Asn Lys Glu Tyr Glu Tyr Ser Val Glu Cys Gln Glu Asp Ser
370 375 380
Ala Cys Pro Ala Ala Glu Glu Ser Leu Pro Ile Glu Val Met Val Asp
385 390 395 400
Ala Val His Lys Leu Lys Tyr Glu Asn Tyr Thr Ser Ser Phe Phe Ile
405 410 415
Arg Asp Ile Ile Lys Pro Asp Pro Pro Lys Asn Leu Gln Leu Lys Pro
420 425 430
Leu Lys Asn Ser Arg Gln Val Glu Val Ser Trp Glu Tyr Pro Asp Thr
435 440 445
Trp Ser Thr Pro His Ser Tyr Phe Ser Leu Thr Phe Cys Val Gln Val
450 455 460
Gln Gly Lys Ser Lys Arg Glu Lys Lys Asp Arg Val Phe Thr Asp Lys
465 470 475 480
Thr Ser Ala Thr Val Ile Cys Arg Lys Asn Ala Ser Ile Ser Val Arg
485 490 495
Ala Gln Asp Arg Tyr Tyr Ser Ser Ser Trp Ser Glu Trp Ala Ser Val
500 505 510
Pro Cys Ser Gly Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Gly
515 520 525
Ser Arg Asn Leu Pro Val Ala Thr Pro Asp Pro Gly Met Phe Pro Cys
530 535 540
Leu His His Ser Gln Asn Leu Leu Arg Ala Val Ser Asn Met Leu Gln
545 550 555 560
Lys Ala Arg Gln Thr Leu Glu Phe Tyr Pro Cys Thr Ser Glu Glu Ile
565 570 575
Asp His Glu Asp Ile Thr Lys Asp Lys Thr Ser Thr Val Glu Ala Cys
580 585 590
Leu Pro Leu Glu Leu Thr Lys Asn Glu Ser Cys Leu Asn Ser Arg Glu
595 600 605
Thr Ser Phe Ile Thr Asn Gly Ser Cys Leu Ala Ser Arg Lys Thr Ser
610 615 620
Phe Met Met Ala Leu Cys Leu Ser Ser Ile Tyr Glu Asp Leu Lys Met
625 630 635 640
Tyr Gln Val Glu Phe Lys Thr Met Asn Ala Lys Leu Leu Met Asp Pro
645 650 655
Lys Arg Gln Ile Phe Leu Asp Gln Asn Met Leu Ala Val Ile Asp Glu
660 665 670
Leu Met Gln Ala Leu Asn Phe Asn Ser Glu Thr Val Pro Gln Lys Ser
675 680 685
Ser Leu Glu Glu Pro Asp Phe Tyr Lys Thr Lys Ile Lys Leu Cys Ile
690 695 700
Leu Leu His Ala Phe Arg Ile Arg Ala Val Thr Ile Asp Arg Val Met
705 710 715 720
Ser Tyr Leu Asn Ala Ser Gly Gly Gly Gly Ser Gly Gly Gly Asp Lys
725 730 735
Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Asp Glu Gly Gly Pro
740 745 750
Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser
755 760 765
Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp
770 775 780
Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn
785 790 795 800
Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val
805 810 815
Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu
820 825 830
Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys
835 840 845
Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Val
850 855 860
Tyr Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr
865 870 875 880
Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu
885 890 895
Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu
900 905 910
Asp Glu Asp Gly Ser Phe Ala Leu Val Ser Lys Leu Thr Val Asp Lys
915 920 925
Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu
930 935 940
Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly
945 950 955 960
Lys
<210> 168
<211> 961
<212> PRT
<213> Homo sapiens (Homo sapiens)
<400> 168
Leu Phe Thr Val Thr Val Pro Lys Glu Leu Tyr Ile Ile Glu His Gly
1 5 10 15
Ser Asn Val Thr Leu Glu Cys Asn Phe Asp Thr Gly Ser His Val Asn
20 25 30
Leu Gly Ala Ile Val Ala Val Leu Leu Lys Val Glu Asn Asp Thr Ser
35 40 45
Pro His Arg Glu Arg Ala Thr Leu Leu Glu Glu Gln Leu Pro Leu Gly
50 55 60
Lys Ala Ser Phe His Ile Pro Gln Val Gln Val Arg Asp Glu Gly Gln
65 70 75 80
Tyr Gln Cys Ile Ile Ile Tyr Gly Val Ala Trp Asp Tyr Lys Tyr Leu
85 90 95
Thr Leu Lys Val Lys Ala Ser Tyr Arg Lys Ile Asn Thr His Ile Leu
100 105 110
Lys Val Pro Glu Thr Asp Glu Val Glu Leu Thr Cys Gln Ala Thr Gly
115 120 125
Tyr Pro Leu Ala Glu Val Ser Trp Pro Asn Val Ser Val Pro Ala Asn
130 135 140
Thr Ser His Ser Arg Thr Pro Glu Gly Leu Tyr Gln Val Thr Ser Val
145 150 155 160
Leu Arg Leu Lys Pro Pro Pro Gly Arg Asn Phe Ser Cys Val Phe Trp
165 170 175
Asn Thr His Val Arg Glu Leu Thr Leu Ala Ser Ile Asp Leu Gln Ser
180 185 190
Gln Met Glu Pro Arg Thr His Pro Thr Gly Gly Gly Gly Ser Gly Gly
195 200 205
Gly Ile Trp Glu Leu Lys Lys Asp Val Tyr Val Val Glu Leu Asp Trp
210 215 220
Tyr Pro Asp Ala Pro Gly Glu Met Val Val Leu Thr Cys Asp Thr Pro
225 230 235 240
Glu Glu Asp Gly Ile Thr Trp Thr Leu Asp Gln Ser Ser Glu Val Leu
245 250 255
Gly Ser Gly Lys Thr Leu Thr Ile Gln Val Lys Ala Ala Gly Asp Ala
260 265 270
Gly Gln Tyr Thr Cys His Lys Gly Gly Glu Val Leu Ser His Ser Leu
275 280 285
Leu Leu Leu His Lys Lys Glu Asp Gly Ile Trp Ser Thr Asp Ile Leu
290 295 300
Lys Asp Gln Lys Glu Pro Lys Asn Lys Thr Phe Leu Arg Cys Glu Ala
305 310 315 320
Lys Asn Tyr Ser Gly Arg Phe Thr Cys Trp Trp Leu Thr Thr Ile Ser
325 330 335
Thr Asp Leu Thr Phe Ser Val Lys Ser Ser Arg Gly Ser Ser Asp Pro
340 345 350
Gln Gly Val Thr Cys Gly Ala Ala Thr Leu Ser Ala Glu Arg Val Arg
355 360 365
Gly Asp Asn Lys Glu Tyr Glu Tyr Ser Val Glu Cys Gln Glu Asp Ser
370 375 380
Ala Cys Pro Ala Ala Glu Glu Ser Leu Pro Ile Glu Val Met Val Asp
385 390 395 400
Ala Val His Lys Leu Lys Tyr Glu Asn Tyr Thr Ser Ser Phe Phe Ile
405 410 415
Arg Asp Ile Ile Lys Pro Asp Pro Pro Lys Asn Leu Gln Leu Lys Pro
420 425 430
Leu Lys Asn Ser Arg Gln Val Glu Val Ser Trp Glu Tyr Pro Asp Thr
435 440 445
Trp Ser Thr Pro His Ser Tyr Phe Ser Leu Thr Phe Cys Val Gln Val
450 455 460
Gln Gly Lys Ser Lys Arg Glu Lys Lys Asp Arg Val Phe Thr Asp Lys
465 470 475 480
Thr Ser Ala Thr Val Ile Cys Arg Lys Asn Ala Ser Ile Ser Val Arg
485 490 495
Ala Gln Asp Arg Tyr Tyr Ser Ser Ser Trp Ser Glu Trp Ala Ser Val
500 505 510
Pro Cys Ser Gly Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Gly
515 520 525
Ser Arg Asn Leu Pro Val Ala Thr Pro Asp Pro Gly Met Phe Pro Cys
530 535 540
Leu His His Ser Gln Asn Leu Leu Arg Ala Val Ser Asn Met Leu Gln
545 550 555 560
Lys Ala Arg Gln Thr Leu Glu Phe Tyr Pro Cys Thr Ser Glu Glu Ile
565 570 575
Asp His Glu Asp Ile Thr Lys Asp Lys Thr Ser Thr Val Glu Ala Cys
580 585 590
Leu Pro Leu Glu Leu Thr Lys Asn Glu Ser Cys Leu Asn Ser Arg Glu
595 600 605
Thr Ser Phe Ile Thr Asn Gly Ser Cys Leu Ala Ser Arg Lys Thr Ser
610 615 620
Phe Met Met Ala Leu Cys Leu Ser Ser Ile Tyr Glu Asp Leu Lys Met
625 630 635 640
Tyr Gln Val Glu Phe Lys Thr Met Asn Ala Lys Leu Leu Met Asp Pro
645 650 655
Lys Arg Gln Ile Phe Leu Asp Gln Asn Met Leu Ala Val Ile Asp Glu
660 665 670
Leu Met Gln Ala Leu Asn Phe Asn Ser Glu Thr Val Pro Gln Lys Ser
675 680 685
Ser Leu Glu Glu Pro Asp Phe Tyr Lys Thr Lys Ile Lys Leu Cys Ile
690 695 700
Leu Leu His Ala Phe Arg Ile Arg Ala Val Thr Ile Asp Arg Val Met
705 710 715 720
Ser Tyr Leu Asn Ala Ser Gly Gly Gly Gly Ser Gly Gly Gly Asp Lys
725 730 735
Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Asp Glu Gly Gly Pro
740 745 750
Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser
755 760 765
Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp
770 775 780
Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn
785 790 795 800
Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val
805 810 815
Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu
820 825 830
Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys
835 840 845
Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Val
850 855 860
Tyr Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr
865 870 875 880
Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu
885 890 895
Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu
900 905 910
Asp Glu Asp Gly Ser Phe Ala Leu Val Ser Lys Leu Thr Val Asp Lys
915 920 925
Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu
930 935 940
Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly
945 950 955 960
Lys
<210> 169
<211> 961
<212> PRT
<213> Homo sapiens (Homo sapiens)
<400> 169
Leu Phe Thr Val Thr Val Pro Lys Glu Leu Tyr Ile Ile Glu His Gly
1 5 10 15
Ser Asn Val Thr Leu Glu Cys Asn Phe Asp Thr Gly Ser His Val Asn
20 25 30
Leu Gly Ala Ile Thr Ala Val Leu Gln Lys Val Glu Asn Asp Thr Ser
35 40 45
Pro His Arg Glu Arg Ala Thr Leu Leu Glu Glu Gln Leu Pro Leu Gly
50 55 60
Lys Ala Ser Phe His Ile Pro Gln Val Gln Val Arg Asp Glu Gly Gln
65 70 75 80
Tyr Gln Cys Ile Ile Ile Tyr Gly Val Ala Trp Asp Tyr Lys Tyr Leu
85 90 95
Thr Leu Lys Val Lys Ala Ser Tyr Arg Lys Ile Asn Thr His Ile Leu
100 105 110
Lys Val Pro Glu Thr Asp Glu Val Glu Leu Thr Cys Gln Ala Thr Gly
115 120 125
Tyr Pro Leu Ala Glu Val Ser Trp Pro Asn Val Ser Val Pro Ala Asn
130 135 140
Thr Ser His Ser Arg Thr Pro Glu Gly Leu Tyr Gln Val Thr Ser Val
145 150 155 160
Leu Arg Leu Lys Pro Pro Pro Gly Arg Asn Phe Ser Cys Val Phe Trp
165 170 175
Asn Thr His Val Arg Glu Leu Thr Leu Ala Ser Ile Asp Leu Gln Ser
180 185 190
Gln Met Glu Pro Arg Thr His Pro Thr Gly Gly Gly Gly Ser Gly Gly
195 200 205
Gly Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Asp Glu
210 215 220
Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu
225 230 235 240
Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser
245 250 255
His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu
260 265 270
Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr
275 280 285
Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn
290 295 300
Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro
305 310 315 320
Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln
325 330 335
Val Tyr Val Tyr Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val
340 345 350
Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val
355 360 365
Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro
370 375 380
Pro Val Leu Asp Glu Asp Gly Ser Phe Ala Leu Val Ser Lys Leu Thr
385 390 395 400
Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val
405 410 415
Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu
420 425 430
Ser Pro Gly Lys Gly Gly Gly Gly Ser Gly Gly Gly Ile Trp Glu Leu
435 440 445
Lys Lys Asp Val Tyr Val Val Glu Leu Asp Trp Tyr Pro Asp Ala Pro
450 455 460
Gly Glu Met Val Val Leu Thr Cys Asp Thr Pro Glu Glu Asp Gly Ile
465 470 475 480
Thr Trp Thr Leu Asp Gln Ser Ser Glu Val Leu Gly Ser Gly Lys Thr
485 490 495
Leu Thr Ile Gln Val Lys Ala Ala Gly Asp Ala Gly Gln Tyr Thr Cys
500 505 510
His Lys Gly Gly Glu Val Leu Ser His Ser Leu Leu Leu Leu His Lys
515 520 525
Lys Glu Asp Gly Ile Trp Ser Thr Asp Ile Leu Lys Asp Gln Lys Glu
530 535 540
Pro Lys Asn Lys Thr Phe Leu Arg Cys Glu Ala Lys Asn Tyr Ser Gly
545 550 555 560
Arg Phe Thr Cys Trp Trp Leu Thr Thr Ile Ser Thr Asp Leu Thr Phe
565 570 575
Ser Val Lys Ser Ser Arg Gly Ser Ser Asp Pro Gln Gly Val Thr Cys
580 585 590
Gly Ala Ala Thr Leu Ser Ala Glu Arg Val Arg Gly Asp Asn Lys Glu
595 600 605
Tyr Glu Tyr Ser Val Glu Cys Gln Glu Asp Ser Ala Cys Pro Ala Ala
610 615 620
Glu Glu Ser Leu Pro Ile Glu Val Met Val Asp Ala Val His Lys Leu
625 630 635 640
Lys Tyr Glu Asn Tyr Thr Ser Ser Phe Phe Ile Arg Asp Ile Ile Lys
645 650 655
Pro Asp Pro Pro Lys Asn Leu Gln Leu Lys Pro Leu Lys Asn Ser Arg
660 665 670
Gln Val Glu Val Ser Trp Glu Tyr Pro Asp Thr Trp Ser Thr Pro His
675 680 685
Ser Tyr Phe Ser Leu Thr Phe Cys Val Gln Val Gln Gly Lys Ser Lys
690 695 700
Arg Glu Lys Lys Asp Arg Val Phe Thr Asp Lys Thr Ser Ala Thr Val
705 710 715 720
Ile Cys Arg Lys Asn Ala Ser Ile Ser Val Arg Ala Gln Asp Arg Tyr
725 730 735
Tyr Ser Ser Ser Trp Ser Glu Trp Ala Ser Val Pro Cys Ser Gly Gly
740 745 750
Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Gly Ser Arg Asn Leu Pro
755 760 765
Val Ala Thr Pro Asp Pro Gly Met Phe Pro Cys Leu His His Ser Gln
770 775 780
Asn Leu Leu Arg Ala Val Ser Asn Met Leu Gln Lys Ala Arg Gln Thr
785 790 795 800
Leu Glu Phe Tyr Pro Cys Thr Ser Glu Glu Ile Asp His Glu Asp Ile
805 810 815
Thr Lys Asp Lys Thr Ser Thr Val Glu Ala Cys Leu Pro Leu Glu Leu
820 825 830
Thr Lys Asn Glu Ser Cys Leu Asn Ser Arg Glu Thr Ser Phe Ile Thr
835 840 845
Asn Gly Ser Cys Leu Ala Ser Arg Lys Thr Ser Phe Met Met Ala Leu
850 855 860
Cys Leu Ser Ser Ile Tyr Glu Asp Leu Lys Met Tyr Gln Val Glu Phe
865 870 875 880
Lys Thr Met Asn Ala Lys Leu Leu Met Asp Pro Lys Arg Gln Ile Phe
885 890 895
Leu Asp Gln Asn Met Leu Ala Val Ile Asp Glu Leu Met Gln Ala Leu
900 905 910
Asn Phe Asn Ser Glu Thr Val Pro Gln Lys Ser Ser Leu Glu Glu Pro
915 920 925
Asp Phe Tyr Lys Thr Lys Ile Lys Leu Cys Ile Leu Leu His Ala Phe
930 935 940
Arg Ile Arg Ala Val Thr Ile Asp Arg Val Met Ser Tyr Leu Asn Ala
945 950 955 960
Ser
<210> 170
<211> 961
<212> PRT
<213> Homo sapiens (Homo sapiens)
<400> 170
Leu Phe Thr Val Thr Val Pro Lys Glu Leu Tyr Ile Ile Glu His Gly
1 5 10 15
Ser Asn Val Thr Leu Glu Cys Asn Phe Asp Thr Gly Ser His Val Asn
20 25 30
Leu Gly Ala Ile Val Ala Val Leu Leu Lys Val Glu Asn Asp Thr Ser
35 40 45
Pro His Arg Glu Arg Ala Thr Leu Leu Glu Glu Gln Leu Pro Leu Gly
50 55 60
Lys Ala Ser Phe His Ile Pro Gln Val Gln Val Arg Asp Glu Gly Gln
65 70 75 80
Tyr Gln Cys Ile Ile Ile Tyr Gly Val Ala Trp Asp Tyr Lys Tyr Leu
85 90 95
Thr Leu Lys Val Lys Ala Ser Tyr Arg Lys Ile Asn Thr His Ile Leu
100 105 110
Lys Val Pro Glu Thr Asp Glu Val Glu Leu Thr Cys Gln Ala Thr Gly
115 120 125
Tyr Pro Leu Ala Glu Val Ser Trp Pro Asn Val Ser Val Pro Ala Asn
130 135 140
Thr Ser His Ser Arg Thr Pro Glu Gly Leu Tyr Gln Val Thr Ser Val
145 150 155 160
Leu Arg Leu Lys Pro Pro Pro Gly Arg Asn Phe Ser Cys Val Phe Trp
165 170 175
Asn Thr His Val Arg Glu Leu Thr Leu Ala Ser Ile Asp Leu Gln Ser
180 185 190
Gln Met Glu Pro Arg Thr His Pro Thr Gly Gly Gly Gly Ser Gly Gly
195 200 205
Gly Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Asp Glu
210 215 220
Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu
225 230 235 240
Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser
245 250 255
His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu
260 265 270
Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr
275 280 285
Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn
290 295 300
Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro
305 310 315 320
Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln
325 330 335
Val Tyr Val Tyr Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val
340 345 350
Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val
355 360 365
Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro
370 375 380
Pro Val Leu Asp Glu Asp Gly Ser Phe Ala Leu Val Ser Lys Leu Thr
385 390 395 400
Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val
405 410 415
Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu
420 425 430
Ser Pro Gly Lys Gly Gly Gly Gly Ser Gly Gly Gly Ile Trp Glu Leu
435 440 445
Lys Lys Asp Val Tyr Val Val Glu Leu Asp Trp Tyr Pro Asp Ala Pro
450 455 460
Gly Glu Met Val Val Leu Thr Cys Asp Thr Pro Glu Glu Asp Gly Ile
465 470 475 480
Thr Trp Thr Leu Asp Gln Ser Ser Glu Val Leu Gly Ser Gly Lys Thr
485 490 495
Leu Thr Ile Gln Val Lys Ala Ala Gly Asp Ala Gly Gln Tyr Thr Cys
500 505 510
His Lys Gly Gly Glu Val Leu Ser His Ser Leu Leu Leu Leu His Lys
515 520 525
Lys Glu Asp Gly Ile Trp Ser Thr Asp Ile Leu Lys Asp Gln Lys Glu
530 535 540
Pro Lys Asn Lys Thr Phe Leu Arg Cys Glu Ala Lys Asn Tyr Ser Gly
545 550 555 560
Arg Phe Thr Cys Trp Trp Leu Thr Thr Ile Ser Thr Asp Leu Thr Phe
565 570 575
Ser Val Lys Ser Ser Arg Gly Ser Ser Asp Pro Gln Gly Val Thr Cys
580 585 590
Gly Ala Ala Thr Leu Ser Ala Glu Arg Val Arg Gly Asp Asn Lys Glu
595 600 605
Tyr Glu Tyr Ser Val Glu Cys Gln Glu Asp Ser Ala Cys Pro Ala Ala
610 615 620
Glu Glu Ser Leu Pro Ile Glu Val Met Val Asp Ala Val His Lys Leu
625 630 635 640
Lys Tyr Glu Asn Tyr Thr Ser Ser Phe Phe Ile Arg Asp Ile Ile Lys
645 650 655
Pro Asp Pro Pro Lys Asn Leu Gln Leu Lys Pro Leu Lys Asn Ser Arg
660 665 670
Gln Val Glu Val Ser Trp Glu Tyr Pro Asp Thr Trp Ser Thr Pro His
675 680 685
Ser Tyr Phe Ser Leu Thr Phe Cys Val Gln Val Gln Gly Lys Ser Lys
690 695 700
Arg Glu Lys Lys Asp Arg Val Phe Thr Asp Lys Thr Ser Ala Thr Val
705 710 715 720
Ile Cys Arg Lys Asn Ala Ser Ile Ser Val Arg Ala Gln Asp Arg Tyr
725 730 735
Tyr Ser Ser Ser Trp Ser Glu Trp Ala Ser Val Pro Cys Ser Gly Gly
740 745 750
Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Gly Ser Arg Asn Leu Pro
755 760 765
Val Ala Thr Pro Asp Pro Gly Met Phe Pro Cys Leu His His Ser Gln
770 775 780
Asn Leu Leu Arg Ala Val Ser Asn Met Leu Gln Lys Ala Arg Gln Thr
785 790 795 800
Leu Glu Phe Tyr Pro Cys Thr Ser Glu Glu Ile Asp His Glu Asp Ile
805 810 815
Thr Lys Asp Lys Thr Ser Thr Val Glu Ala Cys Leu Pro Leu Glu Leu
820 825 830
Thr Lys Asn Glu Ser Cys Leu Asn Ser Arg Glu Thr Ser Phe Ile Thr
835 840 845
Asn Gly Ser Cys Leu Ala Ser Arg Lys Thr Ser Phe Met Met Ala Leu
850 855 860
Cys Leu Ser Ser Ile Tyr Glu Asp Leu Lys Met Tyr Gln Val Glu Phe
865 870 875 880
Lys Thr Met Asn Ala Lys Leu Leu Met Asp Pro Lys Arg Gln Ile Phe
885 890 895
Leu Asp Gln Asn Met Leu Ala Val Ile Asp Glu Leu Met Gln Ala Leu
900 905 910
Asn Phe Asn Ser Glu Thr Val Pro Gln Lys Ser Ser Leu Glu Glu Pro
915 920 925
Asp Phe Tyr Lys Thr Lys Ile Lys Leu Cys Ile Leu Leu His Ala Phe
930 935 940
Arg Ile Arg Ala Val Thr Ile Asp Arg Val Met Ser Tyr Leu Asn Ala
945 950 955 960
Ser
<210> 171
<211> 574
<212> PRT
<213> Homo sapiens (Homo sapiens)
<400> 171
Leu Phe Thr Val Thr Val Pro Lys Glu Leu Tyr Ile Ile Glu His Gly
1 5 10 15
Ser Asn Val Thr Leu Glu Cys Asn Phe Asp Thr Gly Ser His Val Asn
20 25 30
Leu Gly Ala Ile Thr Ala Val Leu Gln Lys Val Glu Asn Asp Thr Ser
35 40 45
Pro His Arg Glu Arg Ala Thr Leu Leu Glu Glu Gln Leu Pro Leu Gly
50 55 60
Lys Ala Ser Phe His Ile Pro Gln Val Gln Val Arg Asp Glu Gly Gln
65 70 75 80
Tyr Gln Cys Ile Ile Ile Tyr Gly Val Ala Trp Asp Tyr Lys Tyr Leu
85 90 95
Thr Leu Lys Val Lys Ala Ser Tyr Arg Lys Ile Asn Thr His Ile Leu
100 105 110
Lys Val Pro Glu Thr Asp Glu Val Glu Leu Thr Cys Gln Ala Thr Gly
115 120 125
Tyr Pro Leu Ala Glu Val Ser Trp Pro Asn Val Ser Val Pro Ala Asn
130 135 140
Thr Ser His Ser Arg Thr Pro Glu Gly Leu Tyr Gln Val Thr Ser Val
145 150 155 160
Leu Arg Leu Lys Pro Pro Pro Gly Arg Asn Phe Ser Cys Val Phe Trp
165 170 175
Asn Thr His Val Arg Glu Leu Thr Leu Ala Ser Ile Asp Leu Gln Ser
180 185 190
Gln Met Glu Pro Arg Thr His Pro Thr Gly Gly Gly Ser Gly Ala Pro
195 200 205
Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu Arg
210 215 220
Leu Asp Leu Glu Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn Pro
225 230 235 240
Lys Leu Thr Asp Met Leu Thr Phe Glu Phe Tyr Met Pro Lys Lys Ala
245 250 255
Thr Glu Leu Lys His Leu Gln Cys Leu Glu Arg Glu Leu Lys Pro Leu
260 265 270
Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg Pro
275 280 285
Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys Gly
290 295 300
Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr Ile
305 310 315 320
Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile Ile Ser
325 330 335
Thr Leu Thr Gly Gly Gly Gly Ser Gly Gly Gly Asp Lys Thr His Thr
340 345 350
Cys Pro Pro Cys Pro Ala Pro Glu Lys Lys Gly Gly Pro Ser Val Phe
355 360 365
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro
370 375 380
Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val
385 390 395 400
Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr
405 410 415
Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val
420 425 430
Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys
435 440 445
Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser
450 455 460
Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Val Leu Pro Pro
465 470 475 480
Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Leu Cys Leu Val
485 490 495
Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly
500 505 510
Gln Pro Glu Asn Arg Tyr Met Thr Trp Pro Pro Val Leu Asp Ser Asp
515 520 525
Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp
530 535 540
Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His
545 550 555 560
Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
565 570
<210> 172
<211> 573
<212> PRT
<213> Homo sapiens (Homo sapiens)
<400> 172
Leu Phe Thr Val Thr Val Pro Lys Glu Leu Tyr Ile Ile Glu His Gly
1 5 10 15
Ser Asn Val Thr Leu Glu Cys Asn Phe Asp Thr Gly Ser His Val Asn
20 25 30
Leu Gly Ala Ile Thr Ala Val Leu Gln Lys Val Glu Asn Asp Thr Ser
35 40 45
Pro His Arg Glu Arg Ala Thr Leu Leu Glu Glu Gln Leu Pro Leu Gly
50 55 60
Lys Ala Ser Phe His Ile Pro Gln Val Gln Val Arg Asp Glu Gly Gln
65 70 75 80
Tyr Gln Cys Ile Ile Ile Tyr Gly Val Ala Trp Asp Tyr Lys Tyr Leu
85 90 95
Thr Leu Lys Val Lys Ala Ser Tyr Arg Lys Ile Asn Thr His Ile Leu
100 105 110
Lys Val Pro Glu Thr Asp Glu Val Glu Leu Thr Cys Gln Ala Thr Gly
115 120 125
Tyr Pro Leu Ala Glu Val Ser Trp Pro Asn Val Ser Val Pro Ala Asn
130 135 140
Thr Ser His Ser Arg Thr Pro Glu Gly Leu Tyr Gln Val Thr Ser Leu
145 150 155 160
Arg Leu Lys Pro Pro Pro Gly Arg Asn Phe Ser Cys Val Phe Trp Asn
165 170 175
Thr His Val Arg Glu Leu Thr Leu Ala Ser Ile Asp Leu Gln Ser Gln
180 185 190
Met Glu Pro Arg Thr His Pro Thr Gly Gly Gly Ser Gly Ala Pro Thr
195 200 205
Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu Leu Leu
210 215 220
Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn Pro Lys
225 230 235 240
Leu Thr Asp Met Leu Thr Phe Glu Phe Tyr Met Pro Lys Lys Ala Thr
245 250 255
Glu Leu Lys His Leu Gln Cys Leu Glu Arg Glu Leu Lys Pro Leu Glu
260 265 270
Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg Pro Arg
275 280 285
Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys Gly Ser
290 295 300
Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr Ile Val
305 310 315 320
Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Thr Ser Ile Ile Ser Thr
325 330 335
Leu Thr Gly Gly Gly Gly Ser Gly Gly Gly Asp Lys Thr His Thr Cys
340 345 350
Pro Pro Cys Pro Ala Pro Glu Lys Lys Gly Gly Pro Ser Val Phe Leu
355 360 365
Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu
370 375 380
Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys
385 390 395 400
Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys
405 410 415
Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu
420 425 430
Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys
435 440 445
Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys
450 455 460
Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Val Leu Pro Pro Ser
465 470 475 480
Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Leu Cys Leu Val Lys
485 490 495
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln
500 505 510
Pro Glu Asn Arg Tyr Met Thr Trp Pro Pro Val Leu Asp Ser Asp Gly
515 520 525
Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln
530 535 540
Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn
545 550 555 560
His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
565 570
<210> 173
<211> 573
<212> PRT
<213> Homo sapiens (Homo sapiens)
<400> 173
Leu Phe Thr Val Thr Val Pro Lys Glu Leu Tyr Ile Ile Glu His Gly
1 5 10 15
Ser Asn Val Thr Leu Glu Cys Asn Phe Asp Thr Gly Ser His Val Asn
20 25 30
Leu Gly Ala Ile Thr Ala Val Leu Gln Lys Val Glu Asn Asp Thr Ser
35 40 45
Pro His Arg Glu Arg Ala Thr Leu Leu Glu Glu Gln Leu Pro Leu Gly
50 55 60
Lys Ala Ser Phe His Ile Pro Gln Val Gln Val Arg Asp Glu Gly Gln
65 70 75 80
Tyr Gln Cys Ile Ile Ile Tyr Gly Val Ala Trp Asp Tyr Lys Tyr Leu
85 90 95
Thr Leu Lys Val Lys Ala Ser Tyr Arg Lys Ile Asn Thr His Ile Leu
100 105 110
Lys Val Pro Glu Thr Asp Glu Val Glu Leu Thr Cys Gln Ala Thr Gly
115 120 125
Tyr Pro Leu Ala Glu Val Ser Trp Pro Asn Val Ser Val Pro Ala Asn
130 135 140
Thr Ser His Ser Arg Thr Pro Glu Gly Leu Tyr Gln Val Thr Ser Leu
145 150 155 160
Arg Leu Lys Pro Pro Pro Gly Arg Asn Phe Ser Cys Val Phe Trp Asn
165 170 175
Thr His Val Arg Glu Leu Thr Leu Ala Ser Ile Asp Leu Gln Ser Gln
180 185 190
Met Glu Pro Arg Thr His Pro Thr Gly Gly Gly Ser Gly Ala Pro Thr
195 200 205
Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu Arg Leu
210 215 220
Asp Leu Glu Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn Pro Lys
225 230 235 240
Leu Thr Asp Met Leu Thr Phe Glu Phe Tyr Met Pro Lys Lys Ala Thr
245 250 255
Glu Leu Lys His Leu Gln Cys Leu Glu Arg Glu Leu Lys Pro Leu Glu
260 265 270
Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg Pro Arg
275 280 285
Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys Gly Ser
290 295 300
Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr Ile Val
305 310 315 320
Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Thr Ser Ile Ile Ser Thr
325 330 335
Leu Thr Gly Gly Gly Gly Ser Gly Gly Gly Asp Lys Thr His Thr Cys
340 345 350
Pro Pro Cys Pro Ala Pro Glu Lys Lys Gly Gly Pro Ser Val Phe Leu
355 360 365
Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu
370 375 380
Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys
385 390 395 400
Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys
405 410 415
Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu
420 425 430
Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys
435 440 445
Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys
450 455 460
Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Val Leu Pro Pro Ser
465 470 475 480
Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Leu Cys Leu Val Lys
485 490 495
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln
500 505 510
Pro Glu Asn Arg Tyr Met Thr Trp Pro Pro Val Leu Asp Ser Asp Gly
515 520 525
Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln
530 535 540
Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn
545 550 555 560
His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
565 570
<210> 174
<211> 573
<212> PRT
<213> Homo sapiens (Homo sapiens)
<400> 174
Leu Phe Thr Val Thr Val Pro Lys Glu Leu Tyr Ile Ile Glu His Gly
1 5 10 15
Ser Asn Val Thr Leu Glu Cys Asn Phe Asp Thr Gly Ser His Val Asn
20 25 30
Leu Gly Ala Ile Thr Ala Val Leu Gln Lys Val Glu Asn Asp Thr Ser
35 40 45
Pro His Arg Glu Arg Ala Thr Leu Leu Glu Glu Gln Leu Pro Leu Gly
50 55 60
Lys Ala Ser Phe His Ile Pro Gln Val Gln Val Arg Asp Glu Gly Gln
65 70 75 80
Tyr Gln Cys Ile Ile Ile Tyr Gly Val Ala Trp Asp Tyr Lys Tyr Leu
85 90 95
Thr Leu Lys Val Lys Ala Ser Tyr Arg Lys Ile Asn Thr His Ile Leu
100 105 110
Lys Val Pro Glu Thr Asp Glu Val Glu Leu Thr Cys Gln Ala Thr Gly
115 120 125
Tyr Pro Leu Ala Glu Val Ser Trp Pro Asn Val Ser Val Pro Ala Asn
130 135 140
Thr Ser His Ser Arg Thr Pro Glu Gly Leu Tyr Gln Val Thr Ser Leu
145 150 155 160
Arg Leu Lys Pro Pro Pro Gly Arg Asn Phe Ser Cys Val Phe Trp Asn
165 170 175
Thr His Val Arg Glu Leu Thr Leu Ala Ser Ile Asp Leu Gln Ser Gln
180 185 190
Met Glu Pro Arg Thr His Pro Thr Gly Gly Gly Ser Gly Ala Pro Thr
195 200 205
Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu Arg Leu
210 215 220
Asp Leu Glu Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn Pro Lys
225 230 235 240
Leu Thr Asp Met Leu Thr Phe Glu Phe Tyr Met Pro Lys Lys Ala Thr
245 250 255
Glu Leu Lys His Leu Gln Cys Leu Glu Arg Glu Leu Lys Pro Leu Glu
260 265 270
Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg Pro Arg
275 280 285
Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys Gly Ser
290 295 300
Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr Ile Val
305 310 315 320
Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Thr Ser Ile Ile Arg Thr
325 330 335
Leu Thr Gly Gly Gly Gly Ser Gly Gly Gly Asp Lys Thr His Thr Cys
340 345 350
Pro Pro Cys Pro Ala Pro Glu Lys Lys Gly Gly Pro Ser Val Phe Leu
355 360 365
Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu
370 375 380
Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys
385 390 395 400
Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys
405 410 415
Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu
420 425 430
Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys
435 440 445
Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys
450 455 460
Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Val Leu Pro Pro Ser
465 470 475 480
Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Leu Cys Leu Val Lys
485 490 495
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln
500 505 510
Pro Glu Asn Arg Tyr Met Thr Trp Pro Pro Val Leu Asp Ser Asp Gly
515 520 525
Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln
530 535 540
Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn
545 550 555 560
His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
565 570
<210> 175
<211> 574
<212> PRT
<213> Homo sapiens (Homo sapiens)
<400> 175
Leu Phe Thr Val Thr Val Pro Lys Glu Leu Tyr Ile Ile Glu His Gly
1 5 10 15
Ser Asn Val Thr Leu Glu Cys Asn Phe Asp Thr Gly Ser His Val Asn
20 25 30
Leu Gly Ala Ile Val Ala Val Leu Leu Lys Val Glu Asn Asp Thr Ser
35 40 45
Pro His Arg Glu Arg Ala Thr Leu Leu Glu Glu Gln Leu Pro Leu Gly
50 55 60
Lys Ala Ser Phe His Ile Pro Gln Val Gln Val Arg Asp Glu Gly Gln
65 70 75 80
Tyr Gln Cys Ile Ile Ile Tyr Gly Val Ala Trp Asp Tyr Lys Tyr Leu
85 90 95
Thr Leu Lys Val Lys Ala Ser Tyr Arg Lys Ile Asn Thr His Ile Leu
100 105 110
Lys Val Pro Glu Thr Asp Glu Val Glu Leu Thr Cys Gln Ala Thr Gly
115 120 125
Tyr Pro Leu Ala Glu Val Ser Trp Pro Asn Val Ser Val Pro Ala Asn
130 135 140
Thr Ser His Ser Arg Thr Pro Glu Gly Leu Tyr Gln Val Thr Ser Val
145 150 155 160
Leu Arg Leu Lys Pro Pro Pro Gly Arg Asn Phe Ser Cys Val Phe Trp
165 170 175
Asn Thr His Val Arg Glu Leu Thr Leu Ala Ser Ile Asp Leu Gln Ser
180 185 190
Gln Met Glu Pro Arg Thr His Pro Thr Gly Gly Gly Ser Gly Ala Pro
195 200 205
Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu Arg
210 215 220
Leu Asp Leu Glu Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn Pro
225 230 235 240
Lys Leu Thr Asp Met Leu Thr Phe Glu Phe Tyr Met Pro Lys Lys Ala
245 250 255
Thr Glu Leu Lys His Leu Gln Cys Leu Glu Arg Glu Leu Lys Pro Leu
260 265 270
Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg Pro
275 280 285
Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys Gly
290 295 300
Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr Ile
305 310 315 320
Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile Ile Ser
325 330 335
Thr Leu Thr Gly Gly Gly Gly Ser Gly Gly Gly Asp Lys Thr His Thr
340 345 350
Cys Pro Pro Cys Pro Ala Pro Glu Lys Lys Gly Gly Pro Ser Val Phe
355 360 365
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro
370 375 380
Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val
385 390 395 400
Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr
405 410 415
Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val
420 425 430
Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys
435 440 445
Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser
450 455 460
Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Val Leu Pro Pro
465 470 475 480
Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Leu Cys Leu Val
485 490 495
Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly
500 505 510
Gln Pro Glu Asn Arg Tyr Met Thr Trp Pro Pro Val Leu Asp Ser Asp
515 520 525
Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp
530 535 540
Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His
545 550 555 560
Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
565 570
<210> 176
<211> 574
<212> PRT
<213> Homo sapiens (Homo sapiens)
<400> 176
Leu Phe Thr Val Thr Val Pro Lys Glu Leu Tyr Ile Ile Glu His Gly
1 5 10 15
Ser Asn Val Thr Leu Glu Cys Asn Phe Asp Thr Gly Ser His Val Asn
20 25 30
Leu Gly Ala Ile Val Ala Val Leu Leu Lys Val Glu Asn Asp Thr Ser
35 40 45
Pro His Arg Glu Arg Ala Thr Leu Leu Glu Glu Gln Leu Pro Leu Gly
50 55 60
Lys Ala Ser Phe His Ile Pro Gln Val Gln Val Arg Asp Glu Gly Gln
65 70 75 80
Tyr Gln Cys Ile Ile Ile Tyr Gly Val Ala Trp Asp Tyr Lys Tyr Leu
85 90 95
Thr Leu Lys Val Lys Ala Ser Tyr Arg Lys Ile Asn Thr His Ile Leu
100 105 110
Lys Val Pro Glu Thr Asp Glu Val Glu Leu Thr Cys Gln Ala Thr Gly
115 120 125
Tyr Pro Leu Ala Glu Val Ser Trp Pro Asn Val Ser Val Pro Ala Asn
130 135 140
Thr Ser His Ser Arg Thr Pro Glu Gly Leu Tyr Gln Val Thr Ser Val
145 150 155 160
Leu Arg Leu Lys Pro Pro Pro Gly Arg Asn Phe Ser Cys Val Phe Trp
165 170 175
Asn Thr His Val Arg Glu Leu Thr Leu Ala Ser Ile Asp Leu Gln Ser
180 185 190
Gln Met Glu Pro Arg Thr His Pro Thr Gly Gly Gly Ser Gly Ala Pro
195 200 205
Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu Leu
210 215 220
Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn Pro
225 230 235 240
Lys Leu Thr Asp Met Leu Thr Phe Glu Phe Tyr Met Pro Lys Lys Ala
245 250 255
Thr Glu Leu Lys His Leu Gln Cys Leu Glu Arg Glu Leu Lys Pro Leu
260 265 270
Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg Pro
275 280 285
Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys Gly
290 295 300
Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr Ile
305 310 315 320
Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Thr Ser Ile Ile Ser
325 330 335
Thr Leu Thr Gly Gly Gly Gly Ser Gly Gly Gly Asp Lys Thr His Thr
340 345 350
Cys Pro Pro Cys Pro Ala Pro Glu Lys Lys Gly Gly Pro Ser Val Phe
355 360 365
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro
370 375 380
Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val
385 390 395 400
Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr
405 410 415
Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val
420 425 430
Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys
435 440 445
Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser
450 455 460
Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Val Leu Pro Pro
465 470 475 480
Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Leu Cys Leu Val
485 490 495
Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly
500 505 510
Gln Pro Glu Asn Arg Tyr Met Thr Trp Pro Pro Val Leu Asp Ser Asp
515 520 525
Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp
530 535 540
Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His
545 550 555 560
Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
565 570
<210> 177
<211> 574
<212> PRT
<213> Homo sapiens (Homo sapiens)
<400> 177
Leu Phe Thr Val Thr Val Pro Lys Glu Leu Tyr Ile Ile Glu His Gly
1 5 10 15
Ser Asn Val Thr Leu Glu Cys Asn Phe Asp Thr Gly Ser His Val Asn
20 25 30
Leu Gly Ala Ile Val Ala Val Leu Leu Lys Val Glu Asn Asp Thr Ser
35 40 45
Pro His Arg Glu Arg Ala Thr Leu Leu Glu Glu Gln Leu Pro Leu Gly
50 55 60
Lys Ala Ser Phe His Ile Pro Gln Val Gln Val Arg Asp Glu Gly Gln
65 70 75 80
Tyr Gln Cys Ile Ile Ile Tyr Gly Val Ala Trp Asp Tyr Lys Tyr Leu
85 90 95
Thr Leu Lys Val Lys Ala Ser Tyr Arg Lys Ile Asn Thr His Ile Leu
100 105 110
Lys Val Pro Glu Thr Asp Glu Val Glu Leu Thr Cys Gln Ala Thr Gly
115 120 125
Tyr Pro Leu Ala Glu Val Ser Trp Pro Asn Val Ser Val Pro Ala Asn
130 135 140
Thr Ser His Ser Arg Thr Pro Glu Gly Leu Tyr Gln Val Thr Ser Val
145 150 155 160
Leu Arg Leu Lys Pro Pro Pro Gly Arg Asn Phe Ser Cys Val Phe Trp
165 170 175
Asn Thr His Val Arg Glu Leu Thr Leu Ala Ser Ile Asp Leu Gln Ser
180 185 190
Gln Met Glu Pro Arg Thr His Pro Thr Gly Gly Gly Ser Gly Ala Pro
195 200 205
Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu Arg
210 215 220
Leu Asp Leu Glu Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn Pro
225 230 235 240
Lys Leu Thr Asp Met Leu Thr Phe Glu Phe Tyr Met Pro Lys Lys Ala
245 250 255
Thr Glu Leu Lys His Leu Gln Cys Leu Glu Arg Glu Leu Lys Pro Leu
260 265 270
Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg Pro
275 280 285
Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys Gly
290 295 300
Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr Ile
305 310 315 320
Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Thr Ser Ile Ile Ser
325 330 335
Thr Leu Thr Gly Gly Gly Gly Ser Gly Gly Gly Asp Lys Thr His Thr
340 345 350
Cys Pro Pro Cys Pro Ala Pro Glu Lys Lys Gly Gly Pro Ser Val Phe
355 360 365
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro
370 375 380
Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val
385 390 395 400
Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr
405 410 415
Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val
420 425 430
Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys
435 440 445
Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser
450 455 460
Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Val Leu Pro Pro
465 470 475 480
Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Leu Cys Leu Val
485 490 495
Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly
500 505 510
Gln Pro Glu Asn Arg Tyr Met Thr Trp Pro Pro Val Leu Asp Ser Asp
515 520 525
Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp
530 535 540
Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His
545 550 555 560
Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
565 570
<210> 178
<211> 574
<212> PRT
<213> Homo sapiens (Homo sapiens)
<400> 178
Leu Phe Thr Val Thr Val Pro Lys Glu Leu Tyr Ile Ile Glu His Gly
1 5 10 15
Ser Asn Val Thr Leu Glu Cys Asn Phe Asp Thr Gly Ser His Val Asn
20 25 30
Leu Gly Ala Ile Val Ala Val Leu Leu Lys Val Glu Asn Asp Thr Ser
35 40 45
Pro His Arg Glu Arg Ala Thr Leu Leu Glu Glu Gln Leu Pro Leu Gly
50 55 60
Lys Ala Ser Phe His Ile Pro Gln Val Gln Val Arg Asp Glu Gly Gln
65 70 75 80
Tyr Gln Cys Ile Ile Ile Tyr Gly Val Ala Trp Asp Tyr Lys Tyr Leu
85 90 95
Thr Leu Lys Val Lys Ala Ser Tyr Arg Lys Ile Asn Thr His Ile Leu
100 105 110
Lys Val Pro Glu Thr Asp Glu Val Glu Leu Thr Cys Gln Ala Thr Gly
115 120 125
Tyr Pro Leu Ala Glu Val Ser Trp Pro Asn Val Ser Val Pro Ala Asn
130 135 140
Thr Ser His Ser Arg Thr Pro Glu Gly Leu Tyr Gln Val Thr Ser Val
145 150 155 160
Leu Arg Leu Lys Pro Pro Pro Gly Arg Asn Phe Ser Cys Val Phe Trp
165 170 175
Asn Thr His Val Arg Glu Leu Thr Leu Ala Ser Ile Asp Leu Gln Ser
180 185 190
Gln Met Glu Pro Arg Thr His Pro Thr Gly Gly Gly Ser Gly Ala Pro
195 200 205
Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu Arg
210 215 220
Leu Asp Leu Glu Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn Pro
225 230 235 240
Lys Leu Thr Asp Met Leu Thr Phe Glu Phe Tyr Met Pro Lys Lys Ala
245 250 255
Thr Glu Leu Lys His Leu Gln Cys Leu Glu Arg Glu Leu Lys Pro Leu
260 265 270
Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg Pro
275 280 285
Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys Gly
290 295 300
Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr Ile
305 310 315 320
Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Thr Ser Ile Ile Arg
