CN109721657B - Fusion protein for blocking PD-1/PD-L1 signal transduction pathway and activating T cells and application thereof - Google Patents

Abstract

The present invention provides a fusion protein that blocks the PD-1/PD-L1 signaling pathway and activates T cells, comprising (i) an antigen-binding fragment derived from an anti-PD-1 antibody and/or an anti-PD-L1 antibody; (ii) an immunoglobulin Fc domain; and (iii) CD80 extracellular domain (ECD). The invention also provides a polynucleotide encoding the fusion protein, a vector comprising the polynucleotide, a host cell comprising the polynucleotide or the vector, and use of the fusion protein in treating, preventing and/or diagnosing diseases related to activation of PD-1/PD-L1 signaling pathway and inhibition of T cell function in an individual.

Description

Fusion protein for blocking PD-1/PD-L1 signal transduction pathway and activating T cells and application thereof

Technical Field

The present invention relates generally to the field of medical biotechnology. In particular, the invention relates to antigen-binding fragments comprising (i) an antibody derived from anti-programmed death protein-1 (PD-1)) antibody and/or anti-programmed death protein ligand 1(PD-L1) antibody; (ii) an immunoglobulin Fc domain; and (iii) a CD80 extracellular domain (ECD), a polynucleotide encoding the fusion protein, a vector comprising the polynucleotide, a host cell comprising the polynucleotide or vector, and the use of the fusion protein in the treatment, prevention and/or diagnosis of a disease associated with activation of the PD-1/PD-L1 signaling pathway and inhibition of T cell function in an individual.

Background

Immune checkpoints (immune checkpoints) are a class of inhibitory signaling molecules present in The immune system that avoid tissue damage by modulating The persistence and intensity of The immune response in peripheral tissues and are involved in maintaining tolerance to self-antigens (pardol DM., The block of immune responses in Cancer immunological. nat Rev Cancer,2012,12(4): 252-264). It has been found that one of the reasons that tumor cells are able to escape the immune system in vivo and proliferate uncontrollably is to utilize the inhibitory signaling pathway of immune checkpoints, thereby inhibiting T lymphocyte activity, rendering T lymphocytes ineffective in exerting a tumor killing effect (Yao S, Zhu Y and Chen l., Advances in targeting cell surface signaling molecules for tumor modulation. nat Rev Drug disease, 2013,12(2): 130-146).

Programmed death protein-1 (PD-1) is an important immune checkpoint protein and is also an important target for tumor immunotherapy at present. PD-1 was first discovered in 1992, and the cloning and expression of its gene suggests that PD-1 is capable of inducing programmed T cell death upon activation. PD-1 protein is found on activated T cells, B cells and myeloid cells. PD-1 is also inducibly expressed in macrophages, dendritic cells and monocytes. No PD-1 expression was observed on the surface of resting lymphocytes.

PD-1 is a 55kDa type I transmembrane protein with a cytoplasmic domain containing an immunoreceptor tyrosine inhibitory motif and homology to CD28 and CTLA-4. Two cell surface glycoprotein ligands have been identified for PD-1, programmed death protein ligand 1(PD-L1) and programmed death protein ligand 2(PD-L2), respectively. Ligand expression of PD-1 has been found on many cancer cells, including human lung, ovarian, colon, and multiple myelomas. In addition, the ligand of PD-1 is highly expressed on the cell surface of various epithelial cancers, hematologic cancers and other malignant tumors. Expression of ligands for PD-1, such as PD-L1, in tumor patients is often associated with poor prognosis of the cancer (Iwai Y et al, Involment of PD-L1on tumor cells in the cancer from the tumor immune system and tumor immunity by PD-L1blockade, PNAS, 2002, 99(19): 12293-7).

Binding of PD-1 to a ligand for PD-1 plays an important role in regulating T lymphocyte activity and maintaining peripheral immune tolerance. The binding of PD-1 to the ligand of PD-1 can lead to T cell apoptosis, immune unresponsiveness, T cell 'exhaustion' and IL-10 secretion, etc. Thus, PD-1 functions to limit T cell activation, inhibit T cell proliferation, and improve tolerance to antigens. Upregulation of the expression of PD-1 on the surface of activated lymphocytes can lead to suppression of acquired or innate immune responses, thereby causing tumor-infiltrating lymphocytes (including T lymphocytes) to have tumor antigen specificity, but the tumor cells can escape the immune system from killing the tumor cells because the binding of the ligand for PD-1 on the tumor cells to PD-1 on the tumor-infiltrating lymphocytes generates a signal that inhibits the activation of the tumor-infiltrating lymphocytes.

Studies have shown that these tumor infiltrating lymphocytes expressing PD-1 are dysfunctional lymphocytes whose biological function can be restored by antibodies that block the binding of PD-1 to the ligand of PD-1. At present, antibodies inhibiting the binding of PD-1 to the ligand of PD-1 mainly include anti-PD-1 monoclonal antibody and anti-PD-L1 monoclonal antibody, but there are also products directed against PD-L2.

Currently, the relatively mature anti-PD-1 antibodies studied were Nivolumab (Nivolumab) of Bevacizumab (BMS) and Pembrolizumab (Pembrolizumab) of Merck (Merck). Nivolumab (trade name)

) Is a fully humanized IgG4 antibody molecule, pembrolizumab (trade name)

) Is a human sourceAn IgG4 antibody molecule was formed. The anti-PD-1 monoclonal antibody can inhibit the binding of PD-1 to the ligands PD-L1 and PD-L2 after binding with PD-1 on T lymphocytes, thereby promoting the activation and proliferation of the T lymphocytes and producing immune activation type cytokines such as IL-2, and relieving the inhibition of the immune monitoring of the T lymphocytes with anti-tumor activity by PD-1. The current approved indications for nivolumab by the U.S. food and drug administration include: melanoma, non-small cell lung cancer, kidney cancer, head and neck tumors, and the like; indications for pembrolizumab include: head and neck tumors, non-small cell lung cancer, melanoma, and the like. With respect to the anti-PD-L1 antibody, atezolizumab developed by Roche (Roche), avelumab developed by cooperation of Merck KGaA (German Merck) and Pfizer (U.S. Perey), durvalumab developed by Aslicon also showed a therapeutic effect on tumors.

Although the anti-PD-1 antibody and the anti-PD-L1 antibody have the treatment effect on tumors, the average treatment efficiency is only about 20 percent, and the five-year survival rate of the lung cancer is only 16 percent. Still a significant fraction of tumor patients do not respond to treatment with anti-PD-1 antibodies, anti-PD-L1 antibodies. Therefore, how to improve the effectiveness of tumor therapy is still a problem to be solved urgently in the field of tumor therapy at present.

On the other hand, activation of T cells requires dual signal stimulation: the first signal is provided by the binding of the T cell antigen receptor (TCR) to the antigenic peptide MHC (major histocompatibility Complex) molecule complex on the Antigen Presenting Cell (APC), and the second signal is provided by the binding of a costimulatory molecule on the APC to a corresponding ligand on the T cell. Among costimulatory molecules involved in T cell activation, the binding of T cell surface CD28 molecules to APC surface corresponding ligands CD80 and CD86 plays an important role. However, low or no expression of CD80 and CD86 in the tumor microenvironment is also one of the important mechanisms responsible for tumor immune evasion.

Both CD80 and CD86 are transmembrane glycoproteins and are members of the immunoglobulin superfamily (IgSF). The mature CD80 molecule consists of 254 amino acids, with an extracellular domain (ECD) of 208 amino acids, a transmembrane domain of 25 amino acids, and an intracellular domain of 21 amino acids. Similarly, the mature CD86 molecule consists of 303 amino acids, with an extracellular domain of 222 amino acids, a transmembrane domain of 20 amino acids, and an intracellular domain of 61 amino acids. The extracellular domains of CD80 and CD86 comprise immunoglobulin v (igv) and immunoglobulin c (igc) regions, and CD80 and CD86 are bound by immunoglobulin v (igv) regions to their ligands CD28 and CTLA-4. In the case of CD80 and CD86 binding to CD28, CD80 and CD86 have important regulatory effects on antigen-induced T cell activation, proliferation and the generation of effector functions, and are positive regulators; whereas in the case of binding of CD80 and CD86 to CTLA-4, CD80 and CD86 down-regulate the immune response, are negative regulators. Thus, CD80 and CD86 are co-stimulatory factors in T lymphocyte activation and play important roles in autoimmune monitoring, humoral immune response, and transplantation response.

In addition, CD80 can also block PD-1/PD-L1 interaction by binding to PD-L1, thereby participating in the activation of the immune system.

As can be seen from the above, the effect of the CD80 protein on T cell responses is complex and cannot be predicted before actual testing (Salomon, B et al, complexes of CD28/B7: CTLA-4 synergistic pathway in autoimmunity and transplantation, annual review of immunology, 2001, 19: 225-.

Since activation of the PD-1/PD-L1 signaling pathway and inhibition of T cell function contribute to the development and progression of tumors, there is a need to develop proteins that have both the activity of interfering with, inhibiting or blocking the PD-1/PD-L1 signaling pathway and activating T cells.

The present inventors have conducted intensive studies to develop a group of fusion proteins that interfere, inhibit or block the PD-1/PD-L1 signaling pathway and activate T cells, which fusion proteins are capable of inhibiting the PD-1/PD-L1 signaling pathway and of inducing T cell activation, and thus can be used for treating, preventing and/or diagnosing diseases associated with the activation of the PD-1/PD-L1 signaling pathway and the inhibition of T cell function in individuals, and particularly can be used for enhancing tumor immune responses in individuals who do not respond or respond weakly to PD-1 antibody and/or PD-L1 antibody therapy.

Summary of The Invention

The invention discloses a novel fusion protein for blocking a PD-1/PD-L1 signal transduction pathway and activating a T cell, a polynucleotide for encoding the fusion protein, a vector containing the polynucleotide, a host cell containing the polynucleotide or the vector, and application of the fusion protein in treating, preventing and/or diagnosing diseases related to activation of the PD-1/PD-L1 signal transduction pathway and inhibition of T cell functions in an individual.

Accordingly, in one aspect, the present invention provides a novel fusion protein that inhibits the PD-1/PD-L1 signaling pathway and is capable of inducing T cell activation, comprising (i) an antigen-binding fragment derived from an anti-PD-1 antibody and/or an anti-PD-L1 antibody; (ii) an immunoglobulin Fc domain; and (iii) CD80 extracellular domain (ECD). In one embodiment, said (i) of said fusion protein is a Fab, Fab ', F (ab') derived from an anti-PD-1 antibody and/or an anti-PD-L1 antibody₂Fv, single-chain Fv. In one embodiment, said (ii) of said fusion protein is a human immunoglobulin Fc domain. In one embodiment, said (iii) of said fusion protein comprises human CD80 ECD.

The (i) antigen-binding fragment contained in the fusion protein may be derived from any anti-PD-1 antibody as long as it is an antibody capable of inhibiting or reducing binding of PD-1 to its ligand, including anti-PD-1 antibodies known in the art and anti-PD-1 antibodies developed in the future. In one embodiment, the antigen-binding fragment comprises a sequence selected from SEQ ID NOs: 1/2, 3/4, 5/6, 7/8, 9/10, 11/12, 13/14, 15/16, 17/18, 19/20, 21/22, 23/24, and 25/26, preferably the antigen binding fragment comprises all 6 heavy chain CDRs and light chain CDRs contained in the heavy chain variable region sequence/light chain variable region sequence selected from the group consisting of SEQ ID NOs: 1/2, 3/4, 5/6, 7/8, 9/10, 11/12, 13/14, 15/16, 17/18, 19/20, 21/22, 23/24, and 25/26, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to said heavy-heavy chain variable region sequence/light chain variable region sequence; more preferably, the antigen-binding fragment comprises a peptide selected from the group consisting of nivolumab, pidilizumab and pembrolizumabThe heavy chain variable region and the light chain variable region of the anti-PD-1 antibody of (1), in particular, the antigen-binding fragment is a Fab, Fab ', F (ab') selected from the group consisting of nivolumab, pidilizumab and pembrolizumab₂Fv, single-chain Fv.

The (i) antigen-binding fragment comprised in the fusion protein may also be derived from any anti-PD-L1 antibody, as long as it is an antibody capable of inhibiting or reducing the binding of PD-L1 to its receptor (e.g. to PD-1 or CD80(B7-1) or to both), including anti-PD-L1 antibodies known in the art and anti-PD-L1 antibodies developed in the future. In one embodiment, the antigen-binding fragment comprises a sequence selected from SEQ ID NOs: 27/28, 29/30, and 31/32, and all 6 heavy and light chain CDRs contained in the heavy/light chain variable region sequences. Preferably, the antigen-binding fragment comprises an amino acid sequence selected from SEQ ID NOs: 27/28, 29/30, and 31/32, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to said heavy chain variable region sequence/light chain variable region sequence; more preferably, the antigen binding fragment is a Fab, Fab ', F (ab') selected from atezolizumab, avelumab and durvalumab₂Fv, single-chain Fv.

In one embodiment, the light chain constant region type in the antigen binding fragment may be of the kappa or lambda type, preferably of the kappa type. The kappa-type light chain constant region amino acid sequence of the exemplary anti-PD-1 antibody is shown in SEQ ID NO. 33. The lambda-type light chain constant region amino acid sequence of an exemplary anti-PD-1 antibody is shown in SEQ ID NO. 34.

The (ii) immunoglobulin Fc domain comprised in the fusion protein may be any immunoglobulin Fc domain, in particular, the (ii) is a human immunoglobulin Fc domain. In one embodiment, the immunoglobulin Fc domain is an Fc domain of an IgG class antibody, particularly an IgG₁Subclass, IgG₂Subclass, IgG₄An Fc domain of a subclass antibody. In a preferred embodiment, the immunoglobulin Fc domain comprised in the fusion protein of the invention is an IgG₄Fc domain of subclass antibodyIn particular human IgG₄An Fc domain of a subclass antibody. In one embodiment, the IgG is₄Subclass antibodies comprise an amino acid substitution at position S228 (EU numbering) in the Fc region, particularly amino acid substitution S228P. Exemplary IgG is shown in SEQ ID NO 35₁Subclass anti-PD-1 antibody heavy chain constant region amino acid sequence. Exemplary IgG is shown in SEQ ID NO 36₂Subclass anti-PD-1 antibody heavy chain constant region amino acid sequence. Exemplary IgG is shown in SEQ ID NO 37₄Subclass anti-PD-1 antibody heavy chain constant region amino acid sequence. Exemplary IgG is shown in SEQ ID NO 38₁Fc domain amino acid sequence of a subclass anti-PD-1 antibody. Exemplary IgG is shown in SEQ ID NO 39₂Fc domain amino acid sequence of a subclass anti-PD-1 antibody. Exemplary IgG is shown in SEQ ID NO 40₄Fc domain amino acid sequence of a subclass anti-PD-1 antibody. In some embodiments, the (ii) immunoglobulin Fc domain comprised in the fusion protein is identical to SEQ ID NO: 38. 39 or 40, or an Fc domain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the amino acid sequence set forth in seq id no.

In one embodiment, the (iii) CD80ECD comprised in the fusion protein is part of the extracellular domain of CD 80. In one embodiment, the CD80ECD comprises a CD80 immunoglobulin v (IgV) region (CD 80-IgV). In one embodiment, the CD80ECD comprises CD80 immunoglobulin V and C regions (CD 80-IgVIgC). In one embodiment, the CD80ECD is a human CD80ECD, preferably the CD80ECD comprises a human CD80 IgV. In a specific embodiment, the CD80-IgV has the amino acid sequence of SEQ ID NO: 41, or an amino acid sequence corresponding to SEQ ID NO: 41 has an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical. In one embodiment, the CD80-IgVIgC has the amino acid sequence of SEQ ID NO: 42, or an amino acid sequence corresponding to SEQ ID NO: 42, has an amino acid sequence of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity.

In one embodiment, said fusion protein further comprises a peptide linker between said (i), (ii) and/or (iii); preferably, the peptide linker comprises one or more amino acids, more preferably at least 5 amino acids, most preferably comprises a sequence selected from the group consisting of SEQ ID NO: 43-71.

In one embodiment, the fusion protein is produced from N-terminus to C-terminus in the order of (i), (ii), and (iii); (iii) the order of (i), (ii) and (iii); or the order of (iii), (ii) and (i) are operably linked.

In one embodiment, the fusion protein comprises

(a) An anti-PD-1 antibody, an anti-PD-L1 antibody, or a bispecific antibody against PD-1 and PD-L1; and a CD80ECD operably linked at the C-terminus of each of the two heavy chains of the antibody;

(b) an anti-PD-1 antibody, an anti-PD-L1 antibody, or a bispecific antibody against PD-1 and PD-L1; a CD80ECD operably linked at the N-terminus of each of the two heavy chains of the antibody; and a CD80ECD operably linked at the N-terminus of each of the two light chains of the antibody; or

(c) A CD80 ECD; an immunoglobulin Fc domain in dimeric form operatively linked at the C-terminus of the CD80 ECD; and an antigen-binding fragment derived from an anti-PD-1 antibody and/or an anti-PD-L1 antibody operably linked at the C-terminus of the immunoglobulin Fc domain in dimeric form;

preferably, the anti-PD-1 antibody is selected from nivolumab, pidilizumab and pembrolizumab; the anti-PD-L1 antibody is selected from the group consisting of atezolizumab, avelumab and durvalumab. Preferably, the antibody is an IgG class antibody, in particular an IgG₁Subclass, IgG₂Subclass, IgG₄Subclass antibody, more particularly IgG₄A subclass antibody; also preferably, the IgG₄Subclass antibodies comprise an amino acid substitution at position S228 in the Fc domain, more preferably amino acid substitution S228P; further preferably, the light chain type of the antibody is of the kappa type or lambda type, preferably of the kappa type.

In a specific embodiment, the fusion protein is a fusion protein comprising a first subunit of the fusion protein of SEQ ID NO:77 and a second subunit of the fusion protein of SEQ ID NO:79, hereinafter referred to as fusion protein BY31.2, comprising an anti-PD-1 antibody (IgG4, kappa, S228P) and a CD80IgV operatively linked to the C-terminus of the antibody heavy chain via a peptide linker.

In a specific embodiment, the fusion protein is a fusion protein comprising a first subunit of the fusion protein of SEQ ID NO:81 and a second subunit of the fusion protein of SEQ ID NO:83, hereinafter referred to as fusion protein BY31.3, comprising an anti-PD-1 antibody (IgG2, kappa) and a CD80IgVIgC operatively linked to the C-terminus of the heavy chain of the antibody BY a peptide linker.

In a specific embodiment, the fusion protein is a fusion protein comprising a first subunit of the fusion protein of SEQ ID NO:85 and a second subunit of the fusion protein of SEQ ID NO:87, hereinafter referred to as fusion protein BY31.7, comprising an anti-PD-1 antibody (IgG4, kappa, S228P) and CD80IgV operatively linked to the N-terminus of each light chain and each heavy chain of the antibody BY a peptide linker.

In a specific embodiment, the fusion protein is a fusion protein comprising a first subunit of the fusion protein of SEQ ID NO 89 comprising, from N-terminus to C-terminus, operably linked CD80IgV, IgG4CH2 and CH3 domains, a peptide linker, an anti-PD-1 antibody light chain, and a second subunit of the fusion protein of from N-terminus to C-terminus, comprising, from N-terminus to C-terminus, the heavy chain variable region of an anti-PD-1 antibody Fab fragment and the CH1 domain, the second subunit being linked BY a disulfide bond to the anti-PD-1 antibody light chain portion of the first subunit of fusion protein BY31.14, and SEQ ID NO 91, hereinafter referred to as fusion protein BY 31.14.

In a specific embodiment, the fusion protein is a fusion protein comprising a first subunit of the fusion protein of SEQ ID NO 93 comprising, from N-terminus to C-terminus, the variable and constant regions of the light chain of the anti-PD-1 antibody and a second subunit of the fusion protein of SEQ ID NO 95 comprising, from N-terminus to C-terminus, operably linked domains of CD80IgV, IgG4CH2 and CH3, a peptide linker, the heavy chain variable region of the Fab fragment of the anti-PD-1 antibody and the CH1 domain, the heavy chain variable region and the CH1 domain of the Fab fragment of the PD-1 antibody in the second subunit being linked to the first subunit BY disulfide bonds, hereinafter referred to as fusion protein BY 31.15.

In a specific embodiment, the fusion protein comprises

(a) An anti-PD-L1 antibody selected from atezolizumab, avelumab and durvalumab; and a CD80 extracellular domain (ECD) operably linked (optionally, operably linked by a peptide linker) at the C-terminus of each of the two heavy chains of the anti-PD-L1 antibody;

(b) an anti-PD-L1 antibody selected from the group consisting of atezolizumab, avelumab and durvalumab, a CD80ECD operably linked (optionally operably linked by a peptide linker) to the N-terminus of each of the two heavy chains of the anti-PD-L1 antibody, and a CD80ECD operably linked (optionally operably linked by a peptide linker) to the N-terminus of each of the two light chains of the anti-PD-L1 antibody; or

(c) A CD80 ECD; an immunoglobulin Fc domain in dimeric form operatively linked at the C-terminus of the CD80 ECD; and an antigen-binding fragment derived from the anti-PD-L1 antibody atezolizumab, avelumab or durvalumab operably linked C-terminal to the dimeric form of the immunoglobulin Fc domain.

Preferably, the CD80ECD is CD80IgV or CD80 IgVIgC.

In one embodiment, the fusion protein specifically targets the PD-1/PD-L1 signaling pathway and activated T cells. The fusion protein of the invention can not only bind to PD-1 and/or PD-L1 with high affinity, but also bind to CD28 constitutively expressed on the surface of T cells with high affinity. The structure of the fusion protein designed by the invention fully ensures the proper physical space distance for the combination of the fusion protein and the target thereof, and the specific combination of the fusion protein and other molecules in the target is not influenced after the fusion protein with the structure is specifically combined with one molecule in the target.

The invention also provides a polynucleotide encoding the fusion protein of the invention, a vector, preferably an expression vector, most preferably a glutamine synthetase expression vector having a dual expression cassette, comprising the polynucleotide encoding the fusion protein of the invention. In another aspect, the invention provides a host cell comprising a polynucleotide or vector of the invention. In one embodiment, the host cell is a CHO, HEK293, or NSO cell. The invention also provides a method for producing a fusion protein of the invention comprising the steps of (i) culturing a host cell of the invention under conditions suitable for expression of the fusion protein of the invention, and (ii) recovering the fusion protein of the invention.

In one aspect, the invention provides diagnostic kits and pharmaceutical compositions comprising the fusion proteins of the invention. Further, the use of the fusion protein, the diagnostic kit or the pharmaceutical composition of the invention is also provided for the treatment, prevention and/or diagnosis of diseases associated with the activation of the PD-1/PD-L1 signaling pathway and the inhibition of T cell function, in particular for the treatment, prevention and/or diagnosis of cancerous diseases (e.g. solid and soft tissue tumors), most particularly for the treatment, prevention and/or diagnosis of melanoma, breast cancer, colon cancer, esophageal cancer, gastrointestinal stromal tumor (GIST), kidney cancer (e.g. renal cell carcinoma), liver cancer, non-small cell lung cancer (NSCLC), ovarian cancer, pancreatic cancer, prostate cancer, head and neck tumors, gastric cancer, hematological malignancies (e.g. lymphomas).

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In addition, the materials, methods, and examples described herein are illustrative only and are not intended to be limiting. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.

Brief Description of Drawings

The preferred embodiments of the present invention described in detail below will be better understood when read in conjunction with the following drawings. For the purpose of illustrating the invention, there is shown in the drawings embodiments which are presently preferred. It should be understood, however, that the invention is not limited to the precise arrangements and instrumentalities of the embodiments shown in the drawings.

FIGS. 1A, B and C provide schematic diagrams of fusion proteins of the invention, wherein FIG. 1A illustrates a schematic structural diagram of a fusion protein comprising, from N-terminus to C-terminus, an antigen-binding fragment of an antibody, an immunoglobulin Fc domain, and a CD80 ECD; FIG. 1B illustrates a schematic structural diagram of a fusion protein comprising, from N-terminus to C-terminus, a CD80ECD, an antigen-binding fragment of an antibody, and an immunoglobulin Fc domain; figure 1C illustrates a schematic structural view of a fusion protein comprising, from N-terminus to C-terminus, a CD80ECD, an immunoglobulin Fc domain, and an antigen-binding fragment of an antibody.

FIG. 2: the results of the fusion protein of the present invention prepared and purified in example 2 after passing through SDS-PAGE in the presence of a reducing agent (5mM 1, 4-dithiothreitol) and staining with Coomassie blue are shown. Lane 1: protein molecular weight standard markers; lane 2: antibody BY 18.1; lane 3: fusion protein BY 31.2; lane 4: fusion protein BY 31.3; lane 5: fusion protein BY 31.7; lane 6: fusion protein BY 31.14; lane 7: the fusion protein BY 31.15.

FIG. 3A: the binding curve of the fusion protein BY31.2 of the present invention prepared and purified in example 2 to human CD28 is illustrated; FIG. 3B: the binding curve of the fusion protein BY31.2 of the present invention prepared and purified in example 2 to human PD-L1 is illustrated; FIG. 3C: the binding curve of the fusion protein BY31.2 of the present invention prepared and purified in example 2 to human CTLA-4 is exemplified.

FIG. 4: the effect of the fusion protein BY31.2 of the invention on the body weight of experimental animals in the animal model of example 6 is shown.

FIG. 5: the in vivo anti-tumor effect of the fusion protein BY31.2 of the present invention in the animal model of example 6 is shown.

FIG. 6: the effect of the fusion protein BY31.2 of the invention on the body weight of experimental animals in the animal model of example 7 is shown.

FIG. 7: a schematic diagram comparing the in vivo anti-tumor effect of the fusion protein BY31.2 of the present invention with anti-PD-L1 mab Avelumab and anti-PD-1 mab Opdivo in the animal model of example 7 is shown.

Detailed Description

The invention provides fusion proteins and pharmaceutical compositions that interfere with, inhibit or block the PD-1/PD-L1 signaling pathway and activate T cells. The invention also provides methods for producing the fusion protein and the use of the fusion protein in treating, preventing and/or diagnosing a disease associated with activation of the PD-1/PD-L1 signaling pathway and inhibition of T cell function in an individual.

