WO2018136725A1 - Protéines de liaison inductibles pour les cellules immunitaires innées et méthodes d'utilisation - Google Patents

Protéines de liaison inductibles pour les cellules immunitaires innées et méthodes d'utilisation Download PDF

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WO2018136725A1
WO2018136725A1 PCT/US2018/014396 US2018014396W WO2018136725A1 WO 2018136725 A1 WO2018136725 A1 WO 2018136725A1 US 2018014396 W US2018014396 W US 2018014396W WO 2018136725 A1 WO2018136725 A1 WO 2018136725A1
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antigen
binding
innate immune
domain
protein
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PCT/US2018/014396
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English (en)
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Patrick Baeuerle
Robert B. Dubridge
Holger Wesche
Luke Evnin
Jeanmarie Guenot
Richard J. Austin
Bryan D. LEMON
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Harpoon Therapeutics, Inc.
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Publication of WO2018136725A1 publication Critical patent/WO2018136725A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2887—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against CD20
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/283—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against Fc-receptors, e.g. CD16, CD32, CD64
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/50—Fusion polypeptide containing protease site

Definitions

  • NK natural killer
  • CTLs cytotoxic T lymphocytes
  • conditional binding protein As nucleic acids, recombinant expression vectors and host cells for making such antigen binding proteins, and methods of use for the treatment of diseases, disorders, or conditions.
  • antigen-binding proteins comprising a single polypeptide chain comprising two or more inactive innate immune cell binding domains, two or more target antigen binding domains, one or more half-life extension domains, and one or more protease cleavage domains; wherein upon protease cleavage of the protease cleavage domain and binding the target antigens by the target antigen binding domains, the innate immune cell binding domain becomes active and binds to an innate immune cell.
  • the innate immune cell binding domains bind to a cell surface antigen selected from CDlc, CD83, CD141, CD209, MHC II, CD123, CD303, CD304, CD16, CD56, CDld, CD160, PLZF, NKG2d, CD94-NKG2A/C/E , CD14, CD64, CD15, 2D7 antigen, CD203c, FcsRIa, CDl lb, CD193, EMR1, and Siglec-8 and activates an innate immune cell selected from dendritic cells, plasmacytoid dendritic cells, natural killer cells, natural killer T cells, monocytes, neutrophils, basophils, and eosinophils, wherein the innate immune cell is not a T cell.
  • a cell surface antigen selected from CDlc, CD83, CD141, CD209, MHC II, CD123, CD303, CD304, CD16, CD56, CDld, CD160, PLZF, NKG2d,
  • the target antigen binding domains bind to the same target antigen. In some embodiments, the target antigen binding domains bind to different target antigens.
  • the protease cleavage domain is cleaved by at least one of a serine protease, a cysteine protease, an aspartate protease, a threonine protease, a glutamic acid protease, a metalloproteinase, a gelatinase, and an asparagine peptide lyase.
  • the protease cleavage domain is cleaved by at least one of a Cathepsin B, a Cathepsin C, a Cathepsin D, a Cathepsin E, a Cathepsin K, a Cathepsin L, a kallikrein, a hKl, a hK10, a hK15, a plasmin, a collagenase, a Type IV collagenase, a stromelysin, a Factor Xa, a chymotryp sin-like protease, a trypsin-like protease, a elastase-like protease, a subtili sin-like protease, an actinidain, a bromelain, a calpain, a caspase, a caspase-3, a Mirl-CP, a papain, a HIV-1 prote
  • the protease cleavage domain is cleaved at the site of a tumor. In some embodiments, the protease is expressed by a cell in a microenvironment of the tumor. In some embodiments, the protease cleavage domain is cleaved in the blood of a subject. In some embodiments, the protein further comprises two or more protease cleavage domains.
  • one or more innate immune cell binding domains comprise a polypeptide derived from a single-chain variable fragment (scFv) specific to a cell surface marker on an innate immune cell, wherein the innate immune cell is not a T cell.
  • one or more innate immune cell binding domains are specific for a dendritic cell.
  • one or more innate immune cell binding domains are specific for a plasmacytoid dendritic cell.
  • one or more innate immune cell binding domains are specific for a natural killer cell.
  • one or more innate immune cell binding domains are specific for a natural killer T cell.
  • one or more innate immune cell binding domains are specific for a monocyte. In some embodiments, one or more innate immune cell binding domains are specific for a neutrophil. In some embodiments, one or more innate immune cell binding domains are specific for a basophil. In some embodiments, one or more innate immune cell binding domains are specific for an eosinophil. In some embodiments, one or more innate immune cell binding domains comprise complementary determining regions (CDRs) selected from the group consisting of Lorvotuzumab, 3C12C, CSL362, 3G8, rMil2, E4, NNC141-0100. In some embodiments, one or more innate immune cell binding domains are humanized.
  • CDRs complementary determining regions
  • one or more activated innate immune cell binding domains have a KD binding 1000 nM or less to innate immune cells. In some embodiments, one or more activated innate immune cell binding domains have a KD binding 100 nM or less to innate immune cell innate immune cells. In some embodiments, one or more activated innate immune cell binding domains have a KD binding 10 nM or less to innate immune cells. In some embodiments, one or more innate immune cell binding domains have crossreactivity with cynomolgus innate immune cells. In some embodiments, one or more innate immune cell binding domains comprise an amino acid sequence provided herein. In some embodiments, one or more half-life extension domains comprise a binding domain to human serum albumin.
  • one or more half-life extension domains comprise a scFv, a variable heavy domain (VH), a variable light domain (VL), a nanobody, a peptide, a ligand, or a small molecule.
  • one or more half-life extension domains comprise a scFv.
  • one or more half-life extension domains comprise a VH domain.
  • one or more half-life extension domains comprise a VL domain.
  • one or more half-life extension domains comprise a nanobody.
  • one or more half-life extension domains comprise a peptide.
  • one or more half-life extension domains comprise a ligand.
  • one or more half-life extension domains comprise an Fc domain.
  • at least one half-life extension domain is at the N-terminus of the protein prior to protease cleavage.
  • at least one half-life extension domain is at the C-terminus of the protein prior to protease cleavage.
  • At least one half-life extension domain is not at the C-terminus or the N-terminus of the protein prior to protease cleavage. In some embodiments, the protease cleavage domain is in the half- life extension domain or the innate immune cell binding domain. In some embodiments, the protease cleavage domain is not in the half-life extension domain or the innate immune cell binding domain.
  • the target antigen binding domains independently comprise a scFv, a VH domain, a VL domain, a non-Ig domain, or a ligand that specifically binds to the target antigen. In some embodiments, at least one target antigen binding domains specifically bind to a cell surface molecule.
  • At least one target antigen binding domains specifically bind to a tumor antigen.
  • the target antigen binding domains specifically and independently bind to an antigen selected from at least one of EpCAM, EGFR, HER-2, HER-3, cMet, CEA, and FoIR.
  • the target antigen binding domains specifically and independently bind to two different antigens, wherein at least one of the antigens is selected from one of EpCAM, EGFR, HER-2, HER-3, cMet, CEA, and FoIR.
  • the protein prior to cleavage of the protease cleavage domain is less than about 100 kDa.
  • the protein after cleavage of the protease cleavage domain is about 25 to about 75 kDa. In some embodiments, the protein prior to protease cleavage has a size that is above the renal threshold for first-pass clearance. In some embodiments, the protein prior to protease cleavage has an elimination half-time of at least about 50 hours. In some embodiments, the protein prior to protease cleavage has an elimination half-time of at least about 100 hours. In some embodiments, the protein has increased tissue penetration or tissue distribution as compared to an IgG to the same target antigen. In some embodiments, the protein has improved pharmacokinetics as compared to an IgG to the same target antigen.
  • the protein has reduced or eliminated target mediated drug disposition through innate immune cell binding as compared to an IgG to an innate immune cell.
  • the protein has a shallower alpha phase and higher exposure in the beta phase as compared to an IgG to the same target antigen.
  • the protein has a larger therapeutic window with smaller peak/trough differences in exposure as compared to an IgG to the same target antigen.
  • polynucleotide encoding an antigen-binding protein according to any one of above embodiments.
  • vectors comprising the above polynucleotides.
  • host cells transformed with the above vectors.
  • compositions comprising (i) the antigen-binding protein according to any one of the above embodiments, the polynucleotide according to the above embodiments, the vector according to the above embodiments, or the host cell according to the above embodiments, and (ii) a pharmaceutically acceptable carrier.