325 330 335
Thr Leu Thr Gly Gly Gly Gly Ser Gly Gly Gly Asp Lys Thr His Thr
340 345 350
Cys Pro Pro Cys Pro Ala Pro Glu Lys Lys Gly Gly Pro Ser Val Phe
355 360 365
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro
370 375 380
Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val
385 390 395 400
Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr
405 410 415
Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val
420 425 430
Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys
435 440 445
Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser
450 455 460
Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Val Leu Pro Pro
465 470 475 480
Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Leu Cys Leu Val
485 490 495
Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly
500 505 510
Gln Pro Glu Asn Arg Tyr Met Thr Trp Pro Pro Val Leu Asp Ser Asp
515 520 525
Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp
530 535 540
Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His
545 550 555 560
Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
565 570
<210> 179
<211> 593
<212> PRT
<213> Homo sapiens (Homo sapiens)
<400> 179
Phe Thr Val Thr Val Pro Lys Asp Leu Tyr Val Val Glu Tyr Gly Ser
1 5 10 15
Asn Met Thr Ile Glu Cys Lys Phe Pro Val Glu Lys Gln Leu Asp Leu
20 25 30
Ala Ala Leu Gln Val Tyr Trp Met Met Glu Asp Lys Asn Ile Ile Gln
35 40 45
Phe Val His Gly Glu Glu Asp Leu Lys Val Gln His Ser Ser Tyr Arg
50 55 60
Gln Arg Ala Arg Leu Leu Lys Asp Gln Leu Ser Leu Gly Asn Ala Ala
65 70 75 80
Leu Gln Ile Thr Asp Val Lys Leu Gln Asp Ala Gly Val Tyr Thr Cys
85 90 95
Leu Ile Ala Tyr Lys Gly Ala Asp Tyr Lys Arg Ile Thr Val Lys Val
100 105 110
Asn Ala Pro Tyr Asn Lys Ile Asn Gln Arg Ile Leu Val Val Asp Pro
115 120 125
Val Thr Ser Glu His Glu Leu Thr Cys Gln Ala Glu Gly Tyr Pro Lys
130 135 140
Ala Glu Val Ile Trp Thr Ser Ser Asp His Gln Val Leu Ser Gly Lys
145 150 155 160
Thr Thr Thr Thr Asn Ser Lys Arg Glu Glu Lys Leu Phe Asn Val Thr
165 170 175
Ser Thr Leu Arg Ile Asn Thr Thr Thr Asn Glu Ile Phe Tyr Cys Thr
180 185 190
Phe Arg Arg Leu Asp Pro Glu Glu Asn His Thr Ala Glu Leu Val Ile
195 200 205
Pro Glu Leu Pro Leu Ala His Pro Pro Asn Glu Arg Gly Gly Gly Ser
210 215 220
Gly Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu
225 230 235 240
His Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr
245 250 255
Lys Asn Pro Lys Leu Thr Asp Met Leu Thr Phe Glu Phe Tyr Met Pro
260 265 270
Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Arg Glu Leu
275 280 285
Lys Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His
290 295 300
Leu Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu
305 310 315 320
Leu Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr
325 330 335
Ala Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser
340 345 350
Ile Ile Ser Thr Leu Thr Gly Gly Gly Gly Ser Gly Gly Gly Asp Lys
355 360 365
Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Lys Lys Gly Gly Pro
370 375 380
Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser
385 390 395 400
Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp
405 410 415
Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn
420 425 430
Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val
435 440 445
Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu
450 455 460
Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys
465 470 475 480
Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Val
485 490 495
Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Leu
500 505 510
Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu
515 520 525
Ser Asn Gly Gln Pro Glu Asn Arg Tyr Met Thr Trp Pro Pro Val Leu
530 535 540
Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys
545 550 555 560
Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu
565 570 575
Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly
580 585 590
Lys
<210> 180
<211> 987
<212> PRT
<213> Homo sapiens (Homo sapiens)
<400> 180
Phe Thr Val Thr Val Pro Lys Asp Leu Tyr Val Val Glu Tyr Gly Ser
1 5 10 15
Asn Met Thr Ile Glu Cys Lys Phe Pro Val Glu Lys Gln Leu Asp Leu
20 25 30
Ala Ala Leu Gln Val Tyr Trp Met Met Glu Asp Lys Asn Ile Ile Gln
35 40 45
Phe Val His Gly Glu Glu Asp Leu Lys Val Gln His Ser Ser Tyr Arg
50 55 60
Gln Arg Ala Arg Leu Leu Lys Asp Gln Leu Ser Leu Gly Asn Ala Ala
65 70 75 80
Leu Gln Ile Thr Asp Val Lys Leu Gln Asp Ala Gly Val Tyr Thr Cys
85 90 95
Leu Ile Ala Tyr Lys Gly Ala Asp Tyr Lys Arg Ile Thr Val Lys Val
100 105 110
Asn Ala Pro Tyr Asn Lys Ile Asn Gln Arg Ile Leu Val Val Asp Pro
115 120 125
Val Thr Ser Glu His Glu Leu Thr Cys Gln Ala Glu Gly Tyr Pro Lys
130 135 140
Ala Glu Val Ile Trp Thr Ser Ser Asp His Gln Val Leu Ser Gly Lys
145 150 155 160
Thr Thr Thr Thr Asn Ser Lys Arg Glu Glu Lys Leu Phe Asn Val Thr
165 170 175
Ser Thr Leu Arg Ile Asn Thr Thr Thr Asn Glu Ile Phe Tyr Cys Thr
180 185 190
Phe Arg Arg Leu Asp Pro Glu Glu Asn His Thr Ala Glu Leu Val Ile
195 200 205
Pro Glu Leu Pro Leu Ala His Pro Pro Asn Glu Arg Gly Gly Gly Gly
210 215 220
Ser Gly Gly Gly Met Trp Glu Leu Glu Lys Asp Val Tyr Val Val Glu
225 230 235 240
Val Asp Trp Thr Pro Asp Ala Pro Gly Glu Thr Val Asn Leu Thr Cys
245 250 255
Asp Thr Pro Glu Glu Asp Asp Ile Thr Trp Thr Ser Asp Gln Arg His
260 265 270
Gly Val Ile Gly Ser Gly Lys Thr Leu Thr Ile Thr Val Lys Ala Ala
275 280 285
Leu Asp Ala Gly Gln Tyr Thr Cys His Lys Gly Gly Glu Thr Leu Ser
290 295 300
His Ser His Leu Leu Leu His Lys Lys Glu Asn Gly Ile Trp Ser Thr
305 310 315 320
Glu Ile Leu Lys Asn Phe Lys Asn Lys Thr Phe Leu Lys Cys Glu Ala
325 330 335
Pro Asn Tyr Ser Gly Arg Phe Thr Cys Ser Trp Leu Val Gln Arg Asn
340 345 350
Met Asp Leu Lys Phe Asn Ile Lys Ser Ser Ser Ser Ser Pro Asp Ser
355 360 365
Arg Ala Val Thr Cys Gly Met Ala Ser Leu Ser Ala Glu Lys Val Thr
370 375 380
Leu Asp Gln Arg Asp Tyr Glu Lys Tyr Ser Val Ser Cys Gln Glu Asp
385 390 395 400
Val Thr Cys Pro Thr Ala Glu Glu Thr Leu Pro Ile Glu Leu Ala Leu
405 410 415
Glu Ala Arg Gln Gln Asn Lys Tyr Glu Asn Tyr Ser Thr Ser Phe Phe
420 425 430
Ile Arg Asp Ile Ile Lys Pro Asp Pro Pro Lys Asn Leu Gln Met Lys
435 440 445
Pro Leu Lys Asn Ser Gln Val Glu Val Ser Trp Glu Tyr Pro Asp Ser
450 455 460
Trp Ser Thr Pro His Ser Tyr Phe Ser Leu Lys Phe Phe Val Arg Ile
465 470 475 480
Gln Arg Lys Lys Glu Lys Met Lys Glu Thr Glu Glu Gly Cys Asn Gln
485 490 495
Lys Gly Ala Phe Leu Val Glu Lys Thr Ser Thr Glu Val Gln Cys Lys
500 505 510
Gly Gly Asn Val Cys Val Gln Ala Gln Asp Arg Tyr Tyr Asn Ser Ser
515 520 525
Cys Ser Lys Trp Ala Cys Val Pro Cys Arg Val Arg Ser Gly Gly Pro
530 535 540
Gly Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Arg
545 550 555 560
Val Ile Pro Val Ser Gly Pro Ala Arg Cys Leu Ser Gln Ser Arg Asn
565 570 575
Leu Leu Lys Thr Thr Asp Asp Met Val Lys Thr Ala Arg Glu Lys Leu
580 585 590
Lys His Tyr Ser Cys Thr Ala Glu Asp Ile Asp His Glu Asp Ile Thr
595 600 605
Arg Asp Gln Thr Ser Thr Leu Lys Thr Cys Leu Pro Leu Glu Leu His
610 615 620
Lys Asn Glu Ser Cys Leu Ala Thr Arg Glu Thr Ser Ser Thr Thr Arg
625 630 635 640
Gly Ser Cys Leu Pro Pro Gln Lys Thr Ser Leu Met Met Thr Leu Cys
645 650 655
Leu Gly Ser Ile Tyr Glu Asp Leu Lys Met Tyr Gln Thr Glu Phe Gln
660 665 670
Ala Ile Asn Ala Ala Leu Gln Asn His Asn His Gln Gln Ile Ile Leu
675 680 685
Asp Lys Gly Met Leu Val Ala Ile Asp Glu Leu Met Gln Ser Leu Asn
690 695 700
His Asn Gly Glu Thr Leu Arg Gln Lys Pro Pro Val Gly Glu Ala Asp
705 710 715 720
Pro Tyr Arg Val Lys Met Lys Leu Cys Ile Leu Leu His Ala Phe Ser
725 730 735
Thr Arg Val Val Thr Ile Asn Arg Val Met Gly Tyr Leu Ser Ser Ala
740 745 750
Gly Gly Gly Gly Ser Gly Gly Gly Asp Lys Thr His Thr Cys Pro Pro
755 760 765
Cys Pro Ala Pro Glu Asp Glu Gly Gly Pro Ser Val Phe Leu Phe Pro
770 775 780
Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr
785 790 795 800
Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn
805 810 815
Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg
820 825 830
Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val
835 840 845
Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser
850 855 860
Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys
865 870 875 880
Gly Gln Pro Arg Glu Pro Gln Val Tyr Val Tyr Pro Pro Ser Arg Glu
885 890 895
Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe
900 905 910
Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu
915 920 925
Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Glu Asp Gly Ser Phe
930 935 940
Ala Leu Val Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly
945 950 955 960
Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr
965 970 975
Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
980 985
<210> 181
<211> 987
<212> PRT
<213> Homo sapiens (Homo sapiens)
<400> 181
Phe Thr Val Thr Val Pro Lys Asp Leu Tyr Val Val Glu Tyr Gly Ser
1 5 10 15
Asn Met Thr Ile Glu Cys Lys Phe Pro Val Glu Lys Gln Leu Asp Leu
20 25 30
Ala Ala Leu Gln Val Tyr Trp Met Met Glu Asp Lys Asn Ile Ile Gln
35 40 45
Phe Val His Gly Glu Glu Asp Leu Lys Val Gln His Ser Ser Tyr Arg
50 55 60
Gln Arg Ala Arg Leu Leu Lys Asp Gln Leu Ser Leu Gly Asn Ala Ala
65 70 75 80
Leu Gln Ile Thr Asp Val Lys Leu Gln Asp Ala Gly Val Tyr Thr Cys
85 90 95
Leu Ile Ala Tyr Lys Gly Ala Asp Tyr Lys Arg Ile Thr Val Lys Val
100 105 110
Asn Ala Pro Tyr Asn Lys Ile Asn Gln Arg Ile Leu Val Val Asp Pro
115 120 125
Val Thr Ser Glu His Glu Leu Thr Cys Gln Ala Glu Gly Tyr Pro Lys
130 135 140
Ala Glu Val Ile Trp Thr Ser Ser Asp His Gln Val Leu Ser Gly Lys
145 150 155 160
Thr Thr Thr Thr Asn Ser Lys Arg Glu Glu Lys Leu Phe Asn Val Thr
165 170 175
Ser Thr Leu Arg Ile Asn Thr Thr Thr Asn Glu Ile Phe Tyr Cys Thr
180 185 190
Phe Arg Arg Leu Asp Pro Glu Glu Asn His Thr Ala Glu Leu Val Ile
195 200 205
Pro Glu Leu Pro Leu Ala His Pro Pro Asn Glu Arg Gly Gly Gly Gly
210 215 220
Ser Gly Gly Gly Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro
225 230 235 240
Glu Asp Glu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys
245 250 255
Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val
260 265 270
Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp
275 280 285
Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr
290 295 300
Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp
305 310 315 320
Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu
325 330 335
Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg
340 345 350
Glu Pro Gln Val Tyr Val Tyr Pro Pro Ser Arg Glu Glu Met Thr Lys
355 360 365
Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp
370 375 380
Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
385 390 395 400
Thr Thr Pro Pro Val Leu Asp Glu Asp Gly Ser Phe Ala Leu Val Ser
405 410 415
Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser
420 425 430
Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser
435 440 445
Leu Ser Leu Ser Pro Gly Lys Gly Gly Gly Gly Ser Gly Gly Gly Met
450 455 460
Trp Glu Leu Glu Lys Asp Val Tyr Val Val Glu Val Asp Trp Thr Pro
465 470 475 480
Asp Ala Pro Gly Glu Thr Val Asn Leu Thr Cys Asp Thr Pro Glu Glu
485 490 495
Asp Asp Ile Thr Trp Thr Ser Asp Gln Arg His Gly Val Ile Gly Ser
500 505 510
Gly Lys Thr Leu Thr Ile Thr Val Lys Ala Ala Leu Asp Ala Gly Gln
515 520 525
Tyr Thr Cys His Lys Gly Gly Glu Thr Leu Ser His Ser His Leu Leu
530 535 540
Leu His Lys Lys Glu Asn Gly Ile Trp Ser Thr Glu Ile Leu Lys Asn
545 550 555 560
Phe Lys Asn Lys Thr Phe Leu Lys Cys Glu Ala Pro Asn Tyr Ser Gly
565 570 575
Arg Phe Thr Cys Ser Trp Leu Val Gln Arg Asn Met Asp Leu Lys Phe
580 585 590
Asn Ile Lys Ser Ser Ser Ser Ser Pro Asp Ser Arg Ala Val Thr Cys
595 600 605
Gly Met Ala Ser Leu Ser Ala Glu Lys Val Thr Leu Asp Gln Arg Asp
610 615 620
Tyr Glu Lys Tyr Ser Val Ser Cys Gln Glu Asp Val Thr Cys Pro Thr
625 630 635 640
Ala Glu Glu Thr Leu Pro Ile Glu Leu Ala Leu Glu Ala Arg Gln Gln
645 650 655
Asn Lys Tyr Glu Asn Tyr Ser Thr Ser Phe Phe Ile Arg Asp Ile Ile
660 665 670
Lys Pro Asp Pro Pro Lys Asn Leu Gln Met Lys Pro Leu Lys Asn Ser
675 680 685
Gln Val Glu Val Ser Trp Glu Tyr Pro Asp Ser Trp Ser Thr Pro His
690 695 700
Ser Tyr Phe Ser Leu Lys Phe Phe Val Arg Ile Gln Arg Lys Lys Glu
705 710 715 720
Lys Met Lys Glu Thr Glu Glu Gly Cys Asn Gln Lys Gly Ala Phe Leu
725 730 735
Val Glu Lys Thr Ser Thr Glu Val Gln Cys Lys Gly Gly Asn Val Cys
740 745 750
Val Gln Ala Gln Asp Arg Tyr Tyr Asn Ser Ser Cys Ser Lys Trp Ala
755 760 765
Cys Val Pro Cys Arg Val Arg Ser Gly Gly Pro Gly Gly Gly Gly Ser
770 775 780
Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Arg Val Ile Pro Val Ser
785 790 795 800
Gly Pro Ala Arg Cys Leu Ser Gln Ser Arg Asn Leu Leu Lys Thr Thr
805 810 815
Asp Asp Met Val Lys Thr Ala Arg Glu Lys Leu Lys His Tyr Ser Cys
820 825 830
Thr Ala Glu Asp Ile Asp His Glu Asp Ile Thr Arg Asp Gln Thr Ser
835 840 845
Thr Leu Lys Thr Cys Leu Pro Leu Glu Leu His Lys Asn Glu Ser Cys
850 855 860
Leu Ala Thr Arg Glu Thr Ser Ser Thr Thr Arg Gly Ser Cys Leu Pro
865 870 875 880
Pro Gln Lys Thr Ser Leu Met Met Thr Leu Cys Leu Gly Ser Ile Tyr
885 890 895
Glu Asp Leu Lys Met Tyr Gln Thr Glu Phe Gln Ala Ile Asn Ala Ala
900 905 910
Leu Gln Asn His Asn His Gln Gln Ile Ile Leu Asp Lys Gly Met Leu
915 920 925
Val Ala Ile Asp Glu Leu Met Gln Ser Leu Asn His Asn Gly Glu Thr
930 935 940
Leu Arg Gln Lys Pro Pro Val Gly Glu Ala Asp Pro Tyr Arg Val Lys
945 950 955 960
Met Lys Leu Cys Ile Leu Leu His Ala Phe Ser Thr Arg Val Val Thr
965 970 975
Ile Asn Arg Val Met Gly Tyr Leu Ser Ser Ala
980 985
<210> 182
<211> 758
<212> PRT
<213> Homo sapiens (Homo sapiens)
<400> 182
Phe Thr Val Thr Val Pro Lys Asp Leu Tyr Val Val Glu Tyr Gly Ser
1 5 10 15
Asn Met Thr Ile Glu Cys Lys Phe Pro Val Glu Lys Gln Leu Asp Leu
20 25 30
Ala Ala Leu Gln Val Tyr Trp Met Met Glu Asp Lys Asn Ile Ile Gln
35 40 45
Phe Val His Gly Glu Glu Asp Leu Lys Val Gln His Ser Ser Tyr Arg
50 55 60
Gln Arg Ala Arg Leu Leu Lys Asp Gln Leu Ser Leu Gly Asn Ala Ala
65 70 75 80
Leu Gln Ile Thr Asp Val Lys Leu Gln Asp Ala Gly Val Tyr Thr Cys
85 90 95
Leu Ile Ala Tyr Lys Gly Ala Asp Tyr Lys Arg Ile Thr Val Lys Val
100 105 110
Asn Ala Pro Tyr Asn Lys Ile Asn Gln Arg Ile Leu Val Val Asp Pro
115 120 125
Val Thr Ser Glu His Glu Leu Thr Cys Gln Ala Glu Gly Tyr Pro Lys
130 135 140
Ala Glu Val Ile Trp Thr Ser Ser Asp His Gln Val Leu Ser Gly Lys
145 150 155 160
Thr Thr Thr Thr Asn Ser Lys Arg Glu Glu Lys Leu Phe Asn Val Thr
165 170 175
Ser Thr Leu Arg Ile Asn Thr Thr Thr Asn Glu Ile Phe Tyr Cys Thr
180 185 190
Phe Arg Arg Leu Asp Pro Glu Glu Asn His Thr Ala Glu Leu Val Ile
195 200 205
Pro Glu Leu Pro Leu Ala His Pro Pro Asn Glu Arg Gly Gly Gly Gly
210 215 220
Ser Gly Gly Gly Gln Asp Pro Tyr Val Lys Glu Ala Glu Asn Leu Lys
225 230 235 240
Lys Tyr Phe Asn Ala Gly His Ser Asp Val Val Asp Asn Gly Thr Leu
245 250 255
Phe Leu Gly Ile Leu Lys Asn Trp Lys Glu Glu Ser Asp Arg Lys Ile
260 265 270
Met Gln Ser Gln Ile Val Ser Phe Tyr Phe Lys Leu Phe Lys Asn Phe
275 280 285
Lys Asp Asp Gln Ser Ile Gln Lys Ser Val Glu Thr Ile Lys Glu Asp
290 295 300
Met Asn Val Lys Phe Phe Asn Ser Asn Lys Lys Lys Arg Asp Asp Phe
305 310 315 320
Glu Lys Leu Thr Asn Tyr Ser Val Thr Asp Leu Asn Val Gln Arg Lys
325 330 335
Ala Ile His Glu Leu Ile Gln Val Met Ala Glu Leu Ser Pro Ala Ala
340 345 350
Lys Thr Gly Lys Arg Lys Arg Ser Gln Met Leu Phe Arg Gly Gly Gly
355 360 365
Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
370 375 380
Ser Gln Asp Pro Tyr Val Lys Glu Ala Glu Asn Leu Lys Lys Tyr Phe
385 390 395 400
Asn Ala Gly His Ser Asp Val Val Asp Asn Gly Thr Leu Phe Leu Gly
405 410 415
Ile Leu Lys Asn Trp Lys Glu Glu Ser Asp Arg Lys Ile Met Gln Ser
420 425 430
Gln Ile Val Ser Phe Tyr Phe Lys Leu Phe Lys Asn Phe Lys Asp Asp
435 440 445
Gln Ser Ile Gln Lys Ser Val Glu Thr Ile Lys Glu Asp Met Asn Val
450 455 460
Lys Phe Phe Asn Ser Asn Lys Lys Lys Arg Asp Asp Phe Glu Lys Leu
465 470 475 480
Thr Asn Tyr Ser Val Thr Asp Leu Asn Val Gln Arg Lys Ala Ile His
485 490 495
Glu Leu Ile Gln Val Met Ala Glu Leu Ser Pro Ala Ala Lys Thr Gly
500 505 510
Lys Arg Lys Arg Ser Gln Met Leu Phe Arg Gly Gly Gly Gly Gly Ser
515 520 525
Gly Gly Gly Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu
530 535 540
Asp Glu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp
545 550 555 560
Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp
565 570 575
Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly
580 585 590
Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn
595 600 605
Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp
610 615 620
Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro
625 630 635 640
Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu
645 650 655
Pro Gln Val Tyr Val Tyr Pro Pro Ser Arg Glu Glu Met Thr Lys Asn
660 665 670
Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile
675 680 685
Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr
690 695 700
Thr Pro Pro Val Leu Asp Glu Asp Gly Ser Phe Ala Leu Val Ser Lys
705 710 715 720
Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys
725 730 735
Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu
740 745 750
Ser Leu Ser Pro Gly Lys
755
<210> 183
<211> 758
<212> PRT
<213> Homo sapiens (Homo sapiens)
<400> 183
Phe Thr Val Thr Val Pro Lys Asp Leu Tyr Val Val Glu Tyr Gly Ser
1 5 10 15
Asn Met Thr Ile Glu Cys Lys Phe Pro Val Glu Lys Gln Leu Asp Leu
20 25 30
Ala Ala Leu Gln Val Tyr Trp Met Met Glu Asp Lys Asn Ile Ile Gln
35 40 45
Phe Val His Gly Glu Glu Asp Leu Lys Val Gln His Ser Ser Tyr Arg
50 55 60
Gln Arg Ala Arg Leu Leu Lys Asp Gln Leu Ser Leu Gly Asn Ala Ala
65 70 75 80
Leu Gln Ile Thr Asp Val Lys Leu Gln Asp Ala Gly Val Tyr Thr Cys
85 90 95
Leu Ile Ala Tyr Gly Gly Ala Asp Tyr Lys Arg Ile Thr Val Lys Val
100 105 110
Asn Ala Pro Tyr Asn Lys Ile Asn Gln Arg Ile Leu Val Val Asp Pro
115 120 125
Val Thr Ser Glu His Glu Leu Thr Cys Gln Ala Glu Gly Tyr Pro Lys
130 135 140
Ala Glu Val Ile Trp Thr Ser Ser Asp His Gln Val Leu Ser Gly Lys
145 150 155 160
Thr Thr Thr Thr Asn Ser Lys Arg Glu Glu Lys Leu Phe Asn Val Thr
165 170 175
Ser Thr Leu Arg Ile Asn Thr Thr Thr Asn Glu Ile Phe Tyr Cys Thr
180 185 190
Phe Arg Arg Leu Asp Pro Glu Glu Asn His Thr Ala Glu Leu Val Ile
195 200 205
Pro Glu Leu Pro Leu Ala His Pro Pro Asn Glu Arg Gly Gly Gly Gly
210 215 220
Ser Gly Gly Gly Gln Asp Pro Tyr Val Lys Glu Ala Glu Asn Leu Lys
225 230 235 240
Lys Tyr Phe Asn Ala Gly His Ser Asp Val Val Asp Asn Gly Thr Leu
245 250 255
Phe Leu Gly Ile Leu Lys Asn Trp Lys Glu Glu Ser Asp Arg Lys Ile
260 265 270
Met Gln Ser Gln Ile Val Ser Phe Tyr Phe Lys Leu Phe Lys Asn Phe
275 280 285
Lys Asp Asp Gln Ser Ile Gln Lys Ser Val Glu Thr Ile Lys Glu Asp
290 295 300
Met Asn Val Lys Phe Phe Asn Ser Asn Lys Lys Lys Arg Asp Asp Phe
305 310 315 320
Glu Lys Leu Thr Asn Tyr Ser Val Thr Asp Leu Asn Val Gln Arg Lys
325 330 335
Ala Ile His Glu Leu Ile Gln Val Met Ala Glu Leu Ser Pro Ala Ala
340 345 350
Lys Thr Gly Lys Arg Lys Arg Ser Gln Met Leu Phe Arg Gly Gly Gly