Unless defined otherwise below, terms in this specification are used as they are commonly used in the art.

I. Definition of

The term "about," when used in conjunction with a numerical value, is intended to encompass a numerical value within a range having a lower limit that is 5% less than the stated numerical value and an upper limit that is 5% greater than the stated numerical value.

As used herein, the term "comprising" or "comprises" is intended to mean including the stated elements, integers or steps, but not excluding any other elements, integers or steps.

"PD-1 pathway" refers to any intracellular signaling pathway initiated by binding to PD-1, including but not limited to the intracellular signaling pathway initiated by binding of PD-1 to PD-L1, or the intracellular signaling pathway initiated by binding of PD-1 to PD-L2, or the intracellular signaling pathway initiated by binding of PD-1 to both PD-L1 and PD-L2.

The "PD-L1 pathway" refers to any intracellular signaling pathway initiated by binding to PD-L1, including but not limited to the intracellular signaling pathway initiated by binding of PD-L1 to PD-1, or the intracellular signaling pathway initiated by binding of PD-L1 to CD80(B7-1), or the intracellular signaling pathway initiated by binding of PD-L1 to both PD-1 and CD80 (B7-1).

As used herein, "interfere with," "inhibit," or "block" the PD-1/PD-L1 signaling pathway, are used interchangeably, and refer to (i) interfering with the interaction between PD-1 and PD-L1; and/or (ii) results in the inhibition of at least one biological function of the PD-1/PD-L1 signaling pathway. The "interference", "inhibition" or "blocking" of the PD-1/PD-L1 signaling pathway resulting from the specific binding of the fusion protein of the invention to PD-1 and/or PD-L1 need not be a complete interference, inhibition or blocking.

As used herein, the term "specific binding" means selective for binding of an antigen or molecule of interest and distinguishable from unwanted or non-specific interactions. The specific binding may be measured by enzyme-linked immunosorbent assay (ELISA) or other techniques familiar to those skilled in the art, such as Surface Plasmon Resonance (SPR) techniques (Analysis on a BIAcore instrument) (Liljebold et al, Analysis of the antibody-IgG in rhematoid reaction using surface plasma resonance, Glyco J.,2000, 17, 323-.

"affinity" or "binding affinity" refers to the inherent binding affinity that reflects the interaction between members of a binding pair. The affinity of a molecule X for its partner Y may be generally determined by the dissociation constant (K)_D) Typically, the dissociation constants are the dissociation and association rate constants (k, respectively)_offAnd k_on) The ratio of (a) to (b). Affinity can be measured by common methods known in the art. One specific method for measuring affinity is Surface Plasmon Resonance (SPR).

The term "antibody" is used herein in the broadest sense and includes, but is not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies) so long as they exhibit the desired antigen binding activity. The antibody can be a whole antibody (e.g., having two full-length light chains and two full-length heavy chains) of any type and subtype (e.g., IgM, IgD, IgG1, IgG2, IgG3, IgG4, IgE, IgA1, and IgA 2).

The terms "whole antibody," "full-length antibody," "full antibody," and "intact antibody" are used interchangeably herein to refer to an antibody having a structure that is substantially similar to a native antibody structure.

The term "antibody heavy chain" refers to the larger of the two types of polypeptide chains present in an antibody molecule in its naturally occurring conformation, which normally determines the class to which an antibody belongs.

The term "antibody light chain" refers to the smaller of the two types of polypeptide chains present in an antibody molecule in its naturally occurring conformation. Kappa and lambda light chains refer to the two major antibody light chain classes.

A "bispecific antibody" is an artificial hybrid antibody having two different heavy/light chain pairs and having two different binding sites. Bispecific antibodies can be prepared by a variety of methods, including hybridoma fusion or ligation of Fab' fragments.

The term "antigen-binding fragment" of an antibody is a portion or section of an antibody or antibody chain having fewer amino acid residues than an intact or complete antibody or antibody chain, which is capable of binding to an antigen or competes for binding to an antigen with an intact antibody (i.e., an intact antibody from which the antigen-binding fragment is derived). Antigen-binding fragments can be prepared by recombinant DNA techniques, or by enzymatic or chemical cleavage of intact antibodies. Antigen binding fragments include, but are not limited to, Fab ', F (ab')₂Fv, single-chain Fv. The Fab fragment is a fragment consisting of V_L、V_H、C_LAnd the CH1 domain, e.g., Fab fragments can be obtained by papain digestion of whole antibodies. In addition, complete antibody production F (ab') by pepsin digestion below the disulfide bond in the hinge region₂It is a dimer of Fab' and is a bivalent fragment. F (ab')₂Can be reduced under neutral conditions by disrupting disulfide bonds in the hinge region, thereby converting F (ab')₂The dimer is converted to Fab' monomer. The Fab' monomer is essentially a Fab fragment with a hinge region (see: basic Immunology, edited by W.E.Paul, Raven Press, N.Y. (1993) for a more detailed description of other antibody fragments). The Fv fragment consists of a V on one arm of an antibody_LAnd V_HDomain composition. In addition, although two domains of the Fv fragment, V_LAnd V_HEncoded by separate genes, but using recombinant methods they can be joined by a synthetic linker that enables the two domains to be produced as a single protein chain in which V is present_LRegion and V_HThe regions pair to form a single chain Fv. The antibody fragment may be obtained by a chemical method, a recombinant DNA method, or a protease digestion method.

The term "immunoglobulin" refers to a protein having the structure of a naturally occurring antibody. For example, immunoglobulins of the IgG class are two light chains and two chains bound by disulfide bondsA heterotetrameric glycoprotein consisting of about 150,000 daltons in heavy chain. From N-terminus to C-terminus, each immunoglobulin heavy chain has one variable region (VH), also known as the variable heavy chain domain or heavy chain variable domain, followed by three constant domains (CH1, CH2, and CH3), also known as heavy chain constant regions. Similarly, from N-terminus to C-terminus, each immunoglobulin light chain has one variable region (VL), also known as a variable light domain or light chain variable domain, followed by one constant light Chain (CL) domain, also known as a light chain constant region. Heavy chains of immunoglobulins can be assigned to one of 5 classes, called α (IgA), δ (IgD), ε (IgE), γ (IgG) or μ (IgM), wherein certain classes can be further divided into subclasses, e.g., γ₁(IgG1)、γ₂(IgG2)、γ₃(IgG₃)、γ₄(IgG₄)、α₁(IgA₁) And alpha₂(IgA₂). The light chains of immunoglobulins can be divided into one of two types, called kappa and lambda, based on the amino acid sequence of their constant domains. An immunoglobulin essentially consists of two Fab molecules and one Fc domain connected by means of an immunoglobulin hinge region.

The term "Fc domain" or "Fc region" is used herein to define a C-terminal region of an immunoglobulin heavy chain that contains at least a portion of a constant region. The term includes native sequence Fc regions and variant Fc regions. A native immunoglobulin "Fc domain" comprises two or three constant domains, namely a CH2 domain, a CH3 domain, and optionally a CH4 domain. For example, in natural antibodies, the immunoglobulin Fc domain comprises the second and third constant domains (CH2 domain and CH3 domain) derived from the two heavy chains of IgG, IgA, and IgD class antibodies; or second, third and fourth constant domains (CH2 domain, CH3 domain and CH4 domain) derived from the two heavy chains of antibodies of the IgM and IgE classes. Unless otherwise indicated herein, the numbering of amino acid residues in the Fc region or heavy chain constant region is according to the EU numbering system (also known as the EU index) as described in Kabat et al, Sequences of Proteins of Immunological Interes, 5 th edition, Public Health Service, National Institutes of Health, Bethesda, MD, 1991.

A "human immunoglobulin" is an immunoglobulin that possesses an amino acid sequence corresponding to an immunoglobulin produced by a human or human cell or is derived from a non-human source using a human immunoglobulin repertoire or other sequence encoding a human immunoglobulin.

"percent (%) identity" of an amino acid sequence refers to the percentage of amino acid residues in the candidate sequence that are identical to the amino acid residues in the specific amino acid sequence shown in the specification, after aligning the candidate sequence with the specific amino acid sequence shown in the specification and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity.

The term "operably linked" means that the specified components are in a relationship that allows them to function in the intended manner.

A "signal sequence" is a sequence of amino acids linked to the N-terminal portion of a protein that promotes secretion of the protein outside the cell. The mature form of the extracellular protein lacks a signal sequence, which is cleaved off during the secretion process.

The term "N-terminal" refers to the last amino acid at the N-terminus, and the term "C-terminal" refers to the last amino acid at the C-terminus.

The term "fusion" refers to the joining of two or more components by peptide bonds either directly or by means of one or more peptide linkers.

The term "host cell" refers to a cell into which an exogenous polynucleotide has been introduced, including progeny of such a cell. Host cells include "transformants" and "transformed cells," which include the primary transformed cell and progeny derived therefrom. Host cells are any type of cell system that can be used to produce the fusion proteins of the invention. Host cells include cultured cells, and also include cells within transgenic animals, transgenic plants, or cultured plant tissues or animal tissues.

The terms "individual" or "subject" are used interchangeably and refer to a mammal. Mammals include, but are not limited to, domesticated animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., human and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In particular, the individual is a human.

The term "treatment" refers to clinical intervention intended to alter the natural course of disease in the individual undergoing treatment. Desirable therapeutic effects include, but are not limited to, preventing the occurrence or recurrence of disease, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of disease progression, ameliorating or palliating the disease state, and alleviating or improving prognosis. In some embodiments, the fusion proteins of the invention are used to delay the progression of a disease or to slow the progression of a disease.

The term "anti-tumor effect" refers to a biological effect that can be exhibited by a variety of means, including, but not limited to, for example, a reduction in tumor volume, a reduction in tumor cell number, a reduction in tumor cell proliferation, or a reduction in tumor cell survival. The terms "tumor" and "cancer" are used interchangeably herein to encompass solid tumors and liquid tumors.

Fusion proteins

The present invention provides a novel fusion protein comprising (i) an antigen-binding fragment derived from an anti-PD-1 antibody and/or an anti-PD-L1 antibody; (ii) an immunoglobulin Fc domain; and (iii) CD80 extracellular domain (ECD).

In some embodiments, said (i), (ii) and/or (iii) are operably linked, optionally via a peptide linker.

In some embodiments, the fusion protein of the invention is a heterotetrameric glycoprotein consisting of a disulfide-bonded first subunit of the fusion protein and a second subunit of the fusion protein.

The fusion protein of the invention blocks the immune checkpoint PD-1/PD-L1 signaling pathway and activates T cells. The fusion protein blocks the immune checkpoint PD-1 pathway which is a signaling pathway mediated by the binding of PD-1 to its ligand. The PD-L1 pathway blocked by the fusion protein is a signal transduction pathway mediated by the binding of PD-L1 and a receptor thereof. The activation of T cells by the fusion protein is realized by blocking an immunosuppressive PD-1/PD-L1 channel and through the positive regulation effect of CD80ECD on the T cells.

In some embodiments, the fusion of the inventionThe synthetic protein is represented by 10^-8M or less, e.g. at 10^-9M to 10^-12Dissociation constant (K) of M_D) Binds to PD-1 or PD-L1; and is numbered 10^-8M or less, e.g. at 10^-9M to 10^-12Dissociation constant (K) of M_D) Specifically binds to the CD28 molecule and has the functions of,

antigen-binding fragments derived from anti-PD-1 antibodies and/or anti-PD-L1 antibodies

The antigen-binding fragment derived from the anti-PD-1 antibody and/or the anti-PD-L1 antibody contained in the fusion protein of the invention is capable of specifically binding to PD-1 and/or PD-L1; or competes with intact anti-PD-1 antibody and/or anti-PD-L1 antibody for binding to PD-1 and/or PD-L1. Such antigen binding fragments include, but are not limited to, Fab ', F (ab')₂Fv, single-chain Fv.

The inclusion of an antigen-binding fragment derived from an anti-PD-1 antibody and/or an anti-PD-L1 antibody in a fusion protein of the invention enables the fusion protein of the invention to be used with high affinity, for example at 10^-8M or less, preferably at 10^-9M to 10^-12K of M_DSpecifically binds to PD-1 and/or PD-L1 and thereby blocks the signaling pathway mediated by PD-1 binding to ligand PD-L1/PD-L2 and/or blocks the signaling pathway mediated by PD-L1 binding to receptor PD-1/CD80 (B7-1).

Examples of heavy chain variable regions (VH) and light chain variable regions (VL) in the antigen-binding fragment of the anti-PD-1 antibody comprised in the fusion protein of the invention are provided herein in table 1A below. In addition, examples of heavy chain variable regions (VH) and light chain variable regions (VL) in the antigen-binding fragment of the anti-PD-L1 antibody included in the fusion protein of the present invention are provided herein in table 1B below. In some embodiments, the antigen-binding fragment in the fusion protein of the invention comprises a sequence that is substantially identical to an amino acid sequence set forth in table 1A and/or table 1B, e.g., a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a heavy chain variable region sequence/light chain variable region sequence set forth in table 1A and/or table 1B.

TABLE 1A. examples of sequences for heavy and light chain variable regions in antigen-binding fragments of anti-PD-1 antibodies

TABLE 1B examples of heavy and light chain variable region sequences in antigen-binding fragments of anti-PD-L1 antibodies

In one embodiment, the antigen binding fragment in the fusion protein of the invention comprises a sequence selected from the group consisting of SEQ ID NOs: 1/2, 3/4, 5/6, 7/8, 9/10, 11/12, 13/14, 15/16, 17/18, 19/20, 21/22, 23/24, and 25/26, and all 6 heavy chain Complementarity Determining Regions (CDRs) and light chain CDRs contained in the heavy chain variable region sequence/light chain variable region sequence. In one embodiment, the antigen binding fragment in the fusion protein of the invention comprises a sequence selected from the group consisting of SEQ ID NOs: 27/28, 29/30, and 31/32, and all 6 heavy and light chain CDRs contained in the heavy/light chain variable region sequences. Methods and techniques for identifying CDRs in the amino acid sequences of heavy chain variable regions and light chain variable regions are known in the art and can be used to identify CDRs in the amino acid sequences of particular heavy chain variable regions and/or light chain variable regions disclosed herein. Exemplary well-known techniques that can be used to identify CDR boundaries include, for example, Kabat, Chothia, and AbM. See, e.g., Kabat, Sequences of Proteins of Immunological Interest, National Institutes of Health, Bethesda, Md. (1991); Al-Lazikani et Al, Standard transformations for the microbiological structures, J.mol.biol.273: 927-; and Martin AC et al, modular antibody hypervariable loops: a combined algorithms, Proc. Natl. Acad. Sci. USA 86: 9268. 9272 (1989).

The anti-PD-1 antibody or anti-PD-L1 antibody that is the source of the antigen-binding fragment in the fusion protein of the present invention may be classified into a kappa type or a lambda type, preferably a kappa type, based on the amino acid sequence of the light chain constant region thereof.

Examples of amino acid sequences of the anti-PD-1 antibody light chain constant regions are provided herein in table 2 below.

TABLE 2 examples of light chain constant region sequences of anti-PD 1 antibodies

The anti-PD-1 antibody or anti-PD-L1 antibody derived as an antigen-binding fragment in the fusion protein of the invention is preferably an antibody of the IgG class, in particular IgG, based on the amino acid sequence of its heavy chain constant region₁Subclass, IgG₂Subclass, IgG₄Subclass antibody, more particularly IgG₄Subclass antibody. Preferably, the IgG is₄Subclass anti-PD-1 antibody or anti-PD-L1 antibody comprises an amino acid substitution at position S228 in the Fc region that prevents the occurrence of an arm exchange, in particular the amino acid substitution S228P.

Examples of amino acid sequences of the anti-PD-1 antibody heavy chain constant region are provided herein in table 3 below.

TABLE 3 examples of heavy chain constant region sequences for anti-PD 1 antibodies

-immunoglobulin Fc domain

The "immunoglobulin Fc domain" in the fusion protein of the present invention comprises all amino acid residues of a naturally occurring immunoglobulin Fc domain or comprises a portion of amino acid residues of a naturally occurring immunoglobulin Fc domain. Immunoglobulin Fc domains provide advantageous pharmacokinetic properties for the fusion proteins of the invention, including but not limited to long serum half-life. In addition, immunoglobulin Fc domains also make it possible to purify the fusion proteins of the invention by, for example, protein a affinity chromatography.

Immunoglobulin Fc domains are typically dimeric molecules, which may be produced by papain digestion or trypsin digestion of a complete (full-length) immunoglobulin or may be produced recombinantly, comprising a CH2 domain, a CH3 domain, and optionally a CH4 domain.

In one embodiment, the IgG Fc region comprises an IgG CH2 domain and an IgG CH3 domain. Preferably, the immunoglobulin Fc domain has the amino acid sequence of SEQ ID NO: 38-40 or an amino acid sequence having an amino acid sequence substantially identical to SEQ ID NO: 38-40, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity.

Table 4 examples of immunoglobulin Fc domain amino acid sequences in fusion proteins

In addition to the amino acid sequence as set forth in SEQ ID NO: 38-40, the IgG Fc region can further comprise a sequence other than the sequences defined for SEQ ID NOs: 38-40, such as the peptide sequence obtained after additional sequence modifications to SEQ ID NO: 38-40 by one or more amino acid substitutions, deletions or derivations. In one embodiment, the IgG Fc region comprises an amino acid substitution at position S228 that prevents the occurrence of an arm exchange (arm-exchange), in particular amino acid substitution S228P.

Extracellular domain (ECD) of CD80

The "extracellular domain (ECD) of CD 80" in the fusion protein of the invention comprises all or a portion of the amino acid residues of a naturally occurring CD80 ECD. In some embodiments, the CD80ECD comprises a CD80IgV, preferably the CD80ECD comprises a human CD80IgV, more preferably the CD80ECD has the amino acid sequence of SEQ ID NO: 41 or 42.

TABLE 5 examples of CD80ECD amino acid sequences in fusion proteins

In addition to the amino acid sequence as set forth in SEQ ID NO: 41 and 42, the CD80ECD may comprise a sequence other than the sequences defined in SEQ ID NOs: 41 and 42, for example, a peptide sequence obtained by additional sequence modification of SEQ ID NO: 41 and 42, as long as they have substantially the same activity or function as the unmodified peptide. The modified peptide will retain the activity or function associated with the unmodified peptide. The modified peptide typically has an amino acid sequence that is substantially homologous to the amino acid sequence of the unmodified sequence, e.g., to the amino acid sequence of SEQ ID NO: 41 or 42, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity.

-peptide linker

The fusion protein of the present invention comprises (i) an antigen-binding fragment derived from an anti-PD-1 antibody and/or an anti-PD-L1 antibody; (ii) an immunoglobulin Fc domain; and (iii) the CD80ECD is optionally operably linked to a "peptide linker" which is a peptide of one or more amino acids, typically about 2-20 amino acids. Peptide linkers are known in the art or described herein.

In some embodiments, the peptide linker comprises at least 5 amino acids, preferably comprises a peptide linker selected from the group consisting of AKTTPKLEEGEFSEAR (SEQ ID NO: 43); AKTTPKLEEGEFSEARV (SEQ ID NO: 44); AKTTPKLGG (SEQ ID NO: 45); SAKTTPKLGG (SEQ ID NO: 46); SAKTTP (SEQ ID NO: 47); RADAAP (SEQ ID NO: 48); RADAAPTVS (SEQ ID NO: 49); RADAAAAGGPGS (SEQ ID NO: 50); RADAAAAA (SEQ ID NO: 51); SAKTTPKLEEGEFSEARV (SEQ ID NO: 52); ADAAP (SEQ ID NO: 53); DAAPTVSIFPP (SEQ ID NO: 54); TVAAP (SEQ ID NO: 55); TVAAPSVFIFPP (SEQ ID NO: 56); QPKAAP (SEQ ID NO: 57); QPKAAPSVTLFPP (SEQ ID NO: 58); AKTTPP (SEQ ID NO: 59); AKTTPPSVTPLAP (SEQ ID NO: 60); AKTTAP (SEQ ID NO: 61); AKTTAPSVYPLAP (SEQ ID NO: 62); ASTKGP (SEQ ID NO: 63); ASTKGPSVFPLAP (SEQ ID NO: 64); GGGGSGGGGSGGGGS (SEQ ID NO: 65); GENKVEYAPALMALS (SEQ ID NO: 66); GPAKELTPLKEAKVS (SEQ ID NO: 67); GHEAAAVMQVQYPAS (SEQ ID NO: 68); GGGGSGGGGSGGGGSA (SEQ ID NO: 69); GQGTKVEIKRGGSGGGGSG (SEQ ID NO: 70) and GQGTLVTVSSGGGGSGGGGS (SEQ ID NO: 71).

-fusion proteins

Provided herein are antigen-binding fragments comprising, in any order, (i) an antigen-binding fragment derived from an anti-PD-1 antibody and/or an anti-PD-L1 antibody; (ii) an immunoglobulin Fc domain; and (iii) a CD80ECD, including but not limited to fusion proteins from N-terminus to C-terminus in the order of (i), (ii), and (iii); (iii) the order of (i), (ii) and (iii); or the order of (iii), (ii) and (i) are operably linked.

In one embodiment, the fusion protein comprises, from N-terminus to C-terminus, an anti-PD-1 antibody, an anti-PD-L1 antibody, or an anti-PD-1 and PD-L1 bispecific antibody; and a CD80ECD operably linked C-terminal to each of the two heavy chains of the antibody.

In another embodiment, the fusion protein comprises an anti-PD-1 antibody, an anti-PD-L1 antibody, or an anti-PD-1 and PD-L1 bispecific antibody; a CD80ECD operably linked at the N-terminus of each of the two heavy chains of the antibody; and a CD80ECD operably linked N-terminal to each of the two light chains of the antibody.

In yet another embodiment, the fusion protein comprises, from N-terminus to C-terminus, CD80 ECD; an immunoglobulin Fc domain in dimeric form operatively linked at the C-terminus of the CD80 ECD; and an antigen-binding fragment derived from an anti-PD-1 antibody and/or an anti-PD-L1 antibody operatively linked at the C-terminus of the immunoglobulin Fc domain in dimeric form.

Production and purification of fusion proteins of the invention

The fusion protein of the present invention can be obtained, for example, by solid-state peptide synthesis (e.g., Merrifield solid phase synthesis) or recombinant production. For recombinant production, the polynucleotide encoding the first subunit of the fusion protein and/or the polynucleotide encoding the second subunit of the fusion protein is isolated and inserted into one or more vectors for further cloning and/or expression in a host cell. The polynucleotides can be readily isolated and sequenced using conventional methods. In one embodiment, a vector, preferably an expression vector, comprising one or more polynucleotides of the invention is provided.

Methods well known to those skilled in the art can be used to construct expression vectors. Expression vectors include, but are not limited to, viruses, plasmids, cosmids, lambda phages, or Yeast Artificial Chromosomes (YACs). In a preferred embodiment, a glutamine synthetase high expression vector having a dual expression cassette is used.

Once an expression vector comprising one or more polynucleotides of the invention has been prepared for expression, the expression vector may be transfected or introduced into a suitable host cell. A variety of techniques can be used to achieve this, for example, protoplast fusion, calcium phosphate precipitation, electroporation, retroviral transduction, viral transfection, gene gun, liposome-based transfection, or other conventional techniques.

In one embodiment, a host cell comprising one or more polynucleotides of the invention is provided. In some embodiments, host cells comprising the expression vectors of the invention are provided. As used herein, the term "host cell" refers to any kind of cellular system that can be engineered to produce a fusion protein of the invention. Host cells suitable for replication and to support expression of the fusion proteins of the invention are well known in the art. Such cells can be transfected or transduced with a particular expression vector, as desired, and a large number of vector-containing cells can be grown for seeding a large-scale fermentor to obtain sufficient quantities of the fusion protein of the invention for clinical use. Suitable host cells include prokaryotic microorganisms such as E.coli, eukaryotic microorganisms such as filamentous fungi or yeast, or various eukaryotic cells such as Chinese hamster ovary Cells (CHO), insect cells, and the like. Mammalian cell lines suitable for suspension culture may be used. Examples of useful mammalian host cell lines include SV40 transformed monkey kidney CV1 line (COS-7); human embryonic kidney lines (HEK 293 or 293F cells), baby hamster kidney cells (BHK), monkey kidney cells (CV1), Vero monkey kidney cells (VERO-76), human cervical cancer cells (HELA), canine kidney cells (MDCK), Bufarro rat liver cells (BRL 3A), human lung cells (W138), human liver cells (Hep G2), CHO cells, NSO cells, myeloma cell lines such as YO, NS0, P3X63, and Sp2/0, and the like. For a review of mammalian host cell lines suitable for protein production see, e.g., Yazaki and Wu, Methods in Molecular Biology, Vol.248 (edited by B.K.C.Lo, Humana Press, Totowa, NJ), pp.255-268 (2003). In a preferred embodiment, the host cell is a CHO, HEK293 or NSO cell.

Standard techniques for expressing foreign genes in these host cell systems are known in the art. In one embodiment, a method of producing a fusion protein of the invention is provided, wherein the method comprises culturing a host cell as provided herein comprising a polynucleotide encoding the fusion protein under conditions suitable for expression of the fusion protein, and recovering the fusion protein from the host cell (or host cell culture medium).

The fusion protein prepared as described herein can be purified by known prior art techniques such as high performance liquid chromatography, ion exchange chromatography, gel electrophoresis, affinity chromatography, size exclusion chromatography, and the like. The actual conditions used to purify a particular protein also depend on factors such as net charge, hydrophobicity, hydrophilicity, and the like, and will be apparent to those skilled in the art.

The purity of the fusion protein of the present invention can be determined by any of a variety of well-known analytical methods, including gel electrophoresis, high performance liquid chromatography, and the like. The physical/chemical properties and/or biological activities of the fusion proteins provided herein can be identified, screened or characterized by a variety of assays known in the art.