  • processes for the production of an antigen-binding protein of any one of the above embodiments comprising culturing a host transformed or transfected with a vector comprising a nucleic acid sequence encoding an antigen-binding protein of any one of the above embodiments under conditions allowing the expression of the protein and recovering and purifying the produced protein from the culture.
  • methods for the treatment or amelioration of a proliferative disease, a tumorous disease, an inflammatory disease, an immunological disorder, an autoimmune disease, an infectious disease, a viral disease, an allergic reaction, a parasitic reaction, a graft-versus-host disease or a host-versus-graft disease comprising the administration of an antigen-binding protein of any one of the above embodiments to a subject in need of such a treatment or amelioration.
  • the subject is a human.
  • the method further comprises administration of an agent in combination with the antigen-binding protein of any one of the above embodiments.
  • antigen-binding proteins wherein the protein comprises a single polypeptide chain comprising a protease cleavage domain (P) separating the chain into a first and second region; wherein the first region comprises an anti- innate immune cell VH binding domain (IVH) and a target antigen binding domain (Tl) and the second region comprises an anti- innate immune cell VL binding domain (IVL) and a target antigen binding domain (T2); wherein the protein optionally comprises a half-life extension domain (H) in the first region, second region, or both regions, and wherein upon activation by protease cleavage of P and binding the target antigen by Tl and T2, the first and second regions associate to form a complete anti- innate immune cell VL/VH binding domain that binds innate immune cells.
  • P protease cleavage domain
  • antigen-binding proteins wherein the protein comprises a single polypeptide chain comprising a protease cleavage domain (P) separating the chain into a first and second region; wherein the first region comprises an anti- innate immune cell VL binding domain (IVL) and a target antigen binding domain (Tl) and the second region comprises an anti- innate immune cell VH binding domain (IVH) and a target antigen binding domain (T2); wherein the protein optionally comprises a half-life extension domain (H) in the first region, second region, or both regions, and wherein upon activation by protease cleavage of P and binding of the target antigen by Tl and T2, the first and second regions associate to form a complete anti- innate immune cell VL/VH binding domain that binds innate immune cells.
  • P protease cleavage domain
  • antigen-binding proteins wherein the protein comprises a single polypeptide chain comprises a first and second region; wherein the first region comprises an anti- innate immune cell VH binding domain (IVH), an inactive anti- innate immune cell VL binding domain (IVLi) which associates with IVH and a target antigen binding domain (Tl); wherein the second region comprises a an anti- innate immune cell VL binding domain (IVL); an inactive anti- innate immune cell VH binding domain (IVHi) which associates with IVH and a target antigen binding domain (T2); wherein the protein optionally comprises a half-life extension domain (H) in the first region, second region, or both regions, and wherein IVLi and IVHi each comprise at least one protease cleavage domains; and wherein upon activation by protease cleavage the protease cleavage domains and binding the target antigen by Tl and T2, the first and second regions associate to form
  • antigen-binding proteins wherein the protein comprises a single polypeptide chain comprises a first and second region; wherein the first region comprises an anti- innate immune cell VL binding domain (IVL), an inactive anti- innate immune cell VH binding domain (IVHi) which associates with IVL and a target antigen binding domain (Tl); wherein the second region comprises a an anti- innate immune cell VH binding domain (IVH); an inactive anti- innate immune cell VL binding domain (IVLi) which associates with IVL and a target antigen binding domain (T2); wherein the protein optionally comprises a half-life extension domain (H) in the first region, second region, or both regions, and wherein IVLi and IVHi each comprise at least one protease cleavage domains; and wherein upon activation by protease cleavage the protease cleavage domains and binding the target antigen by Tl and T2, the first and second regions associate to form
  • Figure 1 shows an exemplary antigen binding protein.
  • the anti-CD16 VH and VL domains are separated by a protease cleavage site which keeps the anti -CD 16 VH and VL domains from folding properly and binding to CD 16 on an innate immune cell.
  • Figure 1 also shows the cleaved antigen binding protein, where the VH and VL domains are folded such that they are able to bind to CD 16 and the anti -target domains are bound to the target antigen on the surface of the target cell.
  • This example also has a half-life extension domain.
  • Figure 2 shows an exemplary dual-target antigen binding protein.
  • the anti-CD 16VH and VL domains are separated by a protease cleavage site which keeps the anti- CD16VH and VL domains from folding properly and binding to CD 16 on an innate immune cell.
  • Figure 2 also shows the cleaved antigen binding protein, where the VH and VL domains are folded such that they are able to bind CD16and each anti -target domain is bound to its target antigen on the surface of the target cell.
  • This example also has a half-life extension domain.
  • Figure 3 shows an exemplary antigen binding protein.
  • the anti- CD 16VH and VL domains are bound to protease cleavable VL and VH domains that together do not bind to CD 16. Once these domains are cleaved, the anti-CD 16VH and VL domains are able to fold and associate with CD16 on an innate immune cell.
  • This example also has two anti- target domains that in some cases bind to two different antigens and in some cases bind to the same antigen. This example also has a half-life extension domain.
  • antigen-binding proteins such as tri-specific, quad-specific antigen, and multi-specific binding proteins, pharmaceutical compositions thereof, as well as nucleic acids, recombinant expression vectors and host cells for making such antigen-binding proteins.
  • methods of using the disclosed antigen-binding proteins in the prevention, and/or treatment of diseases, conditions and disorders are capable of specifically binding to one or more target antigen as well as at least one protein on an innate immune cell, and optionally a half-life extension domain, such as an HSA binding domain. Binding to an innate immune cell is only possible once activated by a protease and binding to the target antigen(s).
  • protease cleavage of the protease cleavage domain occurs before target antigen binding domain binding to the target antigen. It is also to be understood that in some embodiments, protease cleavage of the protease cleavage domain occurs after target antigen binding domain binding to the target antigen.
  • FIGS 1, 2 and 3 depict three non-limiting examples of an antigen-binding protein.
  • the antigen-binding proteins described herein are designed to allow specific targeting of cells expressing a target antigen by recruiting innate immune cells. This improves specificity compared to therapeutics that bind to innate immune cells and a target antigen which may or may not be expressed by a target cell, such as a tumor or cancer cell. In contrast, by activating innate immune cell binding specifically in the microenvironment of the target cell, where the target antigen and proteases are highly expressed, the antigen-binding proteins can crosslink innate immune cells with cells expressing a target antigen in a highly specific fashion, thereby directing the therapeutic potential of the innate immune cell towards the target cell.
  • antigen-binding proteins described herein engage innate immune cells via protease-activated binding to the surface-expressed surface antigens, which are uniquely found on the surface of innate immune cells. Simultaneous binding of several antigen-binding proteins to an innate immune cell and to a target antigen expressed on the surface of particular cells causes innate immune cell activation and mediates the subsequent lysis of the particular target antigen expressing cell. Thus, antigen-binding proteins are contemplated to display strong, specific and efficient target cell killing.
  • the antigen-binding proteins described herein stimulate target cell killing by innate immune cells to eliminate pathogenic cells in protease-rich microenvironments (e.g., tumor cells, virally or bacterially infected cells, autoreactive T cells, etc). In some of such embodiments, cells are eliminated selectively, thereby reducing the potential for toxic side effects.
  • the same polypeptides could be used to enhance the elimination of endogenous cells for therapeutic effect, such as B or T lymphocytes in autoimmune disease, or hematopoietic stem cells (HSCs) for stem cell transplantation.
  • Proteases known to be associated with diseased cells or tissues include but are not limited to serine proteases, cysteine proteases, aspartate proteases, threonine proteases, glutamic acid proteases, metalloproteases, asparagine peptide lyases, serum proteases, cathepsins, Cathepsin B, Cathepsin C, Cathepsin D, Cathepsin E, Cathepsin K, Cathepsin L, kallikreins, hKl, hK10, hK15, plasmin, collagenase, Type IV collagenase, stromelysin, Factor Xa, chymotrypsin-like protease, trypsin-like protease, elastase-like protease, subtili sin-like protease, actinidain, bromelain, calpain, caspases, caspase-3
  • the antigen-binding proteins described herein confer further therapeutic advantages over traditional monoclonal antibodies and other smaller bispecific molecules.
  • Bi-specific molecules are designed to bind to a target cell via a cell-specific marker associated with a pathogenic cell. Toxicities are possible when, in some cases, healthy cells or tissues express the same marker as the pathogenic cell.
  • One benefit to an antigen binding protein is that binding to an innate immune cell is dependent upon activation by a protease expressed by the target cell, such as a tumor cell, and binding of the antigen binding domains to one or more target antigens, for example a tumor antigen.
  • the antigen-binding proteins comprise an inactive innate immune cell binding domain comprising VH and VL domains separated by one or more protease cleavage sites.
  • the protease cleavage sites are cleaved allowing the VH and VL domains to fold properly and bind to an innate immune cell when one or more target antigens are bound.
  • the innate immune cell binding domain is inactive and cannot bind to an innate immune cell.
  • the antigen-binding proteins described herein comprise at least one protease cleavage site comprising an amino acid sequence that is cleaved by at least one protease.
  • the antigen-binding proteins described herein comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more protease cleavage sites that are cleaved by at least one protease.
  • the protease cleavage site comprises an amino acid sequence recognized by a protease is a MMP9 cleavage site comprising a polypeptide having an amino acid sequence LEATA (SEQ ID NO: 4).
  • the antigen-binding proteins described herein confer additional therapeutic advantages over traditional monoclonal antibodies and other smaller bispecific molecules. Generally, the effectiveness of recombinant protein pharmaceuticals depends heavily on the intrinsic pharmacokinetics of the protein itself.
  • One such additional benefit here is that the antigen- binding proteins described herein have extended pharmacokinetic elimination half-time due to having a half-life extension domain, for example a binding domain specific to HSA.
  • the antigen-binding proteins described herein have an extended serum elimination half- time of about two, three, about five, about seven, about 10, about 12, or about 14 days in some embodiments. This contrasts to other binding proteins such as BiTE or DART molecules which have relatively much shorter elimination half-times.
  • the BiTE CD19xCD3 bispecific scFv-scFv fusion molecule requires continuous intravenous infusion (i.v.) drug delivery due to its short elimination half-time.
  • the longer intrinsic half-times of the antigen- binding proteins solve this issue thereby allowing for increased therapeutic potential such as low-dose pharmaceutical formulations, decreased periodic administration and/or novel pharmaceutical compositions.
  • the antigen-binding proteins described herein also have an optimal size for enhanced tissue penetration and distribution and enhanced reduced first pass renal clearance. Because the kidney generally filters out molecules below 50 kDa, efforts to reduce clearance in the design of protein therapeutics have focused on increasing molecular size through protein fusions, glycosylation, or the addition of polyethylene glycol polymers (i.e., PEG). However, while increasing the size of a protein therapeutic may prevent renal clearance, the downside is that the larger size also prevents penetration of the molecule into the target tissues. The antigen-binding proteins described herein avoid this by associating with albumin which will prevent renal clearance while also having a small size that allows enhanced tissue penetration and distribution and optimal efficacy.
  • the antigen-binding proteins described herein in some embodiments have a size of about 50 kD to about 80 kD, about 50 kD to about 75 kD, about 50 kD to about 70 kD, or about 50 kD to about 65 kD.
  • the size of the antigen-binding proteins is advantageous over IgG antibodies which are about 150 kD and the BiTE and DART diabody molecules which are about 55 kD but are not half-life extended and therefore are cleared quickly through the kidney.
  • antigen-binding proteins described herein are of a single-polypeptide design with flexible linkage of their domains. This allows for facile production and manufacturing of the antigen-binding proteins as they can be encoded by single cDNA molecule to be easily incorporated into a vector. Further, because the antigen-binding proteins described herein are a monomeric single polypeptide chain, there are no chain pairing issues or a requirement for dimerization. It is contemplated that the antigen-binding proteins described herein have a reduced tendency to aggregate unlike other reported molecules such as bispecific BiTE proteins.
  • the antigen binding proteins in pre-activated form, comprise a single polypeptide chain comprising a first region and a second region separated by at least one protease cleavage domain (P).
  • the first region comprises an anti-innate immune cell V H binding domain (IV H ) and a target antigen binding domain (Ti).
  • the second region comprises an anti-innate immune cell V L binding domain (IV L ) and a target antigen binding domain (T 2 ).
  • the antigen-binding domain optionally comprises a half-life extension domain (H) in the first region.
  • the antigen-binding domain optionally comprises a half-life extension domain (H) in the second region.
  • the anti- innate immune cell binding domains IV H and IV L are activated to bind to an innate immune cell.
  • the domains in an antigen binding protein are contemplated to be arranged in any order within each region, with a protease cleavage domain (P) in the center of the pre-activated polypeptide. Further, each region may be in any order within the pre-activated polypeptide.
  • exemplary domain order of the antigen-binding proteins include but are not limited to:
  • the antigen-binding proteins in pre-activated form, comprise a single polypeptide chain comprising a first region and a second region.
  • the first region comprises an anti- innate immune cell V H binding domain (IV H ), an inactive anti- innate immune cell V L binding domain (IV L; ) which associates with IV H , and a target antigen binding domain (Ti), wherein IV L; comprises at least one protease cleavage domain (P).
  • the second region comprises an anti- innate immune cell V L binding domain (IV L ), an inactive anti- innate immune cell V H binding domain (IV H ,) which associates with IV L , and a target antigen binding domain (T 2 ), wherein IV comprises at least one protease cleavage domain (P).
  • the antigen-binding domain optionally comprises a half-life extension domain (H) in the first region.
  • the antigen-binding domain optionally comprises a half-life extension domain (H) in the second region.
  • the anti- innate immune cell binding domains IV H and IV L are activated to bind to an innate immune cell.
  • An example of this type of antigen binding protein is described in Figure 3.
  • the domains are linked by internal linkers LI, L2, L3, and L4 where LI links the first and second domain of the antigen-binding proteins, L2 links the second and third domains of the antigen-binding proteins, L3 links the third and fourth domains of the antigen-binding proteins, and L4 links the fourth and fifth domains of the protease activated antigen -binding proteins.
  • Linkers LI, L2, L3, and L4 have an optimized length and/or amino acid composition. In some embodiments, linkers LI, L2, L3, and L4 are the same length and amino acid composition. In other embodiments, LI, L2, L3, and L4 are different.
  • internal linkers LI, L2, L3, and/or L4 are "short", i.e., consist of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 amino acid residues. Thus, in certain instances, the internal linkers consist of about 12 or less amino acid residues. In the case of 0 amino acid residues, the internal linker is a peptide bond. In certain embodiments, internal linkers LI, L2, L3, and/or L4 are "long", i.e., consist of 15, 20 or 25 amino acid residues. In some
  • these internal linkers consist of about 3 to about 15, for example 8, 9 or 10 contiguous amino acid residues.
  • amino acid composition of the internal linkers LI, L2, L3, and L4 peptides are selected with properties that confer flexibility to the antigen- binding proteins, do not interfere with the binding domains as well as resist cleavage from proteases. For example, glycine and serine residues generally provide protease resistance.
  • internal linkers suitable for linking the domains in the antigen-binding proteins include but are not limited to (GS) bias, (GGS) thread, (GGGS) admire, (GGSG) admire, (GGSGG) procur, or (GGGGS) admire, wherein n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.
  • internal linker LI, L2, and/or L3 is (GGGGS) 4 or (GGGGS) 3 .
  • Innate immune cells are a part of the innate immune system. Innate immune cells recognize and respond to pathogens via recognition of pathogens. They play a variety of roles in immunity including but not limited to, recruitment of other immune cells via cytokine secretion, activation of the complement cascade, identification and removal of foreign substances in the body, and presenting antigen to other immune cells. Innate immune cells do not include T cells, such as CD4+ T cells or CD8+ T cells. Innate immune cells include multiple cell types such as dendritic cells, plasmacytoid dendritic cells, natural killer cells, natural killer T cells, monocytes, neutrophils, basophils, and eosinophils.
  • innate immune cells recruitment and activation of innate immune cells to the site of a target antigen, for example at a diseased cell or tissue leads to activation of the immune system at that site.
  • Each innate immune cell is recruited by at least one cell surface marker expressed by the cell, which is specific for the innate immune cell type.
  • Non-limiting examples of innate immune cell surface markers include but are not limited to in dendritic cells: CDlc, CD83, CD141, CD209, and MHC II; plasmacytoid dendritic cells: CD123, CD303, and CD304; natural killer cells: CD16, CD56; natural killer T cells: CDld, CD160, PLZF, NKG2d, and CD94-NKG2A/C/E; monocytes: CD14, CD16, CD47, and CD64; neutrophils: CD15, CD16, and CD89; basophils: 2D7 antigen, CD123, CD203c, and FcsRIa; eosinophils: CDl lb, CD193, EMR1, and Siglec-8.
  • the antigen-binding proteins described herein comprise a domain which specifically binds to an innate immune cell when activated by a protease. In one aspect, the antigen-binding proteins described herein comprise two or more domains which when activated by a protease specifically bind to a human innate immune cell, wherein the innate immune cell is not a T cell. In some embodiments, the antigen-binding proteins described herein comprise two or more domains which when activated by a protease which specifically binds to a dendritic cell.
  • the antigen-binding proteins described herein comprise two or more domains which when activated by a protease specifically bind to a plasmacytoid dendritic cell. In some embodiments, the antigen-binding proteins described herein comprise two or more domains which when activated by a protease specifically bind to a natural killer cell. In some embodiments, the antigen-binding proteins described herein comprise two or more domains which when activated by a protease specifically bind to a natural killer T cell. In some embodiments, the antigen-binding proteins described herein comprise two or more domains which when activated by a protease specifically bind to a monocyte.
  • the antigen-binding proteins described herein comprise two or more domains which when activated by a protease specifically bind to a neutrophil. In some embodiments, the antigen-binding proteins described herein comprise two or more domains which when activated by a protease specifically bind to an eosinophil. In some embodiments, the antigen -binding proteins described herein comprise two or more domains which when activated by a protease specifically bind to a basophil.
  • the protease cleavage site is between the anti- innate immune cell VH and VL domains and keeps them from folding and binding to an innate immune cell. Once the protease cleavage site is cleaved by a protease present at the target cell, the anti- innate immune cell VH and VL domains are able to fold and bind to an innate immune cell. In an alternate embodiment, the protease cleavage site is designed into a non- innate immune cell binding VL and VH domain that binds to the anti- innate immune cell VH and VL domains.
  • Cleavage of the protease cleavage site by a protease present at the target cell removes the non- innate immune cell binding VL and VH domain and allows the anti- innate immune cell VH and VL domain to fold and to bind an innate immune cell.
  • the antigen binding proteins described herein comprise a domain which specifically binds to an innate immune cell when activated by a protease.
  • the domain which specifically binds to an innate immune cell comprises a VH domain and a VL domain separated by at least one protease cleavage site. When the protease cleavage site is cleaved, the VH domain and the VL domain are able to fold and therefore bind to an innate immune cell.
  • the protease cleavage site is in a loop region.
  • the protease cleavage site is within the VH and/or the VL domains and the protease cleavage sites are cleaved revealing the VH and/or the VL domains allowing them to fold and therefore bind to an innate immune cell.
  • the antigen-binding proteins described herein comprise two or more domains which when activated by a protease specifically bind to an innate immune cell surface marker including but are not limited to CDlc, CD83, CD 141, CD209, MHC II, CD 123, CD303, CD304, CD16, CD56, CDld, CD160, PLZF, KG2d, CD94- KG2A/C/E , CD14, CD16, CD47, CD64, CD15, CD16, CD89, 2D7 antigen, CD123, CD203c, FcsRIa, CDl lb, CD193, EMR1, and Siglec-8.
  • the antigen-binding proteins described herein comprise two or more domains which when activated by a protease specifically bind innate immune cell surface marker.
  • the innate immune cell binding domain of the antigen-binding proteins described herein exhibit not only potent innate immune cell binding affinities with human innate immune cells, but show also excellent cross reactivity with the respective cynomolgus monkey innate immune cell proteins.
  • the innate immune cell binding domain of the antigen-binding proteins is cross-reactive with innate immune cells from cynomolgus monkey.
  • human xynomolgous K D ratios for innate immune cell binding are between 5 and 0.2.
  • the innate immune cell binding domain of the antigen binding protein can be any domain that binds to innate immune cell including but not limited to domains from a monoclonal antibody, a polyclonal antibody, a recombinant antibody, a human antibody, a humanized antibody.
  • the antigen-binding domain comprises a humanized or human antibody or an antibody fragment, or a murine antibody or antibody fragment.
  • the humanized or human anti-innate immune cell binding domain comprises one or more (e.g., all three) light chain complementary determining region 1 (LC CDR1), light chain complementary determining region 2 (LC CDR2), and light chain complementary determining region 3 (LC CDR3) of a humanized or human anti- innate immune cell binding domain described herein, and/or one or more (e.g., all three) heavy chain complementary determining region 1 (HC CDR1), heavy chain complementary determining region 2 (HC CDR2), and heavy chain complementary determining region 3 (HC CDR3) of a humanized or human anti -innate immune cell binding domain described herein, e.g., a humanized or human anti-innate immune cell binding domain comprising one or more, e.g., all three, LC CDRs and one or more, e.g., all three, HC CDRs.
  • LC CDR1 light chain complementary determining region 1
  • HC CDR2 light chain complementary determining region 2
  • HC CDR3 light chain complementary
  • the humanized or human anti-innate immune cell binding domain comprises a humanized or human light chain variable region specific to innate immune cell where the light chain variable region specific to innate immune cell comprises human or non- human light chain CDRs in a human light chain framework region.
  • the light chain framework region is a ⁇ (lambda) light chain framework. In other instances, the light chain framework region is a ⁇ (kappa) light chain framework.
  • the humanized or human anti-innate immune cell binding domain comprises a humanized or human heavy chain variable region specific to innate immune cell where the heavy chain variable region specific to innate immune cell comprises human or non- human heavy chain CDRs in a human heavy chain framework region.
  • the complementary determining regions of the heavy chain and/or the light chain are derived from known anti-innate immune cell antibodies, such as, for example, Lorvotuzumab, 3C12C, CSL362, 3G8, rMil2, E4, NNC141-0100.
  • the anti-innate immune cell binding domain is a single chain variable fragment (scFv) comprising a light chain and a heavy chain of an amino acid sequence provided herein.
  • scFv single chain variable fragment
  • single chain variable fragment or “scFv” refers to a antibody fragment comprising a variable region of a light chain and at least one antibody fragment comprising a variable region of a heavy chain, wherein the light and heavy chain variable regions are contiguously linked via a short flexible polypeptide linker, and capable of being expressed as a single polypeptide chain, and wherein the scFv retains the specificity of the intact antibody from which it is derived.
  • the anti-innate immune cell binding domain comprises: a light chain variable region comprising an amino acid sequence having at least one, two or three modifications (e.g., substitutions) but not more than 30, 20 or 10 modifications (e.g., substitutions) of an amino acid sequence of a light chain variable region provided herein, or a sequence with 95-99% identity with an amino acid sequence provided herein; and/or a heavy chain variable region comprising an amino acid sequence having at least one, two or three modifications (e.g., substitutions) but not more than 30, 20 or 10 modifications (e.g., substitutions) of an amino acid sequence of a heavy chain variable region provided herein, or a sequence with 95-99% identity to an amino acid sequence provided herein.
  • a light chain variable region comprising an amino acid sequence having at least one, two or three modifications (e.g., substitutions) but not more than 30, 20 or 10 modifications (e.g., substitutions) of an amino acid sequence of a heavy chain variable region provided herein, or a sequence with 95-99% identity to an
  • the humanized or human anti-innate immune cell binding domain is a scFv, and a light chain variable region comprising an amino acid sequence described herein, is attached to a heavy chain variable region comprising an amino acid sequence described herein, via a scFv linker.
  • the light chain variable region and heavy chain variable region of a scFv can be, e.g., in any of the following orientations: light chain variable region- scFv linker-heavy chain variable region or heavy chain variable region- scFv linker-light chain variable region.
  • scFvs which bind to innate immune cell are prepared according to known methods.
  • scFv molecules can be produced by linking VH and VL regions together using flexible polypeptide linkers.
  • the scFv molecules comprise a scFv linker (e.g., a Ser-Gly linker) with an optimized length and/or amino acid composition.
  • the length of the scFv linker is such that the VH or VL domain can associate intermolecularly with the other variable domain to form the innate immune cell binding site.
  • such scFv linkers are "short", i.e.
  • the scFv linkers consist of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 amino acid residues.
  • the scFv linkers consist of about 12 or less amino acid residues.
  • the scFv linker is a peptide bond.
  • these scFv linkers consist of about 3 to about 15, for example 8, 9 or 10 contiguous amino acid residues.
  • peptides are selected that confer flexibility, do not interfere with the variable domains as well as allow inter-chain folding to bring the two variable domains together to form a functional innate immune cell binding site.
  • scFv linkers comprising glycine and serine residues generally provide protease resistance.
  • linkers in a scFv comprise glycine and serine residues.
  • the amino acid sequence of the scFv linkers can be optimized, for example, by phage-display methods to improve the innate immune cell binding and production yield of the scFv.
  • Examples of peptide scFv linkers suitable for linking a variable light chain domain and a variable heavy chain domain in a scFv include but are not limited to (GS) n , (GGS) n , (GGGS) context, (GGSG) resort, (GGSGG) procur, or (GGGGS) context, wherein n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.
  • the scFv linker can be (GGGGS) 4 or (GGGGS) 3 . Variation in the linker length may retain or enhance activity, giving rise to superior efficacy in activity studies.
  • innate immune cell binding domain of an antigen binding protein has an affinity to innate immune cells with a K D of 1000 nM or less, 100 nM or less, 50 nM or less, 20 nM or less, 10 nM or less, 5 nM or less, 1 nM or less, or 0.5 nM or less.
  • the innate immune cell binding domain of an antigen binding protein has an affinity to CDlc, CD83, CD141, CD209, MHC II, CD123, CD303, CD304, CD16, CD56, CD Id, CD 160, PLZF, KG2d, CD94- KG2A/C/E , CD 14, CD 16, CD64, CD 15 CD 16, 2D7 antigen, CD123, CD203c, FcsRIa, CDl lb, CD193, EMRl, or Siglec-8with a K D of 1000 nM or less, 100 nM or less, 50 nM or less, 20 nM or less, 10 nM or less, 5 nM or less, 1 nM or less, or 0.5 nM or less.
  • innate immune cell binding domain of an antigen binding protein has low affinity to an innate immune cell, i.e., about 100 nM or greater.
  • the affinity to bind to innate immune cell can be determined, for example, by the ability of the antigen binding protein itself or its innate immune cell binding domain to bind to an innate immune cell surface marker coated on an assay plate; displayed on a microbial cell surface; in solution; etc.
  • the binding activity of the antigen binding protein itself or its innate immune cell binding domain of the present disclosure to innate immune cell can be assayed by immobilizing the cell surface marker (e.g., CDlc, CD83, CD141, CD209, MHC II, CD123, CD303, CD304, CD16, CD56, CDld, CD160, PLZF, KG2d, CD94- KG2A/C/E , CD14, CD16, CD64, CD15 CD16, 2D7 antigen, CD123, CD203c, FcsRIa, CDl lb, CD193, EMRl, or Siglec-8) or the antigen binding protein itself or its innate immune cell binding domain, to a bead, substrate, cell, etc.
  • the cell surface marker e.g., CDlc, CD83, CD141, CD209, MHC II, CD123, CD303, CD304, CD16, CD56, CDld, CD160, PLZF, KG2d, CD
  • Agents can be added in an appropriate buffer and the binding partners incubated for a period of time at a given temperature. After washes to remove unbound material, the bound protein can be released with, for example, SDS, buffers with a high pH, and the like and analyzed, for example, by Surface Plasmon Resonance (SPR).
  • SDS Surface Plasmon Resonance
  • Protease cleavage domains are polypeptides having a sequence recognized and cleaved in a sequence-specific manner.
  • Antigen binding proteins contemplated herein comprise a protease cleavage domain recognized in a sequence-specific manner by a matrix metalloprotease (MMP), for example a MMP9.
  • MMP9 matrix metalloprotease 9
  • the protease cleavage domain recognized by a MMP9 comprises a polypeptide having an amino acid sequence
  • the protease cleavage domain recognized by a MMP9 comprises a polypeptide having an amino acid sequence LEATA (SEQ ID NO: 4).
  • the protease cleavage domain is recognized in a sequence-specific manner by a MMP11.
  • the protease cleavage domain recognized by a MMP11 comprises a polypeptide having an amino acid sequence GGAANLVRGG (SEQ IN NO: 3).
  • the protease cleavage domain is recognized by a protease disclosed in Table 1.
  • the protease cleavage domain recognized by a protease disclosed in Table 1 comprises a polypeptide having an amino acid sequence selected from a sequence disclosed in Table 1 (SEQ ID NOS: 1-42).
  • Proteases are proteins that cleave proteins, in some cases, in a sequence-specific manner.
  • Proteases include but are not limited to serine proteases, cysteine proteases, aspartate proteases, threonine proteases, glutamic acid proteases, metalloproteases, asparagine peptide lyases, serum proteases, cathepsins, Cathepsin B, Cathepsin C, Cathepsin D, Cathepsin E, Cathepsin K, Cathepsin L, kallikreins, hKl, hK10, hK15, plasmin, collagenase, Type IV collagenase, stromelysin, Factor Xa, chymotrypsin-like protease, trypsin-like protease, elastase-like protease, subtilisin-like protease, actinidain, bro
  • Table 1 Exemplary Proteases and Protease Cleavage Domain Sequences
  • MMP2, MMP9, MMP 14 EP(Cit)G(Hof)YL 13
  • Proteases are known to be secreted by some diseased cells and tissues, for example tumor or cancer cells, creating a microenvironment that is rich in proteases or a protease-rich microenvironment.
  • the blood of a subject is rich in proteases.
  • cells surrounding the tumor secrete proteases into the tumor microenvironment.
  • Cells surrounding the tumor secreting proteases include but are not limited to the tumor stromal cells,
  • proteases are present in the blood of a subject, for example proteases that target amino acid sequences found in microbial peptides. This feature allows for targeted therapeutics such as antigen-binding proteins to have additional specificity because T cells will not be bound by the antigen binding protein except in the protease rich microenvironment of the targeted cells or tissue.
  • domains which extend the half-life of an antigen-binding domain.
  • Such domains are contemplated to include but are not limited to HSA binding domains, Fc domains, small molecules, and other half-life extension domains known in the art.
  • Fc and albumin binding domains extend half-lives by increasing the size of the peptide drug.
  • Fc and albumin binding domains bind to the neonatal Fc receptor, FcRn, which, at least in some cases prevents degradation of the fusion protein in the endosome.
  • Fc domains, or HSA binding domains improve solubility and stability of the antigen binding domain.
  • HSA Human serum albumin
  • Noncovalent association with albumin extends the elimination half-time of short lived proteins.
  • a recombinant fusion of an albumin binding domain to a Fab fragment resulted in an in vivo clearance of 25- and 58-fold and a half-life extension of 26- and 37-fold when administered intravenously to mice and rabbits respectively as compared to the
  • the antigen-binding proteins described herein comprise a half-life extension domain, for example a domain which specifically binds to HSA.
  • the HSA binding domain of an antigen binding protein can be any domain that binds to HSA including but not limited to domains from a monoclonal antibody, a polyclonal antibody, a recombinant antibody, a human antibody, a humanized antibody.
  • the HSA binding domain is a single chain variable fragments (scFv), single- domain antibody such as a heavy chain variable domain (VH), a light chain variable domain (VL) and a variable domain (VHH) of camelid derived nanobody, peptide, ligand or small molecule specific for HSA.
  • the HSA binding domain is a single- domain antibody.
  • the HSA binding domain is a peptide.
  • the HSA binding domain is a small molecule.
  • the HSA binding domain of an antigen binding protein is fairly small and no more than 25 kD, no more than 20 kD, no more than 15 kD, or no more than 10 kD in some embodiments. In certain instances, the HSA binding is 5 kD or less if it is a peptide or small molecule.
  • the half-life extension domain of an antigen binding protein provides for altered pharmacodynamics and pharmacokinetics of the antigen binding protein itself. As above, the half-life extension domain extends the elimination half-time. The half-life extension domain also alters pharmacodynamic properties including alteration of tissue distribution, penetration, and diffusion of the antigen-binding protein. In some embodiments, the half-life extension domain provides for improved tissue (including tumor) targeting, tissue penetration, tissue distribution, diffusion within the tissue, and enhanced efficacy as compared with a protein without a half-life extension binding domain. In one embodiment, therapeutic methods effectively and efficiently utilize a reduced amount of the antigen-binding protein, resulting in reduced side effects, such as reduced non-tumor cell cytotoxicity.
  • characteristics of the half-life extension domain include the binding affinity of the HSA binding domain for HSA. Affinity of said HSA binding domain can be selected so as to target a specific elimination half-time in a particular antigen-binding protein.
  • the HSA binding domain has a high binding affinity.
  • the HSA binding domain has a medium binding affinity.
  • the HSA binding domain has a low or marginal binding affinity.
  • Exemplary binding affinities include K D concentrations at 10 nM or less (high), between 10 nM and 100 nM (medium), and greater than 100 nM (low). As above, binding affinities to HSA are determined by known methods such as Surface Plasmon Resonance (SPR).
  • SPR Surface Plasmon Resonance
  • the half-life extension domain of an antigen binding protein extends the half-life of an antigen binding protein to at least about two days, about three days, about four days, about five days, about six days, about seven days, about eight days, about nine days, about ten days, or more. In some embodiments, the half-life extension domain extends the half-life of an antigen binding protein to at least 2-3 days, 3-4 days, 4-5, days, 5-6 days, 6-7 days, 7-8 days, 2-5 days, 5-8 days, 2-10 days, or more. In some embodiments, the half-life extension domain extends the half-life of an antigen binding domain to at least about five days. In some embodiments, the half-life extension domain extends the half-life of an antigen binding protein to five or more days.
  • the antigen-binding proteins described herein also comprise at least two more domains that bind to one or more target antigens. It is contemplated herein that an antigen binding protein is cleaved, for example, in a disease-specific microenvironment or in the blood of a subject at the protease cleavage domain and that each target antigen binding domain will bind to a target antigen on a target cell, thereby activating the innate immune cell binding domain to bind an innate immune cell. At least one target antigen is involved in and/or associated with a disease, disorder or condition.
  • a target antigen associated with a proliferative disease a tumorous disease, an inflammatory disease, an immunological disorder, an autoimmune disease, an infectious disease, a viral disease, an allergic reaction, a parasitic reaction, a graft-versus-host disease or a host-versus-graft disease.
  • a target antigen is a tumor antigen expressed on a tumor cell.
  • a target antigen is associated with a pathogen such as a virus or bacterium. At least one target antigen may also be directed against healthy tissue.
  • a target antigen is a cell surface molecule such as a protein, lipid or polysaccharide.
  • a target antigen is a on a tumor cell, virally infected cell, bacterially infected cell, damaged red blood cell, arterial plaque cell, or fibrotic tissue cell. It is contemplated herein that upon binding more than one target antigen, two inactive innate immune cell binding domains are co-localized and form an active innate immune cell binding domain on the surface of the target cell.
  • the antigen binding protein comprises more than one target antigen binding domain to activate an inactive innate immune cell binding domain in the antigen binding protein.
  • the antigen binding protein comprises more than one target antigen binding domain to enhance the strength of binding to the target cell. In some embodiments the antigen binding protein comprises more than one target antigen binding domain to enhance the strength of binding to the target cell. In some embodiments, more than one antigen binding domain comprises the same antigen binding domain. In some embodiments, more than one antigen binding domain comprises different antigen binding domains. For example, two different antigen binding domains known to be dually expressed in a diseased cell or tissue, for example a tumor or cancer cell, can enhance binding or selectivity of an antigen binding protein for a target.
  • Antigen-binding proteins contemplated herein include at least one antigen binding domain, wherein the antigen binding domain binds to at least one target antigen.
  • Target antigens in some cases, are expressed on the surface of a diseased cell or tissue, for example a tumor or a cancer cell.
  • Target antigens include but are not limited to EpCAM, EGFR, HER-2, HER-3, c-Met, FoIR, and CEA.
  • Antigen-binding proteins disclosed herein also include proteins comprising two antigen binding domains that bind to two different target antigens known to be expressed on a diseased cell or tissue. Exemplary pairs of antigen binding domains include but are not limited to EGFR/CEA, EpCAM/CEA, and HER-2/HER-3.
  • the design of the antigen-binding proteins described herein allows the binding domain to one or more target antigens to be flexible in that the binding domain to a target antigen can be any type of binding domain, including but not limited to, domains from a monoclonal antibody, a polyclonal antibody, a recombinant antibody, a human antibody, a humanized antibody.
  • the binding domain to a target antigen is a single chain variable fragment (scFv), single-domain antibody such as a heavy chain variable domain (VH), a light chain variable domain (VL) and a variable domain (VHH) of camelid derived nanobody.
  • the binding domain to a target antigen is a non-Ig binding domain, i.e., antibody mimetic, such as anticalins, affilins, affibody molecules, affimers, affitins, alphabodies, avimers, DARPins, fynomers, kunitz domain peptides, and monobodies.
  • the binding domain to one or more target antigens is a ligand or peptide that binds to or associates with one or more target antigens.
  • antigen-binding proteins described herein have certain advantages that would be recognized by one of skill in the art. For example, antigen-binding proteins described herein have improved pharmacokinetics over traditional antibody therapeutics. Improved
  • pharmacokinetics of antigen-binding proteins herein are attributed to at least the half-life extension domain and the innate immune cell binding domain.
  • Half-life extension domains include various polypeptides including but not limited to Fc domains and polypeptides binding to HSA.
  • Innate immune cell binding domains herein have unique properties which give superior pharmacokinetics.
  • the innate immune cell binding domains herein do not bind to an innate immune cell until they are activated by at least cleavage of at least one protease cleavage domain and binding of the antigen binding domains to target antigens.
  • enhanced pharmacokinetics of antigen binding proteins herein is attributed at least in part to reduced or eliminated target mediated drug disposition through innate immune cell binding in the circulation of a person.
  • Improved pharmacokinetics comprises at least one of a shallower alpha phase and higher exposure in the beta phase.
  • Antigen binding proteins described herein thus have a larger therapeutic window with smaller peak/trough differences in exposure when compared to traditional antibody therapeutics.
  • antigen-binding proteins described herein encompass derivatives or analogs in which (i) an amino acid is substituted with an amino acid residue that is not one encoded by the genetic code, (ii) the mature polypeptide is fused with another compound such as polyethylene glycol, or (iii) additional amino acids are fused to the protein, such as a leader or secretory sequence or a sequence for purification of the protein.
  • Typical modifications include, but are not limited to, acetylation, acylation, ADP- ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphatidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent crosslinks, formation of cystine, formation of pyroglutamate, formylation, gamma carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA mediated addition of amino acids to proteins such as arginylation, and ubiquitination.
  • Modifications are made anywhere in antigen-binding proteins described herein, including the peptide backbone, the amino acid side-chains, and the amino or carboxyl termini.
  • Certain common peptide modifications that are useful for modification of antigen-binding proteins include glycosylation, lipid attachment, sulfation, gamma-carboxylation of glutamic acid residues, hydroxylation, blockage of the amino or carboxyl group in a polypeptide, or both, by a covalent modification, and ADP-ribosylation.
  • polynucleotide molecules encoding an antigen binding protein described herein.
  • the polynucleotide molecules are provided as a DNA construct.
  • the polynucleotide molecules are provided as a messenger RNA transcript.
  • the polynucleotide molecules are constructed by known methods such as by combining the genes encoding the three binding domains either separated by peptide linkers or, in other embodiments, directly linked by a peptide bond, into a single genetic construct operably linked to a suitable promoter, and optionally a suitable transcription terminator, and expressing it in bacteria or other appropriate expression system such as, for example CHO cells.
  • the polynucleotides contain genes encoding the domains that bind to an innate immune cell and the HSA.
  • the polynucleotides contain genes encoding the domains that bind to the innate immune cell and the target antigen.
  • any number of suitable transcription and translation elements including constitutive and inducible promoters, may be used.
  • the promoter is selected such that it drives the expression of the polynucleotide in the respective host cell.
  • the polynucleotide is inserted into a vector, preferably an expression vector, which represents a further embodiment.
  • This recombinant vector can be constructed according to known methods.
  • Vectors of particular interest include plasmids, phagemids, phage derivatives, virii (e.g., retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, lentiviruses, and the like), and cosmids.
  • a variety of expression vector/host systems may be utilized to contain and express the polynucleotide encoding the polypeptide of the described antigen-binding protein.
  • Examples of expression vectors for expression in E.coli are pSKK (Le Gall et al., J Immunol Methods. (2004) 285(1): 111-27) or pcDNA5 (Invitrogen) for expression in mammalian cells.
  • the antigen-binding proteins as described herein are produced by introducing a vector encoding the protein as described above into a host cell and culturing said host cell under conditions whereby the protein domains are expressed, may be isolated and, optionally, further purified.
  • compositions comprising an antigen binding protein described herein, a vector comprising the polynucleotide encoding the polypeptide of the antigen-binding proteins or a host cell transformed by this vector and at least one pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carrier includes, but is not limited to, any carrier that does not interfere with the effectiveness of the biological activity of the ingredients and that is not toxic to the patient to whom it is
  • compositions are sterile. These compositions may also contain adjuvants such as preservative, emulsifying agents and dispersing agents. Prevention of the action of microorganisms may be ensured by the inclusion of various antibacterial and antifungal agents.
  • the antigen binding protein described herein is encapsulated in nanoparticles.
  • the nanoparticles are fullerenes, liquid crystals, liposome, quantum dots, superparamagnetic nanoparticles, dendrimers, or nanorods.
  • the antigen binding protein is attached to liposomes.
  • the antigen binding protein are conjugated to the surface of liposomes.
  • the antigen binding protein are encapsulated within the shell of a liposome.
  • the liposome is a cationic liposome.
  • the antigen-binding proteins described herein are contemplated for use as a medicament.
  • Administration is effected by different ways, e.g. by intravenous, intraperitoneal, subcutaneous, intramuscular, topical or intradermal administration.
  • the route of administration depends on the kind of therapy and the kind of compound contained in the pharmaceutical composition.
  • the dosage regimen will be determined by the attending physician and other clinical factors. Dosages for any one patient depends on many factors, including the patient ' s size, body surface area, age, sex, the particular compound to be administered, time and route of administration, the kind of therapy, general health and other drugs being administered concurrently.
  • An "effective dose” refers to amounts of the active ingredient that are sufficient to affect the course and the severity of the disease, leading to the reduction or remission of such pathology and may be determined using known methods.
  • kits and uses for stimulating the immune system of an individual in need thereof comprising administration of an antigen binding protein described herein.
  • the administration of an antigen binding protein described herein induces and/or sustains cytotoxicity towards a cell expressing a target antigen where the cell expressing the target antigen is in a microenvironment with increased levels of protease activity.
  • the cell expressing a target antigen is a cancer or tumor cell, a virally infected cell, a bacterially infected cell, an autoreactive T or B cell, damaged red blood cells, arterial plaques, or fibrotic tissue.
  • the blood of the subject is rich in proteases.
  • Diseases, disorders or conditions associated with a target antigen include, but are not limited to, viral infection, bacterial infection, autoimmune disease, transplant rejection, atherosclerosis, or fibrosis.
  • the disease, disorder or condition associated with a target antigen is a proliferative disease, a tumorous disease, an inflammatory disease, an immunological disorder, an autoimmune disease, an infectious disease, a viral disease, an allergic reaction, a parasitic reaction, a graft-versus-host disease or a host-versus-graft disease.
  • the disease, disorder or condition associated with a target antigen is cancer.
  • the cancer is a hematological cancer.
  • the cancer is a solid tumor cancer.
  • treatment or “treating” or “treated” refers to therapeutic treatment wherein the object is to slow (lessen) an undesired physiological condition, disorder or disease, or to obtain beneficial or desired clinical results.
  • beneficial or desired clinical results include, but are not limited to, alleviation of symptoms; diminishment of the extent of the condition, disorder or disease; stabilization (i.e., not worsening) of the state of the condition, disorder or disease; delay in onset or slowing of the progression of the condition, disorder or disease; amelioration of the condition, disorder or disease state; and remission (whether partial or total), whether detectable or undetectable, or enhancement or improvement of the condition, disorder or disease.
  • Treatment includes eliciting a clinically significant response without excessive levels of side effects. Treatment also includes prolonging survival as compared to expected survival if not receiving treatment.
  • treatment or “treating” or “treated” refers to prophylactic measures, wherein the object is to delay onset of or reduce severity of an undesired physiological condition, disorder or disease, such as, for example is a person who is predisposed to a disease (e.g., an individual who carries a genetic marker for a disease such as breast cancer).
  • the antigen-binding proteins are administered in combination with an agent for treatment of the particular disease, disorder or condition.
  • Agents include but are not limited to, therapies involving antibodies, small molecules (e.g., chemotherapeutics), hormones (steroidal, peptide, and the like), radiotherapies ( ⁇ -rays, X- rays, and/or the directed delivery of radioisotopes, microwaves, UV radiation and the like), gene therapies (e.g., antisense, retroviral therapy and the like) and other immunotherapies.
  • the antigen-binding proteins are administered in combination with anti -diarrheal agents, anti-emetic agents, analgesics, opioids and/or non-steroidal anti-inflammatory agents. In some embodiments, the antigen-binding proteins are administered before, during, or after surgery.
  • “elimination half-time” is used in its ordinary sense, as is described in Goodman and Gillman's The Pharmaceutical Basis of Therapeutics 21-25 (Alfred Goodman Gilman, Louis S. Goodman, and Alfred Gilman, eds., 6th ed. 1980). Briefly, the term is meant to encompass a quantitative measure of the time course of drug elimination.
  • the elimination of most drugs is exponential (i.e., follows first-order kinetics), since drug concentrations usually do not approach those required for saturation of the elimination process.
  • the rate of an exponential process may be expressed by its rate constant, k, which expresses the fractional change per unit of time, or by its half-time, ti /2 the time required for 50% completion of the process.
  • the units of these two constants are time -1 and time, respectively.
  • the human CD16 canonical sequence is Uniprot Accession No. P08637.
  • Antibodies against CD 16 are generated via known technologies such as affinity maturation. Where murine anti-CD 16 antibodies are used as a starting material, humanization of murine anti-CD 16 antibodies is desired for the clinical setting, where the mouse-specific residues may induce a human-anti -mouse antigen (HAMA) response in subjects who receive treatment of an antigen binding protein described herein. Humanization is accomplished by grafting CDR regions from murine anti-CD 16 antibody onto appropriate human germline acceptor frameworks, optionally including other modifications to CDR and/or framework regions. As provided herein, antibody and antibody fragment residue numbering follows Kabat (Kabat E. A. et al, 1991; Chothia et al, 1987).
  • Human or humanized anti-CD 16 antibodies are therefore used to generate scFv sequences for CD 16 binding domains of an antigen-binding protein.
  • DNA sequences coding for human or humanized VL and VH domains are obtained, and the codons for the constructs are, optionally, optimized for expression in cells from Homo sapiens.
  • a protease cleavage site is included between the VH and VL domains.
  • the order in which the VL and VH domains appear in the scFv is varied (i.e., VL-VH, or VH-VL orientation), and three copies of the "G4S" or "G 4 S" subunit (G 4 S) 3 connect the variable domains to create the scFv domain.
  • Anti-CD 16 scFv plasmid constructs can have optional Flag, His or other affinity tags, and are electroporated into HEK293 or other suitable human or mammalian cell lines and purified.
  • Validation assays include binding analysis by FACS, kinetic analysis using Proteon, and staining of CD16- expressing cells.
  • CD20 is one of the cell surface proteins present on B-lymphocytes.
  • CD20 antigen is found in normal and malignant pre-B and mature B lymphocytes, including those in over 90% of B-cell non-Hodgkin's lymphomas (NHL).
  • the antigen is absent in hematopoietic stem cells, activated B lymphocytes (plasma cells) and normal tissue.
  • hematopoietic stem cells activated B lymphocytes (plasma cells) and normal tissue.
  • a scFv binding domain to CD20 is generated similarly to the above method for generation of a scFv binding domain to CD 16.
  • the anti-CD 16 scFv with protease cleavage site domains are used to construct an antigen binding protein in combination with an anti-CD20 scFv domain and a half-life extension domain (e.g., a HSA binding peptide or VH domain), with the domains organized as shown Figure 1.
  • a half-life extension domain e.g., a HSA binding peptide or VH domain
  • coding sequences of all protein domains are cloned into a mammalian expression vector system.
  • gene sequences encoding the CD 16 binding domain, half-life extension domain, and CD20 binding domain along with peptide linkers LI and L2 are separately synthesized and subcloned.
  • the resulting constructs are then ligated together in the order of 'CD20 binding domain - LI - VH CD 16 binding domain - L2 - protease cleavage domain - L3 - VL CD 16 binding domain - L4 - anti- CD20 scFv - L5 - half-life extension domain to yield a final construct.
  • All expression constructs are designed to contain coding sequences for an N-terminal signal peptide and a C- terminal hexahistidine (6xHis)-tag to facilitate protein secretion and purification, respectively. Expression of antigen-binding proteins in stably transfected CHO cells
  • a CHO cell expression system (Flp-In®, Life Technologies), a derivative of CHO-K1 Chinese Hamster ovary cells (ATCC, CCL-61) (Kao and Puck, Proc. Natl. Acad Sci USA 1968;60(4): 1275-81), is used. Adherent cells are subcultured according to standard cell culture protocols provided by Life Technologies. [0081] For adaption to growth in suspension, cells are detached from tissue culture flasks and placed in serum-free medium. Suspension-adapted cells are cryopreserved in medium with 10% DMSO.
  • Recombinant CHO cell lines stably expressing secreted antigen-binding proteins are generated by transfection of suspension-adapted cells. During selection with the antibiotic Hygromycin B viable cell densities are measured twice a week, and cells are centrifuged and resuspended in fresh selection medium at a maximal density of 0. lxlO 6 viable cells/mL. Cell pools stably expressing antigen-binding proteins are recovered after 2-3 weeks of selection at which point cells are transferred to standard culture medium in shake flasks. Expression of recombinant secreted proteins is confirmed by performing protein gel electrophoresis or flow cytometry. Stable cell pools are cryopreserved in DMSO containing medium.
  • Antigen-binding proteins are produced in 10-day fed-batch cultures of stably transfected CHO cell lines by secretion into the cell culture supernatant.
  • Cell culture supernatants are harvested after 10 days at culture viabilities of typically >75%. Samples are collected from the production cultures every other day and cell density and viability are assessed. On day of harvest, cell culture supernatants are cleared by centrifugation and vacuum filtration before further use.
  • Protein expression titers and product integrity in cell culture supernatants are analyzed by SDS-PAGE.
  • Antigen-binding proteins are purified from CHO cell culture supernatants in a two-step procedure.
  • the constructs are subjected to affinity chromatography in a first step followed by preparative size exclusion chromatography (SEC) on Superdex 200 in a second step.
  • Samples are buffer-exchanged and concentrated by ultrafiltration to a typical concentration of >1 mg/mL.
  • Purity and homogeneity are assessed by SDS PAGE under reducing and non-reducing conditions, followed by immunoblotting using an anti-HSA or anti idiotype antibody as well as by analytical SEC, respectively.
  • Purified proteins are stored at aliquots at -80°C until use.
  • Example 2 Determination of antigen affinity by flow cytometry
  • Example 1 The antigen-binding proteins of Example 1 are tested for their binding affinities to human CD16 + and CD20 + cells and cynomolgus CD16 + and CD20 + cells.
  • CD16 + and CD20 + cells are incubated with 100 ⁇ _, of serial dilutions of the antigen- binding proteins of Example 1 and at least one protease. After washing three times with FACS buffer the cells are incubated with 0.1 mL of 10 ⁇ g/mL mouse monoclonal anti -idiotype antibody in the same buffer for 45 min on ice. After a second washing cycle, the cells are incubated with 0.1 mL of 15 ⁇ / ⁇ . FITC-conjugated goat anti-mouse IgG antibodies under the same conditions as before.
  • Mean fluorescence intensities of the cell samples are calculated using CXP software (Beckman-Coulter, Krefeld, Germany) or Incyte software (Merck Millipore, Schwalbach, Germany). After subtracting the fluorescence intensity values of the cells stained with the secondary and tertiary reagents alone the values are them used for calculation of the K D values with the equation for one-site binding (hyperbola) of the GraphPad Prism (version 6.00 for Windows, GraphPad Software, La Jolla California USA).
  • CD 16 binding affinity and crossreactivity are evaluated in titration and flow cytometric experiments on CD16 + Jurkat cells and the cynomolgus CD16 + HSC-F cell line (JCRB, cat.: JCRBl 164).
  • CD20 binding and crossreactivity are assessed on the human CD20 + tumor cell lines.
  • the K D ratio of crossreactivity is calculated using the K D values determined on the CHO cell lines expressing either recombinant human or recombinant cynomolgus antigens.
  • Example 1 The antigen binding protein of Example 1 is evaluated in vitro on its mediation of T cell dependent cytotoxicity to CD20 + target cells.
  • Fluorescence labeled CD20 + REC-1 cells (a Mantle cell lymphoma cell line, ATCC CRL-3004) are incubated with isolated PBMC of random donors or CB15 T-cells (standardized T-cell line) as effector cells in the presence of the antigen binding protein of Example 1 and at least one protease. After incubation for 4 h at 37°C in a humidified incubator, the release of the fluorescent dye from the target cells into the supernatant is determined in a spectrofluorimeter. Target cells incubated without the antigen binding protein of Example land target cells totally lysed by the addition of saponin at the end of the incubation serve as negative and positive controls, respectively.
  • the percentage of specific cell lysis is calculated according to the following formula: [1 -(number of living targetS (Sam pie ) /number of living targetS( Spont aneous))] x 100%.
  • Sigmoidal dose response curves and EC 50 values are calculated by non-linear regression/4-parameter logistic fit using the GraphPad Software. The lysis values obtained for a given antibody concentration are used to calculate sigmoidal dose- response curves by 4 parameter logistic fit analysis using the Prism software.
  • Example 3 Pharmacokinetics of Antigen-binding Proteins
  • Example 1 The antigen binding protein of Example 1 is evaluated for half-time elimination in animal studies.
  • the antigen binding protein is administered to cynomolgus monkeys as a 0.5 mg/kg bolus injection into the saphenous vein.
  • Another cynomolgus monkey group receives a comparable protein in size with binding domains to CD 16 and CD20, but lacking a half-life extension domain.
  • a third and fourth group receive a protein with CD 16 and half-life extension domains and a protein with CD20 and half-life extension domains respectively, and both comparable in size to the antigen-binding protein.
  • Each test group consists of 5 monkeys.
  • Serum samples are taken at indicated time points, serially diluted, and the concentration of the proteins is determined using a binding ELISA to CD 16 and/or CD20.
  • the data are fit by a standard two-compartment model with bolus input and first-order rate constants for distribution and elimination phases.
  • the a-phase is the initial phase of the clearance and reflects distribution of the protein into all extracellular fluid of the animal, whereas the second or ⁇ -phase portion of the decay curve represents true plasma clearance.
  • Example 1 The antigen binding protein of Example 1 is evaluated in a xenograft model.
  • mice Female immune-deficient NOD/scid mice are sub-lethally irradiated (2 Gy) and subcutaneously inoculated with 4xl0 6 Ramos RA1 cells into the right dorsal flank. When tumors reach 100 to 200 mm 3 , animals are allocated into 3 treatment groups. Groups 2 and 3 (8 animals each) are intraperitoneally injected with 1.5xl0 7 activated human T-cells. Three days later, animals from Group 3 are subsequently treated with a total of 9 intravenous doses of 50 ⁇ g antigen binding protein of Example 1 (qdx9d). Groups 1 and 2 are only treated with vehicle. Body weight and tumor volume are determined for 30 days.
  • Example 5 Proof-of-Concept Clinical Trial Protocol for Administration of the Antigen Binding Protein of Example 1 to B-cell Lymphoma Patients
  • Example 1 as a treatment for B-cell Lymphoma.
  • MTD maximum tolerated dose
  • a subsequent phase II section will be treated at the MTD with a goal of determining if therapy with therapy of antigen binding protein of Example 1 results in at least a 20% response rate.

Abstract

L'invention concerne des protéines de liaison à l'antigène activées de manière conditionnelle comprenant un domaine activé par une protéase se liant à une cellule immunitaire innée, au moins un domaine d'extension de demi-vie, et au moins deux domaines se liant à un ou plusieurs antigènes cibles. L'invention concerne également des compositions pharmaceutiques de celles-ci, ainsi que des acides nucléiques, des vecteurs d'expression de recombinaison et des cellules hôtes permettant d'obtenir de telles protéines de liaison à l'antigène. L'invention concerne également des méthodes d'utilisation desdites protéines de liaison à l'antigène, dans la prévention et/ou le traitement de maladies, d'états pathologiques et de troubles.
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US11535668B2 (en) 2017-02-28 2022-12-27 Harpoon Therapeutics, Inc. Inducible monovalent antigen binding protein
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US10730954B2 (en) 2017-05-12 2020-08-04 Harpoon Therapeutics, Inc. MSLN targeting trispecific proteins and methods of use
US10543271B2 (en) 2017-05-12 2020-01-28 Harpoon Therapeutics, Inc. Mesothelin binding proteins
US10927180B2 (en) 2017-10-13 2021-02-23 Harpoon Therapeutics, Inc. B cell maturation antigen binding proteins
US11136403B2 (en) 2017-10-13 2021-10-05 Harpoon Therapeutics, Inc. Trispecific proteins and methods of use
US11976125B2 (en) 2017-10-13 2024-05-07 Harpoon Therapeutics, Inc. B cell maturation antigen binding proteins
US10696724B2 (en) 2018-05-14 2020-06-30 Werewolf Therapeutics, Inc. Activatable interleukin-2 polypeptides
US10696723B2 (en) 2018-05-14 2020-06-30 Werewolf Therapeutics, Inc. Activatable interleukin 12 polypeptides
US11352403B2 (en) 2018-05-14 2022-06-07 Werewolf Therapeutics, Inc. Activatable interleukin-2 polypeptides and methods of use thereof
US11453710B2 (en) 2018-05-14 2022-09-27 Werewolf Therapeutics, Inc. Activatable interleukin 12 polypeptides and methods of use thereof
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