355 360 365
Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
370 375 380
Ser Gln Asp Pro Tyr Val Lys Glu Ala Glu Asn Leu Lys Lys Tyr Phe
385 390 395 400
Asn Ala Gly His Ser Asp Val Val Asp Asn Gly Thr Leu Phe Leu Gly
405 410 415
Ile Leu Lys Asn Trp Lys Glu Glu Ser Asp Arg Lys Ile Met Gln Ser
420 425 430
Gln Ile Val Ser Phe Tyr Phe Lys Leu Phe Lys Asn Phe Lys Asp Asp
435 440 445
Gln Ser Ile Gln Lys Ser Val Glu Thr Ile Lys Glu Asp Met Asn Val
450 455 460
Lys Phe Phe Asn Ser Asn Lys Lys Lys Arg Asp Asp Phe Glu Lys Leu
465 470 475 480
Thr Asn Tyr Ser Val Thr Asp Leu Asn Val Gln Arg Lys Ala Ile His
485 490 495
Glu Leu Ile Gln Val Met Ala Glu Leu Ser Pro Ala Ala Lys Thr Gly
500 505 510
Lys Arg Lys Arg Ser Gln Met Leu Phe Arg Gly Gly Gly Gly Gly Ser
515 520 525
Gly Gly Gly Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu
530 535 540
Asp Glu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp
545 550 555 560
Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp
565 570 575
Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly
580 585 590
Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn
595 600 605
Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp
610 615 620
Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro
625 630 635 640
Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu
645 650 655
Pro Gln Val Tyr Val Tyr Pro Pro Ser Arg Glu Glu Met Thr Lys Asn
660 665 670
Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile
675 680 685
Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr
690 695 700
Thr Pro Pro Val Leu Asp Glu Asp Gly Ser Phe Ala Leu Val Ser Lys
705 710 715 720
Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys
725 730 735
Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu
740 745 750
Ser Leu Ser Pro Gly Lys
755
<210> 184
<211> 755
<212> PRT
<213> Homo sapiens (Homo sapiens)
<400> 184
Phe Thr Val Thr Val Pro Lys Asp Leu Tyr Val Val Glu Tyr Gly Ser
1 5 10 15
Asn Met Thr Ile Glu Cys Lys Phe Pro Val Glu Lys Gln Leu Asp Leu
20 25 30
Ala Ala Leu Gln Val Tyr Trp Met Met Glu Asp Lys Asn Ile Ile Gln
35 40 45
Phe Val His Gly Glu Glu Asp Leu Lys Val Gln His Ser Ser Tyr Arg
50 55 60
Gln Arg Ala Arg Leu Leu Lys Asp Gln Leu Ser Leu Gly Asn Ala Ala
65 70 75 80
Leu Gln Ile Thr Asp Val Lys Leu Gln Asp Ala Gly Val Tyr Thr Cys
85 90 95
Leu Ile Ala Tyr Lys Gly Ala Asp Tyr Lys Arg Ile Thr Val Lys Val
100 105 110
Asn Ala Pro Tyr Asn Lys Ile Asn Gln Arg Ile Leu Val Val Asp Pro
115 120 125
Val Thr Ser Glu His Glu Leu Thr Cys Gln Ala Glu Gly Tyr Pro Lys
130 135 140
Ala Glu Val Ile Trp Thr Ser Ser Asp His Gln Val Leu Ser Gly Lys
145 150 155 160
Thr Thr Thr Thr Asn Ser Lys Arg Glu Glu Lys Leu Phe Asn Val Thr
165 170 175
Ser Thr Leu Arg Ile Asn Thr Thr Thr Asn Glu Ile Phe Tyr Cys Thr
180 185 190
Phe Arg Arg Leu Asp Pro Glu Glu Asn His Thr Ala Glu Leu Val Ile
195 200 205
Pro Glu Leu Pro Leu Ala His Pro Pro Asn Glu Arg Gly Gly Gly Ser
210 215 220
Gly Gln Asp Pro Tyr Val Lys Glu Ala Glu Asn Leu Lys Lys Tyr Phe
225 230 235 240
Asn Ala Gly His Ser Asp Val Val Asp Asn Gly Thr Leu Phe Leu Gly
245 250 255
Ile Leu Lys Asn Trp Lys Glu Glu Ser Asp Arg Lys Ile Met Gln Ser
260 265 270
Gln Ile Val Ser Phe Tyr Phe Lys Leu Phe Lys Asn Phe Lys Asp Asp
275 280 285
Gln Ser Ile Gln Lys Ser Val Glu Thr Ile Lys Glu Asp Met Asn Val
290 295 300
Lys Phe Phe Asn Ser Asn Lys Lys Lys Arg Asp Asp Phe Glu Lys Leu
305 310 315 320
Thr Asn Tyr Ser Val Thr Asp Leu Asn Val Gln Arg Lys Ala Ile His
325 330 335
Glu Leu Ile Gln Val Met Ala Glu Leu Ser Pro Ala Ala Lys Thr Gly
340 345 350
Lys Arg Lys Arg Ser Gln Met Leu Phe Arg Gly Gly Gly Gly Ser Gly
355 360 365
Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Asp
370 375 380
Pro Tyr Val Lys Glu Ala Glu Asn Leu Lys Lys Tyr Phe Asn Ala Gly
385 390 395 400
His Ser Asp Val Val Asp Asn Gly Thr Leu Phe Leu Gly Ile Leu Lys
405 410 415
Asn Trp Lys Glu Glu Ser Asp Arg Lys Ile Met Gln Ser Gln Ile Val
420 425 430
Ser Phe Tyr Phe Lys Leu Phe Lys Asn Phe Lys Asp Asp Gln Ser Ile
435 440 445
Gln Lys Ser Val Glu Thr Ile Lys Glu Asp Met Asn Val Lys Phe Phe
450 455 460
Asn Ser Asn Lys Lys Lys Arg Asp Asp Phe Glu Lys Leu Thr Asn Tyr
465 470 475 480
Ser Val Thr Asp Leu Asn Val Gln Arg Lys Ala Ile His Glu Leu Ile
485 490 495
Gln Val Met Ala Glu Leu Ser Pro Ala Ala Lys Thr Gly Lys Arg Lys
500 505 510
Arg Ser Gln Met Leu Phe Arg Gly Gly Gly Gly Gly Ser Gly Gly Gly
515 520 525
Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Lys Lys Gly
530 535 540
Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met
545 550 555 560
Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His
565 570 575
Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val
580 585 590
His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr
595 600 605
Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly
610 615 620
Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile
625 630 635 640
Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val
645 650 655
Tyr Val Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser
660 665 670
Leu Leu Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu
675 680 685
Trp Glu Ser Asn Gly Gln Pro Glu Asn Arg Tyr Met Thr Trp Pro Pro
690 695 700
Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val
705 710 715 720
Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met
725 730 735
His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser
740 745 750
Pro Gly Lys
755
<210> 185
<211> 755
<212> PRT
<213> Homo sapiens (Homo sapiens)
<400> 185
Phe Thr Val Thr Val Pro Lys Asp Leu Tyr Val Val Glu Tyr Gly Ser
1 5 10 15
Asn Met Thr Ile Glu Cys Lys Phe Pro Val Glu Lys Gln Leu Asp Leu
20 25 30
Ala Ala Leu Gln Val Tyr Trp Met Met Glu Asp Lys Asn Ile Ile Gln
35 40 45
Phe Val His Gly Glu Glu Asp Leu Lys Val Gln His Ser Ser Tyr Arg
50 55 60
Gln Arg Ala Arg Leu Leu Lys Asp Gln Leu Ser Leu Gly Asn Ala Ala
65 70 75 80
Leu Gln Ile Thr Asp Val Lys Leu Gln Asp Ala Gly Val Tyr Thr Cys
85 90 95
Leu Ile Ala Tyr Gly Gly Ala Asp Tyr Lys Arg Ile Thr Val Lys Val
100 105 110
Asn Ala Pro Tyr Asn Lys Ile Asn Gln Arg Ile Leu Val Val Asp Pro
115 120 125
Val Thr Ser Glu His Glu Leu Thr Cys Gln Ala Glu Gly Tyr Pro Lys
130 135 140
Ala Glu Val Ile Trp Thr Ser Ser Asp His Gln Val Leu Ser Gly Lys
145 150 155 160
Thr Thr Thr Thr Asn Ser Lys Arg Glu Glu Lys Leu Phe Asn Val Thr
165 170 175
Ser Thr Leu Arg Ile Asn Thr Thr Thr Asn Glu Ile Phe Tyr Cys Thr
180 185 190
Phe Arg Arg Leu Asp Pro Glu Glu Asn His Thr Ala Glu Leu Val Ile
195 200 205
Pro Glu Leu Pro Leu Ala His Pro Pro Asn Glu Arg Gly Gly Gly Ser
210 215 220
Gly Gln Asp Pro Tyr Val Lys Glu Ala Glu Asn Leu Lys Lys Tyr Phe
225 230 235 240
Asn Ala Gly His Ser Asp Val Val Asp Asn Gly Thr Leu Phe Leu Gly
245 250 255
Ile Leu Lys Asn Trp Lys Glu Glu Ser Asp Arg Lys Ile Met Gln Ser
260 265 270
Gln Ile Val Ser Phe Tyr Phe Lys Leu Phe Lys Asn Phe Lys Asp Asp
275 280 285
Gln Ser Ile Gln Lys Ser Val Glu Thr Ile Lys Glu Asp Met Asn Val
290 295 300
Lys Phe Phe Asn Ser Asn Lys Lys Lys Arg Asp Asp Phe Glu Lys Leu
305 310 315 320
Thr Asn Tyr Ser Val Thr Asp Leu Asn Val Gln Arg Lys Ala Ile His
325 330 335
Glu Leu Ile Gln Val Met Ala Glu Leu Ser Pro Ala Ala Lys Thr Gly
340 345 350
Lys Arg Lys Arg Ser Gln Met Leu Phe Arg Gly Gly Gly Gly Ser Gly
355 360 365
Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Asp
370 375 380
Pro Tyr Val Lys Glu Ala Glu Asn Leu Lys Lys Tyr Phe Asn Ala Gly
385 390 395 400
His Ser Asp Val Val Asp Asn Gly Thr Leu Phe Leu Gly Ile Leu Lys
405 410 415
Asn Trp Lys Glu Glu Ser Asp Arg Lys Ile Met Gln Ser Gln Ile Val
420 425 430
Ser Phe Tyr Phe Lys Leu Phe Lys Asn Phe Lys Asp Asp Gln Ser Ile
435 440 445
Gln Lys Ser Val Glu Thr Ile Lys Glu Asp Met Asn Val Lys Phe Phe
450 455 460
Asn Ser Asn Lys Lys Lys Arg Asp Asp Phe Glu Lys Leu Thr Asn Tyr
465 470 475 480
Ser Val Thr Asp Leu Asn Val Gln Arg Lys Ala Ile His Glu Leu Ile
485 490 495
Gln Val Met Ala Glu Leu Ser Pro Ala Ala Lys Thr Gly Lys Arg Lys
500 505 510
Arg Ser Gln Met Leu Phe Arg Gly Gly Gly Gly Gly Ser Gly Gly Gly
515 520 525
Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Lys Lys Gly
530 535 540
Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met
545 550 555 560
Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His
565 570 575
Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val
580 585 590
His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr
595 600 605
Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly
610 615 620
Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile
625 630 635 640
Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val
645 650 655
Tyr Val Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser
660 665 670
Leu Leu Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu
675 680 685
Trp Glu Ser Asn Gly Gln Pro Glu Asn Arg Tyr Met Thr Trp Pro Pro
690 695 700
Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val
705 710 715 720
Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met
725 730 735
His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser
740 745 750
Pro Gly Lys
755
<210> 186
<211> 739
<212> PRT
<213> Homo sapiens (Homo sapiens)
<400> 186
Leu Phe Thr Val Thr Val Pro Lys Glu Leu Tyr Ile Ile Glu His Gly
1 5 10 15
Ser Asn Val Thr Leu Glu Cys Asn Phe Asp Thr Gly Ser His Val Asn
20 25 30
Leu Gly Ala Ile Thr Ala Val Leu Gln Lys Val Glu Asn Asp Thr Ser
35 40 45
Pro His Arg Glu Arg Ala Thr Leu Leu Glu Glu Gln Leu Pro Leu Gly
50 55 60
Lys Ala Ser Phe His Ile Pro Gln Val Gln Val Arg Asp Glu Gly Gln
65 70 75 80
Tyr Gln Cys Ile Ile Ile Tyr Gly Val Ala Trp Asp Tyr Lys Tyr Leu
85 90 95
Thr Leu Lys Val Lys Ala Ser Tyr Arg Lys Ile Asn Thr His Ile Leu
100 105 110
Lys Val Pro Glu Thr Asp Glu Val Glu Leu Thr Cys Gln Ala Thr Gly
115 120 125
Tyr Pro Leu Ala Glu Val Ser Trp Pro Asn Val Ser Val Pro Ala Asn
130 135 140
Thr Ser His Ser Arg Thr Pro Glu Gly Leu Tyr Gln Val Thr Ser Val
145 150 155 160
Leu Arg Leu Lys Pro Pro Pro Gly Arg Asn Phe Ser Cys Val Phe Trp
165 170 175
Asn Thr His Val Arg Glu Leu Thr Leu Ala Ser Ile Asp Leu Gln Ser
180 185 190
Gln Met Glu Pro Arg Thr His Pro Thr Gly Gly Gly Gly Ser Gly Gly
195 200 205
Gly Gln Asp Pro Tyr Val Lys Glu Ala Glu Asn Leu Lys Lys Tyr Phe
210 215 220
Asn Ala Gly His Ser Asp Val Val Asp Asn Gly Thr Leu Phe Leu Gly
225 230 235 240
Ile Leu Lys Asn Trp Lys Glu Glu Ser Asp Arg Lys Ile Met Gln Ser
245 250 255
Gln Ile Val Ser Phe Tyr Phe Lys Leu Phe Lys Asn Phe Lys Asp Asp
260 265 270
Gln Ser Ile Gln Lys Ser Val Glu Thr Ile Lys Glu Asp Met Asn Val
275 280 285
Lys Phe Phe Asn Ser Asn Lys Lys Lys Arg Asp Asp Phe Glu Lys Leu
290 295 300
Thr Asn Tyr Ser Val Thr Asp Leu Asn Val Gln Arg Lys Ala Ile His
305 310 315 320
Glu Leu Ile Gln Val Met Ala Glu Leu Ser Pro Ala Ala Lys Thr Gly
325 330 335
Lys Arg Lys Arg Ser Gln Met Leu Phe Arg Gly Gly Gly Gly Ser Gly
340 345 350
Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Asp
355 360 365
Pro Tyr Val Lys Glu Ala Glu Asn Leu Lys Lys Tyr Phe Asn Ala Gly
370 375 380
His Ser Asp Val Val Asp Asn Gly Thr Leu Phe Leu Gly Ile Leu Lys
385 390 395 400
Asn Trp Lys Glu Glu Ser Asp Arg Lys Ile Met Gln Ser Gln Ile Val
405 410 415
Ser Phe Tyr Phe Lys Leu Phe Lys Asn Phe Lys Asp Asp Gln Ser Ile
420 425 430
Gln Lys Ser Val Glu Thr Ile Lys Glu Asp Met Asn Val Lys Phe Phe
435 440 445
Asn Ser Asn Lys Lys Lys Arg Asp Asp Phe Glu Lys Leu Thr Asn Tyr
450 455 460
Ser Val Thr Asp Leu Asn Val Gln Arg Lys Ala Ile His Glu Leu Ile
465 470 475 480
Gln Val Met Ala Glu Leu Ser Pro Ala Ala Lys Thr Gly Lys Arg Lys
485 490 495
Arg Ser Gln Met Leu Phe Arg Gly Gly Gly Gly Gly Ser Gly Gly Gly
500 505 510
Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Asp Glu Gly
515 520 525
Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met
530 535 540
Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His
545 550 555 560
Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val
565 570 575
His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr
580 585 590
Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly
595 600 605
Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile
610 615 620
Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val
625 630 635 640
Tyr Val Tyr Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser
645 650 655
Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu
660 665 670
Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro
675 680 685
Val Leu Asp Glu Asp Gly Ser Phe Ala Leu Val Ser Lys Leu Thr Val
690 695 700
Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met
705 710 715 720
His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser
725 730 735
Pro Gly Lys
<210> 187
<211> 739
<212> PRT
<213> Homo sapiens (Homo sapiens)
<400> 187
Leu Phe Thr Val Thr Val Pro Lys Glu Leu Tyr Ile Ile Glu His Gly
1 5 10 15
Ser Asn Val Thr Leu Glu Cys Asn Phe Asp Thr Gly Ser His Val Asn
20 25 30
Leu Gly Ala Ile Val Ala Val Leu Leu Lys Val Glu Asn Asp Thr Ser
35 40 45
Pro His Arg Glu Arg Ala Thr Leu Leu Glu Glu Gln Leu Pro Leu Gly
50 55 60
Lys Ala Ser Phe His Ile Pro Gln Val Gln Val Arg Asp Glu Gly Gln
65 70 75 80
Tyr Gln Cys Ile Ile Ile Tyr Gly Val Ala Trp Asp Tyr Lys Tyr Leu
85 90 95
Thr Leu Lys Val Lys Ala Ser Tyr Arg Lys Ile Asn Thr His Ile Leu
100 105 110
Lys Val Pro Glu Thr Asp Glu Val Glu Leu Thr Cys Gln Ala Thr Gly
115 120 125
Tyr Pro Leu Ala Glu Val Ser Trp Pro Asn Val Ser Val Pro Ala Asn
130 135 140
Thr Ser His Ser Arg Thr Pro Glu Gly Leu Tyr Gln Val Thr Ser Val
145 150 155 160
Leu Arg Leu Lys Pro Pro Pro Gly Arg Asn Phe Ser Cys Val Phe Trp
165 170 175
Asn Thr His Val Arg Glu Leu Thr Leu Ala Ser Ile Asp Leu Gln Ser
180 185 190
Gln Met Glu Pro Arg Thr His Pro Thr Gly Gly Gly Gly Ser Gly Gly
195 200 205
Gly Gln Asp Pro Tyr Val Lys Glu Ala Glu Asn Leu Lys Lys Tyr Phe
210 215 220
Asn Ala Gly His Ser Asp Val Val Asp Asn Gly Thr Leu Phe Leu Gly
225 230 235 240
Ile Leu Lys Asn Trp Lys Glu Glu Ser Asp Arg Lys Ile Met Gln Ser
245 250 255
Gln Ile Val Ser Phe Tyr Phe Lys Leu Phe Lys Asn Phe Lys Asp Asp
260 265 270
Gln Ser Ile Gln Lys Ser Val Glu Thr Ile Lys Glu Asp Met Asn Val
275 280 285
Lys Phe Phe Asn Ser Asn Lys Lys Lys Arg Asp Asp Phe Glu Lys Leu
290 295 300
Thr Asn Tyr Ser Val Thr Asp Leu Asn Val Gln Arg Lys Ala Ile His
305 310 315 320
Glu Leu Ile Gln Val Met Ala Glu Leu Ser Pro Ala Ala Lys Thr Gly
325 330 335
Lys Arg Lys Arg Ser Gln Met Leu Phe Arg Gly Gly Gly Gly Ser Gly
340 345 350
Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Asp
355 360 365
Pro Tyr Val Lys Glu Ala Glu Asn Leu Lys Lys Tyr Phe Asn Ala Gly
370 375 380
His Ser Asp Val Val Asp Asn Gly Thr Leu Phe Leu Gly Ile Leu Lys
385 390 395 400
Asn Trp Lys Glu Glu Ser Asp Arg Lys Ile Met Gln Ser Gln Ile Val
405 410 415
Ser Phe Tyr Phe Lys Leu Phe Lys Asn Phe Lys Asp Asp Gln Ser Ile
420 425 430
Gln Lys Ser Val Glu Thr Ile Lys Glu Asp Met Asn Val Lys Phe Phe
435 440 445
Asn Ser Asn Lys Lys Lys Arg Asp Asp Phe Glu Lys Leu Thr Asn Tyr
450 455 460
Ser Val Thr Asp Leu Asn Val Gln Arg Lys Ala Ile His Glu Leu Ile
465 470 475 480
Gln Val Met Ala Glu Leu Ser Pro Ala Ala Lys Thr Gly Lys Arg Lys
485 490 495
Arg Ser Gln Met Leu Phe Arg Gly Gly Gly Gly Gly Ser Gly Gly Gly
500 505 510
Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Asp Glu Gly
515 520 525
Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met
530 535 540
Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His
545 550 555 560
Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val
565 570 575
His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr
580 585 590
Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly
595 600 605
Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile
610 615 620
Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val
625 630 635 640
Tyr Val Tyr Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser
645 650 655
Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu
660 665 670
Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro
675 680 685
Val Leu Asp Glu Asp Gly Ser Phe Ala Leu Val Ser Lys Leu Thr Val
690 695 700
Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met
705 710 715 720
His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser
725 730 735
Pro Gly Lys
<210> 188
<211> 736
<212> PRT
<213> Homo sapiens (Homo sapiens)
<400> 188
Leu Phe Thr Val Thr Val Pro Lys Glu Leu Tyr Ile Ile Glu His Gly
1 5 10 15
Ser Asn Val Thr Leu Glu Cys Asn Phe Asp Thr Gly Ser His Val Asn
20 25 30
Leu Gly Ala Ile Thr Ala Val Leu Gln Lys Val Glu Asn Asp Thr Ser
35 40 45
Pro His Arg Glu Arg Ala Thr Leu Leu Glu Glu Gln Leu Pro Leu Gly
50 55 60
Lys Ala Ser Phe His Ile Pro Gln Val Gln Val Arg Asp Glu Gly Gln
65 70 75 80
Tyr Gln Cys Ile Ile Ile Tyr Gly Val Ala Trp Asp Tyr Lys Tyr Leu
85 90 95
Thr Leu Lys Val Lys Ala Ser Tyr Arg Lys Ile Asn Thr His Ile Leu
100 105 110
Lys Val Pro Glu Thr Asp Glu Val Glu Leu Thr Cys Gln Ala Thr Gly
115 120 125
Tyr Pro Leu Ala Glu Val Ser Trp Pro Asn Val Ser Val Pro Ala Asn
130 135 140
Thr Ser His Ser Arg Thr Pro Glu Gly Leu Tyr Gln Val Thr Ser Val
145 150 155 160
Leu Arg Leu Lys Pro Pro Pro Gly Arg Asn Phe Ser Cys Val Phe Trp
165 170 175
Asn Thr His Val Arg Glu Leu Thr Leu Ala Ser Ile Asp Leu Gln Ser
180 185 190
Gln Met Glu Pro Arg Thr His Pro Thr Gly Gly Gly Ser Gly Gln Asp
195 200 205
Pro Tyr Val Lys Glu Ala Glu Asn Leu Lys Lys Tyr Phe Asn Ala Gly
210 215 220
His Ser Asp Val Val Asp Asn Gly Thr Leu Phe Leu Gly Ile Leu Lys
225 230 235 240
Asn Trp Lys Glu Glu Ser Asp Arg Lys Ile Met Gln Ser Gln Ile Val
245 250 255
Ser Phe Tyr Phe Lys Leu Phe Lys Asn Phe Lys Asp Asp Gln Ser Ile
260 265 270
Gln Lys Ser Val Glu Thr Ile Lys Glu Asp Met Asn Val Lys Phe Phe
275 280 285
Asn Ser Asn Lys Lys Lys Arg Asp Asp Phe Glu Lys Leu Thr Asn Tyr
290 295 300
Ser Val Thr Asp Leu Asn Val Gln Arg Lys Ala Ile His Glu Leu Ile
305 310 315 320
Gln Val Met Ala Glu Leu Ser Pro Ala Ala Lys Thr Gly Lys Arg Lys
325 330 335
Arg Ser Gln Met Leu Phe Arg Gly Gly Gly Gly Ser Gly Gly Gly Gly
340 345 350
Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Asp Pro Tyr Val
355 360 365
Lys Glu Ala Glu Asn Leu Lys Lys Tyr Phe Asn Ala Gly His Ser Asp
370 375 380
Val Val Asp Asn Gly Thr Leu Phe Leu Gly Ile Leu Lys Asn Trp Lys
385 390 395 400
Glu Glu Ser Asp Arg Lys Ile Met Gln Ser Gln Ile Val Ser Phe Tyr
405 410 415
Phe Lys Leu Phe Lys Asn Phe Lys Asp Asp Gln Ser Ile Gln Lys Ser
420 425 430
Val Glu Thr Ile Lys Glu Asp Met Asn Val Lys Phe Phe Asn Ser Asn
435 440 445
Lys Lys Lys Arg Asp Asp Phe Glu Lys Leu Thr Asn Tyr Ser Val Thr
450 455 460
Asp Leu Asn Val Gln Arg Lys Ala Ile His Glu Leu Ile Gln Val Met
465 470 475 480
Ala Glu Leu Ser Pro Ala Ala Lys Thr Gly Lys Arg Lys Arg Ser Gln
485 490 495
Met Leu Phe Arg Gly Gly Gly Gly Gly Ser Gly Gly Gly Asp Lys Thr
500 505 510
His Thr Cys Pro Pro Cys Pro Ala Pro Glu Lys Lys Gly Gly Pro Ser
515 520 525
Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg
530 535 540
Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro
545 550 555 560
Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala
565 570 575
Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val
580 585 590
Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr
595 600 605
Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr
610 615 620
Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Val Leu
625 630 635 640
Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Leu Cys
645 650 655
Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser
660 665 670
Asn Gly Gln Pro Glu Asn Arg Tyr Met Thr Trp Pro Pro Val Leu Asp
675 680 685
Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser
690 695 700
Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala
705 710 715 720
Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
725 730 735
<210> 189
<211> 736
<212> PRT
<213> Homo sapiens (Homo sapiens)
<400> 189
Leu Phe Thr Val Thr Val Pro Lys Glu Leu Tyr Ile Ile Glu His Gly
1 5 10 15
Ser Asn Val Thr Leu Glu Cys Asn Phe Asp Thr Gly Ser His Val Asn
20 25 30
Leu Gly Ala Ile Val Ala Val Leu Leu Lys Val Glu Asn Asp Thr Ser
35 40 45
Pro His Arg Glu Arg Ala Thr Leu Leu Glu Glu Gln Leu Pro Leu Gly
50 55 60
Lys Ala Ser Phe His Ile Pro Gln Val Gln Val Arg Asp Glu Gly Gln
65 70 75 80
Tyr Gln Cys Ile Ile Ile Tyr Gly Val Ala Trp Asp Tyr Lys Tyr Leu
85 90 95
Thr Leu Lys Val Lys Ala Ser Tyr Arg Lys Ile Asn Thr His Ile Leu
100 105 110
Lys Val Pro Glu Thr Asp Glu Val Glu Leu Thr Cys Gln Ala Thr Gly
115 120 125
Tyr Pro Leu Ala Glu Val Ser Trp Pro Asn Val Ser Val Pro Ala Asn
130 135 140
Thr Ser His Ser Arg Thr Pro Glu Gly Leu Tyr Gln Val Thr Ser Val
145 150 155 160
Leu Arg Leu Lys Pro Pro Pro Gly Arg Asn Phe Ser Cys Val Phe Trp
165 170 175
Asn Thr His Val Arg Glu Leu Thr Leu Ala Ser Ile Asp Leu Gln Ser
180 185 190
Gln Met Glu Pro Arg Thr His Pro Thr Gly Gly Gly Ser Gly Gln Asp
195 200 205
Pro Tyr Val Lys Glu Ala Glu Asn Leu Lys Lys Tyr Phe Asn Ala Gly
210 215 220
His Ser Asp Val Val Asp Asn Gly Thr Leu Phe Leu Gly Ile Leu Lys
225 230 235 240
Asn Trp Lys Glu Glu Ser Asp Arg Lys Ile Met Gln Ser Gln Ile Val
245 250 255
Ser Phe Tyr Phe Lys Leu Phe Lys Asn Phe Lys Asp Asp Gln Ser Ile
260 265 270
Gln Lys Ser Val Glu Thr Ile Lys Glu Asp Met Asn Val Lys Phe Phe
275 280 285
Asn Ser Asn Lys Lys Lys Arg Asp Asp Phe Glu Lys Leu Thr Asn Tyr
290 295 300
Ser Val Thr Asp Leu Asn Val Gln Arg Lys Ala Ile His Glu Leu Ile
305 310 315 320
Gln Val Met Ala Glu Leu Ser Pro Ala Ala Lys Thr Gly Lys Arg Lys
325 330 335
Arg Ser Gln Met Leu Phe Arg Gly Gly Gly Gly Ser Gly Gly Gly Gly
340 345 350
Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Asp Pro Tyr Val
355 360 365
Lys Glu Ala Glu Asn Leu Lys Lys Tyr Phe Asn Ala Gly His Ser Asp
370 375 380
Val Val Asp Asn Gly Thr Leu Phe Leu Gly Ile Leu Lys Asn Trp Lys
385 390 395 400
Glu Glu Ser Asp Arg Lys Ile Met Gln Ser Gln Ile Val Ser Phe Tyr
405 410 415
Phe Lys Leu Phe Lys Asn Phe Lys Asp Asp Gln Ser Ile Gln Lys Ser
420 425 430
Val Glu Thr Ile Lys Glu Asp Met Asn Val Lys Phe Phe Asn Ser Asn
435 440 445
Lys Lys Lys Arg Asp Asp Phe Glu Lys Leu Thr Asn Tyr Ser Val Thr
450 455 460
Asp Leu Asn Val Gln Arg Lys Ala Ile His Glu Leu Ile Gln Val Met
465 470 475 480
Ala Glu Leu Ser Pro Ala Ala Lys Thr Gly Lys Arg Lys Arg Ser Gln
485 490 495
Met Leu Phe Arg Gly Gly Gly Gly Gly Ser Gly Gly Gly Asp Lys Thr
500 505 510
His Thr Cys Pro Pro Cys Pro Ala Pro Glu Lys Lys Gly Gly Pro Ser
515 520 525
Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg
530 535 540
Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro
545 550 555 560
Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala
565 570 575
Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val
580 585 590
Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr
595 600 605
Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr
610 615 620
Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Val Leu
625 630 635 640
Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Leu Cys
645 650 655
Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser
660 665 670
Asn Gly Gln Pro Glu Asn Arg Tyr Met Thr Trp Pro Pro Val Leu Asp
675 680 685
Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser
690 695 700
Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala
705 710 715 720
Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
725 730 735
<210> 190
<211> 1083
<212> PRT
<213> Homo sapiens (Homo sapiens)
<400> 190
Trp Pro Pro Pro Gly Thr Gly Asp Val Val Val Gln Ala Pro Thr Gln
1 5 10 15
Val Pro Gly Phe Leu Gly Asp Ser Val Thr Leu Pro Cys Tyr Leu Gln
20 25 30
Val Pro Asn Met Glu Val Thr His Val Ser Gln Leu Thr Trp Ala Arg
35 40 45
His Gly Glu Ser Gly Ser Met Ala Val Phe His Gln Thr Gln Gly Pro
50 55 60
Ser Tyr Ser Glu Ser Lys Arg Leu Glu Phe Val Ala Ala Arg Leu Gly
65 70 75 80
Ala Glu Leu Arg Asn Ala Ser Leu Arg Met Phe Gly Leu Arg Val Glu
85 90 95
Asp Glu Gly Asn Tyr Thr Cys Leu Phe Val Thr Phe Pro Gln Gly Ser
100 105 110
Arg Ser Val Asp Ile Trp Leu Arg Val Leu Ala Lys Pro Gln Asn Thr
115 120 125
Ala Glu Val Gln Lys Val Gln Leu Thr Gly Glu Pro Val Pro Met Ala
130 135 140
Arg Cys Val Ser Thr Gly Gly Arg Pro Pro Ala Gln Ile Thr Trp His
145 150 155 160
Ser Asp Leu Gly Gly Met Pro Asn Thr Ser Gln Val Pro Gly Phe Leu
165 170 175
Ser Gly Thr Val Thr Val Thr Ser Leu Trp Ile Leu Val Pro Ser Ser
180 185 190
Gln Val Asp Gly Lys Asn Val Thr Cys Lys Val Glu His Glu Ser Phe
195 200 205
Glu Lys Pro Gln Leu Leu Thr Val Asn Leu Thr Val Tyr Tyr Pro Pro
210 215 220
Glu Val Ser Ile Ser Gly Tyr Asp Asn Asn Trp Tyr Leu Gly Gln Asn
225 230 235 240
Glu Ala Thr Leu Thr Cys Asp Ala Arg Ser Asn Pro Glu Pro Thr Gly
245 250 255
Tyr Asn Trp Ser Thr Thr Met Gly Pro Leu Pro Pro Phe Ala Val Ala
260 265 270
Gln Gly Ala Gln Leu Leu Ile Arg Pro Val Asp Lys Pro Ile Asn Thr
275 280 285
Thr Leu Ile Cys Asn Val Thr Asn Ala Leu Gly Ala Arg Gln Ala Glu
290 295 300
Leu Thr Val Gln Val Lys Glu Gly Pro Pro Ser Glu His Ser Gly Ile
305 310 315 320
Ser Arg Asn Gly Gly Gly Gly Ser Gly Gly Gly Ile Trp Glu Leu Lys
325 330 335
Lys Asp Val Tyr Val Val Glu Leu Asp Trp Tyr Pro Asp Ala Pro Gly
340 345 350
Glu Met Val Val Leu Thr Cys Asp Thr Pro Glu Glu Asp Gly Ile Thr
355 360 365
Trp Thr Leu Asp Gln Ser Ser Glu Val Leu Gly Ser Gly Lys Thr Leu
370 375 380
Thr Ile Gln Val Lys Ala Ala Gly Asp Ala Gly Gln Tyr Thr Cys His
385 390 395 400
Lys Gly Gly Glu Val Leu Ser His Ser Leu Leu Leu Leu His Lys Lys
405 410 415
Glu Asp Gly Ile Trp Ser Thr Asp Ile Leu Lys Asp Gln Lys Glu Pro
420 425 430
Lys Asn Lys Thr Phe Leu Arg Cys Glu Ala Lys Asn Tyr Ser Gly Arg
435 440 445
Phe Thr Cys Trp Trp Leu Thr Thr Ile Ser Thr Asp Leu Thr Phe Ser
450 455 460
Val Lys Ser Ser Arg Gly Ser Ser Asp Pro Gln Gly Val Thr Cys Gly
465 470 475 480
Ala Ala Thr Leu Ser Ala Glu Arg Val Arg Gly Asp Asn Lys Glu Tyr
485 490 495
Glu Tyr Ser Val Glu Cys Gln Glu Asp Ser Ala Cys Pro Ala Ala Glu
500 505 510
Glu Ser Leu Pro Ile Glu Val Met Val Asp Ala Val His Lys Leu Lys
515 520 525
Tyr Glu Asn Tyr Thr Ser Ser Phe Phe Ile Arg Asp Ile Ile Lys Pro
530 535 540
Asp Pro Pro Lys Asn Leu Gln Leu Lys Pro Leu Lys Asn Ser Arg Gln
545 550 555 560
Val Glu Val Ser Trp Glu Tyr Pro Asp Thr Trp Ser Thr Pro His Ser
565 570 575
Tyr Phe Ser Leu Thr Phe Cys Val Gln Val Gln Gly Lys Ser Lys Arg
580 585 590
Glu Lys Lys Asp Arg Val Phe Thr Asp Lys Thr Ser Ala Thr Val Ile
595 600 605
Cys Arg Lys Asn Ala Ser Ile Ser Val Arg Ala Gln Asp Arg Tyr Tyr
610 615 620
Ser Ser Ser Trp Ser Glu Trp Ala Ser Val Pro Cys Ser Gly Gly Gly
625 630 635 640
Gly Ser Gly Gly Gly Ser Gly Gly Gly Gly Ser Arg Asn Leu Pro Val
645 650 655
Ala Thr Pro Asp Pro Gly Met Phe Pro Cys Leu His His Ser Gln Asn
660 665 670
Leu Leu Arg Ala Val Ser Asn Met Leu Gln Lys Ala Arg Gln Thr Leu
675 680 685
Glu Phe Tyr Pro Cys Thr Ser Glu Glu Ile Asp His Glu Asp Ile Thr
690 695 700
Lys Asp Lys Thr Ser Thr Val Glu Ala Cys Leu Pro Leu Glu Leu Thr
705 710 715 720
Lys Asn Glu Ser Cys Leu Asn Ser Arg Glu Thr Ser Phe Ile Thr Asn
725 730 735
Gly Ser Cys Leu Ala Ser Arg Lys Thr Ser Phe Met Met Ala Leu Cys
740 745 750
Leu Ser Ser Ile Tyr Glu Asp Leu Lys Met Tyr Gln Val Glu Phe Lys
755 760 765
Thr Met Asn Ala Lys Leu Leu Met Asp Pro Lys Arg Gln Ile Phe Leu
770 775 780
Asp Gln Asn Met Leu Ala Val Ile Asp Glu Leu Met Gln Ala Leu Asn
785 790 795 800
Phe Asn Ser Glu Thr Val Pro Gln Lys Ser Ser Leu Glu Glu Pro Asp
805 810 815
Phe Tyr Lys Thr Lys Ile Lys Leu Cys Ile Leu Leu His Ala Phe Arg
820 825 830
Ile Arg Ala Val Thr Ile Asp Arg Val Met Ser Tyr Leu Asn Ala Ser
835 840 845
Gly Gly Gly Gly Ser Gly Gly Gly Asp Lys Thr His Thr Cys Pro Pro
850 855 860
Cys Pro Ala Pro Glu Asp Glu Gly Gly Pro Ser Val Phe Leu Phe Pro
865 870 875 880
Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr
885 890 895
Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn
900 905 910
Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg
915 920 925
Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val
930 935 940
Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser
945 950 955 960
Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys
965 970 975
Gly Gln Pro Arg Glu Pro Gln Val Tyr Val Tyr Pro Pro Ser Arg Glu
980 985 990
Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe
995 1000 1005
Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu
1010 1015 1020
Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Glu Asp Gly Ser Phe
1025 1030 1035 1040
Ala Leu Val Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly
1045 1050 1055
Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr
1060 1065 1070
Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
1075 1080
<210> 191
<211> 1083
<212> PRT
<213> Homo sapiens (Homo sapiens)
<400> 191
Trp Pro Pro Pro Gly Thr Gly Asp Val Val Val Gln Ala Pro Thr Gln
1 5 10 15
Val Pro Gly Phe Leu Gly Asp Ser Val Thr Leu Pro Cys Tyr Leu Gln
20 25 30
Val Pro Asn Met Glu Val Thr His Val Ser Gln Leu Thr Trp Ala Arg
35 40 45
His Gly Glu Ser Gly Ser Met Ala Val Phe His Gln Thr Gln Gly Pro
50 55 60
Ser Tyr Ser Glu Ser Lys Arg Leu Glu Phe Val Ala Ala Arg Leu Gly
65 70 75 80
Ala Glu Leu Arg Asn Ala Ser Leu Arg Met Phe Gly Leu Arg Val Glu
85 90 95
Asp Glu Gly Asn Tyr Thr Cys Leu Phe Val Thr Phe Pro Gln Gly Ser
100 105 110
Arg Ser Val Asp Ile Trp Leu Arg Val Leu Ala Lys Pro Gln Asn Thr
115 120 125
Ala Glu Val Gln Lys Val Gln Leu Thr Gly Glu Pro Val Pro Met Ala
130 135 140
Arg Cys Val Ser Thr Gly Gly Arg Pro Pro Ala Gln Ile Thr Trp His
145 150 155 160
Ser Asp Leu Gly Gly Met Pro Asn Thr Ser Gln Val Pro Gly Phe Leu
165 170 175
Ser Gly Thr Val Thr Val Thr Ser Leu Trp Ile Leu Val Pro Ser Ser
180 185 190
Gln Val Asp Gly Lys Asn Val Thr Cys Lys Val Glu His Glu Ser Phe
195 200 205
Glu Lys Pro Gln Leu Leu Thr Val Asn Leu Thr Val Tyr Tyr Pro Pro
210 215 220
Glu Val Ser Ile Ser Gly Tyr Asp Asn Asn Trp Tyr Leu Gly Gln Asn
225 230 235 240
Glu Ala Thr Leu Thr Cys Asp Ala Arg Ser Asn Pro Glu Pro Thr Gly
245 250 255
Tyr Asn Trp Ser Thr Thr Met Gly Pro Leu Pro Pro Phe Ala Val Ala
260 265 270
Gln Gly Ala Gln Leu Leu Ile Arg Pro Val Asp Lys Pro Ile Asn Thr
275 280 285
Thr Leu Ile Cys Asn Val Thr Asn Ala Leu Gly Ala Arg Gln Ala Glu
290 295 300
Leu Thr Val Gln Val Lys Glu Gly Pro Pro Ser Glu His Ser Gly Ile
305 310 315 320
Ser Arg Asn Gly Gly Gly Gly Ser Gly Gly Gly Asp Lys Thr His Thr
325 330 335
Cys Pro Pro Cys Pro Ala Pro Glu Asp Glu Gly Gly Pro Ser Val Phe
340 345 350
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro
355 360 365
Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val
370 375 380
Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr
385 390 395 400
Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val
405 410 415
Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys
420 425 430
Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser
435 440 445
Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Val Tyr Pro Pro
450 455 460
Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val
465 470 475 480
Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly
485 490 495
Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Glu Asp
500 505 510
Gly Ser Phe Ala Leu Val Ser Lys Leu Thr Val Asp Lys Ser Arg Trp
515 520 525
Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His
530 535 540
Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Gly Gly
545 550 555 560
Gly Gly Ser Gly Gly Gly Ile Trp Glu Leu Lys Lys Asp Val Tyr Val
565 570 575
Val Glu Leu Asp Trp Tyr Pro Asp Ala Pro Gly Glu Met Val Val Leu
580 585 590
Thr Cys Asp Thr Pro Glu Glu Asp Gly Ile Thr Trp Thr Leu Asp Gln
595 600 605
Ser Ser Glu Val Leu Gly Ser Gly Lys Thr Leu Thr Ile Gln Val Lys
610 615 620
Ala Ala Gly Asp Ala Gly Gln Tyr Thr Cys His Lys Gly Gly Glu Val
625 630 635 640
Leu Ser His Ser Leu Leu Leu Leu His Lys Lys Glu Asp Gly Ile Trp
645 650 655
Ser Thr Asp Ile Leu Lys Asp Gln Lys Glu Pro Lys Asn Lys Thr Phe
660 665 670
Leu Arg Cys Glu Ala Lys Asn Tyr Ser Gly Arg Phe Thr Cys Trp Trp
675 680 685
Leu Thr Thr Ile Ser Thr Asp Leu Thr Phe Ser Val Lys Ser Ser Arg
690 695 700
Gly Ser Ser Asp Pro Gln Gly Val Thr Cys Gly Ala Ala Thr Leu Ser
705 710 715 720
Ala Glu Arg Val Arg Gly Asp Asn Lys Glu Tyr Glu Tyr Ser Val Glu
725 730 735
Cys Gln Glu Asp Ser Ala Cys Pro Ala Ala Glu Glu Ser Leu Pro Ile
740 745 750
Glu Val Met Val Asp Ala Val His Lys Leu Lys Tyr Glu Asn Tyr Thr
755 760 765
Ser Ser Phe Phe Ile Arg Asp Ile Ile Lys Pro Asp Pro Pro Lys Asn
770 775 780
Leu Gln Leu Lys Pro Leu Lys Asn Ser Arg Gln Val Glu Val Ser Trp
785 790 795 800
Glu Tyr Pro Asp Thr Trp Ser Thr Pro His Ser Tyr Phe Ser Leu Thr
805 810 815
Phe Cys Val Gln Val Gln Gly Lys Ser Lys Arg Glu Lys Lys Asp Arg
820 825 830
Val Phe Thr Asp Lys Thr Ser Ala Thr Val Ile Cys Arg Lys Asn Ala
835 840 845
Ser Ile Ser Val Arg Ala Gln Asp Arg Tyr Tyr Ser Ser Ser Trp Ser
850 855 860
Glu Trp Ala Ser Val Pro Cys Ser Gly Gly Gly Gly Ser Gly Gly Gly
865 870 875 880
Ser Gly Gly Gly Gly Ser Arg Asn Leu Pro Val Ala Thr Pro Asp Pro
885 890 895
Gly Met Phe Pro Cys Leu His His Ser Gln Asn Leu Leu Arg Ala Val
900 905 910
Ser Asn Met Leu Gln Lys Ala Arg Gln Thr Leu Glu Phe Tyr Pro Cys
915 920 925
Thr Ser Glu Glu Ile Asp His Glu Asp Ile Thr Lys Asp Lys Thr Ser
930 935 940
Thr Val Glu Ala Cys Leu Pro Leu Glu Leu Thr Lys Asn Glu Ser Cys
945 950 955 960
Leu Asn Ser Arg Glu Thr Ser Phe Ile Thr Asn Gly Ser Cys Leu Ala
965 970 975
Ser Arg Lys Thr Ser Phe Met Met Ala Leu Cys Leu Ser Ser Ile Tyr
980 985 990
Glu Asp Leu Lys Met Tyr Gln Val Glu Phe Lys Thr Met Asn Ala Lys
995 1000 1005
Leu Leu Met Asp Pro Lys Arg Gln Ile Phe Leu Asp Gln Asn Met Leu
1010 1015 1020
Ala Val Ile Asp Glu Leu Met Gln Ala Leu Asn Phe Asn Ser Glu Thr
1025 1030 1035 1040
Val Pro Gln Lys Ser Ser Leu Glu Glu Pro Asp Phe Tyr Lys Thr Lys
1045 1050 1055
Ile Lys Leu Cys Ile Leu Leu His Ala Phe Arg Ile Arg Ala Val Thr
1060 1065 1070
Ile Asp Arg Val Met Ser Tyr Leu Asn Ala Ser
1075 1080
<210> 192
<211> 861
<212> PRT
<213> Homo sapiens (Homo sapiens)
<400> 192
Trp Pro Pro Pro Gly Thr Gly Asp Val Val Val Gln Ala Pro Thr Gln
1 5 10 15
Val Pro Gly Phe Leu Gly Asp Ser Val Thr Leu Pro Cys Tyr Leu Gln
20 25 30
Val Pro Asn Met Glu Val Thr His Val Ser Gln Leu Thr Trp Ala Arg
35 40 45
His Gly Glu Ser Gly Ser Met Ala Val Phe His Gln Thr Gln Gly Pro
50 55 60
Ser Tyr Ser Glu Ser Lys Arg Leu Glu Phe Val Ala Ala Arg Leu Gly
65 70 75 80
Ala Glu Leu Arg Asn Ala Ser Leu Arg Met Phe Gly Leu Arg Val Glu
85 90 95
Asp Glu Gly Asn Tyr Thr Cys Leu Phe Val Thr Phe Pro Gln Gly Ser
100 105 110
Arg Ser Val Asp Ile Trp Leu Arg Val Leu Ala Lys Pro Gln Asn Thr
115 120 125
Ala Glu Val Gln Lys Val Gln Leu Thr Gly Glu Pro Val Pro Met Ala
130 135 140
Arg Cys Val Ser Thr Gly Gly Arg Pro Pro Ala Gln Ile Thr Trp His
145 150 155 160
Ser Asp Leu Gly Gly Met Pro Asn Thr Ser Gln Val Pro Gly Phe Leu
165 170 175
Ser Gly Thr Val Thr Val Thr Ser Leu Trp Ile Leu Val Pro Ser Ser
180 185 190
Gln Val Asp Gly Lys Asn Val Thr Cys Lys Val Glu His Glu Ser Phe
195 200 205
Glu Lys Pro Gln Leu Leu Thr Val Asn Leu Thr Val Tyr Tyr Pro Pro
210 215 220
Glu Val Ser Ile Ser Gly Tyr Asp Asn Asn Trp Tyr Leu Gly Gln Asn
225 230 235 240
Glu Ala Thr Leu Thr Cys Asp Ala Arg Ser Asn Pro Glu Pro Thr Gly
245 250 255
Tyr Asn Trp Ser Thr Thr Met Gly Pro Leu Pro Pro Phe Ala Val Ala
260 265 270
Gln Gly Ala Gln Leu Leu Ile Arg Pro Val Asp Lys Pro Ile Asn Thr
275 280 285
Thr Leu Ile Cys Asn Val Thr Asn Ala Leu Gly Ala Arg Gln Ala Glu
290 295 300
Leu Thr Val Gln Val Lys Glu Gly Pro Pro Ser Glu His Ser Gly Ile
305 310 315 320
Ser Arg Asn Gly Gly Gly Gly Ser Gly Gly Gly Gln Asp Pro Tyr Val
325 330 335
Lys Glu Ala Glu Asn Leu Lys Lys Tyr Phe Asn Ala Gly His Ser Asp
340 345 350
Val Val Asp Asn Gly Thr Leu Phe Leu Gly Ile Leu Lys Asn Trp Lys
355 360 365
Glu Glu Ser Asp Arg Lys Ile Met Gln Ser Gln Ile Val Ser Phe Tyr
370 375 380
Phe Lys Leu Phe Lys Asn Phe Lys Asp Asp Gln Ser Ile Gln Lys Ser
385 390 395 400
Val Glu Thr Ile Lys Glu Asp Met Asn Val Lys Phe Phe Asn Ser Asn
405 410 415
Lys Lys Lys Arg Asp Asp Phe Glu Lys Leu Thr Asn Tyr Ser Val Thr
420 425 430
Asp Leu Asn Val Gln Arg Lys Ala Ile His Glu Leu Ile Gln Val Met
435 440 445
Ala Glu Leu Ser Pro Ala Ala Lys Thr Gly Lys Arg Lys Arg Ser Gln
450 455 460
Met Leu Phe Arg Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly
465 470 475 480
Gly Gly Ser Gly Gly Gly Gly Ser Gln Asp Pro Tyr Val Lys Glu Ala
485 490 495
Glu Asn Leu Lys Lys Tyr Phe Asn Ala Gly His Ser Asp Val Val Asp
500 505 510
Asn Gly Thr Leu Phe Leu Gly Ile Leu Lys Asn Trp Lys Glu Glu Ser
515 520 525
Asp Arg Lys Ile Met Gln Ser Gln Ile Val Ser Phe Tyr Phe Lys Leu
530 535 540
Phe Lys Asn Phe Lys Asp Asp Gln Ser Ile Gln Lys Ser Val Glu Thr
545 550 555 560
Ile Lys Glu Asp Met Asn Val Lys Phe Phe Asn Ser Asn Lys Lys Lys
565 570 575
Arg Asp Asp Phe Glu Lys Leu Thr Asn Tyr Ser Val Thr Asp Leu Asn
580 585 590
Val Gln Arg Lys Ala Ile His Glu Leu Ile Gln Val Met Ala Glu Leu
595 600 605
Ser Pro Ala Ala Lys Thr Gly Lys Arg Lys Arg Ser Gln Met Leu Phe
610 615 620
Arg Gly Gly Gly Gly Gly Ser Gly Gly Gly Asp Lys Thr His Thr Cys
625 630 635 640
Pro Pro Cys Pro Ala Pro Glu Asp Glu Gly Gly Pro Ser Val Phe Leu
645 650 655
Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu
660 665 670
Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys
675 680 685
Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys
690 695 700
Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu
705 710 715 720
Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys
725 730 735
Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys
740 745 750
Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Val Tyr Pro Pro Ser
755 760 765
Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys
770 775 780
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln
785 790 795 800
Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Glu Asp Gly
805 810 815
Ser Phe Ala Leu Val Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln
820 825 830
Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn
835 840 845
His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
850 855 860
<210> 193
<211> 858
<212> PRT
<213> Homo sapiens (Homo sapiens)
<400> 193
Trp Pro Pro Pro Gly Thr Gly Asp Val Val Val Gln Ala Pro Thr Gln
1 5 10 15
Val Pro Gly Phe Leu Gly Asp Ser Val Thr Leu Pro Cys Tyr Leu Gln
20 25 30
Val Pro Asn Met Glu Val Thr His Val Ser Gln Leu Thr Trp Ala Arg
35 40 45
His Gly Glu Ser Gly Ser Met Ala Val Phe His Gln Thr Gln Gly Pro
50 55 60
Ser Tyr Ser Glu Ser Lys Arg Leu Glu Phe Val Ala Ala Arg Leu Gly
65 70 75 80
Ala Glu Leu Arg Asn Ala Ser Leu Arg Met Phe Gly Leu Arg Val Glu
85 90 95
Asp Glu Gly Asn Tyr Thr Cys Leu Phe Val Thr Phe Pro Gln Gly Ser
100 105 110
Arg Ser Val Asp Ile Trp Leu Arg Val Leu Ala Lys Pro Gln Asn Thr
115 120 125
Ala Glu Val Gln Lys Val Gln Leu Thr Gly Glu Pro Val Pro Met Ala
130 135 140
Arg Cys Val Ser Thr Gly Gly Arg Pro Pro Ala Gln Ile Thr Trp His
145 150 155 160
Ser Asp Leu Gly Gly Met Pro Asn Thr Ser Gln Val Pro Gly Phe Leu
165 170 175
Ser Gly Thr Val Thr Val Thr Ser Leu Trp Ile Leu Val Pro Ser Ser
180 185 190
Gln Val Asp Gly Lys Asn Val Thr Cys Lys Val Glu His Glu Ser Phe
195 200 205
Glu Lys Pro Gln Leu Leu Thr Val Asn Leu Thr Val Tyr Tyr Pro Pro
210 215 220
Glu Val Ser Ile Ser Gly Tyr Asp Asn Asn Trp Tyr Leu Gly Gln Asn
225 230 235 240
Glu Ala Thr Leu Thr Cys Asp Ala Arg Ser Asn Pro Glu Pro Thr Gly
245 250 255
Tyr Asn Trp Ser Thr Thr Met Gly Pro Leu Pro Pro Phe Ala Val Ala
260 265 270
Gln Gly Ala Gln Leu Leu Ile Arg Pro Val Asp Lys Pro Ile Asn Thr
275 280 285
Thr Leu Ile Cys Asn Val Thr Asn Ala Leu Gly Ala Arg Gln Ala Glu
290 295 300
Leu Thr Val Gln Val Lys Glu Gly Pro Pro Ser Glu His Ser Gly Ile
305 310 315 320
Ser Arg Asn Gly Gly Gly Ser Gly Gln Asp Pro Tyr Val Lys Glu Ala
325 330 335
Glu Asn Leu Lys Lys Tyr Phe Asn Ala Gly His Ser Asp Val Val Asp
340 345 350
Asn Gly Thr Leu Phe Leu Gly Ile Leu Lys Asn Trp Lys Glu Glu Ser
355 360 365
Asp Arg Lys Ile Met Gln Ser Gln Ile Val Ser Phe Tyr Phe Lys Leu
370 375 380
Phe Lys Asn Phe Lys Asp Asp Gln Ser Ile Gln Lys Ser Val Glu Thr
385 390 395 400
Ile Lys Glu Asp Met Asn Val Lys Phe Phe Asn Ser Asn Lys Lys Lys
405 410 415
Arg Asp Asp Phe Glu Lys Leu Thr Asn Tyr Ser Val Thr Asp Leu Asn
420 425 430
Val Gln Arg Lys Ala Ile His Glu Leu Ile Gln Val Met Ala Glu Leu
435 440 445
Ser Pro Ala Ala Lys Thr Gly Lys Arg Lys Arg Ser Gln Met Leu Phe
450 455 460
Arg Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
465 470 475 480
Gly Gly Gly Gly Ser Gln Asp Pro Tyr Val Lys Glu Ala Glu Asn Leu
485 490 495
Lys Lys Tyr Phe Asn Ala Gly His Ser Asp Val Val Asp Asn Gly Thr
500 505 510
Leu Phe Leu Gly Ile Leu Lys Asn Trp Lys Glu Glu Ser Asp Arg Lys
515 520 525
Ile Met Gln Ser Gln Ile Val Ser Phe Tyr Phe Lys Leu Phe Lys Asn
530 535 540
Phe Lys Asp Asp Gln Ser Ile Gln Lys Ser Val Glu Thr Ile Lys Glu
545 550 555 560
Asp Met Asn Val Lys Phe Phe Asn Ser Asn Lys Lys Lys Arg Asp Asp
565 570 575
Phe Glu Lys Leu Thr Asn Tyr Ser Val Thr Asp Leu Asn Val Gln Arg
580 585 590
Lys Ala Ile His Glu Leu Ile Gln Val Met Ala Glu Leu Ser Pro Ala
595 600 605
Ala Lys Thr Gly Lys Arg Lys Arg Ser Gln Met Leu Phe Arg Gly Gly
610 615 620
Gly Gly Gly Ser Gly Gly Gly Asp Lys Thr His Thr Cys Pro Pro Cys
625 630 635 640
Pro Ala Pro Glu Lys Lys Gly Gly Pro Ser Val Phe Leu Phe Pro Pro
645 650 655
Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys
660 665 670
Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp
675 680 685
Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu
690 695 700
Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu
705 710 715 720
His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn
725 730 735
Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly
740 745 750
Gln Pro Arg Glu Pro Gln Val Tyr Val Leu Pro Pro Ser Arg Glu Glu
755 760 765
Met Thr Lys Asn Gln Val Ser Leu Leu Cys Leu Val Lys Gly Phe Tyr
770 775 780
Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn
785 790 795 800
Arg Tyr Met Thr Trp Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe
805 810 815
Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn
820 825 830
Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr
835 840 845
Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
850 855
<210> 194
<211> 1
<212> PRT
<213> artificial sequence
<220>
<223> synthetic construct
<220>
<221> variant
<222> 1
<223> can occur repeatedly in any integer
<400> 194
Gly
1
<210> 195
<211> 2
<212> PRT
<213> artificial sequence
<220>
<223> synthetic construct
<220>
<221> variant
<222> (1)..(2)
<223> can occur repeatedly in any integer
<400> 195
Gly Ser
1
<210> 196
<211> 3
<212> PRT
<213> artificial sequence
<220>
<223> synthetic construct
<220>
<221> variant
<222> (1)..(3)
<223> can occur repeatedly in any integer
<400> 196
Gly Gly Ser
1
<210> 197
<211> 4
<212> PRT
<213> artificial sequence
<220>
<223> synthetic construct
<220>
<221> variant
<222> (1)..(4)
<223> can occur repeatedly in any integer
<400> 197
Gly Gly Gly Ser
1
<210> 198
<211> 7
<212> PRT
<213> artificial sequence
<220>
<223> synthetic construct
<220>
<221> variant
<222> (1)..(3)
<223> can occur repeatedly in any integer
<220>
<221> variant
<222> (4)..(7)
<223> can occur repeatedly in any integer
<400> 198
Gly Gly Ser Gly Gly Gly Ser
1 5
<210> 199
<211> 5
<212> PRT
<213> artificial sequence
<220>
<223> synthetic construct
<220>
<221> variant
<222> (1)..(5)
<223> can occur repeatedly in any integer
<400> 199
Gly Ser Gly Gly Ser
1 5
<210> 200
<211> 5
<212> PRT
<213> artificial sequence
<220>
<223> synthetic construct
<220>
<221> variant
<222> (1)..(5)
<223> can occur repeatedly in any integer
<400> 200
Gly Gly Ser Gly Ser
1 5
<210> 201
<211> 5
<212> PRT
<213> artificial sequence
<220>
<223> synthetic construct
<220>
<221> variant
<222> (1)..(5)
<223> can occur repeatedly in any integer
<400> 201
Gly Gly Gly Gly Ser
1 5
<210> 202
<211> 2
<212> PRT
<213> artificial sequence
<220>
<223> synthetic construct
<400> 202
Gly Gly
1
<210> 203
<211> 3
<212> PRT
<213> artificial sequence
<220>
<223> synthetic construct
<400> 203
Gly Ser Gly
1
<210> 204
<211> 4
<212> PRT
<213> artificial sequence
<220>
<223> synthetic construct
<400> 204
Gly Gly Ser Gly
1
<210> 205
<211> 5
<212> PRT
<213> artificial sequence
<220>
<223> synthetic construct
<400> 205
Gly Gly Ser Gly Gly
1 5
<210> 206
<211> 7
<212> PRT
<213> artificial sequence
<220>
<223> synthetic construct
<400> 206
Gly Ser Gly Gly Gly Gly Gly
1 5
<210> 207
<211> 5
<212> PRT
<213> artificial sequence
<220>
<223> synthetic construct
<400> 207
Gly Ser Gly Ser Gly
1 5
<210> 208
<211> 5
<212> PRT
<213> artificial sequence
<220>
<223> synthetic construct
<400> 208
Gly Ser Gly Gly Gly
1 5
<210> 209
<211> 5
<212> PRT
<213> artificial sequence
<220>
<223> synthetic construct
<400> 209
Gly Gly Gly Ser Gly
1 5
<210> 210
<211> 5
<212> PRT
<213> artificial sequence
<220>
<223> synthetic construct
<400> 210
Gly Ser Ser Ser Gly
1 5
<210> 211
<211> 6
<212> PRT
<213> artificial sequence
<220>
<223> synthetic construct
<400> 211
Gly Gly Ser Gly Gly Ser
1 5
<210> 212
<211> 6
<212> PRT
<213> artificial sequence
<220>
<223> synthetic construct
<400> 212
Ser Gly Gly Gly Gly Ser
1 5
<210> 213
<211> 5
<212> PRT
<213> artificial sequence
<220>
<223> synthetic construct
<400> 213
Gly Gly Gly Gly Ser
1 5
<210> 214
<211> 2
<212> PRT
<213> artificial sequence
<220>
<223> synthetic construct
<220>
<221> variant
<222> (1)..(2)
<223> can occur repeatedly in any integer
<400> 214
Gly Ala
1
<210> 215
<211> 12
<212> PRT
<213> artificial sequence
<220>
<223> synthetic construct
<400> 215
Gly Arg Ala Gly Gly Gly Gly Ala Gly Gly Gly Gly
1 5 10
<210> 216
<211> 6
<212> PRT
<213> artificial sequence
<220>
<223> synthetic construct
<400> 216
Gly Arg Ala Gly Gly Gly
1 5
<210> 217
<211> 16
<212> PRT
<213> artificial sequence
<220>
<223> synthetic construct
<400> 217
Gly Ser Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
1 5 10 15
<210> 218
<211> 14
<212> PRT
<213> artificial sequence
<220>
<223> synthetic construct
<400> 218
Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
1 5 10
<210> 219
<211> 10
<212> PRT
<213> artificial sequence
<220>
<223> synthetic construct
<400> 219
Gly Gly Gly Ser Gly Gly Ser Gly Gly Ser
1 5 10
<210> 220
<211> 15
<212> PRT
<213> artificial sequence
<220>
<223> synthetic construct
<400> 220
Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Gly
1 5 10 15
<210> 221
<211> 16
<212> PRT
<213> artificial sequence
<220>
<223> synthetic construct
<400> 221
Gly Gly Ser Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
1 5 10 15
<210> 222
<211> 18
<212> PRT
<213> artificial sequence
<220>
<223> synthetic construct
<400> 222
Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly
1 5 10 15
Gly Ser
<210> 223
<211> 18
<212> PRT
<213> artificial sequence
<220>
<223> synthetic construct
<400> 223
Gly Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
1 5 10 15
Ser Ala
<210> 224
<211> 21
<212> PRT
<213> artificial sequence
<220>
<223> synthetic construct
<400> 224
Gly Ser Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
1 5 10 15
Gly Gly Gly Gly Ser
20
<210> 225
<211> 10
<212> PRT
<213> artificial sequence
<220>
<223> synthetic construct
<400> 225
Lys Thr Gly Gly Gly Ser Gly Gly Gly Ser
1 5 10
<210> 226
<211> 17
<212> PRT
<213> artificial sequence
<220>
<223> synthetic construct
<400> 226
Gly Gly Pro Gly Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Gly
1 5 10 15
Ser
<210> 227
<211> 19
<212> PRT
<213> artificial sequence
<220>
<223> synthetic construct
<400> 227
Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly
1 5 10 15
Gly Gly Ser
<210> 228
<211> 21
<212> PRT
<213> artificial sequence
<220>
<223> synthetic construct
<400> 228
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly
1 5 10 15
Gly Gly Gly Ser Gly
20
<210> 229
<211> 15
<212> PRT
<213> artificial sequence
<220>
<223> synthetic construct
<400> 229
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
1 5 10 15
<210> 230
<211> 6
<212> PRT
<213> artificial sequence
<220>
<223> synthetic construct
<400> 230
Ala Ser Thr Lys Gly Pro
1 5
<210> 231
<211> 3
<212> PRT
<213> artificial sequence
<220>
<223> synthetic construct
<400> 231
Asp Lys Pro
1
<210> 232
<211> 5
<212> PRT
<213> artificial sequence
<220>
<223> synthetic construct
<400> 232
Asp Lys Pro Gly Ser
1 5
<210> 233
<211> 3
<212> PRT
<213> artificial sequence
<220>
<223> synthetic construct
<400> 233
Pro Gly Ser
1
<210> 234
<211> 2
<212> PRT
<213> artificial sequence
<220>
<223> synthetic construct
<400> 234
Gly Ser
1
<210> 235
<211> 6
<212> PRT
<213> artificial sequence
<220>
<223> synthetic construct
<400> 235
Asp Lys Pro Gly Ser Gly
1 5
<210> 236
<211> 4
<212> PRT
<213> artificial sequence
<220>
<223> synthetic construct
<400> 236
Pro Gly Ser Gly
1
<210> 237
<211> 7
<212> PRT
<213> artificial sequence
<220>
<223> synthetic construct
<400> 237
Asp Lys Pro Gly Ser Gly Ser
1 5
<210> 238
<211> 5
<212> PRT
<213> artificial sequence
<220>
<223> synthetic construct
<400> 238
Pro Gly Ser Gly Ser
1 5
<210> 239
<211> 4
<212> PRT
<213> artificial sequence
<220>
<223> synthetic construct
<400> 239
Gly Ser Gly Ser
1
<210> 240
<211> 10
<212> PRT
<213> artificial sequence
<220>
<223> synthetic construct
<400> 240
Asp Lys Pro Gly Ser Gly Gly Gly Gly Gly
1 5 10
<210> 241
<211> 8
<212> PRT
<213> artificial sequence
<220>
<223> synthetic construct
<400> 241
Pro Gly Ser Gly Gly Gly Gly Gly
1 5
<210> 242
<211> 1
<212> PRT
<213> artificial sequence
<220>
<223> synthetic construct
<400> 242
Pro
1
<210> 243
<211> 13
<212> PRT
<213> artificial sequence
<220>
<223> synthetic construct
<400> 243
Gly Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Gly
1 5 10
<210> 244
<211> 8
<212> PRT
<213> artificial sequence
<220>
<223> synthetic construct
<400> 244
Gly Gly Gly Gly Ser Gly Gly Gly
1 5
<210> 245
<211> 16
<212> PRT
<213> artificial sequence
<220>
<223> synthetic construct
<400> 245
Gly Gly Pro Gly Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Gly
1 5 10 15
<210> 246
<211> 14
<212> PRT
<213> artificial sequence
<220>
<223> synthetic construct
<400> 246
Gly Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Gly Ser
1 5 10
<210> 247
<211> 696
<212> PRT
<213> Homo sapiens (Homo sapiens)
<400> 247
Trp Pro Pro Pro Gly Thr Gly Asp Val Val Val Gln Ala Pro Thr Gln
1 5 10 15
Val Pro Gly Phe Leu Gly Asp Ser Val Thr Leu Pro Cys Tyr Leu Gln
20 25 30
Val Pro Asn Met Glu Val Thr His Val Ser Gln Leu Thr Trp Ala Arg
35 40 45
His Gly Glu Ser Gly Ser Met Ala Val Phe His Gln Thr Gln Gly Pro
50 55 60
Ser Tyr Ser Glu Ser Lys Arg Leu Glu Phe Val Ala Ala Arg Leu Gly
65 70 75 80
Ala Glu Leu Arg Asn Ala Ser Leu Arg Met Phe Gly Leu Arg Val Glu
85 90 95
Asp Glu Gly Asn Tyr Thr Cys Leu Phe Val Thr Phe Pro Gln Gly Ser
100 105 110
Arg Ser Val Asp Ile Trp Leu Arg Val Leu Ala Lys Pro Gln Asn Thr
115 120 125
Ala Glu Val Gln Lys Val Gln Leu Thr Gly Glu Pro Val Pro Met Ala
130 135 140
Arg Cys Val Ser Thr Gly Gly Arg Pro Pro Ala Gln Ile Thr Trp His
145 150 155 160
Ser Asp Leu Gly Gly Met Pro Asn Thr Ser Gln Val Pro Gly Phe Leu
165 170 175
Ser Gly Thr Val Thr Val Thr Ser Leu Trp Ile Leu Val Pro Ser Ser
180 185 190
Gln Val Asp Gly Lys Asn Val Thr Cys Lys Val Glu His Glu Ser Phe
195 200 205
Glu Lys Pro Gln Leu Leu Thr Val Asn Leu Thr Val Tyr Tyr Pro Pro
210 215 220
Glu Val Ser Ile Ser Gly Tyr Asp Asn Asn Trp Tyr Leu Gly Gln Asn
225 230 235 240
Glu Ala Thr Leu Thr Cys Asp Ala Arg Ser Asn Pro Glu Pro Thr Gly
245 250 255
Tyr Asn Trp Ser Thr Thr Met Gly Pro Leu Pro Pro Phe Ala Val Ala
260 265 270
Gln Gly Ala Gln Leu Leu Ile Arg Pro Val Asp Lys Pro Ile Asn Thr
275 280 285
Thr Leu Ile Cys Asn Val Thr Asn Ala Leu Gly Ala Arg Gln Ala Glu
290 295 300
Leu Thr Val Gln Val Lys Glu Gly Pro Pro Ser Glu His Ser Gly Ile
305 310 315 320
Ser Arg Asn Gly Gly Gly Ser Gly Ala Pro Thr Ser Ser Ser Thr Lys
325 330 335
Lys Thr Gln Leu Gln Leu Glu His Leu Arg Leu Asp Leu Glu Met Ile
340 345 350
Leu Asn Gly Ile Asn Asn Tyr Lys Asn Pro Lys Leu Thr Asp Met Leu
355 360 365
Thr Phe Glu Phe Tyr Met Pro Lys Lys Ala Thr Glu Leu Lys His Leu
370 375 380
Gln Cys Leu Glu Arg Glu Leu Lys Pro Leu Glu Glu Val Leu Asn Leu
385 390 395 400
Ala Gln Ser Lys Asn Phe His Leu Arg Pro Arg Asp Leu Ile Ser Asn
405 410 415
Ile Asn Val Ile Val Leu Glu Leu Lys Gly Ser Glu Thr Thr Phe Met
420 425 430
Cys Glu Tyr Ala Asp Glu Thr Ala Thr Ile Val Glu Phe Leu Asn Arg
435 440 445
Trp Ile Thr Phe Cys Gln Ser Ile Ile Ser Thr Leu Thr Gly Gly Gly
450 455 460
Gly Ser Gly Gly Gly Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala
465 470 475 480
Pro Glu Lys Lys Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro
485 490 495
Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val
500 505 510
Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val
515 520 525
Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln
530 535 540
Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln
545 550 555 560
Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala
565 570 575
Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro
580 585 590
Arg Glu Pro Gln Val Tyr Val Leu Pro Pro Ser Arg Glu Glu Met Thr
595 600 605
Lys Asn Gln Val Ser Leu Leu Cys Leu Val Lys Gly Phe Tyr Pro Ser
610 615 620
Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Arg Tyr
625 630 635 640
Met Thr Trp Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr
645 650 655
Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe
660 665 670
Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys
675 680 685
Ser Leu Ser Leu Ser Pro Gly Lys
690 695
<210> 248
<211> 696
<212> PRT
<213> Homo sapiens (Homo sapiens)
<400> 248
Trp Pro Pro Pro Gly Thr Gly Asp Val Val Val Gln Ala Pro Thr Gln
1 5 10 15
Val Pro Gly Phe Leu Gly Asp Ser Val Thr Leu Pro Cys Tyr Leu Gln
20 25 30
Val Pro Asn Met Glu Val Thr His Val Ser Gln Leu Thr Trp Ala Arg
35 40 45
His Gly Glu Ser Gly Ser Met Ala Val Phe His Gln Thr Gln Gly Pro
50 55 60
Ser Tyr Ser Glu Ser Lys Arg Leu Glu Phe Val Ala Ala Arg Leu Gly
65 70 75 80
Ala Glu Leu Arg Asn Ala Ser Leu Arg Met Phe Gly Leu Arg Val Glu
85 90 95
Asp Glu Gly Asn Tyr Thr Cys Leu Phe Val Thr Phe Pro Gln Gly Ser
100 105 110
Arg Ser Val Asp Ile Trp Leu Arg Val Leu Ala Lys Pro Gln Asn Thr
115 120 125
Ala Glu Val Gln Lys Val Gln Leu Thr Gly Glu Pro Val Pro Met Ala
130 135 140
Arg Cys Val Ser Thr Gly Gly Arg Pro Pro Ala Gln Ile Thr Trp His
145 150 155 160
Ser Asp Leu Gly Gly Met Pro Asn Thr Ser Gln Val Pro Gly Phe Leu
165 170 175
Ser Gly Thr Val Thr Val Thr Ser Leu Trp Ile Leu Val Pro Ser Ser
180 185 190
Gln Val Asp Gly Lys Asn Val Thr Cys Lys Val Glu His Glu Ser Phe
195 200 205
Glu Lys Pro Gln Leu Leu Thr Val Asn Leu Thr Val Tyr Tyr Pro Pro
210 215 220
Glu Val Ser Ile Ser Gly Tyr Asp Asn Asn Trp Tyr Leu Gly Gln Asn
225 230 235 240
Glu Ala Thr Leu Thr Cys Asp Ala Arg Ser Asn Pro Glu Pro Thr Gly
245 250 255
Tyr Asn Trp Ser Thr Thr Met Gly Pro Leu Pro Pro Phe Ala Val Ala
260 265 270
Gln Gly Ala Gln Leu Leu Ile Arg Pro Val Asp Lys Pro Ile Asn Thr
275 280 285
Thr Leu Ile Cys Asn Val Thr Asn Ala Leu Gly Ala Arg Gln Ala Glu
290 295 300
Leu Thr Val Gln Val Lys Glu Gly Pro Pro Ser Glu His Ser Gly Ile
305 310 315 320
Ser Arg Asn Gly Gly Gly Ser Gly Ala Pro Thr Ser Ser Ser Thr Lys
325 330 335
Lys Thr Gln Leu Gln Leu Glu His Leu Leu Leu Asp Leu Gln Met Ile
340 345 350
Leu Asn Gly Ile Asn Asn Tyr Lys Asn Pro Lys Leu Thr Asp Met Leu
355 360 365
Thr Phe Glu Phe Tyr Met Pro Lys Lys Ala Thr Glu Leu Lys His Leu
370 375 380
Gln Cys Leu Glu Arg Glu Leu Lys Pro Leu Glu Glu Val Leu Asn Leu
385 390 395 400
Ala Gln Ser Lys Asn Phe His Leu Arg Pro Arg Asp Leu Ile Ser Asn
405 410 415
Ile Asn Val Ile Val Leu Glu Leu Lys Gly Ser Glu Thr Thr Phe Met
420 425 430
Cys Glu Tyr Ala Asp Glu Thr Ala Thr Ile Val Glu Phe Leu Asn Arg
435 440 445
Trp Ile Thr Phe Cys Thr Ser Ile Ile Ser Thr Leu Thr Gly Gly Gly
450 455 460
Gly Ser Gly Gly Gly Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala
465 470 475 480
Pro Glu Lys Lys Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro
485 490 495
Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val
500 505 510
Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val
515 520 525
Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln
530 535 540
Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln
545 550 555 560
Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala
565 570 575
Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro
580 585 590
Arg Glu Pro Gln Val Tyr Val Leu Pro Pro Ser Arg Glu Glu Met Thr
595 600 605
Lys Asn Gln Val Ser Leu Leu Cys Leu Val Lys Gly Phe Tyr Pro Ser
610 615 620
Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Arg Tyr
625 630 635 640
Met Thr Trp Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr
645 650 655
Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe
660 665 670
Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys
675 680 685
Ser Leu Ser Leu Ser Pro Gly Lys
690 695
<210> 249
<211> 696
<212> PRT
<213> Homo sapiens (Homo sapiens)
<400> 249
Trp Pro Pro Pro Gly Thr Gly Asp Val Val Val Gln Ala Pro Thr Gln
1 5 10 15
Val Pro Gly Phe Leu Gly Asp Ser Val Thr Leu Pro Cys Tyr Leu Gln
20 25 30
Val Pro Asn Met Glu Val Thr His Val Ser Gln Leu Thr Trp Ala Arg
35 40 45
His Gly Glu Ser Gly Ser Met Ala Val Phe His Gln Thr Gln Gly Pro
50 55 60
Ser Tyr Ser Glu Ser Lys Arg Leu Glu Phe Val Ala Ala Arg Leu Gly
65 70 75 80
Ala Glu Leu Arg Asn Ala Ser Leu Arg Met Phe Gly Leu Arg Val Glu
85 90 95
Asp Glu Gly Asn Tyr Thr Cys Leu Phe Val Thr Phe Pro Gln Gly Ser
100 105 110
Arg Ser Val Asp Ile Trp Leu Arg Val Leu Ala Lys Pro Gln Asn Thr
115 120 125
Ala Glu Val Gln Lys Val Gln Leu Thr Gly Glu Pro Val Pro Met Ala
130 135 140
Arg Cys Val Ser Thr Gly Gly Arg Pro Pro Ala Gln Ile Thr Trp His
145 150 155 160
Ser Asp Leu Gly Gly Met Pro Asn Thr Ser Gln Val Pro Gly Phe Leu
165 170 175
Ser Gly Thr Val Thr Val Thr Ser Leu Trp Ile Leu Val Pro Ser Ser
180 185 190
Gln Val Asp Gly Lys Asn Val Thr Cys Lys Val Glu His Glu Ser Phe
195 200 205
Glu Lys Pro Gln Leu Leu Thr Val Asn Leu Thr Val Tyr Tyr Pro Pro
210 215 220
Glu Val Ser Ile Ser Gly Tyr Asp Asn Asn Trp Tyr Leu Gly Gln Asn
225 230 235 240
Glu Ala Thr Leu Thr Cys Asp Ala Arg Ser Asn Pro Glu Pro Thr Gly
245 250 255
Tyr Asn Trp Ser Thr Thr Met Gly Pro Leu Pro Pro Phe Ala Val Ala
260 265 270
Gln Gly Ala Gln Leu Leu Ile Arg Pro Val Asp Lys Pro Ile Asn Thr
275 280 285
Thr Leu Ile Cys Asn Val Thr Asn Ala Leu Gly Ala Arg Gln Ala Glu
290 295 300
Leu Thr Val Gln Val Lys Glu Gly Pro Pro Ser Glu His Ser Gly Ile
305 310 315 320
Ser Arg Asn Gly Gly Gly Ser Gly Ala Pro Thr Ser Ser Ser Thr Lys
325 330 335
Lys Thr Gln Leu Gln Leu Glu His Leu Arg Leu Asp Leu Glu Met Ile
340 345 350
Leu Asn Gly Ile Asn Asn Tyr Lys Asn Pro Lys Leu Thr Asp Met Leu
355 360 365
Thr Phe Glu Phe Tyr Met Pro Lys Lys Ala Thr Glu Leu Lys His Leu
370 375 380
Gln Cys Leu Glu Arg Glu Leu Lys Pro Leu Glu Glu Val Leu Asn Leu
385 390 395 400
Ala Gln Ser Lys Asn Phe His Leu Arg Pro Arg Asp Leu Ile Ser Asn
405 410 415
Ile Asn Val Ile Val Leu Glu Leu Lys Gly Ser Glu Thr Thr Phe Met
420 425 430
Cys Glu Tyr Ala Asp Glu Thr Ala Thr Ile Val Glu Phe Leu Asn Arg
435 440 445
Trp Ile Thr Phe Cys Thr Ser Ile Ile Ser Thr Leu Thr Gly Gly Gly
450 455 460
Gly Ser Gly Gly Gly Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala
465 470 475 480
Pro Glu Lys Lys Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro
485 490 495
Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val
500 505 510
Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val
515 520 525
Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln
530 535 540
Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln
545 550 555 560
Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala
565 570 575
Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro
580 585 590
Arg Glu Pro Gln Val Tyr Val Leu Pro Pro Ser Arg Glu Glu Met Thr
595 600 605
Lys Asn Gln Val Ser Leu Leu Cys Leu Val Lys Gly Phe Tyr Pro Ser
610 615 620
Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Arg Tyr
625 630 635 640
Met Thr Trp Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr
645 650 655
Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe
660 665 670
Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys
675 680 685
Ser Leu Ser Leu Ser Pro Gly Lys
690 695
<210> 250
<211> 696
<212> PRT
<213> Homo sapiens (Homo sapiens)
<400> 250
Trp Pro Pro Pro Gly Thr Gly Asp Val Val Val Gln Ala Pro Thr Gln
1 5 10 15
Val Pro Gly Phe Leu Gly Asp Ser Val Thr Leu Pro Cys Tyr Leu Gln
20 25 30
Val Pro Asn Met Glu Val Thr His Val Ser Gln Leu Thr Trp Ala Arg
35 40 45
His Gly Glu Ser Gly Ser Met Ala Val Phe His Gln Thr Gln Gly Pro
50 55 60
Ser Tyr Ser Glu Ser Lys Arg Leu Glu Phe Val Ala Ala Arg Leu Gly
65 70 75 80
Ala Glu Leu Arg Asn Ala Ser Leu Arg Met Phe Gly Leu Arg Val Glu
85 90 95
Asp Glu Gly Asn Tyr Thr Cys Leu Phe Val Thr Phe Pro Gln Gly Ser
100 105 110
Arg Ser Val Asp Ile Trp Leu Arg Val Leu Ala Lys Pro Gln Asn Thr
115 120 125
Ala Glu Val Gln Lys Val Gln Leu Thr Gly Glu Pro Val Pro Met Ala
130 135 140
Arg Cys Val Ser Thr Gly Gly Arg Pro Pro Ala Gln Ile Thr Trp His
145 150 155 160
Ser Asp Leu Gly Gly Met Pro Asn Thr Ser Gln Val Pro Gly Phe Leu
165 170 175
Ser Gly Thr Val Thr Val Thr Ser Leu Trp Ile Leu Val Pro Ser Ser
180 185 190
Gln Val Asp Gly Lys Asn Val Thr Cys Lys Val Glu His Glu Ser Phe
195 200 205
Glu Lys Pro Gln Leu Leu Thr Val Asn Leu Thr Val Tyr Tyr Pro Pro
210 215 220
Glu Val Ser Ile Ser Gly Tyr Asp Asn Asn Trp Tyr Leu Gly Gln Asn
225 230 235 240
Glu Ala Thr Leu Thr Cys Asp Ala Arg Ser Asn Pro Glu Pro Thr Gly
245 250 255
Tyr Asn Trp Ser Thr Thr Met Gly Pro Leu Pro Pro Phe Ala Val Ala
260 265 270
Gln Gly Ala Gln Leu Leu Ile Arg Pro Val Asp Lys Pro Ile Asn Thr
275 280 285
Thr Leu Ile Cys Asn Val Thr Asn Ala Leu Gly Ala Arg Gln Ala Glu
290 295 300
Leu Thr Val Gln Val Lys Glu Gly Pro Pro Ser Glu His Ser Gly Ile
305 310 315 320
Ser Arg Asn Gly Gly Gly Ser Gly Ala Pro Thr Ser Ser Ser Thr Lys
325 330 335
Lys Thr Gln Leu Gln Leu Glu His Leu Arg Leu Asp Leu Glu Met Ile
340 345 350
Leu Asn Gly Ile Asn Asn Tyr Lys Asn Pro Lys Leu Thr Asp Met Leu
355 360 365
Thr Phe Glu Phe Tyr Met Pro Lys Lys Ala Thr Glu Leu Lys His Leu
370 375 380
Gln Cys Leu Glu Arg Glu Leu Lys Pro Leu Glu Glu Val Leu Asn Leu
385 390 395 400
Ala Gln Ser Lys Asn Phe His Leu Arg Pro Arg Asp Leu Ile Ser Asn
405 410 415
Ile Asn Val Ile Val Leu Glu Leu Lys Gly Ser Glu Thr Thr Phe Met
420 425 430
Cys Glu Tyr Ala Asp Glu Thr Ala Thr Ile Val Glu Phe Leu Asn Arg
435 440 445
Trp Ile Thr Phe Cys Thr Ser Ile Ile Arg Thr Leu Thr Gly Gly Gly
450 455 460
Gly Ser Gly Gly Gly Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala
465 470 475 480
Pro Glu Lys Lys Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro
485 490 495
Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val
500 505 510
Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val
515 520 525
Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln
530 535 540
Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln
545 550 555 560
Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala
565 570 575
Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro
580 585 590
Arg Glu Pro Gln Val Tyr Val Leu Pro Pro Ser Arg Glu Glu Met Thr
595 600 605
Lys Asn Gln Val Ser Leu Leu Cys Leu Val Lys Gly Phe Tyr Pro Ser
610 615 620
Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Arg Tyr
625 630 635 640
Met Thr Trp Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr
645 650 655
Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe
660 665 670
Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys
675 680 685
Ser Leu Ser Leu Ser Pro Gly Lys
690 695
<210> 251
<211> 295
<212> PRT
<213> artificial sequence
<220>
<223> synthetic construct
<400> 251
Gln Asp Pro Tyr Val Lys Glu Ala Glu Asn Leu Lys Lys Tyr Phe Asn
1 5 10 15
Ala Gly His Ser Asp Val Ala Asp Asn Gly Thr Leu Phe Leu Gly Ile
20 25 30
Leu Lys Asn Trp Lys Glu Glu Ser Asp Arg Lys Ile Met Gln Ser Gln
35 40 45
Ile Val Ser Phe Tyr Phe Lys Leu Phe Lys Asn Phe Lys Asp Asp Gln
50 55 60
Ser Ile Gln Lys Ser Val Glu Thr Ile Lys Glu Asp Met Asn Val Lys
65 70 75 80
Phe Phe Asn Ser Asn Lys Lys Lys Arg Asp Asp Phe Glu Lys Leu Thr
85 90 95
Asn Tyr Ser Val Thr Asp Leu Asn Val Gln Arg Lys Ala Ile His Glu
100 105 110
Leu Ile Gln Val Met Ala Glu Leu Ser Pro Ala Ala Lys Thr Gly Lys
115 120 125
Arg Lys Arg Ser Gln Met Leu Phe Arg Gly Gly Gly Gly Ser Gly Gly
130 135 140
Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Asp Pro
145 150 155 160
Tyr Val Lys Glu Ala Glu Asn Leu Lys Lys Tyr Phe Asn Ala Gly His
165 170 175
Ser Asp Val Ala Asp Asn Gly Thr Leu Phe Leu Gly Ile Leu Lys Asn
180 185 190
Trp Lys Glu Glu Ser Asp Arg Lys Ile Met Gln Ser Gln Ile Val Ser
195 200 205
Phe Tyr Phe Lys Leu Phe Lys Asn Phe Lys Asp Asp Gln Ser Ile Gln
210 215 220
Lys Ser Val Glu Thr Ile Lys Glu Asp Met Asn Val Lys Phe Phe Asn
225 230 235 240
Ser Asn Lys Lys Lys Arg Asp Asp Phe Glu Lys Leu Thr Asn Tyr Ser
245 250 255
Val Thr Asp Leu Asn Val Gln Arg Lys Ala Ile His Glu Leu Ile Gln
260 265 270
Val Met Ala Glu Leu Ser Pro Ala Ala Lys Thr Gly Lys Arg Lys Arg
275 280 285
Ser Gln Met Leu Phe Arg Gly
290 295
<210> 252
<211> 295
<212> PRT
<213> artificial sequence
<220>
<223> synthetic construct
<400> 252
Gln Asp Pro Tyr Val Lys Glu Ala Glu Asn Leu Lys Lys Tyr Phe Asn
1 5 10 15
Ala Gly His Ser Asp Val Val Asp Asn Gly Thr Leu Phe Leu Gly Ile
20 25 30
Leu Lys Asn Trp Lys Glu Glu Ser Asp Arg Lys Ile Met Gln Ser Gln
35 40 45
Ile Val Ser Phe Tyr Phe Lys Leu Phe Lys Asn Phe Lys Asp Asp Gln
50 55 60
Ser Ile Gln Lys Ser Val Glu Thr Ile Lys Glu Asp Met Asn Val Lys
65 70 75 80
Phe Phe Asn Ser Asn Lys Lys Lys Arg Asp Asp Phe Glu Lys Leu Thr
85 90 95
Asn Tyr Ser Val Thr Asp Leu Asn Val Gln Arg Lys Ala Ile His Glu
100 105 110
Leu Ile Gln Val Met Ala Glu Leu Ser Pro Ala Ala Lys Thr Gly Lys
115 120 125
Arg Lys Arg Ser Gln Met Leu Phe Arg Gly Gly Gly Gly Ser Gly Gly
130 135 140
Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Asp Pro
145 150 155 160
Tyr Val Lys Glu Ala Glu Asn Leu Lys Lys Tyr Phe Asn Ala Gly His
165 170 175
Ser Asp Val Val Asp Asn Gly Thr Leu Phe Leu Gly Ile Leu Lys Asn
180 185 190
Trp Lys Glu Glu Ser Asp Arg Lys Ile Met Gln Ser Gln Ile Val Ser
195 200 205
Phe Tyr Phe Lys Leu Phe Lys Asn Phe Lys Asp Asp Gln Ser Ile Gln
210 215 220
Lys Ser Val Glu Thr Ile Lys Glu Asp Met Asn Val Lys Phe Phe Asn
225 230 235 240
Ser Asn Lys Lys Lys Arg Asp Asp Phe Glu Lys Leu Thr Asn Tyr Ser
245 250 255
Val Thr Asp Leu Asn Val Gln Arg Lys Ala Ile His Glu Leu Ile Gln
260 265 270
Val Met Ala Glu Leu Ser Pro Ala Ala Lys Thr Gly Lys Arg Lys Arg
275 280 285
Ser Gln Met Leu Phe Arg Gly
290 295
<210> 253
<211> 517
<212> PRT
<213> artificial sequence
<220>
<223> synthetic construct
<400> 253
Ile Trp Glu Leu Lys Lys Asp Val Tyr Val Val Glu Leu Asp Trp Tyr
1 5 10 15
Pro Asp Ala Pro Gly Glu Met Val Val Leu Thr Cys Asp Thr Pro Glu
20 25 30
Glu Asp Gly Ile Thr Trp Thr Leu Asp Gln Ser Ser Glu Val Leu Gly
35 40 45
Ser Gly Lys Thr Leu Thr Ile Gln Val Lys Glu Phe Gly Asp Ala Gly
50 55 60
Gln Tyr Thr Cys His Lys Gly Gly Glu Val Leu Ser His Ser Leu Leu
65 70 75 80
Leu Leu His Lys Lys Glu Asp Gly Ile Trp Ser Thr Asp Ile Leu Lys
85 90 95
Asp Gln Lys Glu Pro Lys Asn Lys Thr Phe Leu Arg Cys Glu Ala Lys
100 105 110
Asn Tyr Ser Gly Arg Phe Thr Cys Trp Trp Leu Thr Thr Ile Ser Thr
115 120 125
Asp Leu Thr Phe Ser Val Lys Ser Ser Arg Gly Ser Ser Asp Pro Gln
130 135 140
Gly Val Thr Cys Gly Ala Ala Thr Leu Ser Ala Glu Arg Val Arg Gly
145 150 155 160
Asp Asn Lys Glu Tyr Glu Tyr Ser Val Glu Cys Gln Glu Asp Ser Ala
165 170 175
Cys Pro Ala Ala Glu Glu Ser Leu Pro Ile Glu Val Met Val Asp Ala
180 185 190
Val His Lys Leu Lys Tyr Glu Asn Tyr Thr Ser Ser Phe Phe Ile Arg
195 200 205
Asp Ile Ile Lys Pro Asp Pro Pro Lys Asn Leu Gln Leu Lys Pro Leu
210 215 220
Lys Asn Ser Arg Gln Val Glu Val Ser Trp Glu Tyr Pro Asp Thr Trp
225 230 235 240
Ser Thr Pro His Ser Tyr Phe Ser Leu Thr Phe Cys Val Gln Val Gln
245 250 255
Gly Lys Ser Lys Arg Glu Lys Lys Asp Arg Val Phe Thr Asp Lys Thr
260 265 270
Ser Ala Thr Val Ile Cys Arg Lys Asn Ala Ser Ile Ser Val Arg Ala
275 280 285
Gln Asp Arg Tyr Tyr Ser Ser Ser Trp Ser Glu Trp Ala Ser Val Pro
290 295 300
Cys Ser Gly Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Gly Ser
305 310 315 320
Arg Asn Leu Pro Val Ala Thr Pro Asp Pro Gly Met Phe Pro Cys Leu
325 330 335
His His Ser Gln Asn Leu Leu Arg Ala Val Ser Asn Met Leu Gln Lys
340 345 350
Ala Arg Gln Thr Leu Glu Phe Tyr Pro Cys Thr Ser Glu Glu Ile Asp
355 360 365
His Glu Asp Ile Thr Lys Asp Lys Thr Ser Thr Val Glu Ala Cys Leu
370 375 380
Pro Leu Glu Leu Thr Lys Asn Glu Ser Cys Leu Asn Ser Arg Glu Thr
385 390 395 400
Ser Phe Ile Thr Asn Gly Ser Cys Leu Ala Ser Arg Lys Thr Ser Phe
405 410 415
Met Met Ala Leu Cys Leu Ser Ser Ile Tyr Glu Asp Leu Lys Met Tyr
420 425 430
Gln Val Glu Phe Lys Thr Met Asn Ala Lys Leu Leu Met Asp Pro Lys
435 440 445
Arg Gln Ile Phe Leu Asp Gln Asn Met Leu Ala Val Ile Asp Glu Leu
450 455 460
Met Gln Ala Leu Asn Phe Asn Ser Glu Thr Val Pro Gln Lys Ser Ser
465 470 475 480
Leu Glu Glu Pro Asp Phe Tyr Lys Thr Lys Ile Lys Leu Cys Ile Leu
485 490 495
Leu His Ala Phe Arg Ile Arg Ala Val Thr Ile Asp Arg Val Met Ser
500 505 510
Tyr Leu Asn Ala Ser
515
<210> 254
<211> 517
<212> PRT
<213> artificial sequence
<220>
<223> synthetic construct
<400> 254
Ile Trp Glu Leu Lys Lys Asp Val Tyr Val Val Glu Leu Asp Trp Tyr
1 5 10 15
Pro Asp Ala Pro Gly Glu Met Val Val Leu Thr Cys Asp Thr Pro Glu
20 25 30
Glu Asp Gly Ile Thr Trp Thr Leu Asp Gln Ser Ser Glu Val Leu Gly
35 40 45
Ser Gly Lys Thr Leu Thr Ile Gln Val Lys Ala Ala Gly Asp Ala Gly
50 55 60
Gln Tyr Thr Cys His Lys Gly Gly Glu Val Leu Ser His Ser Leu Leu
65 70 75 80
Leu Leu His Lys Lys Glu Asp Gly Ile Trp Ser Thr Asp Ile Leu Lys
85 90 95
Asp Gln Lys Glu Pro Lys Asn Lys Thr Phe Leu Arg Cys Glu Ala Lys
100 105 110
Asn Tyr Ser Gly Arg Phe Thr Cys Trp Trp Leu Thr Thr Ile Ser Thr
115 120 125
Asp Leu Thr Phe Ser Val Lys Ser Ser Arg Gly Ser Ser Asp Pro Gln
130 135 140
Gly Val Thr Cys Gly Ala Ala Thr Leu Ser Ala Glu Arg Val Arg Gly
145 150 155 160
Asp Asn Lys Glu Tyr Glu Tyr Ser Val Glu Cys Gln Glu Asp Ser Ala
165 170 175
Cys Pro Ala Ala Glu Glu Ser Leu Pro Ile Glu Val Met Val Asp Ala
180 185 190
Val His Lys Leu Lys Tyr Glu Asn Tyr Thr Ser Ser Phe Phe Ile Arg
195 200 205
Asp Ile Ile Lys Pro Asp Pro Pro Lys Asn Leu Gln Leu Lys Pro Leu
210 215 220
Lys Asn Ser Arg Gln Val Glu Val Ser Trp Glu Tyr Pro Asp Thr Trp
225 230 235 240
Ser Thr Pro His Ser Tyr Phe Ser Leu Thr Phe Cys Val Gln Val Gln
245 250 255
Gly Lys Ser Lys Arg Glu Lys Lys Asp Arg Val Phe Thr Asp Lys Thr
260 265 270
Ser Ala Thr Val Ile Cys Arg Lys Asn Ala Ser Ile Ser Val Arg Ala
275 280 285
Gln Asp Arg Tyr Tyr Ser Ser Ser Trp Ser Glu Trp Ala Ser Val Pro
290 295 300
Cys Ser Gly Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Gly Ser
305 310 315 320
Arg Asn Leu Pro Val Ala Thr Pro Asp Pro Gly Met Phe Pro Cys Leu
325 330 335
His His Ser Gln Asn Leu Leu Arg Ala Val Ser Asn Met Leu Gln Lys
340 345 350
Ala Arg Gln Thr Leu Glu Phe Tyr Pro Cys Thr Ser Glu Glu Ile Asp
355 360 365
His Glu Asp Ile Thr Lys Asp Lys Thr Ser Thr Val Glu Ala Cys Leu
370 375 380
Pro Leu Glu Leu Thr Lys Asn Glu Ser Cys Leu Asn Ser Arg Glu Thr
385 390 395 400
Ser Phe Ile Thr Asn Gly Ser Cys Leu Ala Ser Arg Lys Thr Ser Phe
405 410 415
Met Met Ala Leu Cys Leu Ser Ser Ile Tyr Glu Asp Leu Lys Met Tyr
420 425 430
Gln Val Glu Phe Lys Thr Met Asn Ala Lys Leu Leu Met Asp Pro Lys
435 440 445
Arg Gln Ile Phe Leu Asp Gln Asn Met Leu Ala Val Ile Asp Glu Leu
450 455 460
Met Gln Ala Leu Asn Phe Asn Ser Glu Thr Val Pro Gln Lys Ser Ser
465 470 475 480
Leu Glu Glu Pro Asp Phe Tyr Lys Thr Lys Ile Lys Leu Cys Ile Leu
485 490 495
Leu His Ala Phe Arg Ile Arg Ala Val Thr Ile Asp Arg Val Met Ser
500 505 510
Tyr Leu Asn Ala Ser
515

Claims (58)

1. An immunomodulatory molecule comprising a first binding domain that specifically recognizes a first target molecule and a second binding domain that specifically recognizes a second target molecule, wherein the first binding domain upregulates an immune response upon binding to the first target molecule, and wherein the second binding domain downregulates an immune response upon binding to the second target molecule.
2. The immunomodulatory molecule of claim 1, wherein the first binding domain, upon binding to the first target molecule, upregulates an immune response by one or more activities ("upregulation activities") selected from the group consisting of: up-regulating the release of immunostimulatory cytokines, down-regulating the release of immunosuppressive cytokines, up-regulating immune cell proliferation, up-regulating immune cell differentiation, up-regulating immune cell activation, up-regulating cytotoxicity to tumor cells, and up-regulating elimination of infectious pathogens.
3. The immunomodulatory molecule of claim 1 or 2, wherein the second binding domain down-regulates an immune response upon binding to the second target molecule by one or more activities ("down-regulating activities") selected from the group consisting of: down-regulating the release of immunostimulatory cytokines, up-regulating the release of immunosuppressive cytokines, down-regulating immune cell proliferation, down-regulating immune cell differentiation, down-regulating immune cell activation, down-regulating cytotoxicity to tumor cells, and down-regulating elimination of infectious pathogens.
4. The immunomodulatory molecule of any of claims 1-3, wherein the first binding domain is an agonist ligand or variant thereof.
5. The immunomodulatory molecule of claim 4, wherein the first binding domain is a variant of an agonist ligand, and wherein the variant of an agonist ligand has increased or decreased binding affinity for the first target molecule compared to the agonist ligand.
6. The immunomodulatory molecule of claim 4 or 5, wherein the second binding domain is an antagonistic antibody or antigen binding fragment thereof.
7. The immunomodulatory molecule of any of claims 1-3, wherein the first target molecule and/or the second target molecule is a receptor for an immunostimulatory cytokine.
8. The immunoregulatory molecule of claim 7, wherein the immunostimulatory cytokine is selected from the group consisting of: IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-12, IL-15, IL-17, IL-18, IL-21, IL-22, IL-23, IL-27, IFN-alpha, IFN-beta, IFN-gamma, TNF-alpha, erythropoietin, thrombopoietin, G-CSF, M-CSF, SCF, and GM-CSF.
9. The immunomodulatory molecule of claim 7 or 8, wherein the first binding domain is the immunostimulatory cytokine or variant thereof.
10. The immunomodulatory molecule of claim 9, wherein the first binding domain is a variant of an immunostimulatory cytokine, and wherein the variant of an immunostimulatory cytokine has increased or decreased binding affinity for the first target molecule as compared to the immunostimulatory cytokine.
11. The immunomodulatory molecule of claim 9 or 10, wherein the first binding domain is IL-12, IL-2, or a variant thereof.
12. The immunomodulatory molecule of any of claims 1-3, wherein the first target molecule and/or the second target molecule is an inhibitory checkpoint molecule.
13. The immunoregulatory molecule of claim 12, wherein the inhibitory checkpoint molecule is selected from the group consisting of: PD-1, PD-L2, CTLA-4, LAG-3, TIM-3, HHA 2, CD47, CXCR4, CD160, CD73, BLTA, B7-H4, TIGIT, siglec7, siglec9 and VISTA.
14. The immunomodulatory molecule of claim 12 or 13, wherein the first binding domain is an antagonist ligand or variant thereof.