Pharmaceutical compositions and kits

In another aspect, the invention provides a composition, e.g., a pharmaceutical composition, comprising a fusion protein described herein formulated with a pharmaceutically acceptable carrier. As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, isotonic and absorption delaying agents, and the like that are physiologically compatible. The pharmaceutical compositions of the invention are suitable for intravenous, intramuscular, subcutaneous, parenteral, rectal, spinal or epidermal administration (e.g., by injection or infusion).

The compositions of the present invention may be in a variety of forms. Such forms include, for example, liquid, semi-solid, and solid dosage forms, such as liquid solutions (e.g., injectable and infusible solutions), dispersions or suspensions, liposomal formulations, and suppositories. The preferred form depends on the intended mode of administration and therapeutic use. The generally preferred compositions are in the form of injectable solutions or infusible solutions. Preferred modes of administration are parenteral (e.g., intravenous, subcutaneous, intraperitoneal (i.p.), intramuscular) injection. In a preferred embodiment, the fusion protein is administered by intravenous infusion or injection. In another preferred embodiment, the fusion protein is administered by intramuscular, intraperitoneal or subcutaneous injection.

The phrases "parenteral administration" and "administered parenterally" as used herein mean modes of administration other than enteral and topical administration, typically by injection, and include, but are not limited to, intravenous, intramuscular, intraarterial, intradermal, intraperitoneal, transtracheal, subcutaneous injection, and infusion.

The therapeutic compositions should generally be sterile and stable under the conditions of manufacture and storage. The compositions may be formulated as solutions, microemulsions, dispersions, liposomes, or lyophilized forms. Sterile injectable solutions can be prepared by incorporating the active compound (i.e., the fusion protein) in the required amount in an appropriate solvent, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and other ingredients. Coating agents such as lecithin and the like may be used. In the case of dispersions, the proper fluidity of solutions can be maintained by the use of surfactants. Prolonged absorption of the injectable compositions can be brought about by including in the composition an absorption delaying substance, for example, monostearate salts and gelatin.

In certain embodiments, a fusion protein of the invention can be administered orally, e.g., with an inert diluent or an edible carrier. The fusion proteins of the invention may also be enclosed in hard or soft shell gelatin capsules, compressed into tablets, or incorporated directly into the diet of a subject. For oral therapeutic administration, the compounds may be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, troches (troche), capsules, elixirs, suspensions, syrups, wafers (wafers), and the like. In order to administer the fusion protein of the present invention by a non-parenteral administration method, it may be necessary to coat the fusion protein with a material that prevents its inactivation or to co-administer it with such a material. Therapeutic compositions may also be administered using medical devices known in the art.

The pharmaceutical composition of the invention may comprise a "therapeutically effective amount" or a "prophylactically effective amount" of the fusion protein of the invention. "therapeutically effective amount" means an amount effective, at dosages and for periods of time as required, to achieve the desired therapeutic result. The therapeutically effective amount may vary depending on factors such as the disease state, age, sex, and weight of the individual. A therapeutically effective amount is any amount that has no toxic or deleterious effect than a therapeutically beneficial effect. A "therapeutically effective amount" preferably inhibits a measurable parameter (e.g., tumor growth rate) by at least about 20%, more preferably by at least about 40%, even more preferably by at least about 60%, and still more preferably by at least about 80%, relative to an untreated subject. The ability of the fusion proteins of the invention to inhibit a measurable parameter (e.g., tumor volume) can be evaluated in an animal model system predictive of efficacy in human tumors.

A "prophylactically effective amount" refers to an amount effective, at dosages and for periods of time as required, to achieve the desired prophylactic result. Typically, a prophylactically effective amount is less than a therapeutically effective amount since a prophylactic dose is used in a subject prior to or at an earlier stage of disease.

Kits comprising the fusion proteins described herein are also within the scope of the invention. The kit may comprise one or more further elements, including for example: instructions for use; other reagents, such as labels or reagents for conjugation; a pharmaceutically acceptable carrier; and a device or other material for administration to a subject.

Use of fusion proteins

The fusion proteins disclosed herein have in vitro and in vivo diagnostic uses as well as therapeutic and prophylactic uses. For example, these molecules can be administered to cultured cells in vitro or ex vivo or to a subject, e.g., a human subject, to treat, prevent and/or diagnose a variety of diseases associated with the activation of the PD-1/PD-L1 signaling pathway and the inhibition of T cell function, e.g., cancer.

In one aspect, the invention provides diagnostic methods for detecting the presence of PD-1, PD-L1, CD28, or CTLA-4 in a biological sample, such as serum, semen, or urine or a tissue biopsy (e.g., from a hyperproliferative or cancerous lesion), in vitro or in vivo. The diagnostic method comprises the following steps: (i) contacting a sample (and optionally a control sample) with a fusion protein as described herein or administering the fusion protein to a subject under conditions that allow an interaction to occur and (ii) detecting the formation of a complex between the fusion protein and the sample (and optionally the control sample). The formation of a complex indicates the presence of PD-1, PD-L1, CD28, or CTLA-4, and may indicate the applicability or need of treatment and/or prevention as described herein.

In some embodiments, PD-1 or PD-L1, CD28, CTLA-4 are detected prior to treatment, e.g., prior to initiation of treatment or prior to some treatment following a treatment interval. Detection methods that may be used include immunohistochemistry, immunocytochemistry, FACS, ELISA assays, PCR-techniques (e.g., RT-PCR) or in vivo imaging techniques. In general, fusion proteins used in vivo and in vitro detection methods are labeled, directly or indirectly, with a detectable substance to facilitate detection of bound or unbound conjugate. Suitable detectable substances include a variety of biologically active enzymes, prosthetic groups, fluorescent materials, luminescent materials, paramagnetic (e.g., nmr active) materials, and radioactive materials.

In some embodiments, the level and/or distribution of PD-1, PD-L1, CD28, or CTLA-4 is determined in vivo, e.g., a fusion protein of the invention that is detectably labeled is determined in a non-invasive manner (e.g., by detection using a suitable imaging technique (e.g., Positron Emission Tomography (PET) scanning), hi one embodiment, e.g., by detection of a protein labeled with a PET agent (e.g.,¹⁸F-Fluorodeoxyglucose (FDG)) detectably labeled fusion proteins of the invention, the level and/or distribution of PD-1, PD-L1, CD28, or CTLA-4 is determined in vivo.

In one embodiment, the invention provides a diagnostic kit comprising a fusion protein as described herein and instructions for use.

In another aspect, the invention relates to the use of the fusion protein for treating or preventing a disease in which enhanced T cell activation and immune response are desired in a subject, thereby inhibiting or reducing the growth or appearance, metastasis or recurrence of an associated disease, such as a cancerous tumor. The fusion protein may be used alone to inhibit the growth of, or prevent the appearance of, cancerous tumors. Alternatively, the fusion protein may be administered in combination with other cancer therapeutic/prophylactic agents. When the fusion protein of the invention is administered in combination with one or more other drugs, such combination may be administered in any order or simultaneously.

Accordingly, in one embodiment, the present invention provides a method of inhibiting tumor cell growth in a subject, the method comprising administering to the subject a therapeutically effective amount of a fusion protein described herein. In another embodiment, the invention provides a method of preventing the appearance or metastasis or recurrence of tumor cells in a subject, comprising administering to the subject a prophylactically effective amount of a fusion protein described herein.

In some embodiments, cancers treated and/or prevented with the fusion protein include, but are not limited to, solid tumors, hematological cancers (e.g., leukemia, lymphoma, myeloma, e.g., multiple myeloma), and metastatic lesions. In one embodiment, the cancer is a solid tumor. Examples of solid tumors include malignancies, e.g., sarcomas and carcinomas of the various organ systems, such as those that affect the lung, breast, ovary, lymphoid, gastrointestinal (e.g., colon), anal, genital, and genitourinary tracts (e.g., kidney, bladder epithelium, bladder cells, prostate), pharynx, CNS (e.g., brain, neural, or glial cells), head and neck, skin (e.g., melanoma), nasopharynx (e.g., differentiated or undifferentiated metastatic or locally recurrent nasopharyngeal carcinoma), and pancreas, and adenocarcinomas, including malignancies, such as colon, rectal, renal cell, liver, non-small cell lung, small intestine, and esophageal cancers. The cancer may be in an early, intermediate or advanced stage or a metastatic cancer.

In some embodiments, the cancer is selected from melanoma, breast cancer, colon cancer, esophageal cancer, gastrointestinal stromal tumors (GIST), renal cancer (e.g., renal cell carcinoma), liver cancer, non-small cell lung cancer (NSCLC), ovarian cancer, pancreatic cancer, prostate cancer, head and neck tumors, gastric cancer, hematologic malignancies (e.g., lymphoma).

The following examples are described to aid in the understanding of the present invention. The examples are not intended to, and should not be construed as, limiting the scope of the invention in any way.

Examples

Example 1 construction of a Glutamine synthetase high expression vector containing a Gene of interest

(1) Synthesis of control anti-PD 1 antibody BY18.1 coding nucleotide and construction of expression vector

The following nucleotide sequence suitable for expression in chinese hamster ovary cancer Cells (CHO) was optimized based on the amino acid sequence data of nivolumab No. 9623 in the International Nproprietary Name (INN) database, and the synthesis of the nucleotide sequence was entrusted to shanghai jieli bioengineering limited. The anti-PD 1 antibody produced upon expression of the nucleotide sequence is denoted herein as antibody BY 18.1.

Light chain (BY18.1L) nucleotide sequence of anti-PD 1 antibody BY18.1 (SEQ ID NO: 72):

CTCGAGGCCACCATGGAGACCGACACACTCCTCCTGTGGGTGCTGCTGCTGTGGGTGCCTGGCTCCACTGGCGAGATTGTGCTGACACAGTCCCCCGCTACTCTGAGCCTGAGCCCTGGCGAGAGGGCTACACTGTCTTGCAGAGCTTCTCAGTCCGTGTCTTCTTACCTCGCTTGGTATCAGCAGAAGCCCGGCCAGGCTCCAAGACTGCTGATCTATGACGCTTCTAACCGCGCTACAGGCATTCCTGCTAGGTTCAGCGGCAGCGGCTCTGGCACCGACTTCACACTCACAATTAGCTCTCTTGAACCTGAGGACTTCGCCGTGTACTACTGCCAGCAGTCTAGCAACTGGCCTAGAACATTCGGCCAGGGCACTAAGGTGGAGATTAAGAGAACCGTGGCCGCCCCCAGCGTGTTCATCTTCCCTCCCAGCGACGAGCAGCTGAAGTCTGGCACCGCCAGCGTGGTGTGCCTGCTGAACAACTTCTACCCCCGCGAGGCCAAGGTGCAGTGGAAGGTGGACAACGCCCTGCAGAGCGGCAACAGCCAGGAGAGCGTGACCGAGCAGGACTCCAAGGACAGCACCTACAGCCTGAGCAGCACCCTGACCCTGAGCAAGGCCGACTACGAGAAGCACAAGGTGTACGCCTGCGAGGTGACCCACCAGGGACTGTCTAGCCCCGTGACCAAGAGCTTCAACCGGGGCGAGTGCTAAGAATTC

light chain (BY18.1L) amino acid sequence of anti-PD 1 antibody BY18.1 (SEQ ID NO: 73):

METDTLLLWVLLLWVPGSTGEIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQSSNWPRTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

heavy chain (BY18.1H) nucleotide sequence of anti-PD 1 antibody BY18.1 (SEQ ID NO: 74):

TCTAGAGCCACCATGGAGACCGACACCCTGCTGCTGTGGGTGCTGCTCCTGTGGGTGCCTGGCTCCACAGGCCAGGTGCAGCTCGTGGAGTCCGGCGGCGGCGTGGTGCAGCCCGGCAGATCCCTCAGACTGGACTGCAAGGCATCCGGCATTACATTCTCTAACTCTGGAATGCACTGGGTGAGACAGGCTCCTGGCAAGGGCCTGGAATGGGTGGCCGTGATTTGGTACGACGGCTCTAAGAGATACTACGCTGACTCCGTGAAGGGCCGGTTCACAATTAGCAGAGACAACTCCAAGAACACTCTGTTCCTCCAGATGAACAGCCTGAGAGCCGAGGACACCGCTGTGTACTACTGCGCCACCAACGACGACTACTGGGGCCAGGGCACCCTCGTGACAGTGTCTTCCGCCTCCACCAAGGGCCCTTCCGTGTTCCCTCTGGCCCCTTGCTCCCGCTCCACCTCCGAGTCCACCGCCGCCCTGGGCTGCCTGGTGAAGGACTACTTCCCTGAGCCTGTGACCGTGTCCTGGAACTCCGGCGCCCTGACCTCCGGCGTGCACACCTTCCCTGCCGTGCTGCAGTCCTCCGGCCTGTACTCCCTGTCCTCCGTGGTGACCGTGCCTTCCTCCTCCCTGGGCACCAAGACCTACACCTGCAACGTGGACCACAAGCCTTCCAACACCAAGGTGGACAAGCGCGTGGAGTCCAAGTACGGCCCTCCTTGCCCTCCTTGCCCTGCCCCTGAGTTCCTGGGCGGCCCTTCCGTGTTCCTGTTCCCTCCTAAGCCTAAGGACACCCTGATGATCTCCCGCACCCCTGAGGTGACCTGCGTGGTGGTGGACGTGTCCCAGGAGGACCCTGAGGTGCAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCACAACGCCAAGACCAAGCCTCGCGAGGAGCAGTTCAACTCCACCTACCGCGTGGTGTCCGTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTGTCCAACAAGGGCCTGCCTTCCTCCATCGAGAAGACCATCTCCAAGGCCAAGGGCCAGCCTCGCGAGCCTCAGGTGTACACCCTGCCTCCTTCCCAGGAGGAGATGACCAAGAACCAGGTGTCCCTGACCTGCCTGGTGAAGGGCTTCTACCCTTCCGACATCGCCGTGGAGTGGGAGTCCAACGGCCAGCCTGAGAACAACTACAAGACCACCCCTCCTGTGCTGGACTCCGACGGCTCCTTCTTCCTGTACTCCCGCCTGACCGTGGACAAGTCCCGCTGGCAGGAGGGCAACGTGTTCTCCTGCTCCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGTCCCTGTCCCTGGGCAAGTAA GTCGAC

heavy chain (BY18.1H) amino acid sequence of anti-PD 1 antibody BY18.1 (SEQ ID NO: 75):METDTLLLWVLLL WVPGSTGQVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKGLEWVAVIWYDGSKRYYADSVKGRFTISRDNSKNTLFLQMNSLRAEDTAVYYCATNDDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

wherein the lower scribed line part "METDTLLLWVLLLWVPGSTG"is a signal peptide sequence.

The BY18.1L-encoding nucleotide sequence and the BY18.1H-encoding nucleotide sequence were synthesized by Shanghai Czeri bioengineering, Inc. Separately double-digesting BY18.1L encoding nucleotides with XhoI-EcoRI, double-digesting glutamine synthetase high-efficiency expression vectors with double expression cassettes (patent grant No: CN104195173B, obtained from Beijing Biyan Biotechnology Co., Ltd.) with XhoI-EcoRI, and then connecting BY18.1L encoding nucleotides double-digested with XhoI-EcoRI to the glutamine synthetase high-efficiency expression vectors with double expression cassettes double-digested with XhoI-EcoRI by ligase to obtain the glutamine synthetase high-efficiency expression vectors with double expression cassettes into which BY18.1L encoding nucleotides have been introduced; then, BY18.1H encoding nucleotides were digested simultaneously with XbaI-SalI, the glutamine synthetase high-efficiency expression vector with double expression cassettes into which BY18.1L encoding nucleotides were introduced was digested simultaneously with XbaI-SalI, the XbaI-SalI digested BY18.1H encoding nucleotides were ligated to the XbaI-SalI digested glutamine synthetase high-efficiency expression vector with double expression cassettes into which BY18.1L encoding nucleotides were introduced, the glutamine synthetase high-efficiency expression vector with double expression cassettes into which BY18.1L encoding nucleotides and BY18.1H encoding nucleotides were introduced was obtained, and the sequences were verified to be correct for expression, thereby obtaining anti-PD 1 antibody BY 18.1.

Alternatively, the antibody BY18.1 can be obtained BY ligating the coding nucleotide BY18.1L to a glutamine synthetase high-efficiency expression vector having a double expression cassette into which the BY18.1H coding nucleotide has been introduced.

(2) Synthesis of encoding nucleotide of fusion protein containing anti-PD-1 antibody and CD80 extracellular domain and construction of expression vector

The amino acid sequence of the heavy and light chain variable regions of the anti-PD-1 antibody in table 1A, the light chain constant region sequence of the antibody in table 2, the heavy chain constant region sequence of the antibody in table 3, the sequence of the extracellular domain of CD80 in table 5, and the amino acid sequence of SEQ ID NO: 43-71, optimized to a nucleotide sequence suitable for expression in Chinese hamster ovary cancer Cells (CHO), and entrusted Shanghai Czeri bioengineering, Inc. to synthesize a polynucleotide sequence as shown by even-numbered SEQ ID NO 76-95.

Nucleotide sequence of first subunit (BY31.2L) of fusion protein BY31.2 (kappa, IgG4) (SEQ ID NO: 76): CTCGAGGCCACCATGGAGACCGACACACTCCTCCTGTGGGTGCTGCTGCTGTGGGTGCCTGGCTCCACTGGCGAGATTGTGCTGACACAGTCCCCCGCTACTCTGAGCCTGAGCCCTGGCGAGAGGGCTACACTGTCTTGCAGAGCTTCTCAGTCCGTGTCTTCTTACCTCGCTTGGTATCAGCAGAAGCCCGGCCAGGCTCCAAGACTGCTGATCTATGACGCTTCTAACCGCGCTACAGGCATTCCTGCTAGGTTCAGCGGCAGCGGCTCTGGCACCGACTTCACACTCACAATTAGCTCTCTTGAACCTGAGGACTTCGCCGTGTACTACTGCCAGCAGTCTAGCAACTGGCCTAGAACATTCGGCCAGGGCACTAAGGTGGAGATTAAGAGAACCGTGGCCGCCCCCAGCGTGTTCATCTTCCCTCCCAGCGACGAGCAGCTGAAGTCTGGCACCGCCAGCGTGGTGTGCCTGCTGAACAACTTCTACCCCCGCGAGGCCAAGGTGCAGTGGAAGGTGGACAACGCCCTGCAGAGCGGCAACAGCCAGGAGAGCGTGACCGAGCAGGACTCCAAGGACAGCACCTACAGCCTGAGCAGCACCCTGACCCTGAGCAAGGCCGACTACGAGAAGCACAAGGTGTACGCCTGCGAGGTGACCCACCAGGGACTGTCTAGCCCCGTGACCAAGAGCTTCAACCGGGGCGAGTGCTAAGAATTC

The amino acid sequence of the first subunit (BY31.2L) of fusion protein BY31.2 (kappa, IgG4) (SEQ ID NO: 77):

METDTLLLWVLLLWVPGSTGEIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQSSNWPRTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

the nucleotide sequence of the second subunit (BY31.2H) of fusion protein BY31.2 (kappa, IgG4) (SEQ ID NO: 78):

TCTAGAGCCACCATGGAGACCGACACCCTGCTGCTGTGGGTGCTGCTCCTGTGGGTGCCTGGCTCCACAGGCCAGGTGCAGCTCGTGGAGTCCGGCGGCGGCGTGGTGCAGCCCGGCAGATCCCTCAGACTGGACTGCAAGGCATCCGGCATTACATTCTCTAACTCTGGAATGCACTGGGTGAGACAGGCTCCTGGCAAGGGCCTGGAATGGGTGGCCGTGATTTGGTACGACGGCTCTAAGAGATACTACGCTGACTCCGTGAAGGGCCGGTTCACAATTAGCAGAGACAACTCCAAGAACACTCTGTTCCTCCAGATGAACAGCCTGAGAGCCGAGGACACCGCTGTGTACTACTGCGCCACCAACGACGACTACTGGGGCCAGGGCACCCTCGTGACAGTGTCTTCCGCCTCCACCAAGGGCCCTTCCGTGTTCCCTCTGGCCCCTTGCTCCCGCTCCACCTCCGAGTCCACCGCCGCCCTGGGCTGCCTGGTGAAGGACTACTTCCCTGAGCCTGTGACCGTGTCCTGGAACTCCGGCGCCCTGACCTCCGGCGTGCACACCTTCCCTGCCGTGCTGCAGTCCTCCGGCCTGTACTCCCTGTCCTCCGTGGTGACCGTGCCTTCCTCCTCCCTGGGCACCAAGACCTACACCTGCAACGTGGACCACAAGCCTTCCAACACCAAGGTGGACAAGCGCGTGGAGTCCAAGTACGGCCCTCCTTGCCCTCCTTGCCCTGCCCCTGAGTTCCTGGGCGGCCCTTCCGTGTTCCTGTTCCCTCCTAAGCCTAAGGACACCCTGATGATCTCCCGCACCCCTGAGGTGACCTGCGTGGTGGTGGACGTGTCCCAGGAGGACCCTGAGGTGCAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCACAACGCCAAGACCAAGCCTCGCGAGGAGCAGTTCAACTCCACCTACCGCGTGGTGTCCGTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTGTCCAACAAGGGCCTGCCTTCCTCCATCGAGAAGACCATCTCCAAGGCCAAGGGCCAGCCTCGCGAGCCTCAGGTGTACACCCTGCCTCCTTCCCAGGAGGAGATGACCAAGAACCAGGTGTCCCTGACCTGCCTGGTGAAGGGCTTCTACCCTTCCGACATCGCCGTGGAGTGGGAGTCCAACGGCCAGCCTGAGAACAACTACAAGACCACCCCTCCTGTGCTGGACTCCGACGGCTCCTTCTTCCTGTACTCCCGCCTGACCGTGGACAAGTCCCGCTGGCAGGAGGGCAACGTGTTCTCCTGCTCCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGTCCCTGTCCCTGGGCGGCGGAGGATCTGGCGGCGGAGGCAGTGGAGGCGGCGGAAGTGTGATCCATGTAACAAAAGAAGTGAAGGAAGTGGCTACACTCTCTTGCGGCCACAACGTGTCCGTGGAGGAACTAGCTCAGACCCGGATCTATTGGCAGAAAGAAAAGAAGATGGTGCTGACCATGATGTCCGGCGACATGAACATTTGGCCAGAGTACAAGAACCGCACAATTTTCGACATTACAAACAACCTCTCTATTGTGATTCTGGCTCTCAGGCCTAGCGACGAGGGCACATACGAGTGCGTGGTGCTCAAGTACGAGAAGGACGCTTTCAAGCGGGAGCACCTCGCTGAGGTGACCCTGTCCGTGAAGGCCGACTTCCCTACTCCATCTTAAGTCGAC

the second subunit (BY31.2H) amino acid sequence of fusion protein BY31.2 (kappa, IgG4) (SEQ ID NO: 79):

METDTLLLWVLLLWVPGSTGQVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKGLEWVAVIWYDGSKRYYADSVKGRFTISRDNSKNTLFLQMNSLRAEDTAVYYCATNDDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGGGGSGGGGSGGGGSVIHVTKEVKEVATLSCGHNVSVEELAQTRIYWQKEKKMVLTMMSGDMNIWPEYKNRTIFDITNNLSIVILALRPSDEGTYECVVLKYEKDAFKREHLAEVTLSVKADFPTPS

nucleotide sequence of first subunit (BY31.3L) of fusion protein BY31.3 (kappa, IgG2) (SEQ ID NO: 80):

CTCGAGGCCACCATGGAGACCGACACACTCCTCCTGTGGGTGCTGCTGCTGTGGGTGCCTGGCTCCACTGGCGAGATCAAGCGGACCGTGGCCGCCCCATCCGTGTTCATTTTCCCACCTTCCGAGATTGTGCTGACACAGTCCCCCGCTACTCTGAGCCTGAGCCCTGGCGAGAGGGCTACACTGTCTTGCAGAGCTTCCAAGGGCGTGAGCACATCCGGCTACTCCTACCTCCACTGGTATCAGCAGAAGCCAGGCCAGGCCCCAAGACTGCTGATATACCTCGCTTCTTACTTAGAGTCTGGCGTGCCCGCTCGGTTCAGCGGCTCCGGCTCTGGCACCGACTTCACCCTGACAATTTCTAGCCTGGAGCCCGAGGACTTCGCCGTGTACTACTGCCAGCACTCTAGGGACCTGCCTCTCACATTCGGCGGCGGCACTAAGGTGGAGATTAAGAGAACCGTGGCCGCCCCCAGCGTGTTCATCTTCCCTCCCAGCGACGAGCAGCTGAAGTCTGGCACCGCCAGCGTGGTGTGCCTGCTGAACAACTTCTACCCCCGCGAGGCCAAGGTGCAGTGGAAGGTGGACAACGCCCTGCAGAGCGGCAACAGCCAGGAGAGCGTGACCGAGCAGGACTCCAAGGACAGCACCTACAGCCTGAGCAGCACCCTGACCCTGAGCAAGGCCGACTACGAGAAGCACAAGGTGTACGCCTGCGAGGTGACCCACCAGGGACTGTCTAGCCCCGTGACCAAGAGCTTCAACCGGGGCGAGTGCTAAGAATTC

the amino acid sequence of the first subunit (BY31.3L) of fusion protein BY31.3 (kappa, IgG2) (SEQ ID NO: 81):

METDTLLLWVLLLWVPGSTGEIVLTQSPATLSLSPGERATLSCRASKGVSTSGYSYLHWYQQKPGQAPRLLIYLASYLESGVPARFSGSGSGTDFTLTISSLEPEDFAVYYCQHSRDLPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

the nucleotide sequence of the second subunit (BY31.3H) of fusion protein BY31.3 (kappa, IgG2) (SEQ ID NO: 82):

TCTAGAGCCACCATGGAGACCGACACCCTGCTGCTGTGGGTGCTGCTCCTGTGGGTGCCTGGCTCCACAGGCCAGGTGCAGCTCGTGCAGTCTGGCGTGGAGGTGAAGAAGCCTGGCGCCTCTGTGAAGGTGTCTTGCAAGGCTTCCGGCTACACTTTCACTAACTACTACATGTACTGGGTGAGACAGGCTCCCGGCCAGGGCCTAGAGTGGATGGGCGGCATTAACCCTAGCAACGGCGGCACAAACTTCAACGAGAAGTTCAAGAACCGCGTGACCCTGACCACAGACTCTAGCACAACAACTGCTTACATGGAGCTGAAGTCTCTCCAGTTCGACGACACCGCTGTGTACTACTGCGCTCGGAGGGACTACAGATTCGACATGGGCTTCGACTACTGGGGCCAGGGCACCACTGTGACAGTGTCTACAGCCTCCACCAAGGGCCCTTCCGTGTTCCCTCTGGCCCCTTGCTCCCGCTCCACCTCCGAGTCCACCGCCGCCCTGGGCTGCCTGGTGAAGGACTACTTCCCTGAGCCTGTGACCGTGTCCTGGAACTCCGGCGCCCTGACCTCCGGCGTGCACACCTTCCCTGCCGTGCTGCAGTCCTCCGGCCTGTACTCCCTGTCCTCCGTGGTGACCGTGCCTTCCTCCAACTTCGGCACCCAGACATACACATGCAACGTGGACCACAAGCCTTCTAACACAAAGGTGGACAAGACCGTGGAGCGGAAGTGCTGCGTGGAGTGCCCACCTTGCCCCGCTCCTCCTGTGGCCGGCCCTTCTGTGTTCCTGTTCCCACCTAAGCCAAAGGACACACTCATGATCAGCAGAACCCCTGAGGTGACCTGCGTGGTGGTGGACGTGAGCCACGAGGACCCCGAGGTGCAGTTCAACTGGTATGTGGACGGCGTGGAGGTGCACAACGCTAAGACCAAGCCTAGAGAAGAACAGTTCAACAGCACATTCAGAGTGGTGTCCGTGCTCACCGTGGTGCACCAGGACTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTGTCCAACAAGGGCCTGCCAGCCCCTATCGAAAAAACAATCAGCAAGACCAAGGGCCAGCCTAGAGAGCCTCAGGTGTACACACTGCCTCCATCTCGGGAAGAAATGACAAAGAACCAGGTGTCCCTCACATGCCTCGTGAAGGGCTTCTACCCATCCGACATCGCTGTGGAGTGGGAGTCTAACGGCCAGCCCGAGAACAACTACAAGACCACCCCTCCTATGCTCGACTCCGACGGCTCTTTCTTCCTGTACTCTAAGCTGACCGTGGACAAGTCCAGATGGCAGCAGGGCAACGTGTTCTCTTGCAGCGTGATGCACGAGGCTCTCCACAACCACTACACCCAGAAGTCCCTGAGCCTGTCTCCAGGCGGCGGAGGATCTGGCGGCGGAGGCAGTGGAGGCGGCGGAAGCGTCATTCACGTCACTAAGGAGGTCAAGGAGGTCGCAACCCTCAGTTGCGGACACAACGTCAGCGTGGAGGAGCTTGCACAGACACGCATCTACTGGCAGAAGGAGAAGAAGATGGTGCTGACCATGATGTCCGGCGATATGAACATTTGGCCAGAGTACAAGAATCGGACCATCTTCGATATTACAAATAACCTGTCCATCGTGATCCTCGCTCTGCGCCCTAGCGACGAGGGAACATACGAGTGTGTGGTGCTGAAGTACGAGAAGGATGCATTTAAGCGCGAGCACCTGGCTGAGGTGACACTCTCCGTCAAGGCCGATTTTCCTACTCCTTCTATCTCCGACTTTGAGATTCCAACATCAAATATTAGGCGCATTATCTGTTCTACATCCGGCGGATTCCCAGAGCCCCACCTCTCTTGGTTGGAGAACGGCGAGGAACTTAATGCTATCAATACAACCGTGTCTCAAGATCCCGAGACTGAGCTGTACGCCGTGTCTAGTAAGCTGGACTTTAACATGACTACCAATCACAGTTTCATGTGCCTGATTAAGTACGGCCACCTGCGGGTGAATCAGACCTTTAATTGGAATACTACCAAGCAGGAGCACTTCCCAGATAACTAAGTCGAC

second subunit (BY31.3H) amino acid sequence of fusion protein BY31.3 (kappa, IgG2) (SEQ ID NO: 83):

METDTLLLWVLLLWVPGSTGQVQLVQSGVEVKKPGASVKVSCKASGYTFTNYYMYWVRQAPGQGLEWMGGINPSNGGTNFNEKFKNRVTLTTDSSTTTAYMELKSLQFDDTAVYYCARRDYRFDMGFDYWGQGTTVTVSTASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGGSGGGGSVIHVTKEVKEVATLSCGHNVSVEELAQTRIYWQKEKKMVLTMMSGDMNIWPEYKNRTIFDITNNLSIVILALRPSDEGTYECVVLKYEKDAFKREHLAEVTLSVKADFPTPSISDFEIPTSNIRRIICSTSGGFPEPHLSWLENGEELNAINTTVSQDPETELYAVSSKLDFNMTTNHSFMCLIKYGHLRVNQTFNWNTTKQEHFPDN

the nucleotide sequence of the first subunit (BY31.7L) of fusion protein BY31.7 (SEQ ID NO: 84):

TCTAGAGCCACCATGGAGACCGACACACTCCTCCTGTGGGTGCTGCTGCTGTGGGTGCCTGGCTCCACTGGCGTCATCCATGTGACCAAAGAGGTCAAAGAAGTCGCAACTCTGTCTTGCGGACACAACGTCTCCGTCGAAGAACTCGCCCAGACTAGAATATATTGGCAGAAGGAAAAGAAGATGGTGCTCACCATGATGTCCGGCGATATGAACATTTGGCCTGAGTACAAGAACCGGACAATCTTCGATATTACTAATAACCTGAGTATCGTGATTCTCGCTCTGCGCCCTAGCGACGAGGGCACATACGAGTGTGTGGTGCTGAAGTACGAGAAGGATGCATTCAAGCGGGAGCACCTCGCAGAGGTGACACTCTCCGTGAAGGCCGACTTCCCAACCCCATCTGGCCAGGGCACTAAGGTGGAGATTAAGAGAGGCGGCTCTGGCGGCGGAGGCAGTGGAGAGATTGTGCTGACACAGTCCCCCGCTACTCTGAGCCTGAGCCCTGGCGAGAGGGCTACACTGTCTTGCAGAGCTTCTCAGTCCGTGTCTTCTTACCTCGCTTGGTATCAGCAGAAGCCCGGCCAGGCTCCAAGACTGCTGATCTATGACGCTTCTAACCGCGCTACAGGCATTCCTGCTAGGTTCAGCGGCAGCGGCTCTGGCACCGACTTCACACTCACAATTAGCTCTCTTGAACCTGAGGACTTCGCCGTGTACTACTGCCAGCAGTCTAGCAACTGGCCTAGAACATTCGGCCAGGGCACTAAGGTGGAGATTAAGAGAACCGTGGCCGCCCCCAGCGTGTTCATCTTCCCTCCCAGCGACGAGCAGCTGAAGTCTGGCACCGCCAGCGTGGTGTGCCTGCTGAACAACTTCTACCCCCGCGAGGCCAAGGTGCAGTGGAAGGTGGACAACGCCCTGCAGAGCGGCAACAGCCAGGAGAGCGTGACCGAGCAGGACTCCAAGGACAGCACCTACAGCCTGAGCAGCACCCTGACCCTGAGCAAGGCCGACTACGAGAAGCACAAGGTGTACGCCTGCGAGGTGACCCACCAGGGACTGTCTAGCCCCGTGACCAAGAGCTTCAACCGGGGCGAGTGCTAA GTCGAC

the amino acid sequence of the first subunit (BY31.7L) of fusion protein BY31.7 (SEQ ID NO: 85):

METDTLLLWVLLLWVPGSTGVIHVTKEVKEVATLSCGHNVSVEELAQTRIYWQKEKKMVLTMMSGDMNIWPEYKNRTIFDITNNLSIVILALRPSDEGTYECVVLKYEKDAFKREHLAEVTLSVKADFPTPSGQGTKVEIKRGGSGGGGSGEIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQSSNWPRTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

the nucleotide sequence of the second subunit (BY31.7H) of fusion protein BY31.7 (SEQ ID NO: 86):

CTCGAGGCCACCATGGAGACCGACACACTCCTCCTGTGGGTGCTGCTGCTGTGGGTGCCTGGCTCCACTGGCGTCATCCATGTGACCAAAGAGGTCAAAGAAGTCGCAACTCTGTCTTGCGGACACAACGTCTCCGTCGAAGAACTCGCCCAGACTAGAATATATTGGCAGAAGGAAAAGAAGATGGTGCTCACCATGATGTCCGGCGATATGAACATTTGGCCTGAGTACAAGAACCGGACAATCTTCGATATTACTAATAACCTGAGTATCGTGATTCTCGCTCTGCGCCCTAGCGACGAGGGCACATACGAGTGTGTGGTGCTGAAGTACGAGAAGGATGCATTCAAGCGGGAGCACCTCGCAGAGGTGACACTCTCCGTGAAGGCCGACTTCCCAACCCCATCTGGCCAGGGCACACTCGTGACCGTGTCTAGCGGCGGCGGAGGCAGTGGCGGCGGAGGCAGTCAGGTGCAGCTCGTGGAGTCCGGCGGCGGCGTGGTGCAGCCCGGCAGATCCCTCAGACTGGACTGCAAGGCATCCGGCATTACATTCTCTAACTCTGGAATGCACTGGGTGAGACAGGCTCCTGGCAAGGGCCTGGAATGGGTGGCCGTGATTTGGTACGACGGCTCTAAGAGATACTACGCTGACTCCGTGAAGGGCCGGTTCACAATTAGCAGAGACAACTCCAAGAACACTCTGTTCCTCCAGATGAACAGCCTGAGAGCCGAGGACACCGCTGTGTACTACTGCGCCACCAACGACGACTACTGGGGCCAGGGCACCCTCGTGACAGTGTCTTCCGCCTCCACCAAGGGCCCTTCCGTGTTCCCTCTGGCCCCTTGCTCCCGCTCCACCTCCGAGTCCACCGCCGCCCTGGGCTGCCTGGTGAAGGACTACTTCCCTGAGCCTGTGACCGTGTCCTGGAACTCCGGCGCCCTGACCTCCGGCGTGCACACCTTCCCTGCCGTGCTGCAGTCCTCCGGCCTGTACTCCCTGTCCTCCGTGGTGACCGTGCCTTCCTCCTCCCTGGGCACCAAGACCTACACCTGCAACGTGGACCACAAGCCTTCCAACACCAAGGTGGACAAGCGCGTGGAGTCCAAGTACGGCCCTCCTTGCCCTCCTTGCCCTGCCCCTGAGTTCCTGGGCGGCCCTTCCGTGTTCCTGTTCCCTCCTAAGCCTAAGGACACCCTGATGATCTCCCGCACCCCTGAGGTGACCTGCGTGGTGGTGGACGTGTCCCAGGAGGACCCTGAGGTGCAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCACAACGCCAAGACCAAGCCTCGCGAGGAGCAGTTCAACTCCACCTACCGCGTGGTGTCCGTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTGTCCAACAAGGGCCTGCCTTCCTCCATCGAGAAGACCATCTCCAAGGCCAAGGGCCAGCCTCGCGAGCCTCAGGTGTACACCCTGCCTCCTTCCCAGGAGGAGATGACCAAGAACCAGGTGTCCCTGACCTGCCTGGTGAAGGGCTTCTACCCTTCCGACATCGCCGTGGAGTGGGAGTCCAACGGCCAGCCTGAGAACAACTACAAGACCACCCCTCCTGTGCTGGACTCCGACGGCTCCTTCTTCCTGTACTCCCGCCTGACCGTGGACAAGTCCCGCTGGCAGGAGGGCAACGTGTTCTCCTGCTCCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGTCCCTGTCCCTGGGCAAGTAAGAATTC

the amino acid sequence of the second subunit (BY31.7H) of fusion protein BY31.7 (SEQ ID NO: 87):

METDTLLLWVLLLWVPGSTGVIHVTKEVKEVATLSCGHNVSVEELAQTRIYWQKEKKMVLTMMSGDMNIWPEYKNRTIFDITNNLSIVILALRPSDEGTYECVVLKYEKDAFKREHLAEVTLSVKADFPTPSGQGTLVTVSSGGGGSGGGGSQVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKGLEWVAVIWYDGSKRYYADSVKGRFTISRDNSKNTLFLQMNSLRAEDTAVYYCATNDDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

nucleotide sequence of the first subunit of fusion protein BY31.14 (linker of PD-1 antibody light chain to C-terminus of CD80-Fc fusion, i.e., BY31.14L) (SEQ ID NO: 88):

CTCGAGGCCACCATGGAGACCGACACACTCCTCCTGTGGGTGCTGCTGCTGTGGGTGCCTGGCTCCACTGGCGTCATCCATGTGACCAAAGAGGTCAAAGAAGTCGCAACTCTGTCTTGCGGACACAACGTCTCCGTCGAAGAACTCGCCCAGACTAGAATATATTGGCAGAAGGAAAAGAAGATGGTGCTCACCATGATGTCCGGCGATATGAACATTTGGCCTGAGTACAAGAACCGGACAATCTTCGATATTACTAATAACCTGAGTATCGTGATTCTCGCTCTGCGCCCTAGCGACGAGGGCACATACGAGTGTGTGGTGCTGAAGTACGAGAAGGATGCATTCAAGCGGGAGCACCTCGCAGAGGTGACACTCTCCGTGAAGGCCGACTTCCCAACCCCATCTGACAAGCGCGTGGAGTCCAAGTACGGCCCTCCTTGCCCTCCTTGCCCTGCCCCTGAGTTCCTGGGCGGCCCTTCCGTGTTCCTGTTCCCTCCTAAGCCTAAGGACACCCTGATGATCTCCCGCACCCCTGAGGTGACCTGCGTGGTGGTGGACGTGTCCCAGGAGGACCCTGAGGTGCAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCACAACGCCAAGACCAAGCCTCGCGAGGAGCAGTTCAACTCCACCTACCGCGTGGTGTCCGTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTGTCCAACAAGGGCCTGCCTTCCTCCATCGAGAAGACCATCTCCAAGGCCAAGGGCCAGCCTCGCGAGCCTCAGGTGTACACCCTGCCTCCTTCCCAGGAGGAGATGACCAAGAACCAGGTGTCCCTGACCTGCCTGGTGAAGGGCTTCTACCCTTCCGACATCGCCGTGGAGTGGGAGTCCAACGGCCAGCCTGAGAACAACTACAAGACCACCCCTCCTGTGCTGGACTCCGACGGCTCCTTCTTCCTGTACTCCCGCCTGACCGTGGACAAGTCCCGCTGGCAGGAGGGCAACGTGTTCTCCTGCTCCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGTCCCTGTCCCTGGGCGGCGGAGGATCTGGCGGCGGAGGCAGTGGAGGCGGCGGAAGTGAGATTGTGCTGACACAGTCCCCCGCTACTCTGAGCCTGAGCCCTGGCGAGAGGGCTACACTGTCTTGCAGAGCTTCTCAGTCCGTGTCTTCTTACCTCGCTTGGTATCAGCAGAAGCCCGGCCAGGCTCCAAGACTGCTGATCTATGACGCTTCTAACCGCGCTACAGGCATTCCTGCTAGGTTCAGCGGCAGCGGCTCTGGCACCGACTTCACACTCACAATTAGCTCTCTTGAACCTGAGGACTTCGCCGTGTACTACTGCCAGCAGTCTAGCAACTGGCCTAGAACATTCGGCCAGGGCACTAAGGTGGAGATTAAGAGAACCGTGGCCGCCCCCAGCGTGTTCATCTTCCCTCCCAGCGACGAGCAGCTGAAGTCTGGCACCGCCAGCGTGGTGTGCCTGCTGAACAACTTCTACCCCCGCGAGGCCAAGGTGCAGTGGAAGGTGGACAACGCCCTGCAGAGCGGCAACAGCCAGGAGAGCGTGACCGAGCAGGACTCCAAGGACAGCACCTACAGCCTGAGCAGCACCCTGACCCTGAGCAAGGCCGACTACGAGAAGCACAAGGTGTACGCCTGCGAGGTGACCCACCAGGGACTGTCTAGCCCCGTGACCAAGAGCTTCAACCGGGGCGAGTGCTAAGAATTC

the amino acid sequence of the first subunit of fusion protein BY31.14 (linker of PD-1 antibody light chain to C-terminus of CD80-Fc fusion, BY31.14L) (SEQ ID NO: 89):

METDTLLLWVLLLWVPGSTGVIHVTKEVKEVATLSCGHNVSVEELAQTRIYWQKEKKMVLTMMSGDMNIWPEYKNRTIFDITNNLSIVILALRPSDEGTYECVVLKYEKDAFKREHLAEVTLSVKADFPTPSDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGGGGSGGGGSGGGGSEIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQSSNWPRTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

nucleotide sequence of the second subunit of fusion protein BY31.14 (heavy chain variable region and CH1 domain of PD-1 antibody Fab fragment, i.e., BY31.14H, disulfide-linked to the PD-1 antibody light chain portion in the first subunit of fusion protein BY 31.14) (SEQ ID NO: 90):

TCTAGAGCCACCATGGAGACCGACACCCTGCTGCTGTGGGTGCTGCTCCTGTGGGTGCCTGGCTCCACAGGCCAGGTGCAGCTCGTGGAGTCCGGCGGCGGCGTGGTGCAGCCCGGCAGATCCCTCAGACTGGACTGCAAGGCATCCGGCATTACATTCTCTAACTCTGGAATGCACTGGGTGAGACAGGCTCCTGGCAAGGGCCTGGAATGGGTGGCCGTGATTTGGTACGACGGCTCTAAGAGATACTACGCTGACTCCGTGAAGGGCCGGTTCACAATTAGCAGAGACAACTCCAAGAACACTCTGTTCCTCCAGATGAACAGCCTGAGAGCCGAGGACACCGCTGTGTACTACTGCGCCACCAACGACGACTACTGGGGCCAGGGCACCCTCGTGACAGTGTCTTCCGCCTCCACCAAGGGCCCTTCCGTGTTCCCTCTGGCCCCTTGCTCCCGCTCCACCTCCGAGTCCACCGCCGCCCTGGGCTGCCTGGTGAAGGACTACTTCCCTGAGCCTGTGACCGTGTCCTGGAACTCCGGCGCCCTGACCTCCGGCGTGCACACCTTCCCTGCCGTGCTGCAGTCCTCCGGCCTGTACTCCCTGTCCTCCGTGGTGACCGTGCCTTCCTCCTCCCTGGGCACCAAGACCTACACCTGCAACGTGGACCACAAGCCTTCCAACACCAAGGTGGACAAGCGCGTGGAGTCCTAAGTCGAC

the amino acid sequence of the second subunit (BY31.14H) of fusion protein BY31.14 (SEQ ID NO: 91):

METDTLLLWVLLLWVPGSTGQVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKGLEWVAVIWYDGSKRYYADSVKGRFTISRDNSKNTLFLQMNSLRAEDTAVYYCATNDDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVES

the nucleotide sequence of the first subunit of fusion protein BY31.15 (BY31.15L, i.e., the light chain of PD-1 antibody) (SEQ ID NO: 92):

TCTAGAGCCACCAATGGAGACCGACACCCTGCTGCTGTGGGTGCTGCTCCTGTGGGTGCCTGGCTCCACAGGCGAGATTGTGCTGACACAGTCCCCCGCTACTCTGAGCCTGAGCCCTGGCGAGAGGGCTACACTGTCTTGCAGAGCTTCTCAGTCCGTGTCTTCTTACCTCGCTTGGTATCAGCAGAAGCCCGGCCAGGCTCCAAGACTGCTGATCTATGACGCTTCTAACCGCGCTACAGGCATTCCTGCTAGGTTCAGCGGCAGCGGCTCTGGCACCGACTTCACACTCACAATTAGCTCTCTTGAACCTGAGGACTTCGCCGTGTACTACTGCCAGCAGTCTAGCAACTGGCCTAGAACATTCGGCCAGGGCACTAAGGTGGAGATTAAGAGAACCGTGGCCGCCCCCAGCGTGTTCATCTTCCCTCCCAGCGACGAGCAGCTGAAGTCTGGCACCGCCAGCGTGGTGTGCCTGCTGAACAACTTCTACCCCCGCGAGGCCAAGGTGCAGTGGAAGGTGGACAACGCCCTGCAGAGCGGCAACAGCCAGGAGAGCGTGACCGAGCAGGACTCCAAGGACAGCACCTACAGCCTGAGCAGCACCCTGACCCTGAGCAAGGCCGACTACGAGAAGCACAAGGTGTACGCCTGCGAGGTGACCCACCAGGGACTGTCTAGCCCCGTGACCAAGAGCTTCAACCGGGGCGAGTGCTAAGTCGAC

the amino acid sequence of the first subunit (BY31.15L) of fusion protein BY31.15 (SEQ ID NO: 93):

METDTLLLWVLLLWVPGSTGEIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQSSNWPRTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

the nucleotide sequence of the second subunit of fusion protein BY31.15 (BY31.15H, i.e., the C-terminus of the CD80-Fc fusion linked to the heavy chain variable region and CH1 domain of the Fab fragment of the PD-1 antibody) (SEQ ID NO: 94):

CTCGAGGCCACCATGGAGACCGACACACTCCTCCTGTGGGTGCTGCTGCTGTGGGTGCCTGGCTCCACTGGCGTCATCCATGTGACCAAAGAGGTCAAAGAAGTCGCAACTCTGTCTTGCGGACACAACGTCTCCGTCGAAGAACTCGCCCAGACTAGAATATATTGGCAGAAGGAAAAGAAGATGGTGCTCACCATGATGTCCGGCGATATGAACATTTGGCCTGAGTACAAGAACCGGACAATCTTCGATATTACTAATAACCTGAGTATCGTGATTCTCGCTCTGCGCCCTAGCGACGAGGGCACATACGAGTGTGTGGTGCTGAAGTACGAGAAGGATGCATTCAAGCGGGAGCACCTCGCAGAGGTGACACTCTCCGTGAAGGCCGACTTCCCAACCCCATCTGACAAGCGCGTGGAGTCCAAGTACGGCCCTCCTTGCCCTCCTTGCCCTGCCCCTGAGTTCCTGGGCGGCCCTTCCGTGTTCCTGTTCCCTCCTAAGCCTAAGGACACCCTGATGATCTCCCGCACCCCTGAGGTGACCTGCGTGGTGGTGGACGTGTCCCAGGAGGACCCTGAGGTGCAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCACAACGCCAAGACCAAGCCTCGCGAGGAGCAGTTCAACTCCACCTACCGCGTGGTGTCCGTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTGTCCAACAAGGGCCTGCCTTCCTCCATCGAGAAGACCATCTCCAAGGCCAAGGGCCAGCCTCGCGAGCCTCAGGTGTACACCCTGCCTCCTTCCCAGGAGGAGATGACCAAGAACCAGGTGTCCCTGACCTGCCTGGTGAAGGGCTTCTACCCTTCCGACATCGCCGTGGAGTGGGAGTCCAACGGCCAGCCTGAGAACAACTACAAGACCACCCCTCCTGTGCTGGACTCCGACGGCTCCTTCTTCCTGTACTCCCGCCTGACCGTGGACAAGTCCCGCTGGCAGGAGGGCAACGTGTTCTCCTGCTCCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGTCCCTGTCCCTGGGCGGCGGAGGATCTGGCGGCGGAGGCAGTGGAGGCGGCGGAAGTCAGGTGCAGCTCGTGGAGTCCGGCGGCGGCGTGGTGCAGCCCGGCAGATCCCTCAGACTGGACTGCAAGGCATCCGGCATTACATTCTCTAACTCTGGAATGCACTGGGTGAGACAGGCTCCTGGCAAGGGCCTGGAATGGGTGGCCGTGATTTGGTACGACGGCTCTAAGAGATACTACGCTGACTCCGTGAAGGGCCGGTTCACAATTAGCAGAGACAACTCCAAGAACACTCTGTTCCTCCAGATGAACAGCCTGAGAGCCGAGGACACCGCTGTGTACTACTGCGCCACCAACGACGACTACTGGGGCCAGGGCACCCTCGTGACAGTGTCTTCCGCCTCCACCAAGGGCCCTTCCGTGTTCCCTCTGGCCCCTTGCTCCCGCTCCACCTCCGAGTCCACCGCCGCCCTGGGCTGCCTGGTGAAGGACTACTTCCCTGAGCCTGTGACCGTGTCCTGGAACTCCGGCGCCCTGACCTCCGGCGTGCACACCTTCCCTGCCGTGCTGCAGTCCTCCGGCCTGTACTCCCTGTCCTCCGTGGTGACCGTGCCTTCCTCCTCCCTGGGCACCAAGACCTACACCTGCAACGTGGACCACAAGCCTTCCAACACCAAGGTGGACAAGCGCGTGGAGTC

the amino acid sequence of the second subunit of fusion protein BY31.15 (BY31.15H, the heavy chain variable region and CH1 domain of the Fab fragment of PD-1 antibody in the second subunit of fusion protein BY31.15 are linked to the first subunit of fusion protein BY31.15 BY a disulfide bond) (SEQ ID NO: 95):

METDTLLLWVLLLWVPGSTGVIHVTKEVKEVATLSCGHNVSVEELAQTRIYWQKEKKMVLTMMSGDMNIWPEYKNRTIFDITNNLSIVILALRPSDEGTYECVVLKYEKDAFKREHLAEVTLSVKADFPTPSDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGGGGSGGGGSGGGGSQVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKGLEWVAVIWYDGSKRYYADSVKGRFTISRDNSKNTLFLQMNSLRAEDTAVYYCATNDDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVES

wherein the amino acid sequence "METDTLLLWVLLLWVPGSTG" is a signal peptide.

The same procedures as in example 1(1) above were used to ligate the coding nucleotides BY31.2L, BY31.3L, BY31.7L, BY31.14L and BY31.15L to the glutamine synthetase high-efficiency expression vector having a double expression cassette by XhoI-EcoRI double digestion, respectively (patent No. CN104195173B, available from Beijing Biyang Biotechnology Co., Ltd.); then cloning BY31.2H, BY31.3H, BY31.7H, BY31.14H and BY31.15H coding nucleotides to a glutamine synthetase high-efficiency expression vector with a double expression cassette which is connected with coding nucleotides of the other subunit of the corresponding fusion protein by XbaI-SalI double digestion; or vice versa. The recombinant vector is used for expression after being sequenced and verified to be correct. The expressed fusion proteins were designated as fusion proteins BY31.2, BY31.3, BY31.7, BY31.14 and BY31.15, respectively.

Example 2 expression and purification of fusion proteins

(1) Transient expression of fusion proteins

293F (from Invitrogen, catalog No. 11625-019) cells were suspension cultured in serum-free CD 293 medium (from Invitrogen, catalog No. 11913-019). The cell culture was centrifuged before transfection to obtain cell pellet, the cells were suspended in fresh serum-free CD 293 medium and the cell concentration was adjusted to 1X 10⁶Individual cells/ml. The cell suspension was placed in a shake flask. Using 100ml of cell suspension as an example, 250ug of each DNA and 500ug of Polyethyleneimine (PEI) (Sigma, catalog # 408727) of each recombinant expression vector plasmid of the fusion proteins BY31.2, BY31.3, BY31.7, BY31.14 and BY31.15 prepared in example 1 were added to 1ml of CD 293 serum-free culture solution and mixed well, and after standing at room temperature for 8 minutes, the PEI/DNA suspension was added dropwise to a flask containing 100ml of cell suspension. Mixing gently, and placing in 5% CO₂Shaking culture (120 rpm) at 37 ℃. Culture supernatants were collected after 5 days.

According to the same method, the antibody BY18.1 was produced as a control BY transient expression.

(2) Purification of expressed proteins

The fusion protein present in the culture supernatant collected in example 2(1) above was purified by using a HiTrap MabSelect SuRe 1ml column (product of GE Healthcare Life Sciences, Cat. No. 11-0034-93) equilibrated with a PBS solution at pH 7.4. Briefly, a HiTrap MabSelect SuRe 1ml column was equilibrated with 10 bed volumes of PBS solution pH7.4 at a flow rate of 0.5 ml/min; the culture supernatant collected in example 2(1) above was filtered through a 0.45 μm filter and loaded on a HiTrap MabSelect SuRe 1ml column equilibrated with a PBS solution having a pH of 7.4; after loading the supernatant, the column was first washed with a PBS solution at pH7.4 at a flow rate of 0.5 ml/min for 5-10 bed volumes and then eluted with 100mM citrate buffer (pH 4.0) at a flow rate of 0.5 ml/min. And collecting an elution peak, wherein the target protein exists in the elution peak.

The purity and molecular weight of the fusion protein were analyzed by SDS-PAGE in the presence of a reducing agent (5mM 1, 4-dithiothreitol) and staining with Coomassie blue. The results are shown in FIG. 2. The theoretical predicted value and the actual measured value of the molecular weight are shown in Table 6. Because of the glycosylation of protein in eukaryotic expression system, the measured value of molecular weight is slightly higher than the theoretical predicted value.

TABLE 6 molecular weight of the purified expressed proteins

Example 3 detection of specific binding Using ELISA method

Recombinant human CD28 (product of Beijing Yiqiao Shenzhou Biotechnology Co., Ltd., catalog number: 50103-M08H), recombinant human PD-L1 (product of Beijing Baiproseis Biotechnology Co., Ltd., catalog number: PD1-H5229), and recombinant human CTLA-4 (product of Beijing Yiqiao Shenzhou Biotechnology Co., Ltd., catalog number: 11159-H08H) were each diluted to 0.5. mu.g/ml, 0.25. mu.g/ml and 1.0. mu.g/ml and coated on 96-well ELISA plates (Corning Co., catalog number: 42592). The fusion proteins BY31.2, BY31.3, BY31.7, BY31.14 and BY31.15 purified in example 2(2) above were diluted to 2000. mu.g/ml, and then 3-fold serial dilutions were performed for 15-16 gradients, and duplicate wells were performed for each concentration gradient. Mu.l of the diluted sample was added to the above-mentioned 96-well plate coated with recombinant human CD28 or recombinant human PD-L1, respectively, and incubated at 37 ℃ for 2 hours. After washing for 3 times, goat anti-human secondary antibody (manufactured by Beijing Zhongxiu gold bridge) labeled with horseradish peroxidase was addedProduct, product number: ZDR-5301), incubated at 37 ℃ for 1 hour. After 3 times of washing, 50. mu.l/well of 3,3',5,5' -Tetramethylbenzidine (TMB) substrate color developing solution (product number: CW0050, Beijing kang, century Biotech Co., Ltd.) was added. After 10 minutes, 2N H was added₂SO₄The color development was terminated. The absorbance OD value of each well was measured at 450nm using an ELISA reader.

ELISA results show that the fusion proteins BY31.2, BY31.3, BY31.7, BY31.14 and BY31.15 of the invention can specifically bind to recombinant human PD-L1 and recombinant human CD 28; can also specifically bind to recombinant human CTLA-4. The binding curves of fusion protein BY31.2 specifically binding to recombinant human CD28, recombinant human PD-L1 and recombinant human CTLA-4 are shown in FIG. 3A, FIG. 3B and FIG. 3C, respectively.

Protein concentrations of the fusion proteins BY31.2, BY31.3, BY31.7, BY31.14 and BY31.15 were plotted against absorbance OD values using GraphPadprism5 software, and the data were fitted to generate half the maximal effective concentration EC for fusion protein-mediated specific binding₅₀The value is obtained. The results are shown in Table 7 below.

TABLE 7 binding of the fusion proteins of the invention to human PD-L1, human CD28 and human CTLA-4

As can be seen from the results in Table 7, the novel fusion proteins BY31.2, BY31.3, BY31.7, BY31.14 and BY31.15 constructed BY the present invention are all capable of specifically binding to human PD-L1, human CD28 and human CTLA-4. The fusion protein BY31.3 has better binding capacity with human CD28, CTLA-4 and PD-L1 than the fusion protein BY31.2 and fusion protein BY31.7, which is probably because the IgC region in the CD80ECD has certain stabilizing effect on the binding of CD80 with CD28, CTLA-4 and PD-L1.

The fusion proteins BY31.14 and BY31.15 have better binding capacity with human PD-L1, human CD28 and human CTLA-4 than BY31.2, BY31.3 and BY 31.7. It is possible that BY31.2, BY31.3 and BY31.7 placed the CD80 extracellular domain (ECD) at the N-terminus or C-terminus of the full length antibody had some effect on binding to human PD-L1, human CD28 and human CTLA-4, respectively.

In addition, EC for specific binding of antibody BY18.1 to antigen PD-1 (product of Beijing Yi Qiao Shen Biotechnology Co., Ltd., catalog No. 10377-H08H) was also detected BY ELISA method₅₀The value was 3.154. mu.g/ml.

Example 4 determination of the affinity of the fusion protein of the invention for PD-1 Using Biacore T100

In that

Surface plasmon resonance measurements were performed on a T100 instrument (GE Healthcare Biosciences AB, Sweden) at 25 ℃.

First, an anti-IgG antibody (GE Healthcare Life Sciences, Cat: BR-1008-39) was covalently immobilized on a CM5 chip by amide coupling. The CM5 chip was activated with 60. mu. l N-ethyl-N' - (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and 60. mu. l N-hydroxysuccinimide (NHS), and then 5. mu.l of the anti-IgG antibody plus 95. mu.l of dilution buffer HBST (0.1M HEPES,1.5MNaCl, pH7.4, plus 0.005% Tween 20) was filtered through a 0.2um filter, and the anti-IgG antibody was covalently immobilized on the CM5 chip by amide coupling, resulting in a capture system of approximately 9000-14000 Resonance Units (RU). The CM5 chip was blocked with 120. mu.l ethanolamine.

Then, the fusion protein of the present invention prepared in example 2 and the antibody BY18.1 were each diluted to 5. mu.g/ml, and the dilution was injected at a flow rate of 10. mu.L/min for 2 minutes, and 1600RU of the fusion protein of the present invention prepared in example 2 and the antibody BY18.1 were non-covalently captured onto the surface of a CM5 chip via the respective Fc regions. The resulting composite was stabilized by crosslinking with EDC/NHS to avoid baseline drift during measurement and regeneration.

Antigen PD-1 (product of Beijing Yi Qiao Shen State Biotechnology Co., Ltd., catalog number 10377-H08H) was formulated as a concentration gradient as follows: 7nM, 22nM, 66nM, 200nM, 600 nM. Binding was measured by injecting each concentration at a flow rate of 30. mu.l/min for 180 seconds, with a dissociation time of 600 seconds. By using 3M MgCl₂The solution was washed at a flow rate of 10. mu.L/min for 30 seconds to regenerate the surface. The samples were evaluated using BIA evaluation software (BIAevaluation 4.1software, from GE Healthcare Biosciences AB,sweden) was performed, and affinity data shown in table 8 below were obtained.

TABLE 8 binding of the respective proteins to PD-1

Name of protein	ka(1/Ms)	kd(1/s)	KD(M)
				Fusion protein BY31.2	6.66E+05	1.58E-03	2.37E-09
Fusion protein BY31.3	5.62E+05	5.66E-04	1.01E-09
				Fusion protein BY31.7	8.56E+05	3.55E-03	4.15E-09
Fusion protein BY31.14	5.04E+05	3.55E-03	7.04E-09
				Fusion protein BY31.15	1.56E+04	8.18E-04	5.24E-08
Antibody BY18.1	1.76E+05	5.18E-04	2.94E-09

As can be seen from the data shown in Table 8, the fusion proteins BY31.2, BY31.3 and BY31.7 of the present invention are all capable of binding PD-1 with high affinity (KD) of 2.37X 10^-9M、1.01×10^-9M and 4.15X 10^-9And M. Antibody BY18.1 at 2.94X 10^-9The KD value of M binds to PD-1.

The fusion proteins BY31.14 and BY31.15 bind to PD-1 less strongly than BY31.2, BY31.3 and BY 31.7. Probably, the antigen binding fragment in the fusion proteins BY31.14 and BY31.15 is a Fab structure positioned at the C terminal, and the binding effect is slightly weakened compared with that of a whole antibody. The fusion protein BY31.14 is stronger in PD-1 binding than the fusion protein BY31.15, and the KD value can reach 10^-9M level.

Example 5 Effect of fusion proteins of the invention on IL-2 and IFN- γ secretion in Mixed Lymphocyte Reaction (MLR)

CD4 available from Beijing Shihe Biotechnology Ltd⁺T lymphocytes and Dendritic Cells (DCs), said CD4⁺T lymphocytes and Dendritic Cells (DCs) are derived from different healthy people. Will CD4⁺T lymphocytes and Dendritic Cells (DCs) were 1X 10, respectively⁵Individual cells/well and 1X 10⁴Individual cells/well were plated in 96-well cell culture plates. The experiment was divided into 6 groups, blank control, BY18.1, BY31.2, BY31.3, BY31.7 and BY31.14, each of 6 duplicate wells. Except for the blank control group, the antibody or the fusion protein was added to the other groups in the amounts shown in Table 9, respectively. Finally, the 1640 culture medium containing 10% fetal calf serum is added to ensure that the final volume is 200 mul。37℃，5％CO₂And (5) culturing.

After 5 days of culture, the clumps of cells formed in each experiment were more abundant and a significant proportion of spindle and adherent cells appeared compared to the control group. Taking the culture supernatant, and detecting the IL-2 and IFN-gamma expression levels of each group by using an IL-2 kit (product number EH002-96) and an IFN-gamma kit (product number EH008-96ELISA) of Ekesai Biotechnology Limited. Compared with the antibody BY18.1 group, the BY31.2 group, the BY31.3 group, the BY31.7 group and the BY31.14 group all significantly improve the expression level of IL-2 and IFN-gamma (P <0.01), wherein the expression level of IL-2 and IFN-gamma is the highest in the supernatant of the BY31.14 group.

TABLE 9 Effect of the fusion proteins on IL-2 and IFN- γ secretion

Name of antibody or protein	μg/ml	IL-2(pg/ml)	IFN-γ(pg/ml)
				Antibody BY18.1	5.0	485±59.4	9552±754
Fusion protein BY31.2	6.0	775±143.6	26431±812
				Fusion protein BY31.7	6.9	903±186.5	21651±1890
Fusion protein BY31.14	6.0	1896±266.2	36378±1531
				Blank control	0	55.4±10.6	63.5±4.8

Example 6 in vivo anti-tumor Effect of the fusion protein of the present invention in B-hPD-1 humanized mouse model

This example investigated the in vivo anti-tumor effect of the fusion protein of the present invention using a B-hPD-1 humanized mouse model.

The B-hPD-1 humanized mouse (product No.: B-CM-001, available from Beijing Baiosai Toxico Gene Biotechnology Co., Ltd.) was a mouse obtained by knocking human PD-1(hPD-1) into the genome of a C57BL/6 mouse. Human PD-1 can be detected in B-hPD-1 humanized mice⁺A cell.

5X 10 in 0.1mL DMEM Medium⁵An individual MC38 murine colon cancer cell (obtained from ATCC, USA) was inoculated subcutaneously into the right anterior flank of an approximately 18g, 6-week-old female B-hPD-1 humanized mouse. Tumors grew in the mice. When the tumor volume reaches about 108mm³The tumor-bearing mice are randomly grouped, 6 mice in each group are divided into 3 groups, and the groups are respectively as follows: PBS solvent control group, fusion protein BY31.2 group (6.0mg/kg) and antibody BY18.1 group (5mg/kg), the dose of each administration group was based on the dose of antibody BY18.1 group, and the dose administered was equivalent in molar amount for the fusion protein BY31.2 group and the antibody BY18.1 group. The time of the first administration was set to day 0. All groups were administered by intraperitoneal (i.p.) injections every three daysThe administration was 1 time, 6 times continuously, and the experiment was terminated 3 days after the last administration. Tumor volume and mouse body weight were measured 2 times per week and mouse body weight and tumor volume were recorded. At the end of the experiment, animals were euthanized, tumors were stripped, weighed, photographed, and tumor growth inhibition rate was calculated (Tumor Growth Inhibition%). The formula used to calculate TGI% is: [ 1- (mean change in tumor volume of administration group/mean change in tumor volume of PBS solvent control group)]x 100%. The experiment was conducted in Beijing Baiosai Tourette Biotechnology, Inc.

All animals had good mental status and no animal death during the whole experimental process. At the end of the experiment (day 21 after the first dose), the animals in each group averaged about 19 g. Compared with the PBS solvent control group, the weight of the animals of the fusion protein BY31.2 group and the antibody BY18.1 group serving as the drug control group has no significant difference (P >0.05), which indicates that the animals are well tolerated BY the fusion protein BY31.2 (figure 4).

At the end of the experiment, the mean tumor volume. + -. standard error of the PBS solvent control group was 1386. + -. 170mm³Fusion protein BY31.2 group mean tumor volume. + -. standard error 953. + -.166 mm³The tumor growth inhibition ratio (TGI%) of the fusion protein BY31.2 group was 31.2%, indicating that the fusion protein BY31.2 has the technical effect of resisting tumors in vivo (fig. 5).

In addition, at the end of the experiment, the mean tumor volume. + -. standard error of the group of antibody BY18.1 as the drug control was 739. + -. 128 and TGI% was 46.9%, indicating that the antibody BY18.1 as the drug control specifically binds to hPD-1 in B-hPD-1 humanized mice⁺The cells exert an anti-tumor effect in vivo. This is consistent with the reports in the prior art that nivolumab is a specific monoclonal antibody directed against a human PD-1 molecule that blocks the inhibitory biological effects mediated by the human PD-1 molecule by specifically binding to the human PD-1 molecule, thereby exerting an anti-tumor effect on human patients expressing PD-1.

The effect of the fusion protein BY31.2 on inhibiting tumor growth was not significantly different from that of the antibody BY18.1 as a drug control, probably because only the anti-PD-1 antibody moiety was able to act in the fusion protein BY31.2, whereas human CD80ECD in the fusion protein BY31.2 did not bind to mouse CD28, mouse CTLA-4, and mouse PD-L1 of the B-hPD-1 humanized mouse; in addition, it is also possible that the fusion protein BY31.2 has a larger molecular weight than the control antibody BY18.1, and that the fusion protein BY31.2 penetrates into the tumor tissue in a smaller amount when administered in an equivalent molar amount (inside the tumor tissue is a hypertonic environment).

Example 7 fusion protein of the invention in human CD34⁺In vivo anti-tumor effects in HSC-transplanted NSG mouse model

This example was prepared by inoculating MDA-MB-231 human triple-negative breast cancer cell line (i.e., a breast cancer cell line that is negative for estrogen receptor, progestin receptor, and HER-2, obtained from ATCC, USA) to human CD34⁺The in vivo anti-tumor effect of the fusion protein of the present invention was studied in an NSG mouse model of HSC transplantation.

Human CD34⁺HSC-transplanted NSG mouse (available from Edmol Biotechnology Ltd. Beijing) is a human CD34⁺Transplantation of Hematopoietic Stem Cells (HSC) to immunodeficient NOD/SCID/IL2 r-gamma^null(NSG) mice, a mouse model humanized in terms of body immunity obtained by reconstituting the human immune system of NSG mice.

This embodiment is on a human CD34⁺On the basis of the HSC-transplanted NSG mouse model, a human-derived tumor cell line, namely MDA-MB-231 human triple-negative breast cancer cell line, is inoculated, and finally, a mouse humanized in both tumor and immunity is obtained. Since such a mouse has both a human immune system and a human tumor tissue, it can truly simulate the process of interaction between the human immune system and a tumor, and is an ideal animal model for evaluating the effectiveness and safety of tumor immunotherapy.

By 5X 10⁶Amount of individual MDA-MB-231 human triple negative breast cancer cells/mouse MDA-MB-231 cells were inoculated into human CD34 weighing approximately 23g, 28-32 weeks old⁺HSC transplanted successful NSG female mice were inoculated at the dorsal right postero-dorsal subcutaneous site. When the tumor volume reaches about 150mm³Randomly grouping tumor-bearing mice into 5 mice each group, and 4 groups in totalRespectively, the following steps: PBS solvent control group, fusion protein BY31.2 group (5.8mg/kg), anti-PD-L1 monoclonal antibody Avelumab group (10mg/kg, manufactured BY Beijing Shanjian Biotechnology Co., Ltd.), and anti-PD-1 monoclonal antibody Opdivo group (10mg/kg, manufactured BY Cassia Tokyo Biotechnology Co., Ltd., batch No.: MB09MA1201), wherein the fusion protein BY31.2 group was administered in a molar amount of 1/2 of the anti-PD-1 monoclonal antibody Opdivo group and the anti-PD-L1 monoclonal antibody Avelumab group. The time of the first administration was set to day 0. All groups were administered by intraperitoneal injection 1 time every 5 days and 4 times continuously. Tumor volume and mouse body weight were measured 3 times per week and mouse body weight and tumor volume were recorded. At the end of the experiment, animals were euthanized, tumors were stripped and weighed, photographed, and tumor growth inhibition (TGI%) calculated. The experiment was performed in Idermer (IDMO) biotechnology limited, beijing.

Figure 6 shows the change in body weight of the animals after administration. On day 25, no significant difference was observed in the body weight comparison of the animals of the fusion protein BY31.2 group of the present invention and the PBS solvent control group (P >0.05), indicating that the fusion protein BY31.2 of the present invention has no significant toxicity to the animals.

Figure 7 shows the tumor volume of the animals as a function of time after administration. Mean tumor volume. + -. standard error of 1287.11. + -. 184.71mm in the PBS solvent control group on day 25³The Avelumab group of the anti-PD-L1 monoclonal antibody is 964.25 +/-20.01 mm³The Opdivo group of the anti-PD-1 monoclonal antibody is 1354.62 +/-126.65 mm³The fusion protein BY31.2 group is 773.14 +/-310.66 mm³。

Relative to the PBS solvent control group, the tumor inhibition ratio (TGI%) of the anti-PD-L1 monoclonal antibody Avelumab group was 25.08%, the TGI% of the anti-PD-1 monoclonal antibody Opdivo group was-5.24%, and the TGI% of the fusion protein BY31.2 group was 39.93%.

From the above experimental results, it can be seen that, compared with the PBS solvent control group, the fusion protein BY31.2 of the present invention can significantly inhibit tumor growth (P <0.05) even when the molar dose used is halved relative to the molar doses of the anti-PD-L1 mab and the anti-PD-1 mab in the prior art.

In addition, unexpectedly, in the fusion protein BY31.2 group, there was a swelling of one mouseThe tumor volume was reduced to only 60mm on day 25³The tumor volume of the PBS solvent control group was up to 1287.11 + -184.71 mm on day 25³This mouse was shown to produce a complete response to the tumor (CR). In both the anti-PD-L1 mAb Avelumab group and the anti-PD-1 mAb Opdivo group, no fully responsive mice were observed.

Since the pharmaceutical efficacy experiment of the antitumor drug was conducted on the mice having both human immune system and human tumor tissue developed by Beijing Addermer (IDMO) Biotechnology Ltd, there are very few cases where the mice completely responded to the tumor to be tested. The fusion protein of the present invention still enables observation of complete response to tumor when the molar dose used is halved relative to the molar doses of anti-PD-L1 mab and anti-PD-1 mab of the prior art, thereby achieving unexpected technical effects.

The anti-PD-1 mAb Opdivo group had no anti-tumor effect at all in this mouse model. The anti-PD-L1 monoclonal antibody Avelumab has certain tumor inhibition effect in the mouse model (the tumor volume of the PBS solvent control group is 1287.11 +/-184.71 mm)³The anti-PD-L1 monoclonal antibody Avelumab group tumor volume is 964.25 +/-20.01 mm³) However, the antitumor effect was significant without the fusion protein BY31.2 of the present invention (the tumor volume of the fusion protein BY31.2 group was 773.14. + -. 310.66mm³). This shows that the fusion protein BY31.2 of the present invention can generate very significant anti-tumor effect in a mouse model that truly simulates the interaction process between the human immune system and the tumor.

Sequence listing

<110> Beijing Biyang Biotechnology Co., Ltd

<120> fusion protein blocking PD-1/PD-L1 signaling pathway and activating T cell and use thereof

<130>

<160> 95

<170> PatentIn version 3.3

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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

100 105

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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

100

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Gln Val Gln Leu Val Gln Ser Gly Val Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr

20 25 30

Tyr Met Tyr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Gly Ile Asn Pro Ser Asn Gly Gly Thr Asn Phe Asn Glu Lys Phe

50 55 60

Lys Asn Arg Val Thr Leu Thr Thr Asp Ser Ser Thr Thr Thr Ala Tyr

65 70 75 80

Met Glu Leu Lys Ser Leu Gln Phe Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Arg Asp Tyr Arg Phe Asp Met Gly Phe Asp Tyr Trp Gly Gln

100 105 110

Gly

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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 Lys Gly Val Ser Thr Ser

20 25 30

Gly Tyr Ser Tyr Leu His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro

35 40 45

Arg Leu Leu Ile Tyr Leu Ala Ser Tyr Leu Glu Ser Gly Val Pro Ala

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser

65 70 75 80

Ser Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln His Ser Arg

85 90 95

Asp Leu Pro Leu Thr Phe Gly Gly Gly

100 105

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Gln Val Gln Leu Val Gln Ser Gly Ser Glu Leu Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr

20 25 30

Gly Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Gln Trp Met

35 40 45

Gly Trp Ile Asn Thr Asp Ser Gly Glu Ser Thr Tyr Ala Glu Glu Phe

50 55 60

Lys Gly Arg Phe Val Phe Ser Leu Asp Thr Ser Val Asn Thr Ala Tyr

65 70 75 80

Leu Gln Ile Thr Ser Leu Thr Ala Glu Asp Thr Gly Met Tyr Phe Cys

85 90 95

Val Arg Val Gly Tyr Asp Ala Leu Asp Tyr Trp Gly Gln Gly

100 105 110

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Glu Ile Val Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Ser Ala Arg Ser Ser Val Ser Tyr Met

20 25 30

His Trp Phe Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Trp Ile Tyr

35 40 45

Arg Thr Ser Asn Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Ser Tyr Cys Leu Thr Ile Asn Ser Leu Gln Pro Glu

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Arg Ser Ser Phe Pro Leu Thr

85 90 95

Phe Gly Gly Gly

100

<210> 7

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Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Met Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Thr Ile Ser Gly Gly Gly Ala Asn Thr Tyr Tyr Pro Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gln Leu Tyr Tyr Phe Asp Tyr Trp Gly Gln Gly

100 105

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Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Leu Ala Ser Gln Thr Ile Gly Thr Trp

20 25 30

Leu Thr Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Thr Ala Thr Ser Leu Ala Asp Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Val Tyr Ser Ile Pro Trp

85 90 95

Thr Phe Gly Gly Gly

100

<210> 9

<211> 114

<212> PRT

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Gln Val Thr Leu Lys Glu Ser Gly Pro Ala Leu Val Lys Pro Thr Gln

1 5 10 15

Thr Leu Thr Leu Thr Cys Thr Phe Ser Gly Phe Ser Leu Ser Thr Ser

20 25 30

Gly Thr Cys Val Ser Trp Ile Arg Gln Pro Pro Gly Lys Ala Leu Glu

35 40 45

Trp Leu Ala Thr Ile Cys Trp Glu Asp Ser Lys Gly Tyr Asn Pro Ser

50 55 60

Leu Lys Ser Arg Leu Thr Ile Ser Lys Asp Thr Ser Lys Asn Gln Ala

65 70 75 80

Val Leu Thr Met Thr Asn Met Asp Pro Val Asp Thr Ala Thr Tyr Tyr

85 90 95

Cys Ala Arg Arg Glu Asp Ser Gly Tyr Phe Trp Phe Pro Tyr Trp Gly

100 105 110

Gln Gly

<210> 10

<211> 100

<212> PRT

<213> Artificial sequence

<400> 10

Asn Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ala Gly Gln Asn Val Asn Asn Tyr

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Val Leu Ile

35 40 45

Phe Asn Ala Asn Ser Leu Gln Thr Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Ser Trp Thr Thr

85 90 95

Phe Gly Gly Gly

100

<210> 11

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Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Ser Tyr

20 25 30

Gly Val His Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Leu

35 40 45

Gly Val Ile Trp Ala Gly Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys

50 55 60

Ser Arg Leu Thr Ile Ser Lys Asp Asn Ser Lys Ser Gln Val Ser Leu

65 70 75 80

Lys Met Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Ala Tyr Gly Asn Tyr Trp Tyr Ile Asp Val Trp Gly Gln Gly

100 105 110

<210> 12

<211> 101

<212> PRT

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<400> 12

Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Glu Arg Ala Thr Ile Asn Cys Lys Ala Ser Gln Ser Val Ser Asn Asp

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile

35 40 45

Asn Tyr Ala Phe His Arg Phe Thr Gly Val Pro Asp Arg Phe Ser Gly

50 55 60

Ser Gly Tyr Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Ala

65 70 75 80

Glu Asp Val Ala Val Tyr Tyr Cys His Gln Ala Tyr Ser Ser Pro Tyr

85 90 95

Thr Phe Gly Gly Gly

100

<210> 13

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<212> PRT

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Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg Ser

1 5 10 15

Leu Arg Leu Asp Cys Lys Ala Ser Gly Ile Thr Phe Ser Asn Tyr Gly

20 25 30

Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala

35 40 45

Val Ile Trp Tyr Asp Ser Ser Arg Lys Tyr Tyr Ala Asp Ser Val Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Phe Leu

65 70 75 80

Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Thr Asn Asn Asp Tyr Trp Gly Gln Gly

100 105

<210> 14

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<212> PRT

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<400> 14

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Val Ser Asn Tyr

20 25 30

Leu Asp Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Asp Ala Ser Thr Arg Ala Thr Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Asn Met Gln Leu Pro Leu

85 90 95

Thr Phe Gly Gln Gly

100

<210> 15

<211> 113

<212> PRT

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<400> 15

Asp Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala Ser Val

1 5 10 15

Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr Gly Ile

20 25 30

Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met Gly Trp

35 40 45

Ile Ser Ala Tyr Asn Gly Asn Thr Asn Tyr Ala Gln Lys Leu Gln Gly

50 55 60

Arg Val Thr Met Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr Met Glu

65 70 75 80

Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys Ala Arg

85 90 95

Gly Arg Gly Tyr Ser Tyr Gly Ile Asp Ala Phe Asp Ile Trp Gly Gln

100 105 110

Gly

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Leu Ser Tyr Val Leu Thr Gln Pro Pro Ser Val Ser Val Ser Pro Gly

1 5 10 15

Gln Thr Ala Arg Ile Thr Cys Ser Gly Asp Ala Leu Pro Lys Gln Tyr

20 25 30

Ala Tyr Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Ile

35 40 45

Tyr Lys Asp Ser Glu Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly

50 55 60

Ser Ser Ser Gly Thr Thr Val Thr Leu Thr Ile Ser Gly Val Gln Ala

65 70 75 80

Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Ala Asp Ser Ser Gly Thr

85 90 95

Tyr Val Val Phe Gly Gly Gly

100

<210> 17

<211> 118

<212> PRT

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Gln Gly Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr

20 25 30

Glu Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Val Ile Glu Ser Glu Thr Gly Gly Thr Ala Tyr Asn Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Thr Arg Glu Gly Ile Thr Thr Val Ala Thr Thr Tyr Tyr Trp Tyr Phe

100 105 110

Asp Val Trp Gly Gln Gly

115

<210> 18

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Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Leu Gly

1 5 10 15

Gln Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile Val His Ser

20 25 30

Asn Gly Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser

35 40 45

Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Phe Gln Gly

85 90 95

Ser His Val Pro Leu Thr Phe Gly Gln Gly

100 105

<210> 19

<211> 111

<212> PRT

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<400> 19

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Thr Ile Ser Gly Gly Gly Ser Asp Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gln Leu Asn Tyr Ala Trp Phe Ala Tyr Trp Gly Gln Gly

100 105 110

<210> 20

<211> 105

<212> PRT

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<400> 20

Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Pro Gly

1 5 10 15

Gln Arg Ala Thr Ile Thr Cys Arg Ala Ser Glu Ser Val Asp Asn Tyr

20 25 30

Gly Ile Ser Phe Met Asn Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro

35 40 45

Lys Leu Leu Ile Tyr Thr Ser Ser Asn Lys Asp Thr Gly Val Pro Ala

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Asn

65 70 75 80

Pro Met Glu Ala Glu Asp Thr Ala Val Tyr Tyr Cys Gln Gln Ser Lys

85 90 95

Glu Val Pro Trp Thr Phe Gly Gly Gly

100 105

<210> 21

<211> 113

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<400> 21

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Ser Tyr

20 25 30

Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Leu Ile Ile Pro Met Phe Asp Thr Ala Gly Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Ala Ile Thr Val Asp Glu Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Ala Glu His Ser Ser Thr Gly Thr Phe Asp Tyr Trp Gly Gln

100 105 110

Gly

<210> 22

<211> 101

<212> PRT

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<400> 22

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser Trp

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Ser Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ala Asn His Leu Pro Phe

85 90 95

Thr Phe Gly Gly Gly

100

<210> 23

<211> 116

<212> PRT

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<400> 23

Gln Leu Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu

1 5 10 15

Thr Leu Thr Leu Thr Cys Thr Val Ser Ala Asp Ser Ile Ser Ser Thr

20 25 30

Thr Tyr Tyr Trp Val Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu

35 40 45

Trp Ile Gly Ser Ile Ser Tyr Ser Gly Ser Thr Tyr Tyr Asn Pro Ser

50 55 60

Leu Lys Ser Arg Val Thr Val Ser Val Asp Thr Ser Lys Asn Gln Phe

65 70 75 80

Ser Leu Lys Leu Asn Ser Val Ala Ala Thr Asp Thr Ala Leu Tyr Tyr

85 90 95

Cys Ala Arg His Leu Gly Tyr Asn Gly Arg Tyr Leu Pro Phe Asp Tyr

100 105 110

Trp Gly Gln Gly

115

<210> 24

<211> 104

<212> PRT

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<400> 24

Gln Ser Ala Leu Thr Gln Pro Ala Ser Val Ser Gly Ser Pro Gly Gln

1 5 10 15

Ser Ile Thr Ile Ser Cys Thr Gly Thr Ser Ser Asp Val Gly Phe Tyr

20 25 30

Asn Tyr Val Ser Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Glu Leu

35 40 45

Met Ile Tyr Asp Val Ser Asn Arg Pro Ser Gly Val Ser Asp Arg Phe

50 55 60

Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Ile Ser Gly Leu

65 70 75 80

Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Ser Ser Tyr Thr Asn Ile

85 90 95

Ser Thr Trp Val Phe Gly Gly Gly

100

<210> 25

<211> 106

<212> PRT

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<400> 25

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Thr Cys Lys Ala Ser Gly Leu Thr Phe Ser Ser 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

100 105

<210> 26

<211> 102

<212> PRT

<213> Artificial sequence

<400> 26

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 Thr 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 Tyr Ser Asn Trp Pro Arg

85 90 95

Thr Phe Gly Gln Gly Thr

100

<210> 27

<211> 111

<212> PRT

<213> Artificial sequence

<400> 27

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Ser

20 25 30

Trp Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Trp Ile Ser Pro Tyr Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Arg His Trp Pro Gly Gly Phe Asp Tyr Trp Gly Gln Gly

100 105 110

<210> 28

<211> 101

<212> PRT

<213> Artificial sequence

<400> 28

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Ser Thr Ala

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Leu Tyr His Pro Ala

85 90 95

Thr Phe Gly Gln Gly

100

<210> 29

<211> 114

<212> PRT

<213> Artificial sequence

<400> 29

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Arg Tyr

20 25 30

Trp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Asn Ile Lys Gln Asp Gly Ser Glu Lys Tyr Tyr Val Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Glu Gly Gly Trp Phe Gly Glu Leu Ala Phe Asp Tyr Trp Gly

100 105 110

Gln Gly

<210> 30

<211> 102

<212> PRT

<213> Artificial sequence

<400> 30

Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Arg Val Ser Ser Ser

20 25 30

Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu

35 40 45

Ile Tyr Asp Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser

50 55 60

Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu

65 70 75 80

Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Gly Ser Leu Pro

85 90 95

Trp Thr Phe Gly Gln Gly

100

<210> 31

<211> 113

<212> PRT

<213> Artificial sequence

<400> 31

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Ile Met Met Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ser Ile Tyr Pro Ser Gly Gly Ile Thr Phe Tyr Ala Asp Thr Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Ile Lys Leu Gly Thr Val Thr Thr Val Asp Tyr Trp Gly Gln

100 105 110

Gly

<210> 32

<211> 104

<212> PRT

<213> Artificial sequence

<400> 32

Gln Ser Ala Leu Thr Gln Pro Ala Ser Val Ser Gly Ser Pro Gly Gln

1 5 10 15

Ser Ile Thr Ile Ser Cys Thr Gly Thr Ser Ser Asp Val Gly Gly Tyr

20 25 30

Asn Tyr Val Ser Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Leu

35 40 45

Met Ile Tyr Asp Val Ser Asn Arg Pro Ser Gly Val Ser Asn Arg Phe

50 55 60

Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Ile Ser Gly Leu

65 70 75 80

Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Ser Ser Tyr Thr Ser Ser

85 90 95

Ser Thr Arg Val Phe Gly Thr Gly

100

<210> 33

<211> 113

<212> PRT

<213> Artificial sequence

<400> 33

Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile

1 5 10 15

Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val

20 25 30

Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys

35 40 45

Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu

50 55 60

Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu

65 70 75 80

Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr

85 90 95

His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu

100 105 110

Cys

<210> 34

<211> 112

<212> PRT

<213> Artificial sequence

<400> 34

Thr Lys Leu Thr Val Leu Gly Gln Pro Lys Ala Ala Pro Ser Val Thr

1 5 10 15

Leu Phe Pro Pro Ser Ser Glu Glu Leu Gln Ala Asn Lys Ala Thr Leu

20 25 30

Val Cys Leu Ile Ser Asp Phe Tyr Pro Gly Ala Val Thr Val Ala Trp

35 40 45

Lys Ala Asp Ser Ser Pro Val Lys Ala Gly Val Glu Thr Thr Thr Pro

50 55 60

Ser Lys Gln Ser Asn Asn Lys Tyr Ala Ala Ser Ser Tyr Leu Ser Leu

65 70 75 80

Thr Pro Glu Gln Trp Lys Ser His Arg Ser Tyr Ser Cys Gln Val Thr

85 90 95

His Glu Gly Ser Thr Val Glu Lys Thr Val Ala Pro Thr Glu Cys Ser

100 105 110

<210> 35

<211> 337

<212> PRT

<213> Artificial sequence

<400> 35

Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe

1 5 10 15

Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu

20 25 30

Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp

35 40 45

Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu

50 55 60

Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser

65 70 75 80

Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro

85 90 95

Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys

100 105 110

Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro

115 120 125

Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser

130 135 140

Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp

145 150 155 160

Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn

165 170 175

Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val

180 185 190

Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu

195 200 205

Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys

210 215 220

Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr

225 230 235 240

Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr

245 250 255

Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu

260 265 270

Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu

275 280 285

Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys

290 295 300

Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu

305 310 315 320

Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

325 330 335

Lys

<210> 36

<211> 333

<212> PRT

<213> Artificial sequence

<400> 36

Thr Thr Val Thr Val Ser Thr Ala Ser Thr Lys Gly Pro Ser Val Phe

1 5 10 15

Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu

20 25 30

Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp

35 40 45

Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu

50 55 60

Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser

65 70 75 80

Ser Asn Phe Gly Thr Gln Thr Tyr Thr Cys Asn Val Asp His Lys Pro

85 90 95

Ser Asn Thr Lys Val Asp Lys Thr Val Glu Arg Lys Cys Cys Val Glu

100 105 110

Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val Phe Leu

115 120 125

Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu

130 135 140

Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Gln

145 150 155 160

Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys

165 170 175

Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Val Val Ser Val Leu

180 185 190

Thr Val Val His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys

195 200 205

Val Ser Asn Lys Gly Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys

210 215 220

Thr Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser

225 230 235 240

Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys

245 250 255

Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln

260 265 270

Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Met Leu Asp Ser Asp Gly

275 280 285

Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln

290 295 300

Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn

305 310 315 320

His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

325 330

<210> 37

<211> 334

<212> PRT

<213> Artificial sequence

<400> 37

Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe

1 5 10 15

Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu

20 25 30

Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp

35 40 45

Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu

50 55 60

Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser

65 70 75 80

Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro

85 90 95

Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro

100 105 110

Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe

115 120 125

Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro

130 135 140

Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val

145 150 155 160

Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr

165 170 175

Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val

180 185 190

Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys

195 200 205

Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser

210 215 220

Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro

225 230 235 240

Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val

245 250 255

Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly

260 265 270

Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp

275 280 285

Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp

290 295 300

Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His

305 310 315 320

Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys

325 330

<210> 38

<211> 232

<212> PRT

<213> Artificial sequence

<400> 38

Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro

1 5 10 15

Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe

20 25 30

Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val

35 40 45

Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe

50 55 60

Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro

65 70 75 80

Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr

85 90 95

Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val

100 105 110

Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala

115 120 125

Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg

130 135 140

Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly

145 150 155 160

Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro

165 170 175

Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser

180 185 190

Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln

195 200 205

Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His

210 215 220

Tyr Thr Gln Lys Ser Leu Ser Leu

225 230

<210> 39

<211> 228

<212> PRT

<213> Artificial sequence

<400> 39

Asp Lys Thr Val Glu Arg Lys Cys Cys Val Glu Cys Pro Pro Cys Pro

1 5 10 15

Ala Pro Pro Val Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro

20 25 30

Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val

35 40 45

Val Asp Val Ser His Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val

50 55 60

Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln

65 70 75 80

Phe Asn Ser Thr Phe Arg Val Val Ser Val Leu Thr Val Val His Gln

85 90 95

Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly

100 105 110

Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln Pro

115 120 125

Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr

130 135 140

Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser

145 150 155 160

Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr

165 170 175

Lys Thr Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr

180 185 190

Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe

195 200 205

Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys

210 215 220

Ser Leu Ser Leu

225

<210> 40

<211> 231

<212> PRT

<213> Artificial sequence

<400> 40

Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro

1 5 10 15

Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys

20 25 30

Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val

35 40 45

Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr

50 55 60

Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu

65 70 75 80

Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His

85 90 95

Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys

100 105 110

Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln

115 120 125

Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met

130 135 140

Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro

145 150 155 160

Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn

165 170 175

Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu

180 185 190

Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val

195 200 205

Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln

210 215 220

Lys Ser Leu Ser Leu Ser Leu

225 230

<210> 41

<211> 112

<212> PRT

<213> Artificial sequence

<400> 41

Val Ile His Val Thr Lys Glu Val Lys Glu Val Ala Thr Leu Ser Cys

1 5 10 15

Gly His Asn Val Ser Val Glu Glu Leu Ala Gln Thr Arg Ile Tyr Trp

20 25 30

Gln Lys Glu Lys Lys Met Val Leu Thr Met Met Ser Gly Asp Met Asn

35 40 45

Ile Trp Pro Glu Tyr Lys Asn Arg Thr Ile Phe Asp Ile Thr Asn Asn

50 55 60

Leu Ser Ile Val Ile Leu Ala Leu Arg Pro Ser Asp Glu Gly Thr Tyr

65 70 75 80

Glu Cys Val Val Leu Lys Tyr Glu Lys Asp Ala Phe Lys Arg Glu His

85 90 95

Leu Ala Glu Val Thr Leu Ser Val Lys Ala Asp Phe Pro Thr Pro Ser

100 105 110

<210> 42

<211> 208

<212> PRT

<213> Artificial sequence

<400> 42

Val Ile His Val Thr Lys Glu Val Lys Glu Val Ala Thr Leu Ser Cys

1 5 10 15

Gly His Asn Val Ser Val Glu Glu Leu Ala Gln Thr Arg Ile Tyr Trp

20 25 30

Gln Lys Glu Lys Lys Met Val Leu Thr Met Met Ser Gly Asp Met Asn

35 40 45

Ile Trp Pro Glu Tyr Lys Asn Arg Thr Ile Phe Asp Ile Thr Asn Asn

50 55 60

Leu Ser Ile Val Ile Leu Ala Leu Arg Pro Ser Asp Glu Gly Thr Tyr

65 70 75 80

Glu Cys Val Val Leu Lys Tyr Glu Lys Asp Ala Phe Lys Arg Glu His

85 90 95

Leu Ala Glu Val Thr Leu Ser Val Lys Ala Asp Phe Pro Thr Pro Ser

100 105 110

Ile Ser Asp Phe Glu Ile Pro Thr Ser Asn Ile Arg Arg Ile Ile Cys

115 120 125

Ser Thr Ser Gly Gly Phe Pro Glu Pro His Leu Ser Trp Leu Glu Asn

130 135 140

Gly Glu Glu Leu Asn Ala Ile Asn Thr Thr Val Ser Gln Asp Pro Glu

145 150 155 160

Thr Glu Leu Tyr Ala Val Ser Ser Lys Leu Asp Phe Asn Met Thr Thr

165 170 175

Asn His Ser Phe Met Cys Leu Ile Lys Tyr Gly His Leu Arg Val Asn

180 185 190

Gln Thr Phe Asn Trp Asn Thr Thr Lys Gln Glu His Phe Pro Asp Asn

195 200 205

<210> 43

<211> 16

<212> PRT

<213> Artificial sequence

<400> 43

Ala Lys Thr Thr Pro Lys Leu Glu Glu Gly Glu Phe Ser Glu Ala Arg

1 5 10 15

<210> 44

<211> 17

<212> PRT

<213> Artificial sequence

<400> 44

Ala Lys Thr Thr Pro Lys Leu Glu Glu Gly Glu Phe Ser Glu Ala Arg

1 5 10 15

Val

<210> 45

<211> 9

<212> PRT

<213> Artificial sequence

<400> 45

Ala Lys Thr Thr Pro Lys Leu Gly Gly

1 5

<210> 46

<211> 10

<212> PRT

<213> Artificial sequence

<400> 46

Ser Ala Lys Thr Thr Pro Lys Leu Gly Gly

1 5 10

<210> 47

<211> 6

<212> PRT

<213> Artificial sequence

<400> 47

Ser Ala Lys Thr Thr Pro

1 5

<210> 48

<211> 6

<212> PRT

<213> Artificial sequence

<400> 48

Arg Ala Asp Ala Ala Pro

1 5

<210> 49

<211> 9

<212> PRT

<213> Artificial sequence

<400> 49

Arg Ala Asp Ala Ala Pro Thr Val Ser

1 5

<210> 50

<211> 12

<212> PRT

<213> Artificial sequence

<400> 50

Arg Ala Asp Ala Ala Ala Ala Gly Gly Pro Gly Ser

1 5 10

<210> 51

<211> 7

<212> PRT

<213> Artificial sequence

<400> 51

Arg Ala Asp Ala Ala Ala Ala

1 5

<210> 52

<211> 18

<212> PRT

<213> Artificial sequence

<400> 52

Ser Ala Lys Thr Thr Pro Lys Leu Glu Glu Gly Glu Phe Ser Glu Ala

1 5 10 15

Arg Val

<210> 53

<211> 5

<212> PRT

<213> Artificial sequence

<400> 53

Ala Asp Ala Ala Pro

1 5

<210> 54

<211> 11

<212> PRT

<213> Artificial sequence

<400> 54

Asp Ala Ala Pro Thr Val Ser Ile Phe Pro Pro

1 5 10

<210> 55

<211> 5

<212> PRT

<213> Artificial sequence

<400> 55

Thr Val Ala Ala Pro

1 5

<210> 56

<211> 12

<212> PRT

<213> Artificial sequence

<400> 56

Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro

1 5 10

<210> 57

<211> 6

<212> PRT

<213> Artificial sequence

<400> 57

Gln Pro Lys Ala Ala Pro

1 5

<210> 58

<211> 13

<212> PRT

<213> Artificial sequence

<400> 58

Gln Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro

1 5 10

<210> 59

<211> 6

<212> PRT

<213> Artificial sequence

<400> 59

Ala Lys Thr Thr Pro Pro

1 5

<210> 60

<211> 13

<212> PRT

<213> Artificial sequence

<400> 60

Ala Lys Thr Thr Pro Pro Ser Val Thr Pro Leu Ala Pro

1 5 10

<210> 61

<211> 6

<212> PRT

<213> Artificial sequence

<400> 61

Ala Lys Thr Thr Ala Pro

1 5

<210> 62

<211> 13

<212> PRT

<213> Artificial sequence

<400> 62

Ala Lys Thr Thr Ala Pro Ser Val Tyr Pro Leu Ala Pro

1 5 10

<210> 63

<211> 6

<212> PRT

<213> Artificial sequence

<400> 63

Ala Ser Thr Lys Gly Pro

1 5

<210> 64

<211> 13

<212> PRT

<213> Artificial sequence

<400> 64

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro

1 5 10

<210> 65

<211> 15

<212> PRT

<213> Artificial sequence

<400> 65

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

1 5 10 15

<210> 66

<211> 15

<212> PRT

<213> Artificial sequence

<400> 66

Gly Glu Asn Lys Val Glu Tyr Ala Pro Ala Leu Met Ala Leu Ser

1 5 10 15

<210> 67

<211> 15

<212> PRT

<213> Artificial sequence

<400> 67

Gly Pro Ala Lys Glu Leu Thr Pro Leu Lys Glu Ala Lys Val Ser

1 5 10 15

<210> 68

<211> 15

<212> PRT

<213> Artificial sequence

<400> 68

Gly His Glu Ala Ala Ala Val Met Gln Val Gln Tyr Pro Ala Ser

1 5 10 15

<210> 69

<211> 16

<212> PRT

<213> Artificial sequence

<400> 69

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala

1 5 10 15

<210> 70

<211> 19

<212> PRT

<213> Artificial sequence

<400> 70

Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Gly Gly Ser Gly Gly Gly

1 5 10 15

Gly Ser Gly

<210> 71

<211> 20

<212> PRT

<213> Artificial sequence

<400> 71

Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly

1 5 10 15

Gly Gly Gly Ser

20

<210> 72

<211> 723

<212> DNA

<213> Artificial sequence

<400> 72

ctcgaggcca ccatggagac cgacacactc ctcctgtggg tgctgctgct gtgggtgcct 60

ggctccactg gcgagattgt gctgacacag tcccccgcta ctctgagcct gagccctggc 120

gagagggcta cactgtcttg cagagcttct cagtccgtgt cttcttacct cgcttggtat 180

cagcagaagc ccggccaggc tccaagactg ctgatctatg acgcttctaa ccgcgctaca 240

ggcattcctg ctaggttcag cggcagcggc tctggcaccg acttcacact cacaattagc 300

tctcttgaac ctgaggactt cgccgtgtac tactgccagc agtctagcaa ctggcctaga 360

acattcggcc agggcactaa ggtggagatt aagagaaccg tggccgcccc cagcgtgttc 420

atcttccctc ccagcgacga gcagctgaag tctggcaccg ccagcgtggt gtgcctgctg 480

aacaacttct acccccgcga ggccaaggtg cagtggaagg tggacaacgc cctgcagagc 540

ggcaacagcc aggagagcgt gaccgagcag gactccaagg acagcaccta cagcctgagc 600

agcaccctga ccctgagcaa ggccgactac gagaagcaca aggtgtacgc ctgcgaggtg 660

acccaccagg gactgtctag ccccgtgacc aagagcttca accggggcga gtgctaagaa 720

ttc 723

<210> 73

<211> 234

<212> PRT

<213> Artificial sequence

<400> 73

Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro

1 5 10 15

Gly Ser Thr Gly Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser

20 25 30

Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser

35 40 45

Val Ser Ser Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro

50 55 60

Arg Leu Leu Ile Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala

65 70 75 80

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser

85 90 95

Ser Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Ser Ser

100 105 110

Asn Trp Pro Arg Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg

115 120 125

Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln

130 135 140

Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr

145 150 155 160

Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser

165 170 175

Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr

180 185 190

Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys

195 200 205

His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro

210 215 220

Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

225 230

<210> 74

<211> 1401

<212> DNA

<213> Artificial sequence

<400> 74

tctagagcca ccatggagac cgacaccctg ctgctgtggg tgctgctcct gtgggtgcct 60

ggctccacag gccaggtgca gctcgtggag tccggcggcg gcgtggtgca gcccggcaga 120

tccctcagac tggactgcaa ggcatccggc attacattct ctaactctgg aatgcactgg 180

gtgagacagg ctcctggcaa gggcctggaa tgggtggccg tgatttggta cgacggctct 240

aagagatact acgctgactc cgtgaagggc cggttcacaa ttagcagaga caactccaag 300

aacactctgt tcctccagat gaacagcctg agagccgagg acaccgctgt gtactactgc 360

gccaccaacg acgactactg gggccagggc accctcgtga cagtgtcttc cgcctccacc 420

aagggccctt ccgtgttccc tctggcccct tgctcccgct ccacctccga gtccaccgcc 480

gccctgggct gcctggtgaa ggactacttc cctgagcctg tgaccgtgtc ctggaactcc 540

ggcgccctga cctccggcgt gcacaccttc cctgccgtgc tgcagtcctc cggcctgtac 600

tccctgtcct ccgtggtgac cgtgccttcc tcctccctgg gcaccaagac ctacacctgc 660

aacgtggacc acaagccttc caacaccaag gtggacaagc gcgtggagtc caagtacggc 720

cctccttgcc ctccttgccc tgcccctgag ttcctgggcg gcccttccgt gttcctgttc 780

cctcctaagc ctaaggacac cctgatgatc tcccgcaccc ctgaggtgac ctgcgtggtg 840

gtggacgtgt cccaggagga ccctgaggtg cagttcaact ggtacgtgga cggcgtggag 900

gtgcacaacg ccaagaccaa gcctcgcgag gagcagttca actccaccta ccgcgtggtg 960

tccgtgctga ccgtgctgca ccaggactgg ctgaacggca aggagtacaa gtgcaaggtg 1020

tccaacaagg gcctgccttc ctccatcgag aagaccatct ccaaggccaa gggccagcct 1080

cgcgagcctc aggtgtacac cctgcctcct tcccaggagg agatgaccaa gaaccaggtg 1140

tccctgacct gcctggtgaa gggcttctac ccttccgaca tcgccgtgga gtgggagtcc 1200

aacggccagc ctgagaacaa ctacaagacc acccctcctg tgctggactc cgacggctcc 1260

ttcttcctgt actcccgcct gaccgtggac aagtcccgct ggcaggaggg caacgtgttc 1320

tcctgctccg tgatgcacga ggccctgcac aaccactaca cccagaagtc cctgtccctg 1380

tccctgggca agtaagtcga c 1401

<210> 75

<211> 460

<212> PRT

<213> Artificial sequence

<400> 75

Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro

1 5 10 15

Gly Ser Thr Gly Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val

20 25 30

Gln Pro Gly Arg Ser Leu Arg Leu Asp Cys Lys Ala Ser Gly Ile Thr

35 40 45

Phe Ser Asn Ser Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly

50 55 60

Leu Glu Trp Val Ala Val Ile Trp Tyr Asp Gly Ser Lys Arg Tyr Tyr

65 70 75 80

Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys

85 90 95

Asn Thr Leu Phe Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala

100 105 110

Val Tyr Tyr Cys Ala Thr Asn Asp Asp Tyr Trp Gly Gln Gly Thr Leu

115 120 125

Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu

130 135 140

Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys

145 150 155 160

Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser

165 170 175

Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser

180 185 190

Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser

195 200 205

Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn

210 215 220

Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro

225 230 235 240

Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe

245 250 255

Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val

260 265 270

Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe

275 280 285

Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro

290 295 300

Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr

305 310 315 320

Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val

325 330 335

Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala

340 345 350

Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln

355 360 365

Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly

370 375 380

Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro

385 390 395 400

Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser

405 410 415

Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu

420 425 430

Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His

435 440 445

Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys

450 455 460

<210> 76

<211> 723

<212> DNA

<213> Artificial sequence

<400> 76

ctcgaggcca ccatggagac cgacacactc ctcctgtggg tgctgctgct gtgggtgcct 60

ggctccactg gcgagattgt gctgacacag tcccccgcta ctctgagcct gagccctggc 120

gagagggcta cactgtcttg cagagcttct cagtccgtgt cttcttacct cgcttggtat 180

cagcagaagc ccggccaggc tccaagactg ctgatctatg acgcttctaa ccgcgctaca 240

ggcattcctg ctaggttcag cggcagcggc tctggcaccg acttcacact cacaattagc 300

tctcttgaac ctgaggactt cgccgtgtac tactgccagc agtctagcaa ctggcctaga 360

acattcggcc agggcactaa ggtggagatt aagagaaccg tggccgcccc cagcgtgttc 420

atcttccctc ccagcgacga gcagctgaag tctggcaccg ccagcgtggt gtgcctgctg 480

aacaacttct acccccgcga ggccaaggtg cagtggaagg tggacaacgc cctgcagagc 540

ggcaacagcc aggagagcgt gaccgagcag gactccaagg acagcaccta cagcctgagc 600

agcaccctga ccctgagcaa ggccgactac gagaagcaca aggtgtacgc ctgcgaggtg 660

acccaccagg gactgtctag ccccgtgacc aagagcttca accggggcga gtgctaagaa 720

ttc 723

<210> 77

<211> 234

<212> PRT

<213> Artificial sequence

<400> 77

Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro

1 5 10 15

Gly Ser Thr Gly Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser

20 25 30

Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser

35 40 45

Val Ser Ser Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro

50 55 60

Arg Leu Leu Ile Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala

65 70 75 80

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser

85 90 95

Ser Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Ser Ser

100 105 110

Asn Trp Pro Arg Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg

115 120 125

Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln

130 135 140

Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr

145 150 155 160

Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser

165 170 175

Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr

180 185 190

Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys

195 200 205

His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro

210 215 220

Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

225 230

<210> 78

<211> 1776

<212> DNA

<213> Artificial sequence

<400> 78

tctagagcca ccatggagac cgacaccctg ctgctgtggg tgctgctcct gtgggtgcct 60

ggctccacag gccaggtgca gctcgtggag tccggcggcg gcgtggtgca gcccggcaga 120

tccctcagac tggactgcaa ggcatccggc attacattct ctaactctgg aatgcactgg 180

gtgagacagg ctcctggcaa gggcctggaa tgggtggccg tgatttggta cgacggctct 240

aagagatact acgctgactc cgtgaagggc cggttcacaa ttagcagaga caactccaag 300

aacactctgt tcctccagat gaacagcctg agagccgagg acaccgctgt gtactactgc 360

gccaccaacg acgactactg gggccagggc accctcgtga cagtgtcttc cgcctccacc 420

aagggccctt ccgtgttccc tctggcccct tgctcccgct ccacctccga gtccaccgcc 480

gccctgggct gcctggtgaa ggactacttc cctgagcctg tgaccgtgtc ctggaactcc 540

ggcgccctga cctccggcgt gcacaccttc cctgccgtgc tgcagtcctc cggcctgtac 600

tccctgtcct ccgtggtgac cgtgccttcc tcctccctgg gcaccaagac ctacacctgc 660

aacgtggacc acaagccttc caacaccaag gtggacaagc gcgtggagtc caagtacggc 720

cctccttgcc ctccttgccc tgcccctgag ttcctgggcg gcccttccgt gttcctgttc 780

cctcctaagc ctaaggacac cctgatgatc tcccgcaccc ctgaggtgac ctgcgtggtg 840

gtggacgtgt cccaggagga ccctgaggtg cagttcaact ggtacgtgga cggcgtggag 900

gtgcacaacg ccaagaccaa gcctcgcgag gagcagttca actccaccta ccgcgtggtg 960

tccgtgctga ccgtgctgca ccaggactgg ctgaacggca aggagtacaa gtgcaaggtg 1020

tccaacaagg gcctgccttc ctccatcgag aagaccatct ccaaggccaa gggccagcct 1080

cgcgagcctc aggtgtacac cctgcctcct tcccaggagg agatgaccaa gaaccaggtg 1140

tccctgacct gcctggtgaa gggcttctac ccttccgaca tcgccgtgga gtgggagtcc 1200

aacggccagc ctgagaacaa ctacaagacc acccctcctg tgctggactc cgacggctcc 1260

ttcttcctgt actcccgcct gaccgtggac aagtcccgct ggcaggaggg caacgtgttc 1320

tcctgctccg tgatgcacga ggccctgcac aaccactaca cccagaagtc cctgtccctg 1380

tccctgggcg gcggaggatc tggcggcgga ggcagtggag gcggcggaag tgtgatccat 1440

gtaacaaaag aagtgaagga agtggctaca ctctcttgcg gccacaacgt gtccgtggag 1500

gaactagctc agacccggat ctattggcag aaagaaaaga agatggtgct gaccatgatg 1560

tccggcgaca tgaacatttg gccagagtac aagaaccgca caattttcga cattacaaac 1620

aacctctcta ttgtgattct ggctctcagg cctagcgacg agggcacata cgagtgcgtg 1680

gtgctcaagt acgagaagga cgctttcaag cgggagcacc tcgctgaggt gaccctgtcc 1740

gtgaaggccg acttccctac tccatcttaa gtcgac 1776

<210> 79

<211> 585

<212> PRT

<213> Artificial sequence

<400> 79

Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro

1 5 10 15

Gly Ser Thr Gly Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val

20 25 30

Gln Pro Gly Arg Ser Leu Arg Leu Asp Cys Lys Ala Ser Gly Ile Thr

35 40 45

Phe Ser Asn Ser Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly

50 55 60

Leu Glu Trp Val Ala Val Ile Trp Tyr Asp Gly Ser Lys Arg Tyr Tyr

65 70 75 80

Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys

85 90 95

Asn Thr Leu Phe Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala

100 105 110

Val Tyr Tyr Cys Ala Thr Asn Asp Asp Tyr Trp Gly Gln Gly Thr Leu

115 120 125

Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu

130 135 140

Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys

145 150 155 160

Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser

165 170 175

Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser

180 185 190

Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser

195 200 205

Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn

210 215 220

Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro

225 230 235 240

Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe

245 250 255

Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val

260 265 270

Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe

275 280 285

Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro

290 295 300

Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr

305 310 315 320

Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val

325 330 335

Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala

340 345 350

Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln

355 360 365

Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly

370 375 380

Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro

385 390 395 400

Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser

405 410 415

Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu

420 425 430

Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His

435 440 445

Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Gly Gly Gly Ser Gly

450 455 460

Gly Gly Gly Ser Gly Gly Gly Gly Ser Val Ile His Val Thr Lys Glu

465 470 475 480

Val Lys Glu Val Ala Thr Leu Ser Cys Gly His Asn Val Ser Val Glu

485 490 495

Glu Leu Ala Gln Thr Arg Ile Tyr Trp Gln Lys Glu Lys Lys Met Val

500 505 510

Leu Thr Met Met Ser Gly Asp Met Asn Ile Trp Pro Glu Tyr Lys Asn

515 520 525

Arg Thr Ile Phe Asp Ile Thr Asn Asn Leu Ser Ile Val Ile Leu Ala

530 535 540

Leu Arg Pro Ser Asp Glu Gly Thr Tyr Glu Cys Val Val Leu Lys Tyr

545 550 555 560

Glu Lys Asp Ala Phe Lys Arg Glu His Leu Ala Glu Val Thr Leu Ser

565 570 575

Val Lys Ala Asp Phe Pro Thr Pro Ser

580 585

<210> 80

<211> 786

<212> DNA

<213> Artificial sequence

<400> 80

ctcgaggcca ccatggagac cgacacactc ctcctgtggg tgctgctgct gtgggtgcct 60

ggctccactg gcgagatcaa gcggaccgtg gccgccccat ccgtgttcat tttcccacct 120

tccgagattg tgctgacaca gtcccccgct actctgagcc tgagccctgg cgagagggct 180

acactgtctt gcagagcttc caagggcgtg agcacatccg gctactccta cctccactgg 240

tatcagcaga agccaggcca ggccccaaga ctgctgatat acctcgcttc ttacttagag 300

tctggcgtgc ccgctcggtt cagcggctcc ggctctggca ccgacttcac cctgacaatt 360

tctagcctgg agcccgagga cttcgccgtg tactactgcc agcactctag ggacctgcct 420

ctcacattcg gcggcggcac taaggtggag attaagagaa ccgtggccgc ccccagcgtg 480

ttcatcttcc ctcccagcga cgagcagctg aagtctggca ccgccagcgt ggtgtgcctg 540

ctgaacaact tctacccccg cgaggccaag gtgcagtgga aggtggacaa cgccctgcag 600

agcggcaaca gccaggagag cgtgaccgag caggactcca aggacagcac ctacagcctg 660

agcagcaccc tgaccctgag caaggccgac tacgagaagc acaaggtgta cgcctgcgag 720

gtgacccacc agggactgtc tagccccgtg accaagagct tcaaccgggg cgagtgctaa 780

gaattc 786

<210> 81

<211> 238

<212> PRT

<213> Artificial sequence

<400> 81

Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro

1 5 10 15

Gly Ser Thr Gly Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser

20 25 30

Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Lys Gly

35 40 45

Val Ser Thr Ser Gly Tyr Ser Tyr Leu His Trp Tyr Gln Gln Lys Pro

50 55 60

Gly Gln Ala Pro Arg Leu Leu Ile Tyr Leu Ala Ser Tyr Leu Glu Ser

65 70 75 80

Gly Val Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr

85 90 95

Leu Thr Ile Ser Ser Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys

100 105 110

Gln His Ser Arg Asp Leu Pro Leu Thr Phe Gly Gly Gly Thr Lys Val

115 120 125

Glu Ile Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro

130 135 140

Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu

145 150 155 160

Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn

165 170 175

Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser

180 185 190

Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala

195 200 205

Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly

210 215 220

Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

225 230 235

<210> 82

<211> 2082

<212> DNA

<213> Artificial sequence

<400> 82

tctagagcca ccatggagac cgacaccctg ctgctgtggg tgctgctcct gtgggtgcct 60

ggctccacag gccaggtgca gctcgtgcag tctggcgtgg aggtgaagaa gcctggcgcc 120

tctgtgaagg tgtcttgcaa ggcttccggc tacactttca ctaactacta catgtactgg 180

gtgagacagg ctcccggcca gggcctagag tggatgggcg gcattaaccc tagcaacggc 240

ggcacaaact tcaacgagaa gttcaagaac cgcgtgaccc tgaccacaga ctctagcaca 300

acaactgctt acatggagct gaagtctctc cagttcgacg acaccgctgt gtactactgc 360

gctcggaggg actacagatt cgacatgggc ttcgactact ggggccaggg caccactgtg 420

acagtgtcta cagcctccac caagggccct tccgtgttcc ctctggcccc ttgctcccgc 480

tccacctccg agtccaccgc cgccctgggc tgcctggtga aggactactt ccctgagcct 540

gtgaccgtgt cctggaactc cggcgccctg acctccggcg tgcacacctt ccctgccgtg 600

ctgcagtcct ccggcctgta ctccctgtcc tccgtggtga ccgtgccttc ctccaacttc 660

ggcacccaga catacacatg caacgtggac cacaagcctt ctaacacaaa ggtggacaag 720

accgtggagc ggaagtgctg cgtggagtgc ccaccttgcc ccgctcctcc tgtggccggc 780

ccttctgtgt tcctgttccc acctaagcca aaggacacac tcatgatcag cagaacccct 840

gaggtgacct gcgtggtggt ggacgtgagc cacgaggacc ccgaggtgca gttcaactgg 900

tatgtggacg gcgtggaggt gcacaacgct aagaccaagc ctagagaaga acagttcaac 960

agcacattca gagtggtgtc cgtgctcacc gtggtgcacc aggactggct gaacggcaaa 1020

gagtacaagt gcaaggtgtc caacaagggc ctgccagccc ctatcgaaaa aacaatcagc 1080

aagaccaagg gccagcctag agagcctcag gtgtacacac tgcctccatc tcgggaagaa 1140

atgacaaaga accaggtgtc cctcacatgc ctcgtgaagg gcttctaccc atccgacatc 1200

gctgtggagt gggagtctaa cggccagccc gagaacaact acaagaccac ccctcctatg 1260

ctcgactccg acggctcttt cttcctgtac tctaagctga ccgtggacaa gtccagatgg 1320

cagcagggca acgtgttctc ttgcagcgtg atgcacgagg ctctccacaa ccactacacc 1380

cagaagtccc tgagcctgtc tccaggcggc ggaggatctg gcggcggagg cagtggaggc 1440

ggcggaagcg tcattcacgt cactaaggag gtcaaggagg tcgcaaccct cagttgcgga 1500

cacaacgtca gcgtggagga gcttgcacag acacgcatct actggcagaa ggagaagaag 1560

atggtgctga ccatgatgtc cggcgatatg aacatttggc cagagtacaa gaatcggacc 1620

atcttcgata ttacaaataa cctgtccatc gtgatcctcg ctctgcgccc tagcgacgag 1680

ggaacatacg agtgtgtggt gctgaagtac gagaaggatg catttaagcg cgagcacctg 1740

gctgaggtga cactctccgt caaggccgat tttcctactc cttctatctc cgactttgag 1800

attccaacat caaatattag gcgcattatc tgttctacat ccggcggatt cccagagccc 1860

cacctctctt ggttggagaa cggcgaggaa cttaatgcta tcaatacaac cgtgtctcaa 1920

gatcccgaga ctgagctgta cgccgtgtct agtaagctgg actttaacat gactaccaat 1980

cacagtttca tgtgcctgat taagtacggc cacctgcggg tgaatcagac ctttaattgg 2040

aatactacca agcaggagca cttcccagat aactaagtcg ac 2082

<210> 83

<211> 687

<212> PRT

<213> Artificial sequence

<400> 83

Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro

1 5 10 15

Gly Ser Thr Gly Gln Val Gln Leu Val Gln Ser Gly Val Glu Val Lys

20 25 30

Lys Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr

35 40 45

Phe Thr Asn Tyr Tyr Met Tyr Trp Val Arg Gln Ala Pro Gly Gln Gly

50 55 60

Leu Glu Trp Met Gly Gly Ile Asn Pro Ser Asn Gly Gly Thr Asn Phe

65 70 75 80

Asn Glu Lys Phe Lys Asn Arg Val Thr Leu Thr Thr Asp Ser Ser Thr

85 90 95

Thr Thr Ala Tyr Met Glu Leu Lys Ser Leu Gln Phe Asp Asp Thr Ala

100 105 110

Val Tyr Tyr Cys Ala Arg Arg Asp Tyr Arg Phe Asp Met Gly Phe Asp

115 120 125

Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Thr Ala Ser Thr Lys

130 135 140

Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu

145 150 155 160

Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro

165 170 175

Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr

180 185 190

Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val

195 200 205

Val Thr Val Pro Ser Ser Asn Phe Gly Thr Gln Thr Tyr Thr Cys Asn

210 215 220

Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys Thr Val Glu Arg

225 230 235 240

Lys Cys Cys Val Glu Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly

245 250 255

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

260 265 270

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

275 280 285

Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

290 295 300

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg

305 310 315 320

Val Val Ser Val Leu Thr Val Val His Gln Asp Trp Leu Asn Gly Lys

325 330 335

Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ala Pro Ile Glu

340 345 350

Lys Thr Ile Ser Lys Thr Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

355 360 365

Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu

370 375 380

Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

385 390 395 400

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Met

405 410 415

Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp

420 425 430

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

435 440 445

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

450 455 460

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Val

465 470 475 480

Ile His Val Thr Lys Glu Val Lys Glu Val Ala Thr Leu Ser Cys Gly

485 490 495

His Asn Val Ser Val Glu Glu Leu Ala Gln Thr Arg Ile Tyr Trp Gln

500 505 510

Lys Glu Lys Lys Met Val Leu Thr Met Met Ser Gly Asp Met Asn Ile

515 520 525

Trp Pro Glu Tyr Lys Asn Arg Thr Ile Phe Asp Ile Thr Asn Asn Leu

530 535 540

Ser Ile Val Ile Leu Ala Leu Arg Pro Ser Asp Glu Gly Thr Tyr Glu

545 550 555 560

Cys Val Val Leu Lys Tyr Glu Lys Asp Ala Phe Lys Arg Glu His Leu

565 570 575

Ala Glu Val Thr Leu Ser Val Lys Ala Asp Phe Pro Thr Pro Ser Ile

580 585 590

Ser Asp Phe Glu Ile Pro Thr Ser Asn Ile Arg Arg Ile Ile Cys Ser

595 600 605

Thr Ser Gly Gly Phe Pro Glu Pro His Leu Ser Trp Leu Glu Asn Gly

610 615 620

Glu Glu Leu Asn Ala Ile Asn Thr Thr Val Ser Gln Asp Pro Glu Thr

625 630 635 640

Glu Leu Tyr Ala Val Ser Ser Lys Leu Asp Phe Asn Met Thr Thr Asn

645 650 655

His Ser Phe Met Cys Leu Ile Lys Tyr Gly His Leu Arg Val Asn Gln

660 665 670

Thr Phe Asn Trp Asn Thr Thr Lys Gln Glu His Phe Pro Asp Asn

675 680 685

<210> 84

<211> 2232

<212> DNA

<213> Artificial sequence

<400> 84

tctagagcca ccatggagac cgacacactc ctcctgtggg tgctgctgct gtgggtgcct 60

ggctccactg gcgtcatcca tgtgaccaaa gaggtcaaag aagtcgcaac tctgtcttgc 120

ggacacaacg tctccgtcga agaactcgcc cagactagaa tatattggca gaaggaaaag 180

aagatggtgc tcaccatgat gtccggcgat atgaacattt ggcctgagta caagaaccgg 240

acaatcttcg atattactaa taacctgagt atcgtgattc tcgctctgcg ccctagcgac 300

gagggcacat acgagtgtgt ggtgctgaag tacgagaagg atgcattcaa gcgggagcac 360

ctcgcagagg tgacactctc cgtgaaggcc gacttcccaa ccccatctgg ccagggcact 420

aaggtggaga ttaagagagg cggctctggc ggcggaggca gtggagagat tgtgctgaca 480

cagtcccccg ctactctgag cctgagccct ggcgagaggg ctacactgtc ttgcagagct 540

tctcagtccg tgtcttctta cctcgcttgg tatcagcaga agcccggcca ggctccaaga 600

ctgctgatct atgacgcttc taaccgcgct acaggcattc ctgctaggtt cagcggcagc 660

ggctctggca ccgacttcac actcacaatt agctctcttg aacctgagga cttcgccgtg 720

tactactgcc agcagtctag caactggcct agaacattcg gccagggcac taaggtggag 780

attaagagaa ccgtggccgc ccccagcgtg ttcatcttcc ctcccagcga cgagcagctg 840

aagtctggca ccgccagcgt ggtgtgcctg ctgaacaact tctacccccg cgaggccaag 900

gtgcagtgga aggtggacaa cgccctgcag agcggcaaca gccaggagag cgtgaccgag 960

caggactcca aggacagcac ctacagcctg agcagcaccc tgaccctgag caaggccgac 1020

tacgagaagc acaaggtgta cgcctgcgag gtgacccacc agggactgtc tagccccgtg 1080

accaagagct tcaaccgggg cgagtgctaa gtcgactcta gagccaccat ggagaccgac 1140

acactcctcc tgtgggtgct gctgctgtgg gtgcctggct ccactggcgt catccatgtg 1200

accaaagagg tcaaagaagt cgcaactctg tcttgcggac acaacgtctc cgtcgaagaa 1260

ctcgcccaga ctagaatata ttggcagaag gaaaagaaga tggtgctcac catgatgtcc 1320

ggcgatatga acatttggcc tgagtacaag aaccggacaa tcttcgatat tactaataac 1380

ctgagtatcg tgattctcgc tctgcgccct agcgacgagg gcacatacga gtgtgtggtg 1440

ctgaagtacg agaaggatgc attcaagcgg gagcacctcg cagaggtgac actctccgtg 1500

aaggccgact tcccaacccc atctggccag ggcactaagg tggagattaa gagaggcggc 1560

tctggcggcg gaggcagtgg agagattgtg ctgacacagt cccccgctac tctgagcctg 1620

agccctggcg agagggctac actgtcttgc agagcttctc agtccgtgtc ttcttacctc 1680

gcttggtatc agcagaagcc cggccaggct ccaagactgc tgatctatga cgcttctaac 1740

cgcgctacag gcattcctgc taggttcagc ggcagcggct ctggcaccga cttcacactc 1800

acaattagct ctcttgaacc tgaggacttc gccgtgtact actgccagca gtctagcaac 1860

tggcctagaa cattcggcca gggcactaag gtggagatta agagaaccgt ggccgccccc 1920

agcgtgttca tcttccctcc cagcgacgag cagctgaagt ctggcaccgc cagcgtggtg 1980

tgcctgctga acaacttcta cccccgcgag gccaaggtgc agtggaaggt ggacaacgcc 2040

ctgcagagcg gcaacagcca ggagagcgtg accgagcagg actccaagga cagcacctac 2100

agcctgagca gcaccctgac cctgagcaag gccgactacg agaagcacaa ggtgtacgcc 2160

tgcgaggtga cccaccaggg actgtctagc cccgtgacca agagcttcaa ccggggcgag 2220

tgctaagtcg ac 2232

<210> 85

<211> 365

<212> PRT

<213> Artificial sequence

<400> 85

Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro

1 5 10 15

Gly Ser Thr Gly Val Ile His Val Thr Lys Glu Val Lys Glu Val Ala

20 25 30

Thr Leu Ser Cys Gly His Asn Val Ser Val Glu Glu Leu Ala Gln Thr

35 40 45

Arg Ile Tyr Trp Gln Lys Glu Lys Lys Met Val Leu Thr Met Met Ser

50 55 60

Gly Asp Met Asn Ile Trp Pro Glu Tyr Lys Asn Arg Thr Ile Phe Asp

65 70 75 80

Ile Thr Asn Asn Leu Ser Ile Val Ile Leu Ala Leu Arg Pro Ser Asp

85 90 95

Glu Gly Thr Tyr Glu Cys Val Val Leu Lys Tyr Glu Lys Asp Ala Phe

100 105 110

Lys Arg Glu His Leu Ala Glu Val Thr Leu Ser Val Lys Ala Asp Phe

115 120 125

Pro Thr Pro Ser Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Gly Gly

130 135 140

Ser Gly Gly Gly Gly Ser Gly Glu Ile Val Leu Thr Gln Ser Pro Ala

145 150 155 160

Thr Leu Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala

165 170 175

Ser Gln Ser Val Ser Ser Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly

180 185 190

Gln Ala Pro Arg Leu Leu Ile Tyr Asp Ala Ser Asn Arg Ala Thr Gly

195 200 205

Ile Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu

210 215 220

Thr Ile Ser Ser Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln

225 230 235 240

Gln Ser Ser Asn Trp Pro Arg Thr Phe Gly Gln Gly Thr Lys Val Glu

245 250 255

Ile Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser

260 265 270

Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn

275 280 285

Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala

290 295 300

Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys

305 310 315 320

Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp

325 330 335

Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu

340 345 350

Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

355 360 365

<210> 86

<211> 1797

<212> DNA

<213> Artificial sequence

<400> 86

ctcgaggcca ccatggagac cgacacactc ctcctgtggg tgctgctgct gtgggtgcct 60

ggctccactg gcgtcatcca tgtgaccaaa gaggtcaaag aagtcgcaac tctgtcttgc 120

ggacacaacg tctccgtcga agaactcgcc cagactagaa tatattggca gaaggaaaag 180

aagatggtgc tcaccatgat gtccggcgat atgaacattt ggcctgagta caagaaccgg 240

acaatcttcg atattactaa taacctgagt atcgtgattc tcgctctgcg ccctagcgac 300

gagggcacat acgagtgtgt ggtgctgaag tacgagaagg atgcattcaa gcgggagcac 360

ctcgcagagg tgacactctc cgtgaaggcc gacttcccaa ccccatctgg ccagggcaca 420

ctcgtgaccg tgtctagcgg cggcggaggc agtggcggcg gaggcagtca ggtgcagctc 480

gtggagtccg gcggcggcgt ggtgcagccc ggcagatccc tcagactgga ctgcaaggca 540

tccggcatta cattctctaa ctctggaatg cactgggtga gacaggctcc tggcaagggc 600

ctggaatggg tggccgtgat ttggtacgac ggctctaaga gatactacgc tgactccgtg 660

aagggccggt tcacaattag cagagacaac tccaagaaca ctctgttcct ccagatgaac 720

agcctgagag ccgaggacac cgctgtgtac tactgcgcca ccaacgacga ctactggggc 780

cagggcaccc tcgtgacagt gtcttccgcc tccaccaagg gcccttccgt gttccctctg 840

gccccttgct cccgctccac ctccgagtcc accgccgccc tgggctgcct ggtgaaggac 900

tacttccctg agcctgtgac cgtgtcctgg aactccggcg ccctgacctc cggcgtgcac 960

accttccctg ccgtgctgca gtcctccggc ctgtactccc tgtcctccgt ggtgaccgtg 1020

ccttcctcct ccctgggcac caagacctac acctgcaacg tggaccacaa gccttccaac 1080

accaaggtgg acaagcgcgt ggagtccaag tacggccctc cttgccctcc ttgccctgcc 1140

cctgagttcc tgggcggccc ttccgtgttc ctgttccctc ctaagcctaa ggacaccctg 1200

atgatctccc gcacccctga ggtgacctgc gtggtggtgg acgtgtccca ggaggaccct 1260

gaggtgcagt tcaactggta cgtggacggc gtggaggtgc acaacgccaa gaccaagcct 1320

cgcgaggagc agttcaactc cacctaccgc gtggtgtccg tgctgaccgt gctgcaccag 1380

gactggctga acggcaagga gtacaagtgc aaggtgtcca acaagggcct gccttcctcc 1440

atcgagaaga ccatctccaa ggccaagggc cagcctcgcg agcctcaggt gtacaccctg 1500

cctccttccc aggaggagat gaccaagaac caggtgtccc tgacctgcct ggtgaagggc 1560

ttctaccctt ccgacatcgc cgtggagtgg gagtccaacg gccagcctga gaacaactac 1620

aagaccaccc ctcctgtgct ggactccgac ggctccttct tcctgtactc ccgcctgacc 1680

gtggacaagt cccgctggca ggagggcaac gtgttctcct gctccgtgat gcacgaggcc 1740

ctgcacaacc actacaccca gaagtccctg tccctgtccc tgggcaagta agaattc 1797

<210> 87

<211> 592

<212> PRT

<213> Artificial sequence

<400> 87

Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro

1 5 10 15

Gly Ser Thr Gly Val Ile His Val Thr Lys Glu Val Lys Glu Val Ala

20 25 30

Thr Leu Ser Cys Gly His Asn Val Ser Val Glu Glu Leu Ala Gln Thr

35 40 45

Arg Ile Tyr Trp Gln Lys Glu Lys Lys Met Val Leu Thr Met Met Ser

50 55 60

Gly Asp Met Asn Ile Trp Pro Glu Tyr Lys Asn Arg Thr Ile Phe Asp

65 70 75 80

Ile Thr Asn Asn Leu Ser Ile Val Ile Leu Ala Leu Arg Pro Ser Asp

85 90 95

Glu Gly Thr Tyr Glu Cys Val Val Leu Lys Tyr Glu Lys Asp Ala Phe

100 105 110

Lys Arg Glu His Leu Ala Glu Val Thr Leu Ser Val Lys Ala Asp Phe

115 120 125

Pro Thr Pro Ser Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly

130 135 140

Gly Gly Ser Gly Gly Gly Gly Ser Gln Val Gln Leu Val Glu Ser Gly

145 150 155 160

Gly Gly Val Val Gln Pro Gly Arg Ser Leu Arg Leu Asp Cys Lys Ala

165 170 175

Ser Gly Ile Thr Phe Ser Asn Ser Gly Met His Trp Val Arg Gln Ala

180 185 190

Pro Gly Lys Gly Leu Glu Trp Val Ala Val Ile Trp Tyr Asp Gly Ser

195 200 205

Lys Arg Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg

210 215 220

Asp Asn Ser Lys Asn Thr Leu Phe Leu Gln Met Asn Ser Leu Arg Ala

225 230 235 240

Glu Asp Thr Ala Val Tyr Tyr Cys Ala Thr Asn Asp Asp Tyr Trp Gly

245 250 255

Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser

260 265 270

Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala

275 280 285

Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val

290 295 300

Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala

305 310 315 320

Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val

325 330 335

Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His

340 345 350

Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly

355 360 365

Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser

370 375 380

Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg

385 390 395 400

Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro

405 410 415

Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala

420 425 430

Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val

435 440 445

Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr

450 455 460

Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr

465 470 475 480

Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu

485 490 495

Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys

500 505 510

Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser

515 520 525

Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp

530 535 540

Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser

545 550 555 560

Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala

565 570 575

Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys

580 585 590

<210> 88

<211> 1797

<212> DNA

<213> Artificial sequence

<400> 88

ctcgaggcca ccatggagac cgacacactc ctcctgtggg tgctgctgct gtgggtgcct 60

ggctccactg gcgtcatcca tgtgaccaaa gaggtcaaag aagtcgcaac tctgtcttgc 120

ggacacaacg tctccgtcga agaactcgcc cagactagaa tatattggca gaaggaaaag 180

aagatggtgc tcaccatgat gtccggcgat atgaacattt ggcctgagta caagaaccgg 240

acaatcttcg atattactaa taacctgagt atcgtgattc tcgctctgcg ccctagcgac 300

gagggcacat acgagtgtgt ggtgctgaag tacgagaagg atgcattcaa gcgggagcac 360

ctcgcagagg tgacactctc cgtgaaggcc gacttcccaa ccccatctga caagcgcgtg 420

gagtccaagt acggccctcc ttgccctcct tgccctgccc ctgagttcct gggcggccct 480

tccgtgttcc tgttccctcc taagcctaag gacaccctga tgatctcccg cacccctgag 540

gtgacctgcg tggtggtgga cgtgtcccag gaggaccctg aggtgcagtt caactggtac 600

gtggacggcg tggaggtgca caacgccaag accaagcctc gcgaggagca gttcaactcc 660

acctaccgcg tggtgtccgt gctgaccgtg ctgcaccagg actggctgaa cggcaaggag 720

tacaagtgca aggtgtccaa caagggcctg ccttcctcca tcgagaagac catctccaag 780

gccaagggcc agcctcgcga gcctcaggtg tacaccctgc ctccttccca ggaggagatg 840

accaagaacc aggtgtccct gacctgcctg gtgaagggct tctacccttc cgacatcgcc 900

gtggagtggg agtccaacgg ccagcctgag aacaactaca agaccacccc tcctgtgctg 960

gactccgacg gctccttctt cctgtactcc cgcctgaccg tggacaagtc ccgctggcag 1020

gagggcaacg tgttctcctg ctccgtgatg cacgaggccc tgcacaacca ctacacccag 1080

aagtccctgt ccctgtccct gggcggcgga ggatctggcg gcggaggcag tggaggcggc 1140

ggaagtgaga ttgtgctgac acagtccccc gctactctga gcctgagccc tggcgagagg 1200

gctacactgt cttgcagagc ttctcagtcc gtgtcttctt acctcgcttg gtatcagcag 1260

aagcccggcc aggctccaag actgctgatc tatgacgctt ctaaccgcgc tacaggcatt 1320

cctgctaggt tcagcggcag cggctctggc accgacttca cactcacaat tagctctctt 1380

gaacctgagg acttcgccgt gtactactgc cagcagtcta gcaactggcc tagaacattc 1440

ggccagggca ctaaggtgga gattaagaga accgtggccg cccccagcgt gttcatcttc 1500

cctcccagcg acgagcagct gaagtctggc accgccagcg tggtgtgcct gctgaacaac 1560

ttctaccccc gcgaggccaa ggtgcagtgg aaggtggaca acgccctgca gagcggcaac 1620

agccaggaga gcgtgaccga gcaggactcc aaggacagca cctacagcct gagcagcacc 1680

ctgaccctga gcaaggccga ctacgagaag cacaaggtgt acgcctgcga ggtgacccac 1740

cagggactgt ctagccccgt gaccaagagc ttcaaccggg gcgagtgcta agaattc 1797

<210> 89

<211> 592

<212> PRT

<213> Artificial sequence

<400> 89

Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro

1 5 10 15

Gly Ser Thr Gly Val Ile His Val Thr Lys Glu Val Lys Glu Val Ala

20 25 30

Thr Leu Ser Cys Gly His Asn Val Ser Val Glu Glu Leu Ala Gln Thr

35 40 45

Arg Ile Tyr Trp Gln Lys Glu Lys Lys Met Val Leu Thr Met Met Ser

50 55 60

Gly Asp Met Asn Ile Trp Pro Glu Tyr Lys Asn Arg Thr Ile Phe Asp

65 70 75 80

Ile Thr Asn Asn Leu Ser Ile Val Ile Leu Ala Leu Arg Pro Ser Asp

85 90 95

Glu Gly Thr Tyr Glu Cys Val Val Leu Lys Tyr Glu Lys Asp Ala Phe

100 105 110

Lys Arg Glu His Leu Ala Glu Val Thr Leu Ser Val Lys Ala Asp Phe

115 120 125

Pro Thr Pro Ser Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys

130 135 140

Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu

145 150 155 160

Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu

165 170 175

Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln

180 185 190

Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys

195 200 205

Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu

210 215 220

Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys

225 230 235 240

Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys

245 250 255

Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser

260 265 270

Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys

275 280 285

Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln

290 295 300

Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly

305 310 315 320

Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln

325 330 335

Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn

340 345 350

His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Gly Gly Gly Ser

355 360 365

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Ile Val Leu Thr Gln

370 375 380

Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser

385 390 395 400

Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr Leu Ala Trp Tyr Gln Gln

405 410 415

Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile Tyr Asp Ala Ser Asn Arg

420 425 430

Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp

435 440 445

Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro Glu Asp Phe Ala Val Tyr

450 455 460

Tyr Cys Gln Gln Ser Ser Asn Trp Pro Arg Thr Phe Gly Gln Gly Thr

465 470 475 480

Lys Val Glu Ile Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe

485 490 495

Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys

500 505 510

Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val

515 520 525

Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln

530 535 540

Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser

545 550 555 560

Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His

565 570 575

Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

580 585 590

<210> 90

<211> 720

<212> DNA

<213> Artificial sequence

<400> 90

tctagagcca ccatggagac cgacaccctg ctgctgtggg tgctgctcct gtgggtgcct 60

ggctccacag gccaggtgca gctcgtggag tccggcggcg gcgtggtgca gcccggcaga 120

tccctcagac tggactgcaa ggcatccggc attacattct ctaactctgg aatgcactgg 180

gtgagacagg ctcctggcaa gggcctggaa tgggtggccg tgatttggta cgacggctct 240

aagagatact acgctgactc cgtgaagggc cggttcacaa ttagcagaga caactccaag 300

aacactctgt tcctccagat gaacagcctg agagccgagg acaccgctgt gtactactgc 360

gccaccaacg acgactactg gggccagggc accctcgtga cagtgtcttc cgcctccacc 420

aagggccctt ccgtgttccc tctggcccct tgctcccgct ccacctccga gtccaccgcc 480

gccctgggct gcctggtgaa ggactacttc cctgagcctg tgaccgtgtc ctggaactcc 540

ggcgccctga cctccggcgt gcacaccttc cctgccgtgc tgcagtcctc cggcctgtac 600

tccctgtcct ccgtggtgac cgtgccttcc tcctccctgg gcaccaagac ctacacctgc 660

aacgtggacc acaagccttc caacaccaag gtggacaagc gcgtggagtc ctaagtcgac 720

<210> 91

<211> 233

<212> PRT

<213> Artificial sequence

<400> 91

Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro

1 5 10 15

Gly Ser Thr Gly Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val

20 25 30

Gln Pro Gly Arg Ser Leu Arg Leu Asp Cys Lys Ala Ser Gly Ile Thr

35 40 45

Phe Ser Asn Ser Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly

50 55 60

Leu Glu Trp Val Ala Val Ile Trp Tyr Asp Gly Ser Lys Arg Tyr Tyr

65 70 75 80

Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys

85 90 95

Asn Thr Leu Phe Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala

100 105 110

Val Tyr Tyr Cys Ala Thr Asn Asp Asp Tyr Trp Gly Gln Gly Thr Leu

115 120 125

Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu

130 135 140

Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys

145 150 155 160

Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser

165 170 175

Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser

180 185 190

Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser

195 200 205

Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn

210 215 220

Thr Lys Val Asp Lys Arg Val Glu Ser

225 230

<210> 92

<211> 724

<212> DNA

<213> Artificial sequence

<400> 92

tctagagcca ccaatggaga ccgacaccct gctgctgtgg gtgctgctcc tgtgggtgcc 60

tggctccaca ggcgagattg tgctgacaca gtcccccgct actctgagcc tgagccctgg 120

cgagagggct acactgtctt gcagagcttc tcagtccgtg tcttcttacc tcgcttggta 180

tcagcagaag cccggccagg ctccaagact gctgatctat gacgcttcta accgcgctac 240

aggcattcct gctaggttca gcggcagcgg ctctggcacc gacttcacac tcacaattag 300

ctctcttgaa cctgaggact tcgccgtgta ctactgccag cagtctagca actggcctag 360

aacattcggc cagggcacta aggtggagat taagagaacc gtggccgccc ccagcgtgtt 420

catcttccct cccagcgacg agcagctgaa gtctggcacc gccagcgtgg tgtgcctgct 480

gaacaacttc tacccccgcg aggccaaggt gcagtggaag gtggacaacg ccctgcagag 540

cggcaacagc caggagagcg tgaccgagca ggactccaag gacagcacct acagcctgag 600

cagcaccctg accctgagca aggccgacta cgagaagcac aaggtgtacg cctgcgaggt 660

gacccaccag ggactgtcta gccccgtgac caagagcttc aaccggggcg agtgctaagt 720

cgac 724

<210> 93

<211> 234

<212> PRT

<213> Artificial sequence

<400> 93

Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro

1 5 10 15

Gly Ser Thr Gly Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser

20 25 30

Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser

35 40 45

Val Ser Ser Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro

50 55 60

Arg Leu Leu Ile Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala

65 70 75 80

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser

85 90 95

Ser Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Ser Ser

100 105 110

Asn Trp Pro Arg Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg

115 120 125

Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln

130 135 140

Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr

145 150 155 160

Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser

165 170 175

Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr

180 185 190

Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys

195 200 205

His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro

210 215 220

Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

225 230

<210> 94

<211> 1784

<212> DNA

<213> Artificial sequence

<400> 94

ctcgaggcca ccatggagac cgacacactc ctcctgtggg tgctgctgct gtgggtgcct 60

ggctccactg gcgtcatcca tgtgaccaaa gaggtcaaag aagtcgcaac tctgtcttgc 120

ggacacaacg tctccgtcga agaactcgcc cagactagaa tatattggca gaaggaaaag 180

aagatggtgc tcaccatgat gtccggcgat atgaacattt ggcctgagta caagaaccgg 240

acaatcttcg atattactaa taacctgagt atcgtgattc tcgctctgcg ccctagcgac 300

gagggcacat acgagtgtgt ggtgctgaag tacgagaagg atgcattcaa gcgggagcac 360

ctcgcagagg tgacactctc cgtgaaggcc gacttcccaa ccccatctga caagcgcgtg 420

gagtccaagt acggccctcc ttgccctcct tgccctgccc ctgagttcct gggcggccct 480

tccgtgttcc tgttccctcc taagcctaag gacaccctga tgatctcccg cacccctgag 540

gtgacctgcg tggtggtgga cgtgtcccag gaggaccctg aggtgcagtt caactggtac 600

gtggacggcg tggaggtgca caacgccaag accaagcctc gcgaggagca gttcaactcc 660

acctaccgcg tggtgtccgt gctgaccgtg ctgcaccagg actggctgaa cggcaaggag 720

tacaagtgca aggtgtccaa caagggcctg ccttcctcca tcgagaagac catctccaag 780

gccaagggcc agcctcgcga gcctcaggtg tacaccctgc ctccttccca ggaggagatg 840

accaagaacc aggtgtccct gacctgcctg gtgaagggct tctacccttc cgacatcgcc 900

gtggagtggg agtccaacgg ccagcctgag aacaactaca agaccacccc tcctgtgctg 960

gactccgacg gctccttctt cctgtactcc cgcctgaccg tggacaagtc ccgctggcag 1020

gagggcaacg tgttctcctg ctccgtgatg cacgaggccc tgcacaacca ctacacccag 1080

aagtccctgt ccctgtccct gggcggcgga ggatctggcg gcggaggcag tggaggcggc 1140

ggaagtcagg tgcagctcgt ggagtccggc ggcggcgtgg tgcagcccgg cagatccctc 1200

agactggact gcaaggcatc cggcattaca ttctctaact ctggaatgca ctgggtgaga 1260

caggctcctg gcaagggcct ggaatgggtg gccgtgattt ggtacgacgg ctctaagaga 1320

tactacgctg actccgtgaa gggccggttc acaattagca gagacaactc caagaacact 1380

ctgttcctcc agatgaacag cctgagagcc gaggacaccg ctgtgtacta ctgcgccacc 1440

aacgacgact actggggcca gggcaccctc gtgacagtgt cttccgcctc caccaagggc 1500

ccttccgtgt tccctctggc cccttgctcc cgctccacct ccgagtccac cgccgccctg 1560

ggctgcctgg tgaaggacta cttccctgag cctgtgaccg tgtcctggaa ctccggcgcc 1620

ctgacctccg gcgtgcacac cttccctgcc gtgctgcagt cctccggcct gtactccctg 1680

tcctccgtgg tgaccgtgcc ttcctcctcc ctgggcacca agacctacac ctgcaacgtg 1740

gaccacaagc cttccaacac caaggtggac aagcgcgtgg agtc 1784

<210> 95

<211> 591

<212> PRT

<213> Artificial sequence

<400> 95

Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro

1 5 10 15

Gly Ser Thr Gly Val Ile His Val Thr Lys Glu Val Lys Glu Val Ala

20 25 30

Thr Leu Ser Cys Gly His Asn Val Ser Val Glu Glu Leu Ala Gln Thr

35 40 45

Arg Ile Tyr Trp Gln Lys Glu Lys Lys Met Val Leu Thr Met Met Ser

50 55 60

Gly Asp Met Asn Ile Trp Pro Glu Tyr Lys Asn Arg Thr Ile Phe Asp

65 70 75 80

Ile Thr Asn Asn Leu Ser Ile Val Ile Leu Ala Leu Arg Pro Ser Asp

85 90 95

Glu Gly Thr Tyr Glu Cys Val Val Leu Lys Tyr Glu Lys Asp Ala Phe

100 105 110

Lys Arg Glu His Leu Ala Glu Val Thr Leu Ser Val Lys Ala Asp Phe

115 120 125

Pro Thr Pro Ser Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys

130 135 140

Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu

145 150 155 160

Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu

165 170 175

Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln

180 185 190

Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys

195 200 205

Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu

210 215 220

Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys

225 230 235 240

Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys

245 250 255

Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser

260 265 270

Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys

275 280 285

Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln

290 295 300

Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly

305 310 315 320

Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln

325 330 335

Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn

340 345 350

His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Gly Gly Gly Ser

355 360 365

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val Gln Leu Val Glu

370 375 380

Ser Gly Gly Gly Val Val Gln Pro Gly Arg Ser Leu Arg Leu Asp Cys

385 390 395 400

Lys Ala Ser Gly Ile Thr Phe Ser Asn Ser Gly Met His Trp Val Arg

405 410 415

Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Val Ile Trp Tyr Asp

420 425 430

Gly Ser Lys Arg Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile

435 440 445

Ser Arg Asp Asn Ser Lys Asn Thr Leu Phe Leu Gln Met Asn Ser Leu

450 455 460

Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Thr Asn Asp Asp Tyr

465 470 475 480

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly

485 490 495

Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser

500 505 510

Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val

515 520 525

Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe

530 535 540

Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val

545 550 555 560

Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val

565 570 575

Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser

580 585 590

Claims (32)

1. A fusion protein that blocks the PD-1/PD-L1 signaling pathway and activates T cells, comprising (i) an antigen binding fragment derived from an anti-PD-1 antibody that is Fab, Fab ', F (ab') derived from an anti-PD-1 antibody₂Fv, single chain Fv comprising SEQ ID NO: 1/2 for the heavy chain variable region sequence/the light chain variable region sequence comprises all 6 heavy chain CDRs and light chain CDRs; (ii) an immunoglobulin Fc domain; and (iii) a CD80 extracellular domain, also abbreviated as CD80ECD, which is SEQ ID NO: 41 or 42, wherein the fusion protein is from N-terminus to C-terminus in the order of (i), (ii), and (iii); (iii) the order of (i), (ii) and (iii); or the order of (iii), (ii) and (i) are operably linked and the peptide linker between said (i), (ii) and/or (iii) is selected from the group consisting of SEQ ID NO: 43-71.

2. the fusion protein of claim 1, wherein said (i) comprises the heavy chain variable region and the light chain variable region of nivolumab.

3. The fusion protein of claim 1, wherein said (ii) is a human immunoglobulin Fc domain.

4. The fusion protein of claim 3, wherein the (ii) is an Fc domain of human IgG1, IgG2, IgG3, or IgG 4.

5. The fusion protein of claim 4, wherein the (ii) polypeptide comprises the amino acid sequence of SEQ ID NO: 38. 39 or 40.

6. The fusion protein of claim 1, comprising

(a) An anti-PD-1 antibody; and a CD80ECD operably linked C-terminal to each of the two heavy chains of the antibody;

(b) an anti-PD-1 antibody; a CD80ECD operably linked at the N-terminus of each of the two heavy chains of the antibody; and a CD80ECD operably linked N-terminal to each of the two light chains of the antibody; or

(c) CD80 ECD; an immunoglobulin Fc domain in dimeric form operatively linked at the C-terminus of the CD80 ECD; and an antigen-binding fragment derived from an anti-PD-1 antibody operatively linked at the C-terminus of the immunoglobulin Fc domain in dimeric form.

7. The fusion protein of claim 6, wherein the antibody is an IgG class antibody.

8. The fusion protein of claim 7, wherein the antibody is an IgG₁Subclass, IgG₂Subclass, or IgG₄Subclass antibody.

9. The fusion protein of claim 8, wherein the antibody is an IgG₄A subclass of antibody, and comprises at position S228 in the Fc domainAnd (3) amino acid substitution.

10. The fusion protein of claim 9, wherein the antibody is an IgG₄Subclass antibody, and comprises the amino acid substitution S228P at position S228 in the Fc domain.

11. The fusion protein of claim 6, wherein the light chain type of the antibody is kappa type or lambda type.

12. The fusion protein of claim 6, wherein the anti-PD-1 antibody is nivolumab.

13. The fusion protein of claim 6 selected from

(1) A fusion protein comprising a first subunit of the fusion protein of SEQ ID NO. 77 and a second subunit of the fusion protein of SEQ ID NO. 79;

(2) a fusion protein comprising a first subunit of the fusion protein of SEQ ID NO. 81 and a second subunit of the fusion protein of SEQ ID NO. 83;

(3) a fusion protein comprising a first subunit of the fusion protein of SEQ ID NO. 85 and a second subunit of the fusion protein of SEQ ID NO. 87;

(4) a fusion protein comprising a first subunit of the fusion protein of SEQ ID NO. 89 and a second subunit of the fusion protein of SEQ ID NO. 91; or

(5) A fusion protein comprising a first subunit of the fusion protein of SEQ ID NO 93 and a second subunit of the fusion protein of SEQ ID NO 95.

14. A polynucleotide encoding the fusion protein of any one of claims 1-13.

15. A vector comprising the polynucleotide of claim 14.

16. The vector of claim 15, which is an expression vector.

17. The vector of claim 16, wherein the expression vector is a glutamine synthetase expression vector having a dual expression cassette.

18. A host cell comprising the polynucleotide of claim 14 or the vector of any one of claims 15-17.

19. The host cell of claim 18, which is a CHO, HEK293 or NSO cell.

20. A method for producing the fusion protein of any one of claims 1-13, the method comprising the steps of (i) culturing the host cell of claim 18 under conditions suitable for expression of the fusion protein, and (ii) recovering the fusion protein.

21. A pharmaceutical composition comprising the fusion protein of any one of claims 1-13 and a pharmaceutically acceptable carrier.

22. Use of the fusion protein of any one of claims 1-13, the pharmaceutical composition of claim 21, for the manufacture of a medicament for treating or preventing a cancerous disease associated with PD-1 activity, PD-L1 activity, and CD28 activity in an individual, wherein the cancerous disease is melanoma, breast cancer, colon cancer, esophageal cancer, gastrointestinal stromal tumor, kidney cancer, liver cancer, non-small cell lung cancer, ovarian cancer, pancreatic cancer, prostate cancer, head and neck tumors, stomach cancer, hematologic malignancies, and the individual is a mammal.

23. The use of claim 22, wherein the renal cancer is renal cell carcinoma; the hematological malignancy is lymphoma.

24. The use of claim 22, wherein the breast cancer is triple negative breast cancer.

25. The use of claim 22, wherein the subject is a human.

26. A diagnostic kit comprising the fusion protein of any one of claims 1-13 and optionally a label or a reagent for conjugation.

27. The diagnostic kit of claim 26, comprising the fusion protein of any one of claims 1-13 labeled with a positron emission tomography detectable label.

28. The diagnostic kit of claim 27, wherein the marker is¹⁸F-fluorodeoxyglucose.

29. Use of a diagnostic kit of any one of claims 26-28 for the manufacture of a reagent for diagnosing a cancerous disease associated with PD-1 activity, PD-L1 activity and CD28 activity in an individual, wherein the cancerous disease is melanoma, breast cancer, colon cancer, esophageal cancer, gastrointestinal stromal tumor, kidney cancer, liver cancer, non-small cell lung cancer, ovarian cancer, pancreatic cancer, prostate cancer, head and neck tumors, gastric cancer, hematological malignancies, and the individual is a mammal.

30. The use of claim 29, wherein the renal cancer is renal cell carcinoma; the hematological malignancy is lymphoma.

31. The use of claim 29, wherein the breast cancer is triple negative breast cancer.

32. The use of claim 29, wherein the subject is a human.

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