15. The immunoregulatory molecule of any one of claim 12-14,
(i) Wherein the second target molecule is PD-1, and wherein the second binding domain is PD-L1, PD-L2, or a variant thereof;
(ii) Wherein the second target molecule is TIGIT, and wherein the second binding domain is CD112, CD155, or a variant thereof;
(iii) Wherein the second target molecule is LAG-3, and wherein the second binding domain is MHC II, lseclin, or a variant thereof;
(iv) Wherein the second target molecule is TIM-3, and wherein the second binding domain is galectin-9, caecam-1, HMGB-1, phosphatidylserine or a variant thereof; or (b)
(v) Wherein the second target molecule is CTLA-4, and wherein the second binding domain is CD80, CD86, or variant thereof.
16. The immunomodulatory molecule of any of claims 1-3, wherein the first binding domain is IL-12 or a variant thereof, and wherein the second binding domain is PD-L2 or a variant thereof.
17. The immunomodulatory molecule of claim 16, wherein the second binding domain is a variant of PD-L2, and wherein the variant of PD-L2 has increased or decreased binding affinity for the second target molecule compared to PD-L2.
18. The immunomodulatory molecule of claim 16 or 17, wherein the first binding domain is a variant of IL-12, and wherein the variant of IL-12 has increased or decreased binding affinity for the first target molecule compared to IL-12.
19. The immunomodulatory molecule of any of claims 1-3, wherein the first binding domain is IL-2 or a variant thereof, and wherein the second binding domain is an agonist antibody or antigen-binding fragment thereof that specifically recognizes PD-1.
20. The immunomodulatory molecule of any of claims 1-3, wherein the first binding domain is IL-2 or a variant thereof, and wherein the second binding domain is PD-L1 or a variant thereof.
21. The immunomodulatory molecule of claim 20, wherein the second binding domain is a variant of PD-L1, wherein the variant of PD-L1 has increased or decreased binding affinity for the second target molecule compared to PD-L1.
22. The immunomodulatory molecule of any of claims 1-3, wherein the first binding domain is IL-2 or a variant thereof, and wherein the second binding domain is PD-L2 or a variant thereof.
23. The immunomodulatory molecule of claim 22, wherein the second binding domain is a variant of PD-L2, and wherein the variant of PD-L2 has increased or decreased binding affinity for the second target molecule compared to PD-L2.
24. The immunomodulatory molecule of any of claims 19-23, wherein the first binding domain is a variant of IL-2, and wherein the variant of IL-2 has increased or decreased binding affinity for the first target molecule compared to IL-2.
25. The immunomodulatory molecule of any of claims 1-24, wherein the immunomodulatory molecule comprises: i) An antigen binding protein comprising an antigen binding polypeptide; and ii) the first binding domain, wherein the antigen binding polypeptide comprises, from N-terminus to C-terminus: the second binding domain or portion thereof, a hinge region, and an FC domain subunit or portion thereof, and wherein the first binding domain is located at the hinge region.
26. The immunomodulatory molecule of claim 25, wherein the first binding domain is an immunostimulatory cytokine or variant thereof and is selected from the group consisting of: IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-12, IL-15, IL-17, IL-18, IL-21, IL-22, IL-23, IL-27, IFN-alpha, IFN-beta, IFN-gamma, TNF-alpha, erythropoietin, thrombopoietin, G-CSF, M-CSF, SCF, and GM-CSF.
27. The immunomodulatory molecule of claim 26, wherein the immunostimulatory cytokine or variant thereof is IL-2 or variant thereof.
28. The immunoregulatory molecule of claim 26, wherein the IL-2 variant comprises one or more mutations relative to wild-type IL-2 selected from the group consisting of: F24A, R D, K3543, E, E R and P65L.
29. The immunoregulatory molecule of claim 27 or 28, wherein the IL-2 variant comprises an R38D/K43E/E61R mutation relative to wild-type IL-2.
30. The immunomodulatory molecule of claim 26, wherein the immunostimulatory cytokine or variant thereof is IL-12 or variant thereof.
31. The immunomodulatory molecule of claim 30, wherein the IL-12 variant comprises one or more mutations within the p40 subunit relative to a wild-type p40 subunit selected from the group consisting of: Q56A, V57A, K A, E3559A, F60A, G61A, D A, A63S, G a and Q65A.
32. The immunoregulatory molecule of claim 30 or 31, wherein the IL-12 variant comprises an E59A/F60A mutation within the p40 subunit relative to a wild-type p40 subunit.
33. The immunoregulatory molecule of claim 30 or 31, wherein the IL-12 variant comprises an F60A mutation within the p40 subunit relative to a wild-type p40 subunit.
34. The immunomodulatory molecule of any of claims 30-33, wherein the p40 subunit and the p35 subunit of the IL-12 or variant thereof are connected by a linker.
35. The immunomodulatory molecule of any of claims 25-34, wherein the second binding domain is an agonist ligand of an inhibitory checkpoint molecule or a variant thereof.
36. The immunoregulatory molecule of claim 35, wherein the inhibitory checkpoint molecule is selected from the group consisting of: PD-1, PD-L2, CTLA-4, LAG-3, TIM-3, HHA 2, CD47, CXCR4, CD160, CD73, BLTA, B7-H4, TIGIT, siglec7, siglec9 and VISTA.
37. The immunomodulatory molecule of claim 35 or 36, wherein the second binding domain is PD-L1 or a variant thereof.
38. The immunomodulatory molecule of claim 37, wherein the PD-L1 variant comprises one or more mutations relative to wild-type PD-L1 selected from the group consisting of: I54Q, Y56F, E58M, R113T, M115L, S a and G119K.
39. The immunoregulatory molecule of claim 37 or 38, wherein the PD-L1 variant comprises an I54Q/Y56F/E58M/R113T/M115L/S117A/G119K mutation relative to wild-type PD-L1.
40. The immunomodulatory molecule of claim 35 or 36, wherein the second binding domain is PD-L2 or a variant thereof.
41. The immunoregulatory molecule of any one of claims 25-40, comprising:
(i) A first antigen binding polypeptide comprising, from N-terminus to C-terminus: a first PD-L2 or PD-L1 or variant thereof, a second PD-L2 or PD-L1 or variant thereof, a p35 subunit and a p40 subunit in tandem of IL-12 or variant thereof at the first hinge region, and a first subunit of an Fc domain or portion thereof; a second antigen binding polypeptide comprising, from N-terminus to C-terminus: VH, optionally CH1, a second hinge region, and a second subunit of the Fc domain, or a portion thereof; and a third antigen binding polypeptide comprising, from N-terminus to C-terminus: VL and optionally CL; wherein said VH and said VL, and optionally said CH1 and said CL, form a third binding domain that specifically recognizes a third target molecule;
(ii) A first antigen binding polypeptide comprising, from N-terminus to C-terminus: a first VH, optionally a first CH1, a p35 subunit and a p40 subunit in tandem with IL-12 or variant thereof at a first hinge region, and a first subunit of an Fc domain or portion thereof; a second antigen binding polypeptide comprising, from N-terminus to C-terminus: a second VH, optionally a second CH1, a second hinge region, and a second subunit of the Fc domain, or portion thereof; a third antigen binding polypeptide comprising, from N-terminus to C-terminus: a first VL and optionally a first CL; and a fourth antigen binding polypeptide comprising, from N-terminus to C-terminus: a second VL and optionally a second CL, wherein the first VH and the first VL, and optionally the first CH1 and the first CL, form the second binding domain that is an agonist antigen-binding fragment that specifically recognizes PD-1, and wherein the second VH and the second VL, and optionally the second CH1 and the second CL, form a third binding domain that specifically recognizes a third target molecule;
(iii) A first antigen binding polypeptide comprising, from N-terminus to C-terminus: a first PD-L2 or PD-L1 or variant thereof, a p35 subunit and a p40 subunit in tandem with IL-12 or variant thereof at the first hinge region, and a first subunit of an Fc domain or portion thereof; and a second antigen binding polypeptide comprising, from N-terminus to C-terminus: a second PD-L2 or PD-L1 or variant thereof, a second hinge region, and a second subunit of an Fc domain or portion thereof;
(iv) A first antigen binding polypeptide comprising, from N-terminus to C-terminus: a first PD-L2 or PD-L1 or variant thereof, a second PD-L2 or PD-L1 or variant thereof, a p35 subunit and a p40 subunit in tandem of IL-12 or variant thereof at the first hinge region, and a first subunit of an Fc domain or portion thereof; and a second antigen binding polypeptide comprising, from N-terminus to C-terminus: a third PD-L2 or PD-L1 or variant thereof, a fourth PD-L2 or PD-L1 or variant thereof, a second hinge region, and a second subunit of the Fc domain or a portion thereof;
(v) A first antigen binding polypeptide comprising, from N-terminus to C-terminus: a first PD-L2 or PD-L1 or variant thereof, a p35 subunit of IL-12 or variant thereof located at the first hinge region, and a first subunit of an Fc domain or portion thereof; and a second antigen binding polypeptide comprising, from N-terminus to C-terminus: a second PD-L2 or PD-L1 or variant thereof, a p40 subunit of IL-12 or variant thereof located in a second hinge region, and a second subunit of the Fc domain or a portion thereof;
(vi) A first antigen binding polypeptide comprising, from N-terminus to C-terminus: a p35 subunit or p40 subunit of IL-12 or a variant thereof located in the first hinge region and a first subunit of an Fc domain or a portion thereof; and a second antigen binding polypeptide comprising, from N-terminus to C-terminus: a first PD-L2 or PD-L1 or variant thereof, a second PD-L2 or PD-L1 or variant thereof, a p40 subunit or p35 subunit of IL-12 or variant thereof located at a second hinge region, and a second subunit of the Fc domain or portion thereof; or (b)
(vii) A first antigen binding polypeptide comprising, from N-terminus to C-terminus: a first VH, optionally a first CH1, a p35 subunit or a p40 subunit of IL-12 or a variant thereof located at a first hinge region, and a first subunit of an Fc domain or portion thereof; a second antigen binding polypeptide comprising, from N-terminus to C-terminus: a second VH, an optional second CH1, a p40 subunit or a p35 subunit of IL-12 or a variant thereof located in a second hinge region, and a second subunit of the Fc domain or a portion thereof; a third antigen binding polypeptide comprising, from N-terminus to C-terminus: a first VL and optionally a first CL; and a fourth antigen binding polypeptide comprising, from N-terminus to C-terminus: a second VL and optionally a second CL, wherein the first VH and the first VL, and optionally the first CH1 and the first CL, form the second binding domain that is an agonist antigen-binding fragment that specifically recognizes PD-1, and wherein the second VH and the second VL, and optionally the second CH1 and the second CL, form a third binding domain that specifically recognizes a third target molecule.
42. The immunomodulatory molecule of any of claims 1-41, wherein the immunomodulatory molecule comprises an antigen-binding protein comprising an antigen-binding polypeptide, wherein the antigen-binding polypeptide comprises, from N-terminus to C-terminus: the first binding domain or portion thereof, the second binding domain or portion thereof, an optional hinge region, and an Fc domain subunit or portion thereof.
43. The immunoregulatory molecule of any one of claim 42, comprising:
(i) A first antigen binding polypeptide comprising, from N-terminus to C-terminus: a p35 subunit and a p40 subunit of IL-12 or a variant thereof, optionally a first CH1, a first hinge region, and a first subunit of an Fc domain, or a portion thereof, fused in tandem; a second antigen binding polypeptide comprising, from N-terminus to C-terminus: a second VH, optionally a second CH1, a second hinge region, and a second subunit of an Fc domain, or portion thereof; a third antigen binding polypeptide comprising, from N-terminus to C-terminus: a first VL and optionally a first CL; and a fourth antigen binding polypeptide comprising, from N-terminus to C-terminus: a second VL and optionally a second CL, wherein the first VH and the first VL, and optionally the first CH1 and the first CL, form the second binding domain that is an agonist antigen-binding fragment that specifically recognizes PD-1, and wherein the second VH and the second VL, and optionally the second CH1 and the second CL, form a third binding domain that specifically recognizes a third target molecule;
(ii) A first antigen binding polypeptide comprising, from N-terminus to C-terminus: a p35 subunit and a p40 subunit of IL-12 or a variant thereof, a first PD-L2 or PD-L1 or variant thereof, a second PD-L2 or PD-L1 or variant thereof, a first hinge region, and a first subunit of an Fc domain or portion thereof fused in tandem; and a second antigen binding polypeptide comprising, from N-terminus to C-terminus: a third PD-L2 or PD-L1 or variant thereof, a fourth PD-L2 or PD-L1 or variant thereof, a second hinge region, and a second subunit of an Fc domain or portion thereof;
(iii) A first antigen binding polypeptide comprising, from N-terminus to C-terminus: a p35 subunit and a p40 subunit of IL-12 or a variant thereof, a first PD-L2 or PD-L1 or variant thereof, a second PD-L2 or PD-L1 or variant thereof, a first hinge region, and a first subunit of an Fc domain or portion thereof fused in tandem; a second antigen binding polypeptide comprising, from N-terminus to C-terminus: VH, optionally CH1, a second hinge region, and a second subunit of an Fc domain, or portion thereof; and a third antigen binding polypeptide comprising, from N-terminus to C-terminus: VL and optionally CL, wherein the VH and the VL, and optionally the CH1 and the CL, form a third binding domain that specifically recognizes a third target molecule; or (b)
(iv) A first antigen binding polypeptide comprising, from N-terminus to C-terminus: a p35 subunit and a p40 subunit of IL-12 or variant thereof, VH, optionally CH1, a first hinge region, and a first subunit of an Fc domain, or portion thereof, fused in tandem; a second antigen binding polypeptide comprising, from N-terminus to C-terminus: a first PD-L2 or PD-L1 or variant thereof, a second hinge region, and a second subunit of an Fc domain or portion thereof; and a third antigen binding polypeptide comprising, from N-terminus to C-terminus: VL and optionally CL, wherein the VH and the VL, and optionally the CH1 and the CL, form the second binding domain, which is an agonist antigen-binding fragment that specifically recognizes PD-1.
44. The immunoregulatory molecule of any one of claims 1-43, comprising:
(i) A first antigen binding polypeptide comprising, from N-terminus to C-terminus: a first VH, optionally a first CH1, a first hinge region, and a first subunit of an Fc domain, or portion thereof; a second antigen binding polypeptide comprising, from N-terminus to C-terminus: a second VH, optionally a second CH1, a second hinge region, and a second subunit of an Fc domain, or portion thereof; a third antigen binding polypeptide comprising, from N-terminus to C-terminus: a p35 subunit and a p40 subunit of IL-12, or a variant thereof, fused in tandem, a first VL, and optionally a first CL; and a fourth antigen binding polypeptide comprising, from N-terminus to C-terminus: a second VL and optionally a second CL, wherein the first VH and the first VL, and optionally the first CH1 and the first CL, form a second binding domain that is an agonist antigen-binding fragment that specifically recognizes PD-1, and wherein the second VH and the second VL, and optionally the second CH1 and the second CL, form a third binding domain that specifically recognizes a third target molecule;
(ii) A first antigen binding polypeptide comprising, from N-terminus to C-terminus: VH, optionally CH1, a first hinge region, and a first subunit of an Fc domain, or portion thereof; a second antigen binding polypeptide comprising, from N-terminus to C-terminus: a first PD-L2 or PD-L1 or variant thereof, a second hinge region, and a second subunit of an Fc domain or portion thereof; and a third antigen binding polypeptide comprising, from N-terminus to C-terminus: in a tandem fusion of the p35 subunit and the p40 subunit of IL-12 or a variant thereof, a VL, and optionally a CL, wherein the VH and the VL, and optionally the CH1 and the CL, form the second binding domain, which is an agonist antigen-binding fragment that specifically recognizes PD-1.
45. The immunomodulatory molecule of any of claims 1-44, wherein the immunomodulatory molecule comprises an antigen-binding protein comprising a first antigen-binding polypeptide and a second antigen-binding polypeptide, wherein the first antigen-binding polypeptide comprises from N-terminus to C-terminus: the second antigen binding domain or portion thereof, the first hinge domain, and the first subunit of the Fc domain or portion thereof; wherein the second antigen binding polypeptide comprises, from N-terminus to C-terminus: the first antigen binding domain or portion thereof, the second hinge domain, and the second subunit of the Fc domain or portion thereof.
46. The immunoregulatory molecule of claim 45, wherein said second binding domain is an agonist Fab or agonist scFv that specifically recognizes an inhibitory checkpoint molecule, or an agonist ligand of an inhibitory checkpoint molecule or a variant thereof.
47. The immunoregulatory molecule of claim 45, wherein the second binding domain is PD-L1 or PD-L2 or a variant thereof.
48. The immunomodulatory molecule of any of claims 45-47, wherein the first binding domain is an immunostimulatory cytokine or variant thereof.
49. The immunoregulatory molecule of claim 48, wherein said immunostimulatory cytokine or variant thereof is IL-2 or IL-12 or variant thereof.
50. The immunoregulatory molecule of any one of claims 45-49, comprising:
(i) A first antigen binding polypeptide comprising, from N-terminus to C-terminus: VH, optionally CH1, a first hinge region, and a first subunit of an Fc domain, or portion thereof; a second antigen binding polypeptide comprising, from N-terminus to C-terminus: a p35 subunit and a p40 subunit of IL-12 or a variant thereof, a second hinge region, and a second subunit of an Fc domain, or a portion thereof, fused in tandem; and a third antigen binding polypeptide comprising, from N-terminus to C-terminus: VL and optionally CL, wherein the VH and the VL, and optionally the CH1 and the CL, form the second binding domain, which is an agonist antigen-binding fragment that specifically recognizes PD-1; or (b)
(ii) A first antigen binding polypeptide comprising, from N-terminus to C-terminus: a first PD-L2 or PD-L1 or variant thereof, a second PD-L2 or PD-L1 or variant thereof, a first hinge region, and a first subunit of an Fc domain or portion thereof; and a second antigen binding polypeptide comprising, from N-terminus to C-terminus: the p35 subunit and the p40 subunit of IL-12 or variant thereof, the second hinge region, and the second subunit of the Fc domain, or a portion thereof, are fused in tandem.
51. The immunomodulatory molecule of any of claims 1-50, wherein the immunomodulatory molecule comprises an antigen-binding protein comprising an antigen-binding polypeptide, wherein the antigen-binding polypeptide comprises from N-terminus to C-terminus: the second binding domain or portion thereof, an optional hinge region, an Fc domain subunit or portion thereof, and the first binding domain or portion thereof.
52. The immunoregulatory molecule of claim 51, wherein the second binding domain is an agonist Fab or agonist scFv that specifically recognizes an inhibitory checkpoint molecule, or an agonist ligand of an inhibitory checkpoint molecule or a variant thereof.
53. The immunoregulatory molecule of claim 51 or 52, wherein said first binding domain is an immunostimulatory cytokine or variant thereof.
54. The immunoregulatory molecule of claim 53, wherein said immunostimulatory cytokine or variant thereof is IL-2 or IL-12 or variant thereof.
55. The immunoregulatory molecule of any one of claims 51-54, comprising:
(i) A first antigen binding polypeptide comprising, from N-terminus to C-terminus: a first PD-L2 or PD-L1 or variant thereof, a first hinge region, a first subunit of an Fc domain or portion thereof, and a p35 subunit and a p40 subunit of IL-12 or variant thereof fused in tandem; and a second antigen binding polypeptide comprising, from N-terminus to C-terminus: a second PD-L2 or PD-L1 or variant thereof, a second hinge region, and a second subunit of the Fc domain or a portion thereof;
(ii) A first antigen binding polypeptide comprising, from N-terminus to C-terminus: a first VH, optionally a first CH1, a first hinge region, a first subunit of an Fc domain or portion thereof, and a p35 subunit and a p40 subunit of IL-12 or variant thereof fused in tandem; a second antigen binding polypeptide comprising, from N-terminus to C-terminus: a second VH, optionally a second CH1, a second hinge region, and a second subunit of the Fc domain, or portion thereof; a third antigen binding polypeptide comprising, from N-terminus to C-terminus: a first VL and optionally a first CL; and a fourth antigen binding polypeptide comprising, from N-terminus to C-terminus: a second VL and optionally a second CL, wherein the first VH and the first VL, and optionally the first CH1 and the first CL, form the second binding domain that is an agonist antigen-binding fragment that specifically recognizes PD-1, and wherein the second VH and the second VL, and optionally the second CH1 and the second CL, form a third binding domain that specifically recognizes a third target molecule;
(iii) A first antigen binding polypeptide comprising, from N-terminus to C-terminus: VH, optionally CH1, a first hinge region, a first subunit of an Fc domain or portion thereof, and a p35 subunit and a p40 subunit of IL-12 or variant thereof fused in tandem; a second antigen binding polypeptide comprising, from N-terminus to C-terminus: a first PD-L2 or PD-L1 or variant thereof, a second hinge region, and a second subunit of an Fc domain or portion thereof; and a third antigen binding polypeptide comprising, from N-terminus to C-terminus: VL and optionally CL, wherein the VH and the VL, and optionally the CH1 and the CL, form the second binding domain, which is an agonist antigen-binding fragment that specifically recognizes PD-1;
(iv) A first antigen binding polypeptide comprising, from N-terminus to C-terminus: a first PD-L2 or PD-L1 or variant thereof, a first hinge region, a first subunit of an Fc domain or portion thereof, and a p35 subunit or p40 subunit of IL-12 or variant thereof; and a second antigen binding polypeptide comprising, from N-terminus to C-terminus: a second PD-L2 or PD-L1 or variant thereof, a second hinge region, a second subunit of an Fc domain or portion thereof, and a p35 subunit or p40 subunit of IL-12 or variant thereof; or (b)
(v) A first antigen binding polypeptide comprising, from N-terminus to C-terminus: a first VH, optionally a first CH1, a first hinge region, a first subunit of an Fc domain or portion thereof, and a p35 subunit or p40 subunit of IL-12 or variant thereof; a second antigen binding polypeptide comprising, from N-terminus to C-terminus: a second VH, optionally a second CH1, a second hinge region, a second subunit of an Fc domain or portion thereof, and a p40 subunit or p35 subunit of IL-12 or variant thereof; a third antigen binding polypeptide comprising, from N-terminus to C-terminus: a first VL and optionally a first CL; and a fourth antigen binding polypeptide comprising, from N-terminus to C-terminus: a second VL and optionally a second CL, wherein the first VH and the first VL, and optionally the first CH1 and the first CL, form the second binding domain that is an agonist antigen-binding fragment that specifically recognizes PD-1, and wherein the second VH and the second VL, and optionally the second CH1 and the second CL, form a third binding domain that specifically recognizes a third target molecule.
56. A pharmaceutical composition comprising the immunoregulatory molecule of any one of claims 1-55, and optionally a pharmaceutically acceptable carrier.
57. A method of treating a disease or disorder in an individual comprising administering to the individual an effective amount of the immunomodulatory molecule of any one of claims 1-55, or the pharmaceutical composition of claim 56.
58. The method of claim 57, wherein the disease or disorder is cancer.
CN202280019509.3A 2021-03-10 2022-03-10 Immunomodulatory molecules and uses thereof Pending CN117377767A (en)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US63/159,441 2021-03-10
USPCT/US2021/073107 2021-12-23
PCT/US2021/073107 WO2022140797A1 (en) 2020-12-23 2021-12-23 Immunocytokines and uses thereof
PCT/US2022/071077 WO2022192898A2 (en) 2021-03-10 2022-03-10 Immunomodulatory molecules and uses thereof

Publications (1)

Publication Number Publication Date
CN117377767A true CN117377767A (en) 2024-01-09

Family

ID=89391485

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202280019509.3A Pending CN117377767A (en) 2021-03-10 2022-03-10 Immunomodulatory molecules and uses thereof

Country Status (1)

Country Link
CN (1) CN117377767A (en)

Similar Documents

Publication Publication Date Title
JP7369127B2 (en) Single domain antibodies against TIGIT and variants thereof
JP7379561B2 (en) Chimeric antigens and T cell receptors and methods of use
WO2018068201A1 (en) Single-domain antibodies and variants thereof against ctla-4
CN111527109A (en) Fusion protein dimer with antibody Fc region as framework and application thereof
US20240092855A1 (en) Engineered proteins to enhance sensitivity of a cell to il-2
JP7360440B2 (en) Antibody molecules that bind to PD-L1 and CD137
BR112020002706A2 (en) bonding agents and use thereof
TWI802633B (en) Single-domain antibodies and variants thereof against pd-1
JP7346790B2 (en) Single domain antibodies against LAG-3 and their uses
WO2022140797A1 (en) Immunocytokines and uses thereof
EP3568150A1 (en) Combination tumor treatment with an integrin-binding-fc fusion protein and immune modulator
BR112021000396A2 (en) MESOTELINE AND CD137 CONNECTION MOLECULES
US20230167164A1 (en) Immunocytokines and uses thereof
CN117377767A (en) Immunomodulatory molecules and uses thereof
RU2818371C1 (en) Immunocytokines and their use
KR20230148226A (en) Immunomodulatory Molecules and Their Uses
IL298449A (en) Improved antigen binding receptors
TW202413396A (en) Masking polypeptides, activatable novel prodrugs and methods of use thereof
JP2023516595A (en) Dimeric antigen receptor (DAR) that binds CD20

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination