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WO2024148336A1 - Molécules de liaison multimères costimulatrices de lymphocytes t et utilisations associées - Google Patents

Molécules de liaison multimères costimulatrices de lymphocytes t et utilisations associées Download PDF

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Publication number
WO2024148336A1
WO2024148336A1 PCT/US2024/010591 US2024010591W WO2024148336A1 WO 2024148336 A1 WO2024148336 A1 WO 2024148336A1 US 2024010591 W US2024010591 W US 2024010591W WO 2024148336 A1 WO2024148336 A1 WO 2024148336A1
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seq
binding molecule
amino acid
antibody
igm
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Liqin Liu
Deepal PANDYA
Beatrice Tien-Yi WANG
Hyewon PHEE
Keyu LI
Umesh S. Muchhal
Angus SINCLAIR
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IGM Biosciences Inc
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IGM Biosciences Inc
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • 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
    • 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
    • C07K16/2809—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 the T-cell receptor (TcR)-CD3 complex
    • 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
    • C07K16/2818—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 CD28 or CD152
    • 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
    • C07K16/2878—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 NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
    • 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
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • 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
    • C07K2317/60—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)

Definitions

  • T cell-engaging bispecific antibodies that can bind to a tumor antigen and simultaneously target cytotoxic T cells, e.g., by binding to CD3 to promote killing of tumor cells are a valuable and rapidly expanding tool in the treatment of various malignancies. See, e.g., Einsele, H., etal., Cancer 126'. 3192-3201 (2020). While the advancements made in T cell engager (TCE) therapeutics has been significant, a need remains for improved therapeutic results, especially in solid tumor indications, where the tumor microenvironment can pose a significant barrier to T cell infiltration. See, e.g., Lim, A.R., et al., eLife 9. doi: 10.7554/eLife.55185 (2020).
  • T cell activation typically involves two signals — activation through MHC-antigen engagement with a T cell receptor (TCR) (“signal 1”) and coactivation through a costimulatory molecule (“signal 2”).
  • TCR T cell receptor
  • costimulatory molecules are receptors expressed on T cells that upon coactivation can, e.g., induce signaling to activate T cells more fully upon T cell receptor engagement, thereby enhancing signaling and cytokine stimulation. This co-signaling can further modulate T cell differentiation, effector function, and survival. See, e.g., Baeuerle, P.A., and H. Wesche, Curr. Opin. Oncol. 34:552-558 (2022).
  • Exemplary T cell costimulatory molecules include, without limitation, CD28 and 4- IBB. See, e.g., Jeong, S., and S-H. Park, Immune Netw. doi: 10.4110/in.2020.20. e3 (2020).
  • CD3 engagement by a TCE can replace the conventional signal 1 but lacks the signal 2 coactivation.
  • Combining a bispecific TCE that engages CD3 with a tumor-targeted bispecific antibody that engages and coactivates a T cell costimulatory molecule has shown promise in preclinical studies. See, e.g., Skokos, D., et al., Sci. Transl. Med. /2(525 ):caaw7888. doi: 10.1126/scitranslmed.aaw7888 (2020).
  • Binding molecules e.g., antibodies that can multimerize, such as IgA and IgM antibodies, have emerged as promising drug candidates in the fields of, e.g., immunooncology and infectious diseases, allowing for improved specificity, improved avidity, and the ability to bind to multiple targets. See, e.g., U.S. Patent Nos. 9,951,134, 9,938,347, 10,351,631, 10,400,038, 10,570,191, 1,0604,559, 10,689,449, 10,787,520, 10,899,835, 11,555,075, 10,618,978, and 11,639,389, U.S. Patent Application Publication Nos.
  • imvotamab (IGM-2323), a CD20 x CD3 IgM bispecific antibody, has shown promise in early clinical studies. See, e.g., Budde, E., etal., Blood 138 (Suppl. 1): 132 doi: 10.1182/blood-2021-153355 (2021).
  • a multimeric binding molecule comprising five, four, or two bivalent binding units and a modified J chain, wherein each binding unit comprises two antibody heavy chains, each comprising an IgM or IgA heavy chain constant region or multimerizing variant or fragment thereof each associated with an antigen-binding domain that specifically binds to a target antigen (a “target antigen-binding domain”), and wherein the modified J chain comprises (a) a J chain or a functional fragment or variant thereof (“J”), (b) an anti-CD3 scFv (“C”), and (c) a heterologous polypeptide (“H”), wherein H comprises a polypeptide agonist of a T cell costimulatory molecule, wherein the J, C, and H are associated as a fusion protein.
  • the T cell costimulatory molecule comprises CD28 or 4-1BB.
  • H comprises an anti-CD28 antibody or antigen-binding fragment thereof or a 4- IBB ligand
  • the modified J chain comprises, in the N-terminal to C- terminal direction, C-J-H or H-J-C.
  • C, J, and H are fused via amino acid linkers which can be the same or different.
  • each linker consists, independently, of 5 to 25 amino acids.
  • each linker consists, independently, of the amino acid sequence (GGGGS)n (SEQ ID NO: 279), wherein n is an integer between 2 and 5, GGGGSGGGGS (SEQ ID NO: 10), GGGGSGGGGSGGGGS (SEQ ID NO: 11), GKPGSGKPGSGKPGSGKPGS (SEQ ID NO: 14), or GGGGSGGGGSGGGG (SEQ ID NO: 235).
  • the linker consists of GGGGSGGGGS (SEQ ID NO: 10).
  • the linker consists of GGGGSGGGGSGGGGS (SEQ ID NO: 11).
  • the linker consists of GKPGSGKPGSGKPGSGKPGS (SEQ ID NO: 14).
  • the antigen-binding fragment of an antibody is an anti-CD28 single domain heavy chain variable region (sdVH) or an anti-CD28 single domain light chain variable region (sdVL).
  • the anti-CD28 sdVH comprises complementarity-determining regions CDR1, CDR2, and CDR3, wherein the CDR1, CDR2, and CDR3 comprise, respectively, the amino acid sequences SEQ ID NO: 183, SEQ ID NO: 187, and SEQ ID NO: 191; SEQ ID NO: 184, SEQ ID NO: 188, and SEQ ID NO: 192; or SEQ ID NO: 185, SEQ ID NO: 189, and SEQ ID NO: 193 ; or wherein the anti-CD28 sdVL comprises complementarity-determining regions CDR1, CDR2, and CDR3, wherein the CDR1, CDR2, and CDR3 comprise, respectively, the amino acid sequences SEQ ID NO: 186, SEQ ID NO: 190, and
  • H comprises a 4-1BBL trimer.
  • the 4- 1BBL trimer comprises three 4-1BBL monomers.
  • each 4-1BBL monomer is a soluble fragment of human 4-1BBL comprising amino acids X to 254 of SEQ ID NO: 15, wherein X is an integer from 50 to 99.
  • the soluble fragments of 4-1BBL each comprise amino acids 71 to 254 of SEQ ID NO: 15 (SEQ ID NO: 197).
  • the soluble fragments of 4-1BBL each comprise amino acids 58 to 254 of SEQ ID NO: 15 (SEQ ID NO: 196).
  • the VH and VL of C comprise an amino acid sequence at least 80%, 85%, 90%, 95%, or 100% identical to the amino acid sequence of SEQ ID NO: 16 and SEQ ID NO: 20; SEQ ID NO: 24 and SEQ ID NO: 28; SEQ ID NO: 24 and SEQ ID NO: 32; SEQ ID NO: 34 and SEQ ID NO: 38; SEQ ID NO: 42 and SEQ ID NO: 44; or SEQ ID NO: 46 and SEQ ID NO: 48, respectively.
  • the C comprises the VH and VL amino acid sequences SEQ ID NO: 24 and SEQ ID NO: 28, respectively.
  • the VH and VL are fused via an amino acid linker.
  • the linker consists of the amino acid sequence (GGGGS)n (SEQ ID NO: 279), wherein n is an integer between 2 and 5, GGGGSGGGGS (SEQ ID NO: 10), GGGGSGGGGSGGGGS (SEQ ID NO: 11), GKPGSGKPGSGKPGSGKPGS (SEQ ID NO: 14), or GGGGSGGGGSGGGG (SEQ ID NO: 235).
  • the linker consists of GGGGSGGGGS (SEQ ID NO: 10).
  • the linker consists of GGGGS GGGGSGGGGS (SEQ ID NO: 11).
  • the linker consists of GKPGSGKPGSGKPGSGKPGS (SEQ ID NO 14)
  • J comprises SEQ ID NO: 7 or a functional fragment or variant thereof.
  • J comprises an alanine substitution at the amino acid position corresponding to amino acid Y102 of the mature wild-type human J chain (SEQ ID NO: 7).
  • J comprises the amino acid sequence SEQ ID NO: 8 (“J*”).
  • the modified J chain comprises the amino acid sequence SEQ ID NO: 231, SEQ ID NO: 232, SEQ ID NO: 233, or SEQ ID NO: 234
  • the modified J chain comprises the amino acid sequence SEQ ID NO: 213 or SEQ ID NO: 214.
  • a multimeric binding molecule comprising five, four, or two bivalent binding units and a modified J chain, wherein each binding unit comprises two antibody heavy chains, each comprising an IgM or IgA heavy chain constant region or multimerizing variant or fragment thereof, and wherein the modified J chain comprises a J chain or a functional fragment or variant thereof and a 4-1BBL trimer associated as a fusion protein, wherein the 4-1BBL trimer comprises three 4-1BBL monomers each comprising a soluble fragment of 4-1BBL.
  • each 4-1BBL monomer is a soluble fragment of human 4- 1BBL comprising amino acids X to 254 of SEQ ID NO: 15, wherein X is an integer from 50 to 99.
  • the soluble fragments of 4-1BBL each comprise amino acids 71 to 254 of SEQ ID NO: 15 (SEQ ID NO: 197).
  • the soluble fragments of 4-1BBL each comprise amino acids 58 to 254 of SEQ ID NO: 15 (SEQ ID NO: 196).
  • the 4-1BBL monomers are fused via amino acid linkers which can the same or different.
  • each linker consists, independently, of 5 to 25 amino acids.
  • each linker consists, independently, of the amino acid sequence (GGGGS)n (SEQ ID NO: 279), wherein n is an integer between 2 and 5, GGGGSGGGGS (SEQ ID NO: 10), GGGGS GGGGS GGGGS (SEQ ID NO: 11), GKPGSGKPGSGKPGSGKPGS (SEQ ID NO: 14), or GGGGSGGGGSGGGG (SEQ ID NO: 235).
  • the linker consists of GGGGSGGGGS (SEQ ID NO: 10).
  • the linker consists of GGGGSGGGGSGGGGS (SEQ ID NO: 11).
  • the tumor-associated target antigen is CD20. In some embodiments, the tumor-associated target antigen is mesothelin (MSLN). In some embodiments, the tumor- associated target antigen is CD38. In some embodiments the tumor-associated target antigen is tumor-associated calcium signal transducer 2 (TROP-2). In some embodiments, the multimeric binding molecule conditionally activates T cells in the presence of the tumor-associated target antigen.
  • Also provided herein is a vector comprising a composition disclosed herein.
  • Also provided herein is a method of producing a multimeric binding molecule disclosed herein, comprising culturing the host cell disclosed herein, and recovering the multimeric binding molecule.
  • FIGs. 53A-53C are graphs of tumor volume (mm 3 ) over time (days) for different antibody treatment groups (Tl: TROP-2 x CD3 x CD28 IgM at doses of 0.3 mg/kg, 1 mg/kg, and 3 mg/kg; T3: TROP-2 x CD3 IgM at doses of 0.3 mg/kg, 1 mg/kg, and 3 mg/kg) and vehicle, evaluated in a humanized HEK293/TROP-2 xenograft model as described in Example 26.
  • FIG. 53A provides data for the 0.3 mg/kg treatment groups
  • FIG. 53B provides data for the 1 mg/kg treatment groups
  • FIG. 53C provides data for the 3 mg/kg treatment groups.
  • FIGs. 59A-C are bar graphs showing the ability of various combinations of anti- CD28 and/or anti CD3 IgM or IgG4 antibodies to activate T cells and induce cytokine release in a plate-based assay as described in Example 29.
  • the results of one of two exemplary PMBC donors (PBMC Donor 1) is shown.
  • the various antibody and antibody combinations are detailed in Table 27.
  • FIG. 59A shows CD4+ T cell activation.
  • FIG. 59B shows CD8+ T cell activation.
  • FIG. 59C shows IL-2 expression.
  • MSLN anti-mesothelin
  • MSLN anti-CD3 x anti-CD28 IgM antibody
  • polypeptide encompasses a singular “polypeptide” as well as plural “polypeptides,” and refers to a molecule composed of monomers (amino acids) linearly linked by amide bonds (also known as peptide bonds).
  • polypeptide refers to any chain or chains of two or more amino acids and does not refer to a specific length of the product.
  • peptides, dipeptides, tripeptides, oligopeptides, “protein,” “amino acid chain,” or any other term used to refer to a chain or chains of two or more amino acids are included within the definition of "polypeptide,” and the term “polypeptide” can be used instead of any of these terms.
  • a binding unit need not include full-length antibody heavy and light chains, but will typically be bivalent, i.e., will include two “antigen-binding domains,” e.g., “target antigen-binding domains,” as defined above.
  • certain binding molecules provided in this disclosure are “pentameric” and include five bivalent binding units that include IgM constant regions or multimerizing fragments or variants thereof and a J chain or functional fragment or variant thereof.
  • a binding molecule e.g., an antibody, comprising two or more, e.g., two, four, or five binding units, is referred to herein as a “multimeric binding molecule.”
  • J chain refers to the J chain of IgM or IgA antibodies of any animal species, any functional fragment thereof, derivative thereof, and/or variant thereof, including a mature human J chain, the amino acid sequence of which is presented as SEQ ID NO: 7.
  • Various J chain variants and modified J chain derivatives are available, e.g., in U.S. Patent No. 10,899,835.
  • a functional fragment or “a functional variant” includes those fragments and variants that can associate with IgM heavy chain constant regions to form a pentameric IgM antibody.
  • modified J chain is used herein to refer to a derivative of a J chain polypeptide comprising a heterologous moiety, e.g., a heterologous polypeptide, e.g., an extraneous antigen-binding domain or functional domain (e.g., a ligand that binds to an effector cell, e.g., a T cell receptor or an immunostimulatory molecule) introduced into or attached to the J chain sequence.
  • a heterologous polypeptide e.g., an extraneous antigen-binding domain or functional domain (e.g., a ligand that binds to an effector cell, e.g., a T cell receptor or an immunostimulatory molecule) introduced into or attached to the J chain sequence.
  • the introduction can be achieved by any means, including direct or indirect fusion of the heterologous polypeptide or other moiety or by attachment through a peptide or chemical linker.
  • valency refers to the number of binding domains, e.g., anti gen -binding domains, e.g., target antigenbinding domains, in a given antibody or in a given binding unit.
  • binding domains e.g., anti gen -binding domains, e.g., target antigenbinding domains
  • the terms “bivalent,” “tetravalent,” and “hexavalent” in reference to a given antibody denote the presence of two antigen-binding domains, four antigen-binding domains, and six antigenbinding domains, respectively.
  • a typical pentameric IgM antibody, where each binding unit is bivalent has 10 or more valencies, e.g., target antigen-binding domains.
  • target is used in the broadest sense to include substances that can be bound by a binding molecule, e.g., a multimeric binding molecule as provided herein.
  • a target can be, e.g., a polypeptide, a nucleic acid, a carbohydrate, a lipid, or other molecule.
  • a “target” can, for example, be a cell, an organ, or an organism that comprises an epitope that can be bound by a binding molecule, e.g., a multimeric binding molecule as provided herein.
  • Both the light and heavy chain of a binding molecule are divided into regions of structural and functional homology.
  • the terms "constant” and “variable” are used functionally.
  • the variable domains of both the variable light (VL) and variable heavy (VH) chain portions determine antigen recognition and specificity.
  • the constant region domains of the light chain (CL) and the heavy chain e.g., CHI, CH2, CH3, or CH4) confer biological properties such as secretion, transplacental mobility, Fc receptor binding, complement binding, and the like.
  • the N-terminal portion is a variable region and at the C-terminal portion is a constant region; the CH3 (or CH4-ptp, e.g., in the case of IgM) and CL domains comprise the carboxy-terminus of the heavy and light chain, respectively.
  • a “full length IgM antibody heavy chain” is a polypeptide that includes, in N- terminal to C terminal direction, an antibody heavy chain variable domain (VH), an antibody heavy chain constant domain 1 (CM1 or Cpl), an antibody heavy chain constant domain 2 (CM2 or Cp2), an antibody heavy chain constant domain 3 (CM3 or Cp3), and an antibody heavy chain constant domain 4 (CM4 or Cp4) that can include a p tailpiece.
  • VH antibody heavy chain variable domain
  • CM1 or Cpl an antibody heavy chain constant domain 1
  • CM2 or Cp2 an antibody heavy chain constant domain 2
  • CM3 or Cp3 an antibody heavy chain constant domain 3
  • CM4 or Cp4 an antibody heavy chain constant domain 4
  • IgM-like antibody refers to a binding molecule that includes at least a multimerizing fragment or variant of an IgM heavy chain constant region that retains the ability to form hexamers or pentamers, e.g., in association with a J chain.
  • An IgM-like antibody typically includes at least the Cp4 and IgM tailpiece (ptp) domains of an IgM constant region but can include heavy chain constant region domains from other antibody isotypes, e.g., IgG, from the same species or from a different species.
  • an IgM- like antibody can likewise be an antibody fragment in which one or more constant region domains are deleted, as long as the IgM-like antibody is capable of multimerizing into a hexamer and/or a pentamer.
  • an IgM-like antibody can be, e.g., a hybrid IgM/IgG antibody or can be a “multimerizing fragment” of an IgM antibody.
  • a “full length IgA antibody heavy chain” is a polypeptide that includes, in N- terminal to C terminal direction, an antibody heavy chain variable domain (VH), an antibody constant heavy chain constant domain 1 (CAI or Cal), an antibody heavy chain constant domain 2 (CA2 or Ca2), and an antibody heavy chain constant domain 3 (CA3 or Ca3) that can include an a tailpiece.
  • VH antibody heavy chain variable domain
  • CAI or Cal antibody constant heavy chain constant domain 1
  • CA2 or Ca2 an antibody heavy chain constant domain 2
  • CA3 or Ca3 an antibody heavy chain constant domain 3
  • IgA-like antibody refers to a binding molecule that includes at least a multimerizing fragment or variant of an IgA heavy chain constant region that retains the ability to form dimers or tetramers, e.g., in association with a I chain.
  • variable region(s) allow a binding molecule, e.g., a multimeric binding molecule as provided herein, to selectively recognize and specifically bind epitopes on target antigens.
  • a binding molecule e.g., a multimeric binding molecule as provided herein
  • CDRs complementarity determining regions
  • an antigenbinding domain can be defined by the six CDRs on the VH and VL chains, or the three CDRs on an sdVH or sdVL chain, respectively. Certain antibodies form larger structures.
  • IgA can form a molecule that includes two or four H2L2 binding units and a J chain covalently connected via disulfide bonds, which can be further associated with a secretory component
  • IgM can form a pentameric molecule that includes five H2L2 binding units and a J chain covalently connected via disulfide bonds.
  • CDRs complementarity determining regions
  • the six (or in the case of single domain antibodies, three) “complementarity determining regions” or “CDRs” present in an antibody antigen-binding domain are short, non-contiguous sequences of amino acids that are specifically positioned to form the antigen-binding domain as the antibody assumes its three-dimensional configuration in an aqueous environment.
  • the remainder of the amino acids in the antigen-binding domain referred to as "framework" regions, show less inter-molecular variability.
  • the framework regions largely adopt a P-sheet conformation and the CDRs form loops which connect, and in some cases form part of, the 3 -sheet structure.
  • framework regions act to form a scaffold that provides for positioning the CDRs in correct orientation by inter-chain, non- covalent interactions.
  • the antigen-binding domain formed by the positioned CDRs defines a surface complementary to the epitope on the target antigen. This complementary surface promotes the non-covalent binding of the antibody to its cognate epitope.
  • the amino acids that make up the CDRs and the framework regions, respectively, can be readily identified for any given heavy or light chain variable region by one of ordinary skill in the art, since they have been defined in various different ways (see, "Sequences of Proteins of Immunological Interest, " Kabat, E., et al., U.S. Department of Health and Human Services, (1983); and Chothia and Lesk, J. Mol. Biol., 196:901-917 (1987), which are incorporated herein by reference in their entireties).
  • CDR complementarity determining region
  • the Kabat and Chothia definitions include overlapping or subsets of amino acids when compared against each other. Other overlapping CDR definitions can be found, e.g., in Al-Lazikani B. et al., J. Mol. Biol. 273'. 927-948 (1997); MacCallum et al., J. Mol. Biol. 262'.732- 5 (1996); Abhinandan and Martin, Mol. Immunol. 45'. 3832- 3839 (2008); Lefranc M. P. et al., Dev. Comp. Immunol. 27'. 55-77 (2003); and Honegger and Pliickthun, J. Mol. Biol.
  • Antibody variable domains can also be analyzed, e.g., using the IMGT information system (imgt_dot_cines_dot_fr/) (IMGT®/V-Quest) to identify variable region segments, including CDRs.
  • IMGT information system imgt_dot_cines_dot_fr/
  • IMGT®/V-Quest IMGT information system
  • IgM constant regions can be numbered sequentially (i.e., amino acid #1 starting with the first amino acid of the constant region, or by using the Kabat numbering scheme.
  • SEQ ID NO: 1 allele IGHM*03
  • SEQ ID NO: 2 allele IGHM*04
  • underlined amino acid residues are not accounted for in the Kabat system (“X,” double underlined below, can be serine (S) (SEQ ID NO: 1) or glycine (G) (SEQ ID NO: 2)): equential ( SEQ ID NO : 1 or SEQ ID NO : 2 ) /KABAT numbering key for gM heavy chain
  • a binding molecule e.g., an antibody
  • binds to an epitope via its antigen-binding domain and that the binding entails some complementarity between the antigen-binding domain and the epitope.
  • a binding molecule e.g., antibody
  • binds to an epitope when it binds to that epitope via its antigen-binding domain more readily than it would bind to a random, unrelated epitope.
  • the term "specificity" is used herein to qualify the relative affinity by which a certain binding molecule binds to a certain epitope.
  • binding molecule "A” can be deemed to have a higher specificity for a given epitope than binding molecule "B,” or binding molecule “A” can be said to bind to epitope “C” with a higher specificity than it has for related epitope "D.”
  • the term "affinity” refers to a measure of the strength of the binding of an individual epitope with one or more antigen-binding domains, e.g., of an immunoglobulin molecule. See, e.g., Greenfield, E.A. (ed.), Antibodies: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, 2014.
  • the term “avidity” refers to the overall stability of the complex between a population of antigen-binding domains and an antigen. Avidity is related to both the affinity of individual antigen-binding domains in the population with specific epitopes, and the valencies of the immunoglobulins and the antigen.
  • Antigen-binding antibody fragments including single-chain antibodies or other antigen-binding domains can exist alone or in combination with one or more of the following: hinge region, CHI, CH2, CH3, or CH4 domains, J chain, or secretory component. Also included are antigen-binding fragments that can include any combination of variable region(s) with one or more of a hinge region, CHI, CH2, CH3, or CH4 domains, a J chain, or a secretory component.
  • Antibodies can be from any animal origin including fish, birds, and mammals. The antibodies can be human, murine, donkey, rabbit, goat, guinea pig, camel, llama, horse, or chicken antibodies.
  • variable region can be condricthoid in origin e.g., from sharks).
  • "human” antibodies include antibodies having the amino acid sequence of a human immunoglobulin and include antibodies isolated from human immunoglobulin libraries or from animals transgenic for one or more human immunoglobulins and can in some instances express endogenous immunoglobulins and some not, as described infra and, for example in, U.S. Pat. No. 5,939,598 by Kucherlapati et al.
  • multimeric binding molecule e.g., an IgM antibody as described herein can include an antigen-binding fragment of an antibody, e.g., a scFv fragment, so long as the IgM antibody is able to form a multimer, e.g., a hexamer or a pentamer
  • an IgA antibody as provided herein can include an antigen-binding fragment of an antibody, e.g., a scFv fragment, so long as the IgA antibody is able to form a multimer, e.g., a dimer or a tetramer.
  • a binding molecule e.g., an antibody can include, in addition to a VH domain, a CH3 domain and a CH4 domain; or a CH3 domain, a CH4 domain, an IgM tailpiece, and a J chain.
  • a binding molecule e.g., an antibody can lack certain constant region portions, e.g., all or part of a CH2 domain. It will be understood by one of ordinary skill in the art that these domains (e.g., the heavy chain subunit) can be modified such that they vary in amino acid sequence from the original immunoglobulin molecule.
  • a multimeric binding molecule e.g., a pentameric IgM antibody, IgM- like antibody, or other IgM-derived binding molecule or a dimeric or tetrameric IgA antibody, IgA-like antibody, or other IgA-derived binding molecule as provided herein includes sufficient portions of an IgM or IgA heavy chain constant region to allow the pentameric IgM antibody, IgM-like antibody, or other IgM-derived binding molecule, or dimeric or tetrameric IgA antibody, IgA-like antibody, or other IgA-derived binding molecule to form a multimer, e.g., a pentamer, a tetramer, or a dimer.
  • a heavy chain constant region fragment comprises a “multimerizing fragment.”
  • the term “light chain subunit” includes amino acid sequences derived from an immunoglobulin light chain.
  • the light chain subunit includes at least a VL, and can further include a CL (e.g., CK or C7.) domain.
  • CL e.g., CK or C7.
  • Binding molecules e.g., antibodies
  • Binding molecules can be described or specified in terms of the epitope(s) or portion(s) of a target, e.g., a target antigen that they recognize or specifically bind.
  • the portion of a target antigen that specifically interacts with the target antigenbinding domain of an antibody is an "epitope," or an "antigenic determinant.”
  • a target antigen can comprise a single epitope or at least two epitopes, and can include any number of epitopes, depending on the size, conformation, and type of antigen.
  • chimeric antibody refers to an antibody in which the immunoreactive region or site is obtained or derived from a first species and the constant region (which can be intact, partial, or modified) is obtained from a second species.
  • the target binding region or site will be from a non-human source (e.g., mouse or primate) and the constant region is human.
  • multispecific antibody or “bispecific antibody” refer to an antibody, which has antigen-binding domains for two or more different epitopes within a single antibody molecule.
  • Other binding molecules in addition to the canonical antibody structure can be constructed with two binding specificities.
  • Epitope binding by bispecific or multispecific antibodies can be simultaneous or sequential.
  • Triomas and hybrid hybridomas are two examples of cell lines that can secrete bispecific antibodies
  • Bispecific antibodies can also be constructed by recombinant means. (Strbhlein and Heiss, Future Oncol. 6:1387-94 (2010); Mabry and Snavely, IDrugs. 13:543-9 (2010)).
  • a bispecific antibody can also be a diabody.
  • the term "engineered antibody” refers to an antibody in which a variable domain, constant region, and/or J chain is altered by at least partial deletion, addition, or replacement of one or more amino acids.
  • entire CDRs from an antibody of known specificity can be grafted into the framework regions of a heterologous antibody.
  • alternate CDRs can be derived from an antibody of the same class or even subclass as the antibody from which the framework regions are derived, CDRs can also be derived from an antibody of different class, e.g., from an antibody from a different species.
  • in-frame fusion refers to the joining of two or more polynucleotide open reading frames (ORFs) to form a continuous longer ORF, in a manner that maintains the translational reading frame of the original ORFs.
  • a recombinant fusion protein is a single protein containing two or more segments that correspond to polypeptides encoded by the original ORFs (which segments are not normally so joined in nature.) Although the reading frame is thus made continuous throughout the fused segments, the segments can be physically or spatially separated by, for example, and inframe linker sequence.
  • polynucleotides encoding the CDRs of an immunoglobulin variable region can be fused, in-frame, but be separated by a polynucleotide encoding at least one immunoglobulin framework region or additional CDR regions, as long as the "fused" CDRs are co-translated as part of a continuous polypeptide.
  • Two half-lives can be described: the alpha half-life, a half-life, or tl/2a, which is the rate of decline in plasma concentrations due to the process of drug redistribution from the central compartment, e.g., the blood in the case of intravenous delivery, to a peripheral compartment (e.g., a tissue or organ), and the beta half-life, halflife, or tl/2p which is the rate of decline due to the processes of excretion or metabolism.
  • the alpha half-life, a half-life, or tl/2a which is the rate of decline in plasma concentrations due to the process of drug redistribution from the central compartment, e.g., the blood in the case of intravenous delivery, to a peripheral compartment (e.g., a tissue or organ)
  • the beta half-life, halflife, or tl/2p which is the rate of decline due to the processes of excretion or metabolism.
  • the term “area under the plasma drug concentration-time curve” or “AUC” reflects the actual body exposure to drug after administration of a dose of the drug and is expressed in mg*h/L. This area under the curve can be measured, e.g., from time 0 (tO) to infinity (co) and is dependent on the rate of elimination of the drug from the body and the dose administered.
  • the term “mean residence time” or “MRT” refers to the average length of time the drug remains in the body.
  • the human Cpl region ranges from about amino acid 5 to about amino acid 102 of SEQ ID NO: 1 or SEQ ID NO: 2; the human Cp2 region ranges from about amino acid 114 to about amino acid 205 of SEQ ID NO: 1 or SEQ ID NO: 2, the human Cp3 region ranges from about amino acid 224 to about amino acid 319 of SEQ ID NO: 1 or SEQ ID NO: 2, the Cp4 region ranges from about amino acid 329 to about amino acid 430 of SEQ ID NO: 1 or SEQ ID NO: 2, and the ptp ranges from about amino acid 431 to about amino acid 453 of SEQ ID NO: 1 or SEQ ID NO: 2.
  • An IgM heavy chain constant region can additionally include a Cp3 domain or a fragment thereof, a Cp2 domain or a fragment thereof, a C p 1 domain or a fragment thereof, and/or other IgM heavy chain domains.
  • an IgM antibody as provided herein can include a complete IgM heavy (p) chain constant domain, e.g., SEQ ID NO: 1 or SEQ ID NO: 2, or a variant, derivative, or analog thereof, e.g., as provided herein.
  • the two IgM heavy chain constant regions included in each binding unit are human heavy chain constant regions.
  • the heavy chains are glycosylated.
  • the heavy chains can be mutated to affect glycosylation. See, e.g., U.S. Patent Application Publication No. US2022/0306760 Al, which is incorporated herein by reference in its entirety.
  • the IgM antibody provided in this disclosure is pentameric
  • the IgM antibody further includes a modified J chain or variant thereof that further comprises one or more heterologous moieties attached to a J chain, as described elsewhere herein.
  • the I chain can be mutated to affect, e.g., enhance, the serum half-life of the IgM antibody provided herein, as discussed elsewhere in this disclosure.
  • the J chain can be mutated to affect glycosylation, as discussed elsewhere in this disclosure.
  • the human Cal domain extends from about amino acid 6 to about amino acid 98 of SEQ ID NO: 3; the human IgAl hinge region extends from about amino acid 102 to about amino acid 124 of SEQ ID NO: 3, the human Ca3 domain extends from about amino acid 228 to about amino acid 330 of SEQ ID NO: 3, and the atp extends from about amino acid 331 to about amino acid 352 of SEQ ID NO: 3.
  • the human IgA2 constant region typically includes the amino acid sequence SEQ ID NO: 4.
  • Two or four IgA binding units can form a complex with two additional polypeptide chains, the J chain (e.g., SEQ ID NO: 7 or SEQ ID NO: 8) and the secretory component (precursor, SEQ ID NO: 5, mature: amino acids 19 to 603 of SEQ ID NO: 5) to form a secretory IgA (slgA) antibody.
  • the assembly of IgA binding units into a dimeric or tetrameric slgA antibody is thought to involve the Ca3 and atp domains (also referred to herein collectively as the Ca3-tp domain).
  • a dimeric or tetrameric slgA antibody provided in this disclosure typically includes IgA constant regions that include at least the Ca3 and atp domains.
  • An IgA heavy chain constant region can additionally include a Ca2 domain or a fragment thereof, an IgA hinge region, a Cal domain or a fragment thereof, and/or other IgA heavy chain domains.
  • an IgA antibody or IgA-like binding molecule as provided herein can include a complete IgA heavy (a) chain constant domain (e.g., SEQ ID NO: 3 or SEQ ID NO: 4), or a variant thereof.
  • each IgA heavy chain constant region or multimerizing fragment thereof is a human IgA constant region.
  • the binding units of the IgA antibody, IgA-like antibody, or other IgA-derived binding molecule comprise two light chains.
  • the light chains are kappa light chains.
  • the light chains are lambda light chains.
  • each binding unit comprises two immunoglobulin light chains each comprising a VL situated amino terminal to an immunoglobulin light chain constant region.
  • a tetrameric or dimeric IgA antibody or IgA-like antibody as provided herein can possess binding characteristics or biological activity that can be distinguished from a binding molecule comprising five binding units, e.g., a pentameric IgM antibody.
  • a dimeric binding molecule would be smaller, and could, for example, achieve better tissue penetration in certain solid tumors.
  • An IgA heavy chain constant region can include one or more of a Cal domain, an IgA hinge region, a Ca2 domain, a Ca3 domain, and/or an atp, provided that the constant region can serve a desired function in the binding molecule, e.g., associate with a light chain constant region to facilitate formation of a target antigen-binding domain or associate with another IgA binding unit to form a dimeric binding molecule
  • the two IgA heavy chain constant regions or fragments thereof within an individual binding unit each comprise a Ca3 domain or multimerizing fragment thereof, a tailpiece (atp) or multimerizing fragment thereof, or any combination of a Ca3 domain, a tp, or multimerizing fragment thereof.
  • the two IgA heavy chain constant regions or fragments thereof within an individual binding unit each further comprise a Ca2 domain or fragment thereof, a Cal domain or fragment thereof, an IgA hinge region, or a Cal domain or fragment thereof, an IgA hinge region, and a Ca2 domain or fragment thereof.
  • each of the two IgA heavy chain constant regions in a given binding unit is associated with a target antigen-binding domain, for example an Fv portion of an antibody, e.g., a VH and a VL of a human or murine antibody, where the VL can be associated with a light chain constant region.
  • a multimeric binding molecule as provided herein comprises at least three target antigen-binding domains that can specifically bind to a target antigen, e.g., a tumor-associated or tumor-specific target antigen.
  • Certain multimeric binding molecules provided herein include a modified J chain or functional fragment or variant thereof.
  • the multimeric binding molecule provided herein is pentameric and comprises a modified I chain or functional fragment or variant thereof.
  • the binding molecule provided herein is dimeric or tetrameric and comprises a modified J chain or functional fragment or variant thereof.
  • the dimeric, tetrameric, or pentameric binding molecule includes a modified J chain comprising a naturally occurring J chain sequence, such as a mature human J chain sequence (e.g., SEQ ID NO: 7).
  • the dimeric, tetrameric, or pentameric binding molecule includes a modified J chain comprising a variant J chain sequence, such as a variant sequence described herein with reduced glycosylation and/or reduced binding to a polymeric Ig receptor (e.g., plgR, Fc alpha-mu receptor (FcapR), or Fc mu receptor (FcpR)).
  • a polymeric Ig receptor e.g., plgR, Fc alpha-mu receptor (FcapR), or Fc mu receptor (FcpR)
  • the modified J chain of the dimeric, tetrameric, or pentameric binding molecule can comprise a functional fragment of a naturally occurring or variant J chain.
  • a functional fragment or a “functional variant” in this context includes those fragments and variants that can associate with binding units, e.g., IgM or IgA heavy chain constant regions, to form a pentameric IgM antibody, IgM-like antibody, or IgM-derived binding molecule or a dimeric or tetrameric IgA antibody, IgA-like antibody, or IgA-derived binding molecule, and/or can associate with certain immunoglobulin receptors, e.g., plgR.
  • binding units e.g., IgM or IgA heavy chain constant regions
  • the J chain is modified, e.g., by introduction of a heterologous moiety, or two or more heterologous moieties, e.g., polypeptides, without interfering with the ability of binding molecule to assemble and bind to its binding target(s).
  • a heterologous moiety or two or more heterologous moieties, e.g., polypeptides, without interfering with the ability of binding molecule to assemble and bind to its binding target(s).
  • a multimeric binding molecule comprises a modified J chain or functional fragment or variant thereof comprising a heterologous moiety, e.g., a heterologous polypeptide, attached, e.g., fused or chemically conjugated, into the J chain or fragment or variant thereof.
  • a heterologous polypeptide can be fused to the N-terminus of the J chain or functional fragment or variant thereof, or the C-terminus of the J chain or functional fragment or variant thereof.
  • a heterologous moiety e.g., a heterologous polypeptide
  • the heterologous polypeptide can be fused internally within the J chain or functional fragment or variant thereof.
  • the heterologous polypeptide can be introduced into the J chain at or near a glycosylation site.
  • the heterologous polypeptide can be introduced into the J chain within about 10 amino acid residues from the C-terminus, or within about 10 amino acids from the N- terminus.
  • the heterologous moiety can be a peptide or polypeptide sequence fused in frame to the J chain or chemically conjugated to the J chain or fragment or variant thereof.
  • the heterologous polypeptide is fused to the J chain or functional fragment thereof via an amino acid linker.
  • Any suitable linker can be used, for example the amino acid linker can include at least 5 amino acids, at least ten amino acids, and least 20 amino acids, at least 30 amino acids or more, and so on.
  • the amino acid linker includes least 5 amino acids, but no more than 25 amino acids.
  • the amino acid linker can consist of 5 amino acids, 10 amino acids, 15 amino acids, 20 amino acids, or 25 amino acids.
  • the amino acid linker consists of the amino acid sequence (GGGGS) n (SEQ ID NO: 279), where n is an integer between 2 and 5.
  • the amino acid linker consists of GGGGS (SEQ ID NO: 9), GGGGSGGGGS (SEQ ID NO: 10), GGGGSGGGGSGGGGS (SEQ ID NO: 11), GGGGS GGGGS GGGGS GGGGS (SEQ ID NO: 12), GGGGSGGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 13), GKPGSGKPGSGKPGSGKPGS (SEQ ID NO: 14), or GGGGS GGGGS GGGG (SEQ ID NO: 235).
  • the heterologous moiety can be a chemical moiety conjugated to the J chain.
  • Heterologous moieties to be attached to a J chain can include, without limitation, a binding moiety, e.g., an antibody or antigen-binding fragment thereof, e.g., a single chain Fv (scFv) molecule, a cytokine, e.g., IL-2 or IL-15 (see, e.g., PCT Publication No.
  • a stabilizing peptide that can increase the half-life of the binding molecule, e.g., human serum albumin (HSA) or an HSA binding molecule, or a heterologous chemical moiety such as a polymer or a cytotoxin.
  • the heterologous moiety includes a polypeptide agonist of a T cell costimulatory molecule.
  • a modified J chain of a multimeric binding molecule as provided herein comprises two heterologous moieties, an anti-CD3 scFv and a polypeptide agonist of a T cell costimulatory molecule.
  • the T cell costimulatory molecule is CD28. In certain embodiments, the T cell costimulatory molecule is 4-1BB. In certain embodiments, the polypeptide agonist of the T cell costimulatoiy molecule is an anti-CD28 antibody or antigen-binding fragment thereof. In certain embodiments, the polypeptide agonist of a T cell costimulatory molecule is a 4- IBB ligand (4-1BBL) trimer. [0184] In some embodiments, the modified J chain comprises an antigen-binding domain.
  • an antigen-binding domain associated with a modified J chain is an antibody or an antigen-binding fragment thereof
  • the antigenbinding domain can be a scFv antigen-binding domain or a single-chain antigen-binding domain derived, e.g., from a camelid or condricthoid antibody (sdVH) or from an antibody light chain (sdVL).
  • an antigen-binding domain binds to a T cell, e.g., a cytotoxic T cell.
  • An antigen-binding domain can be introduced into the J chain at any location that allows the binding of the antigen-binding domain to its antigen without interfering with J chain function or the function of an associated multimeric binding molecule, e.g., a pentameric IgM or dimeric IgA antibody. Insertion locations include but are not limited to at or near the C-terminus, at or near the N-terminus or at an internal location that, based on the three-dimensional structure of the J chain, is accessible.
  • the modified J chain of a multimeric binding molecule as provided herein comprises a functional variant J chain that includes one or more single amino acid substitutions, deletions, or insertions relative to a reference J chain identical to the variant J chain except for the one or more single amino acid substitutions, deletions, or insertions.
  • certain amino acid substitutions, deletions, or insertions can result in the IgM-derived multimeric binding molecule exhibiting an increased serum half-life upon administration to a subject animal relative to a reference IgM-derived binding molecule that is identical except for the one or more single amino acid substitutions, deletions, or insertions in the variant modified J chain, and is administered using the same method to the same animal species.
  • the variant modified J chain can include one, two, three, or four single amino acid substitutions, deletions, or insertions relative to the reference J chain.
  • the modified J chain comprises an amino acid substitution at the amino acid position corresponding to amino acid Y102 of the mature wild-type human J chain (SEQ ID NO: 7).
  • an amino acid corresponding to amino acid Y102 of the mature wild-type human J chain is meant the amino acid in the sequence of the J chain, which is homologous to Y102 in the human J chain.
  • the position corresponding to Y102 in SEQ ID NO: 7 is conserved in the J chain amino acid sequences of at least 43 other species. See FIG. 4 ofU.S. Patent No. 9,951,134, which is incorporated by reference.
  • Certain mutations at the position corresponding to Y102 of SEQ ID NO: 7 can inhibit the binding of certain immunoglobulin receptors, e.g., the human or murine Fcap receptor, the murine Fcp receptor, and/or the human or murine polymeric Ig receptor (plgR) to an IgM pentamer comprising the variant J chain.
  • certain immunoglobulin receptors e.g., the human or murine Fcap receptor, the murine Fcp receptor, and/or the human or murine polymeric Ig receptor (plgR) to an IgM pentamer comprising the variant J chain.
  • a multimeric binding molecule comprising a modified I chain with a mutation at the amino acid corresponding to Y102 of SEQ ID NO: 7 has an improved serum half-life when administered to an animal than a corresponding multimeric binding molecule that is identical except for the substitution, and which is administered to the same species in the same manner.
  • the amino acid corresponding to Y102 of SEQ ID NO: 7 can be substituted with any amino acid.
  • the amino acid corresponding to Y 102 of SEQ ID NO: 7 can be substituted with alanine (A), serine (S) or arginine (R).
  • the amino acid corresponding to Y102 of SEQ ID NO: 7 can be substituted with alanine.
  • the modified J chain or functional fragment or variant thereof comprises a variant human J chain referred to herein as “J*,” comprising the amino acid sequence SEQ ID NO: 8.
  • Wild-type J chains typically include one N-linked glycosylation site.
  • a modified I chain comprising a variant J chain or functional fragment thereof of a multimeric binding molecule as provided herein includes a mutation within the asparagine(N)-linked glycosylation motif N-Xl-S/T, e.g., starting at the amino acid position corresponding to amino acid 49 (motif N6) of the mature human I chain (SEQ ID NO: 7) or J* (SEQ ID NO: 8), where N is asparagine, XI is any amino acid except proline, and S/T is serine or threonine, and where the mutation prevents glycosylation at that motif.
  • N asparagine
  • XI is any amino acid except proline
  • S/T is serine or threonine
  • mutations preventing glycosylation at this site can result in the multimeric binding molecule as provided herein, exhibiting an increased serum half-life upon administration to a subject animal relative to a reference multimeric binding molecule that is identical except for the mutation or mutations preventing glycosylation in the variant J chain, and is administered in the same way to the same animal species.
  • a modified J chain comprising a variant J chain or functional fragment thereof of a pentameric IgM-derived or dimeric IgA-derived binding molecule as provided herein can include an amino acid substitution at the amino acid position corresponding to amino acid N49 or amino acid S51 of SEQ ID NO: 7 or SEQ ID NO: 8, provided that the amino acid corresponding to S51 is not substituted with threonine (T), or where the variant J chain comprises amino acid substitutions at the amino acid positions corresponding to both amino acids N49 and S51 of SEQ ID NO: 7 or SEQ ID NO: 8.
  • T threonine
  • the position corresponding to N49 of SEQ ID NO: 7 or SEQ ID NO: 8 is substituted with any amino acid, e.g., alanine (A), glycine (G), threonine (T), serine (S) or aspartic acid (D).
  • alanine A
  • G glycine
  • T threonine
  • S serine
  • D aspartic acid
  • the position corresponding to N49 of SEQ ID NO: 7 or SEQ ID NO: 8 can be substituted with alanine (A).
  • the position corresponding to N49 of SEQ ID NO: 7 or SEQ ID NO: 8 can be substituted with aspartic acid (D).
  • IgM heavy chain constant regions of a multimeric binding molecule as provided herein can be engineered to confer certain desirable properties to the multimeric binding molecules provided herein.
  • IgM heavy chain constant regions can be engineered to confer enhanced serum half-life to multimeric binding molecules as provided herein.
  • Exemplary IgM heavy chain constant region mutations that can enhance serum half-life of an IgM-derived binding molecule are disclosed in US Patent No. 10,899,835, which is incorporated by reference herein in its entirety.
  • an amino acid corresponding to amino acid S401, E402, E403, R344, and/or E345 of a wild-type human IgM constant region is meant the amino acid in the sequence of the IgM constant region of any species which is homologous to S401, E402, E403, R344, and/or E345 in the human IgM constant region.
  • the amino acid corresponding to S401, E402, E403, R344, and/or E345 of SEQ ID NO: 1 or SEQ ID NO: 2 can be substituted with any amino acid, e.g., alanine.
  • an IgM antibody, IgM-like antibody, or other IgM-derived binding molecule as provided herein can be engineered to exhibit reduced complementdependent cytotoxic (CDC) activity to cells in the presence of complement, relative to a reference IgM antibody, IgM-like antibody, or other IgM-derived binding molecule with corresponding reference human IgM constant regions identical, except for the mutations conferring reduced CDC activity.
  • CDC complementdependent cytotoxic
  • corresponding reference human IgM constant region is meant a human IgM constant region that is identical to the variant IgM constant region except for the modification or modifications in the constant region affecting CDC activity.
  • the variant human IgM constant region includes one or more amino acid substitutions, e.g., in the Cp3 domain, relative to a wild-type human IgM constant region as described, e.g., in US Patent No. 11,401,337, which is incorporated herein by reference in its entirety.
  • Assays for measuring CDC are well known to those of ordinary skill in the art, and exemplary assays are described e.g., in US Patent No. 11,401,337.
  • a variant human IgM constant region conferring reduced CDC activity includes an amino acid substitution corresponding to the wild-type human IgM constant region at position L310, P311, P313, and/or K315 of SEQ ID NO: 1 (human IgM constant region allele IGHM*03) or SEQ ID NO: 2 (human IgM constant region allele IGHM*04).
  • a variant human IgM constant region conferring reduced CDC activity includes an amino acid substitution corresponding to the wild-type human IgM constant region at position P311 of SEQ ID NO: 1 or SEQ ID NO: 2.
  • the variant IgM constant region as provided herein contains an amino acid substitution corresponding to the wild-type human IgM constant region at position P313 of SEQ ID NO: 1 or SEQ ID NO: 2.
  • the variant IgM constant region as provided herein contains a combination of substitutions corresponding to the wild-type human IgM constant region at positions P311 of SEQ ID NO: 1 or SEQ ID NO: 2 and P313 of SEQ ID NO: 1 or SEQ ID NO: 2.
  • These proline residues can be independently substituted with any amino acid, e.g., with alanine, serine, or glycine.
  • a variant human IgM constant region conferring reduced CDC activity includes an amino acid substitution corresponding to the wild-type human IgM constant region at position K315 of SEQ ID NO: 1 or SEQ ID NO: 2.
  • the lysine residue can be independently substituted with any amino acid, e.g., with alanine, serine, glycine, or aspartic acid.
  • a variant human IgM constant region conferring reduced CDC activity includes an amino acid substitution corresponding to the wild-type human IgM constant region at position K315 of SEQ ID NO: 1 or SEQ ID NO: 2 , which can be substituted, e.g., with aspartic acid.
  • N-linked glycosylation motif comprises or consists of the amino acid sequence N- Xl-S/T, where N is asparagine, XI is any amino acid except proline (P), and S/T is serine (S) or threonine (T).
  • the glycan is attached to the nitrogen atom of the asparagine residue. See, e.g., Drickamer K, Taylor ME (2006), Introduction to Gfycobiology (2nd ed.). Oxford University Press, USA.
  • N-linked glycosylation motifs occur in the human IgM heavy chain constant regions of SEQ ID NO: 1 or SEQ ID NO: 2 starting at positions 46 (“Nl”), 209 (“N2”), 272 (“N3”), 279 (“N4”), and 440 (“N5”). These five motifs are conserved in nonhuman primate IgM heavy chain constant regions, and four of the five are conserved in the mouse IgM heavy chain constant region. Accordingly, in some embodiments, IgM heavy chain constant regions of a multimeric binding molecule as provided herein comprise 5 N- linked glycosylation motifs: Nl, N2, N3, N4, and N5. In some embodiments, at least three of the N-linked glycosylation motifs (e.g., Nl, N2, and N3) on each IgM heavy chain constant region are occupied by a complex glycan.
  • At least one, at least two, at least three, or at least four of the N- Xl-S/T motifs can include an amino acid insertion, deletion, or substitution that prevents glycosylation at that motif.
  • the IgM-derived multimeric binding molecule can include an amino acid insertion, deletion, or substitution at motif Nl, motif N2, motif N3, motif N5, or any combination of two or more, three or more, or all four of motifs Nl, N2, N3, or N5, where the amino acid insertion, deletion, or substitution prevents glycosylation at that motif.
  • the signal 1 and signal 2 targeting is accomplished, e.g., through dual T cell engagement via a “bidentate” modified J chain that comprises a J chain or a functional fragment or variant thereof, an scFv that specifically binds to CD3, and a polypeptide agonist of a T cell costimulatory molecule.
  • the T cell costimulatory molecule can be, e.g., CD28 or 4-1BB
  • the polypeptide agonist can be, e.g., an scFv of an anti-CD28 agonist antibody, or a trimer of the receptor-binding portion of the 4-1BB ligand (4-1BBL).
  • Costimulatory molecules are receptors expressed on T cells that upon coactivation can, e.g., induce signaling to more fully activate T cells upon T cell receptor (TCR) engagement, thereby enhancing signaling and cytokine stimulation. This co-signaling can further modulate T cell differentiation, effector function, and survival. See, e.g., Baeuerle, P.A., and H. Wesche, Curr. Opin. Oncol. 3 :552-558 (2022).
  • Exemplary T cell costimulatory molecules include, without limitation, CD28 and 4-1BB. See, e.g., Jeong, S., and S-H. Park, Immune Netw. doi: 10.4110/in.2020.20. e3 (2020).
  • the transmembrane domain of human CD28 extends from about amino acid 153 to about amino acid 179 of SEQ ID NO: 236.
  • the cytoplasmic domain of human CD28 extends from about amino acid 180 to amino acid 220 of SEQ ID NO: 236.
  • 4-1BB also known as CD137 or TNFRSF9, is a tumor necrosis factor superfamily receptor (TNFSFR) expressed, e.g., on activated T cells.
  • TNFSFR tumor necrosis factor superfamily receptor
  • 4- IBB is expressed on both activated CD4+ and CD8+ T cells (Bartkowiak, T, and MA Curran, Front Oncol. 5 doi: 10.3389/fonc.2015.00117 (2015); Vinay, DS, and BS Kwon, Mol Cancer Ther. 11: DOI: 10.1158/1535-7163. MCT-11-0677 (2012)).
  • CD8+ effector T cells upregulate 4-1BB expression, and 4-1BB signaling promotes survival, T cell proliferation, and enhanced effector function (Bartkowiak, T, and MA Curran, Front Oncol. 5: doi: 10.3389/fonc.2015.00117 (2015)).
  • Interaction with its trimeric ligand (4-1BBL, TNFSF9) expressed on activated antigen-presenting cells (APCs), e.g., macrophages and dendritic cells (DC) provides enhanced costimulatory proliferation, survival, and effector functions in CD8+ effector T cells (Moran, AE, et al., Curr Opin Immunol. 25: 10.1016/j.coi.2013.01.004 (2013)).
  • Human 4-1BB a type 1 membrane protein, is presented as SEQ ID NO: 195.
  • the signal peptide of human 4- IBB extends from amino acid 1 to about amino acid 23 of SEQ ID NO: 195.
  • the extracellular domain of human 4- 1BB extends from about amino acid 24 to about amino acid 186 of SEQ ID NO: 195.
  • the transmembrane domain of human 4- IBB extends from about amino acid 187 to about amino acid 213 of SEQ ID NO: 195.
  • the cytoplasmic domain of human 4- IBB extends from about amino acid 214 to amino acid 255 of SEQ ID NO: 195.
  • the ligand for 4-1BB (4-1BBL) is a type 2 transmembrane glycoprotein receptor that is found on antigen presenting cells (APCs). Gramaglia et al., Eur. J. Immunol. 30(2):392-402, 2000. 4-1BBL is the high affinity ligand of 4-1BB. Chin et al., Nature Comm. 9:4679, 2018. 4-1BBL induces the proliferation of activated peripheral blood T cells and may have a role in activation-induced cell death (AICD). Vinay etal., BMB Rep. 47(3): 122-129, 2014.
  • AICD activation-induced cell death
  • 4-1BBL provides T cells co-stimulatory signals for survival, proliferation, and differentiation and may play a role in cognate interactions between T cells and B cells/macrophages.
  • Human 4-1BBL a type 2 membrane protein, is presented as SEQ ID NO: 15.
  • the cytoplasmic domain of human 4-1BBL extends from amino acid 1 to about amino acid 28 of SEQ ID NO: 15.
  • the transmembrane domain of human 4-1BBL extends from about amino acid 29 to about amino acid 49 of SEQ ID NO: 15.
  • the extracellular domain of human 4-1BBL extends from about amino acid 50 to amino acid 254 of SEQ ID NO: 15.
  • the 4-1BB/4-1BBL complex typically consists of three monomeric 4-lBBs bound to a trimeric 4-1BBL. Won et al., J. Biol. Chem. 255:9202-9210 (2010); Rabu et al., J. Biol. Chem. 250(50):41472-41481 (2005). Each 4-1BB monomer binds to two 4-lBBLs via cysteine-rich domains (CRDs). Bitra et al., J. Biol. Chem. 293(4): 1317-1329 (2016).
  • An exemplary T cell-engaging multimeric binding molecule includes a modified J chain, wherein the modified J chain comprises (a) a J chain or a functional fragment or variant thereof (“J”), (b) an anti-CD3 scFv (“C”), and (c) a heterologous polypeptide (“H”), wherein H comprises a polypeptide agonist of a T cell costimulatory molecule, where the J, C, and H are associated as a fusion protein, such as in the N-terminal to C-terminal direction, C-J-H or H-J-C.
  • J J chain or a functional fragment or variant thereof
  • C an anti-CD3 scFv
  • H heterologous polypeptide
  • the VH that includes the three VHCDRs as noted above comprises an amino acid sequence at least 80%, 85%, 90%, 95%, or 100% identical to a VH amino acid sequence listed in Table 2.
  • the VL that includes the three VLCDRs as noted above comprises an amino acid sequence at least 80%, 85%, 90%, 95%, or 100% identical to a VL amino acid sequence listed in Table 2.
  • the VH comprises a VH amino acid sequence listed in Table 2 and the VL comprises a VL amino acid sequence listed in Table 2.
  • the anti-CD28 sdVH that includes the CDRs noted above comprises an amino acid sequence at least 80%, 85%, 90%, 95%, or 100% identical to the amino acid sequence SEQ ID NO: 179, SEQ ID NO: 180, or SEQ ID NO: 181, or the anti - CD28 sdVL that includes the CDRs noted above comprises an amino acid sequence at least 80%, 85%, 90%, 95%, or 100% identical to the amino acid sequence SEQ ID NO: 182.
  • the anti-CD28 sdVH comprises SEQ ID NO: 179, SEQ ID NO: 180, or SEQ ID NO: 181, or the anti-CD28 sdVL comprises SEQ ID NO: 182.
  • the VH and VL of C comprise an amino acid sequence at least 80%, 85%, 90%, 95%, or 100% identical to the amino acid sequence of SEQ ID NO: 16 and SEQ ID NO: 20; SEQ ID NO: 24 and SEQ ID NO: 28; SEQ ID NO: 24 and SEQ ID NO: 32; SEQ ID NO: 34 and SEQ ID NO: 38; SEQ ID NO: 42 and SEQ ID NO: 44; or SEQ ID NO: 46 and SEQ ID NO: 48, respectively.
  • this disclosure provides a multimeric binding molecule, e.g., an IgM, IgM-like, IgA, and IgA-like binding molecule comprising two, four, or ten binding units collectively comprising three, four, five, six, seven, eight, nine, or ten “target-antigen binding domains,” i.e., antigen-binding domains that specifically bind to a target antigen, e.g., a tumor-associated or tumor-specific target antigen.
  • a target antigen e.g., a tumor-associated or tumor-specific target antigen.
  • cancer refers to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth.
  • cancers include but are not limited to, carcinoma including adenocarcinomas, lymphomas, blastomas, melanomas, sarcomas, and leukemias.
  • the subject to be treated can be any animal, e.g., mammal, in need of treatment, in certain embodiments, subject is a human subject.
  • the method provided herein is a method for inhibiting, delaying, or reducing malignant cell growth in a subject with cancer, where the cancer is a hematologic cancer or a solid tumor.
  • the cancer is a hematologic cancer, such as acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia, hairy cell leukemia, Hodgkin lymphoma, non-Hodgkin lymphoma, multiple myeloma, any metastases thereof, or any combination thereof
  • the cancer is a solid tumor, such as bladder cancer, colorectal cancer, sarcoma (e.g., fibrosarcoma), gastric cancer, lung cancer (e.g., non-small cell lung cancer (NSCLC)), or pancreatic cancer.
  • AML acute myeloid leukemia
  • CML chronic myeloid leukemia
  • ALL acute lymphocytic leukemia
  • a pharmaceutically effective amount of a multimeric binding molecule such as a dimeric or pentameric binding molecule as provided herein means an amount sufficient to achieve effective binding to a target and to achieve a therapeutic benefit.
  • a pharmaceutically effective amount of a cancer therapy as provided herein means an amount sufficient to achieve a therapeutic benefit. Suitable formulations are described in Remington: The Science and Practice of Pharmacy (Elsevier Science) 23rd ed. (2020). [0239] In keeping with the scope of the present disclosure, a multimeric binding molecule as provided herein can be administered to a subject in need of therapy in an amount sufficient to produce a therapeutic effect.
  • a multimeric binding molecule as provided herein can be administered to the subject in a conventional dosage form prepared by combining the multimeric binding molecule of the disclosure with a conventional pharmaceutically acceptable carrier or diluent according to known techniques.
  • a conventional pharmaceutically acceptable carrier or diluent according to known techniques.
  • the form and character of the pharmaceutically acceptable carrier or diluent can be dictated by the amount of active ingredient with which it is to be combined, the route of administration and other well-known variables.
  • Embodiment 1 A multimeric binding molecule comprising five, four, or two bivalent binding units and a modified J chain, wherein each binding unit comprises two antibody heavy chains, each comprising an IgM or IgA heavy chain constant region or multimerizing variant or fragment thereof each associated with a target antigen-binding domain, and wherein the modified J chain comprises (a) a J chain or a functional fragment or variant thereof (“J”), (b) an anti-CD3 scFv (“C”), and (c) a heterologous polypeptide (“El”), wherein H comprises a polypeptide agonist of a T cell costimulatory molecule, wherein the J, C, and H are associated as a fusion protein.
  • J J chain or a functional fragment or variant thereof
  • C an anti-CD3 scFv
  • El a heterologous polypeptide
  • H comprises a polypeptide agonist of a T cell costimulatory molecule, wherein the J, C, and H are associated as a fusion
  • Embodiment 2 The multimeric binding molecule of embodiment 1, wherein the
  • T cell costimulatory molecule comprises CD28 or 4-1BB.
  • Embodiment s The multimeric binding molecule of embodiment 1 or embodiment 2, wherein H comprises an anti-CD28 antibody or antigen-binding fragment thereof or a 4-1BB ligand (“4-1BBL”) trimer.
  • Embodiment 4 The multimeric binding molecule of any one of embodiments 1 to 3, wherein the modified J chain comprises, in the N-terminal to C-terminal direction, C- J-H or H-J-C
  • Embodiment 5 The multimeric binding molecule of embodiment 4, wherein C, J, and H are fused via amino acid linkers which can be the same or different.
  • Embodiment 7 The multimeric binding molecule of embodiment 6, wherein each linker consists, independently, of the amino acid sequence (GGGGS)n (SEQ ID NO: 279), wherein n is an integer between 2 and 5, GGGGSGGGGS (SEQ ID NO: 10), GGGGSGGGGSGGGGS (SEQ ID NO: 11), GKPGSGKPGSGKPGSGKPGS (SEQ ID NO: 14), or GGGGSGGGGSGGGG (SEQ ID NO: 235)
  • Embodiment 8 The multimeric binding molecule of embodiment 7, wherein the linker consists of GGGGSGGGGS (SEQ ID NO: 10).
  • Embodiment 10 The multimeric binding molecule of embodiment 7, wherein the linker consists of GKPGSGKPGSGKPGSGKPGS (SEQ ID NO: 14).
  • Embodiment 11 The multimeric binding molecule of any one of embodiments 3 to 10, wherein H comprises an antigen-binding fragment of an anti-CD28 antibody.
  • Embodiment 12 The multimeric binding molecule of embodiment 11, wherein the antigen-binding fragment of the anti-CD28 antibody comprises a single-chain Fv (scFv) fragment.
  • scFv single-chain Fv
  • Embodiment 13 The multimeric binding molecule of embodiment 12, wherein the anti-CD28 scFv fragment comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises VH complementarity-determining regions VHCDR1, VHCDR2, and VHCDR3 and the VL comprises VL complementaritydetermining regions VLCDR1, VLCDR2, and VLCDR3, wherein the VHCDR1, VHCDR2, VHCDR3, VLCDR1, VLCDR2, and VLCDR3 comprise, respectively, the amino acid sequences SEQ ID NO: 100, SEQ ID NO: 120, SEQ ID NO: 127, SEQ ID NO: 146, SEQ ID NO: 153, and SEQ ID NO: 172; SEQ ID NO: 95, SEQ ID NO: 105, SEQ ID NO: 133, SEQ ID NO: 138, SEQ ID NO: 164, and SEQ ID NO: 165; SEQ ID NO: 98, SEQ ID NO: 106, SEQ ID NO:
  • Embodiment 16 The multimeric binding molecule of embodiment 15, wherein the linker consists of the amino acid sequence (GGGGS)n (SEQ ID NO: 279), wherein n is an integer between 2 and 5, GGGGSGGGGS (SEQ ID NO: 10), GGGGSGGGGSGGGGS (SEQ ID NO: 11), GKPGSGKPGSGKPGSGKPGS (SEQ ID NO: 14), or GGGGSGGGGSGGGG (SEQ ID NO: 235)
  • Embodiment 17 The multimeric binding molecule of embodiment 15, wherein the linker consists of GGGGSGGGGS (SEQ ID NO: 10).
  • Embodiment 18 The multimeric binding molecule of embodiment 15, wherein the linker consists of GGGGSGGGGSGGGGS (SEQ ID NO: 11).
  • Embodiment 19 The multimeric binding molecule of embodiment 15, wherein the linker consists of GKPGSGKPGSGKPGSGKPGS (SEQ ID NO: 14).
  • Embodiment 20 The multimeric binding molecule of embodiment 11, wherein the antigen-binding fragment of an antibody is an anti-CD28 single domain heavy chain variable region (sdVH) or an anti-CD28 single domain light chain variable region (sdVL).
  • sdVH anti-CD28 single domain heavy chain variable region
  • sdVL anti-CD28 single domain light chain variable region
  • Embodiment 21 The multimeric binding molecule of embodiment 20, wherein the anti-CD28 sdVH comprises complementarity-determining regions CDR1, CDR2, and CDR3, wherein the CDR1, CDR2, and CDR3 comprise, respectively, the amino acid sequences SEQ ID NO: 183, SEQ ID NO: 187, and SEQ ID NO: 191; SEQ ID NO: 184, SEQ ID NO: 188, and SEQ ID NO: 192; or SEQ ID NO: 185, SEQ ID NO: 189, and SEQ ID NO: 193; or wherein the anti-CD28 sdVL comprises complementarity-determining regions CDR1, CDR2, and CDR3, wherein the CDR1, CDR2, and CDR3 comprise, respectively, the amino acid sequences SEQ ID NO: 186, SEQ ID NO: 190, and SEQ ID NO: 194.
  • Embodiment 22 The multimeric binding molecule of embodiment 21, wherein the anti-CD28 sdVH comprises an amino acid sequence at least 80%, 85%, 90%, 95%, or 100% identical to the amino acid sequence SEQ ID NO: 179, SEQ ID NO: 180, or SEQ ID NO: 181, or wherein the anti-CD28 sdVL comprises an amino acid sequence at least 80%, 85%, 90%, 95%, or 100% identical to the amino acid sequence SEQ ID NO: 182.
  • Embodiment 23 The multimeric binding molecule of any one of embodiments 3 to 10, wherein H comprises a 4-1BBL trimer.
  • Embodiment 24 The multimeric binding molecule of embodiment 23, wherein the 4-1BBL trimer comprises three 4-1BBL monomers.
  • Embodiment 25 The multimeric binding molecule of embodiment 24, wherein each 4-1BBL monomer is a soluble fragment of human 4-1BBL comprising amino acids X to 254 of SEQ ID NO: 15, wherein X is an integer from 50 to 99.
  • Embodiment 27 The multimeric binding molecule of embodiment 25 or embodiment 26, wherein the soluble fragments of 4-1BBL each comprise amino acids 58 to 254 of SEQ ID NO: 15 (SEQ ID NO: 196).
  • Embodiment 28 The multimeric binding molecule of any one of embodiments 24 to 27, wherein the 4-1BBL monomers are fused via amino acid linkers which can the same or different.
  • Embodiment 29 The multimeric binding molecule of embodiment 28, wherein each linker consists, independently, of 5 to 25 amino acids.
  • Embodiment 31 The multimeric binding molecule of embodiment 30, wherein the linker consists of GGGGSGGGGS (SEQ ID NO: 10)
  • Embodiment 32 The multimeric binding molecule of embodiment 30, wherein the linker consists of GGGGSGGGGSGGGGS (SEQ ID NO: 11).
  • Embodiment 33 The multimeric binding molecule of embodiment 30, wherein the linker consists of GKPGSGKPGSGKPGSGKPGS (SEQ ID NO: 14).
  • Embodiment 34 The multimeric binding molecule of any one of embodiments 23 to 33, wherein H comprises the amino acid sequence SEQ ID NO: 198, SEQ ID NO: 199, SEQ ID NO: 200, or SEQ ID NO: 201.
  • Embodiment 35 The multimeric binding molecule of any one of embodiments 1 to 34, wherein the C comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH of C comprises VH complementarity-determining regions VHCDR1, VHCDR2, and VHCDR3 and the VL of C comprises VL complementarity-determining regions VLCDR1, VLCDR2, and VLCDR3, wherein the VHCDR1, VHCDR2, VHCDR3, VLCDR1, VLCDR2, and VLCDR3 comprise, respectively, the amino acid sequences SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 30, and SEQ ID NO: 31; SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 22, and SEQ ID NO: 23; SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 30, and SEQ ID NO:
  • Embodiment 36 The multimeric binding molecule of embodiment 35, wherein the VHCDR1, VHCDR2, VHCDR3, VLCDR1, VLCDR2, and VLCDR3 of C comprise, respectively, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 30, and SEQ ID NO: 31.
  • Embodiment 37 The multimeric binding molecule of embodiment 35, wherein the VH and VL of C comprise an amino acid sequence at least 80%, 85%, 90%, 95%, or 100% identical to the amino acid sequence of SEQ ID NO: 24 and SEQ ID NO: 28; SEQ ID NO: 16 and SEQ ID NO: 20; SEQ ID NO: 24 and SEQ ID NO: 32; SEQ ID NO: 34 and SEQ ID NO: 38; SEQ ID NO: 42 and SEQ ID NO: 44; or SEQ ID NO: 46 and SEQ ID NO: 48, respectively.
  • Embodiment 38 The multimeric binding molecule of embodiment 37, wherein the C comprises the VH and VL amino acid sequences SEQ ID NO: 24 and SEQ ID NO: 28, respectively.
  • Embodiment 40 The multimeric binding molecule of embodiment 39, wherein the linker consists of the amino acid sequence (GGGGS)n (SEQ ID NO: 279), wherein n is an integer between 2 and 5, GGGGSGGGGS (SEQ ID NO: 10), GGGGSGGGGSGGGGS (SEQ ID NO: 11), GKPGSGKPGSGKPGSGKPGS (SEQ ID NO: 14), or GGGGSGGGGSGGGG (SEQ ID NO: 235) [0282] Embodiment 41. The multimeric binding molecule of embodiment 40, wherein the linker consists of GGGGSGGGGS (SEQ ID NO: 10).
  • Embodiment 42 The multimeric binding molecule of embodiment 40, wherein the linker consists of GGGGSGGGGSGGGGS (SEQ ID NO: 11).
  • Embodiment 45 The multimeric binding molecule of any one of embodiments 1 to 44, wherein J comprises a variant J chain with an alanine substitution at the amino acid position corresponding to amino acid Y102 of the mature wild-type human J chain (SEQ ID NO: 7).
  • Embodiment 47 The multimeric binding molecule of any one of embodiments 1 to 22 or 35 to 46, wherein the modified J chain comprises the amino acid sequence SEQ ID NO: 233, SEQ ID NO: 231, SEQ ID NO: 232, or SEQ ID NO: 234
  • Embodiment 48 The multimeric binding molecule of any one of embodiments 1 to 10 or 23 to 46, wherein the modified J chain comprises the amino acid sequence SEQ ID NO: 213 or SEQ ID NO: 214.
  • Embodiment 50 The multimeric binding molecule of embodiment 49, wherein each 4-1BBL monomer is a soluble fragment of human 4-1BBL comprising amino acids X to 254 of SEQ ID NO: 15, wherein X is an integer from 50 to 99.
  • Embodiment 51 The multimeric binding molecule of embodiment 50, wherein the soluble fragments of 4-1BBL each comprise amino acids 71 to 254 of SEQ ID NO: 15 (SEQ ID NO: 197).
  • Embodiment 52 The multimeric binding molecule of embodiment 50 or embodiment 51, wherein the soluble fragments of 4-1BBL each comprise amino acids 58 to 254 of SEQ ID NO: 15 (SEQ ID NO: 196)
  • Embodiment 53 The multimeric binding molecule of any one of embodiments 49 to 52, wherein the 4-1BBL monomers are fused via amino acid linkers which can the same or different.
  • Embodiment 54 The multimeric binding molecule of embodiment 53, wherein each linker consists, independently, of 5 to 25 amino acids.
  • Embodiment 55 The multimeric binding molecule of embodiment 54, wherein each linker consists, independently, of the amino acid sequence (GGGGS)n (SEQ ID NO: 279), wherein n is an integer between 2 and 5, GGGGSGGGGS (SEQ ID NO: 10), GGGGSGGGGSGGGGS (SEQ ID NO: 11), GKPGSGKPGSGKPGSGKPGS (SEQ ID NO: 14), or GGGGSGGGGSGGGG (SEQ ID NO: 235)
  • Embodiment 56 The multimeric binding molecule of embodiment 55, wherein the linker consists of GGGGSGGGGS (SEQ ID NO: 10).
  • Embodiment 57 The multimeric binding molecule of embodiment 55, wherein the linker consists of GGGGSGGGGSGGGGS (SEQ ID NO: 11).
  • Embodiment 58 The multimeric binding molecule of embodiment 55, wherein the linker consists of GKPGSGKPGSGKPGSGKPGS (SEQ ID NO: 14).
  • Embodiment 59 The multimeric binding molecule of any one of embodiments 49 to 58, wherein the 4-1BB trimer comprises the amino acid sequence SEQ ID NO: 198, SEQ ID NO: 199, SEQ ID NO: 200, or SEQ ID NO: 201.
  • Embodiment 60 The multimeric binding molecule of any one of embodiments 49 to 58, wherein the 4-1BBL trimer is fused to the N terminus of the J chain or fragment or variant thereof, or fused to the C terminus of the J chain or fragment or variant thereof, or wherein a copy of the 4- IBB trimer is fused to both the N terminus and the C terminus of the J chain or fragment or variant thereof.
  • Embodiment 61 The multimeric binding molecule of any one of embodiments 49 to 60 wherein the J chain or a functional fragment or variant thereof comprises the mature human J chain SEQ ID NO: 7 or a functional fragment or variant thereof.
  • Embodiment 67 The multimeric binding molecule of embodiment 65 or embodiment 66, wherein the IgM heavy chain constant regions or multimerizing fragments or variants thereof are human IgM constant regions.
  • Embodiment 70 The multimeric binding molecule of embodiment 69, wherein each variant human IgM constant region comprises an amino acid substitution corresponding to position L310 of SEQ ID NO: 1 or SEQ ID NO: 2, an amino acid substitution corresponding to position P311 of SEQ ID NO: 1 or SEQ ID NO: 2, an amino acid substitution corresponding to position P313 of SEQ ID NO: 1 or SEQ ID NO: 2, an amino acid substitution corresponding to position K315 of SEQ ID NO: 1 or SEQ ID NO: 2, or any combination thereof.
  • Embodiment 71 Embodiment 71.
  • Embodiment 76 The multimeric binding molecule of any one of embodiments 1 to 75, wherein each target antigen-binding domain comprises a heavy chain variable region (VH) and a light chain variable region (VL), or a single-domain heavy chain variable region (sdVH).
  • VH heavy chain variable region
  • VL light chain variable region
  • sdVH single-domain heavy chain variable region
  • Embodiment 88 The multimeric binding molecule of any one of embodiments 81 to 87, wherein the multimeric binding molecule activates T cells after 7 days in the presence of the target antigen.
  • Embodiment 97 A polynucleotide comprising a nucleic acid sequence that encodes modified J chain of the multimeric binding molecule of any one of embodiments 1 to 95.
  • Embodiment 98 A vector comprising the polynucleotide of embodiment 97.
  • Embodiment 99 A composition comprising the polynucleotide of embodiment
  • Embodiment 100 The composition of embodiment 99, further comprising a polynucleotide encoding a light chain of the multimeric binding molecule of any one of embodiments 1 to 95.
  • Embodiment 101 A vector comprising the composition of embodiment 99 or embodiment 100.
  • Embodiment 105 The multimeric binding molecule of any one of embodiments 1 to 95, for use in treating cancer.
  • Embodiment 106 Use of the multimeric binding molecule of any one of embodiments 1 to 95 in the preparation of a medicament for the treatment of cancer.
  • This disclosure employs, unless otherwise indicated, conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology, which are within the skill of the art. Such techniques are explained fully in the literature. See, for example, Green and Sambrook, ed. (2012) Molecular Cloning A Laboratory Manual (4th ed.; Cold Spring Harbor Laboratory Press); Sambrook et al., ed. (1992) Molecular Cloning: A Laboratory Manual, (Cold Springs Harbor Laboratory, NY); D. N. Glover and B.D. Hames, eds., (1995) DNA Cloning 2d Edition (IRL Press), Volumes 1-4; Gait, ed.
  • modified J chains comprising a 4-1BBL trimer with three copies of either the “58” fragment (SEQ ID NO: 196) or “71” fragment (SEQ ID NO: 197) of 4-1BBL on the N and/or C terminus of a J chain comprising a Y to A amino acid substitution at position 102 (“Y102A” or “J*,” amino acid sequence of the variant presented as SEQ ID NO: 8) that enhances serum half-life of IgM pentamers that comprise the J chain variant were prepared. See U.S. Patent No. 10,899,835.
  • the 4-1BBL monomers were connected by a ten or fifteen-amino acid long linker (SEQ ID NOS: 10 and 11, respectively) and the trimers were also connected to the J chain by these linkers. Some molecules also comprise an anti-CD3 scFv (SEQ ID NO: 219).
  • the IgM antibodies produced comprise target antigen-binding domains that bind to exemplary tumor-specific or tumor-associated target antigens CD20 or mesothelin.
  • MSLN is mesothelin.
  • Additional IgM antibodies were generated with modified J chains comprising one of four anti-CD28 scFvs: W (VH: SEQ ID NO: 69, VL: SEQ ID NO: 78, scFv: SEQ ID NO: 284), X (VH: SEQ ID NO: 58, VL: SEQ ID NO: 83, scFv: SEQ ID NO: 225), Y (VH: SEQ ID NO: 59, VL: SEQ ID NO: 83, scFv: SEQ ID NO: 226), or Z (VH: SEQ ID NO: 61, VL: SEQ ID NO: 81, scFv: SEQ ID NO: 227), and optionally an anti-CD3 scFv (SEQ ID NO: 219).
  • a list of molecules made comprising anti-CD28 scFvs is provided in Table 12. Table 12: Antibodies with anti-CD28 on J* chain
  • FIGS. 2A-2G Schematics of exemplary IgM antibodies made and contemplated are shown in FIGS. 2A-2G
  • VH heavy chain variable regions
  • VL light chain variable regions
  • Assay plates were then incubated on ice for 45 minutes, and cells were washed three times using stain buffer. 1,000-fold diluted 7-aminoactinomycin D (7-AAD) viability dye was then added to the cells, and fluorescence was analyzed using an INTELLICYT® IQUE® flow cytometer. Data was plotted as antibody concentration (x-axis) and relative fluorescence as a percentage of live cells (y-axis), using GraphPad Prism 8 software.
  • the binding affinities and avidity of 4-1BBL comprising antibodies, A5, A6, or A10, or 4-1BBL trimer fused to an Fc, D2 (Acrobiosystems # 41L-H5269), were determined by biolayer interferometry (BLI) on an Octet-384 (Sartorius/ForteBio, NY, USA) using Anti- Penta-His biosensors (Sartorius/Fortebio Cat#18-5120).
  • PBST lxPBS+ l%BSA + 0.05% Tween-20
  • the experiment followed a six-step sequential assay at 24°C. First, biosensors were hydrated for 10 minutes.
  • Adherent cell line 293T/huCD137+ cells (Crown Bio#C2012 lot# 160331- IGM042020) was cultured in DMEM+10% FBS+ 1 pg/ml Puromycin and dissociated using Trypsin-free Cell Dissociation buffer. The viable cell density was adjusted to 6.0 x 10 5 cells/ml (30,000 cells/well). Then, 50 pl cells were added per well to both CD20- coated and non-coated plates.
  • DMEM+10% FBS+ 1 pg/ml Puromycin was added for conditions with crosslinking, and 10 pg/mL of anti -human Fc antibody was added to the cells.
  • Jurkat human 4-1BB positive Luciferase reporter cells (Promega #JA2351) cells were thawed at 37 °C for 2 mins, washed once in complete RPML1640+ 10% heat inactivated FBS buffer. Jurkat cell density was adjusted to 6.25 x 10 5 viable cells/mL. 20 pl of 4-1BB+ Reporter cells were added to the wells.
  • BIO-GLOTM Promega # G7940
  • Luminescence (RLU) was measured using a Perkin Elmer ENVISION® reader. Graphs were plotted using antibody concentration on the x-axis and RLU on the y-axis using GraphPad Prism 8 software.
  • FIGS. 5A and 5B The luminescence measured at each concentration for A5, A6, A8, and A9 with CD20 coating or without CD20 coating is shown in FIGS. 5A and 5B, respectively.
  • FIG. 5C shows the luminescence measured at the highest concentration tested for A5, A6, A8, A9, DI, D2 (with and without crosslinking), D3, and D4 (with and without crosslinking) in the presence or absence of CD20.
  • the anti-CD20 IgM antibodies comprising 4-1BBL trimer (A5, A6, A8, and A9) on the J chain showed greater agonist activity than the anti-4-lBB IgM antibody (D3), the anti-4-lBB IgG antibody (D4) with or without crosslinking, the 4-1 BBL trimer fused to an Fc (D2) with or without crosslinking, or the anti-CD20 IgM without the 4-1BBL trimer (DI).
  • Test molecules Al, A2, A3, A4, A5, A6, A8, A9, DI, or D2 were diluted to 20 nM and then serially diluted in assay buffer. 10 pl of test molecule was added into 384-well opaque plates followed by 10 pL of CD20+ and CD20- cells. The wells were incubated at room temperature for 15 minutes. Finally, 20 pL of 4-1BB+ reporter cells were added to the wells and the wells were incubated at 37°C for 16 hours. 20 pL per well ofBIO-GLOTM Luciferase reagent (Promega #G7941) was added to each well and luminescence (RLU) was read on ENVISION® (Perkin Elmer 2103). Graphs were plotted using antibody concentration on the x-axis and RLU on the y-axis using GraphPad Prism 8 software.
  • All anti-CD20 IgM antibodies comprising 4-1BBL trimer on the J chain showed CD20-level dependent agonist activity.
  • Antibodies with two copies of the 4-1BBL trimer showed higher levels of agonist activity; antibodies with an anti-CD3 scFv and a 4-1BBL trimer showed the highest levels of agonist activity.
  • B1-B4 comprise 4-1BBL trimer on the J chain
  • D5 comprises an unmodified J chain
  • D6 comprises an anti-CD3 scFv on the J chain.
  • Binding to 4- IBB was then measured in ELISA assays as follows. 96-well white polystyrene ELISA plates (Pierce 15042) were coated with 100 pL per well of 1.0 pg/mL recombinant target 4- IBB overnight at 4°C. Plates were then washed 5 times with 0.05% PBS-Tween and blocked with 2% BSA-PBS.
  • Example 8 Luciferase Reporter Assay Using Mesothelin-Expressing Cells
  • T cell dependent cellular cytotoxicity (TDCC), T cell proliferation and cytokine release was assessed in an in vitro model with human PBMC and Ramos tumor cells.
  • Antitumor activity of the exemplary IgM antibodies C3 (the anti-CD20 x anti-CD28
  • FIGs. 32A-C Graphs of tumor volumes versus days post-Ramos implant i.e., time, in days) for each of the treatment groups are provided in FIGs. 32A-C.
  • Tumor volumes over time for each group are provided in FIG. 32A; tumor volumes over time for the 10 mg/kg treatment groups (groups 2, 3, and 5) are shown in FIG. 32B; tumor volumes for the 1 mg/kg C7 and D7 treatment groups, and for the 10 mg/kg control group are shown in FIG. 32C.
  • Tumor volume over time was lower for the C7 (anti-CD20 x anti-CD3 x anti-CD28) treatment groups at dosages of both 1 mg/kg and 10 mg/kg than for any other treatment group.
  • Adherent target tumor cells were added to the 96-well E-plates at the specified seeding densities.
  • the E-plates were loaded to RTCA inside a humidified cell incubator with 5% CO2 at 37 °C for 24 hours to allow target tumor cells to settle and attach to the bottom of the plates.
  • Effector cells PBMC or T cells
  • test articles were then added to the wells before resuming the culture and real time cell index monitoring.
  • Cell index data were exported and analyzed with GraphPad Prism.
  • supernatant samples were collected at specified time points for cytokine analysis using the Human Thl/Th2 Cytokine Cytometric Bead Array (CBA) Kit II from BD Biosciences (catalog # 551809).
  • CBA Human Thl/Th2 Cytokine Cytometric Bead Array
  • T cell activation can be evaluated by collecting cell samples from the E-plates for flow cytometry analysis of various biomarkers (CD3, CD4, CD8 and CD25).
  • Cytokine Release To measure the cytokine release mediated by the test articles, supernatant samples from the 72-hour time point of the RTCA assays shown in FIGS. 38 and 41 were thawed and analyzed by flow cytometry, using the Human Thl/Th2 Cytokine Cytometric Bead Array (CBA) Kit II from BD Biosciences (catalog # 551809) based on manufacture’s recommendation, except using smaller culture volume to accommodate a plate-based format. The flow cytometry analysis was performed on the INTELLICYT® IQUE® Screen er PLUS and data was analyzed using the INTELLICYT® IQUE FORECYT® software, including standard curve fitting and cytokine concentration calculations.
  • CBA Human Thl/Th2 Cytokine Cytometric Bead Array
  • the levels of IL-2, IL-4, IL-6, IL-10, IFN-y, and TNFa are shown for the anti-mesothelin constructs in FIGs. 39A-39F, respectively, and for the anti-TROP-2 constructs in FIGS. 42A-42F, respectively.
  • T-cell Activation T cell numbers and the extent of T cell activation for both CD4+ and CD8+ T cell subsets at the 96-hour time point of the RTCA assays shown in FIGS. 38 and 41 were measured as follows. Cells from each well were collected by pipetting up and down multiple times and transferred to a new U-bottom 96-well plate. Cells were stained for FACS analysis with the following staining panel: anti-CD3 PerCP-Cy5.5, anti- CD8 BV785, anti-CD25 APC, and anti-CD4 BV605 from Biolegend, and the Fixable viability dye EFLUOR® 780 (ThermoFisher, 65-0865-14).
  • the number of different T cell subsets in each well were compared to spiked-in counting beads to calculate T cell number and T cell activation status were evaluated based on CD25 staining result.
  • the data was then analyzed and plotted with GraphPad Prism.
  • the results for the mesothelin-targeting constructs are shown in FIGS. 40A-40D, and the results for the TROP-2-targeting constructs are shown in FIGS. 43A-43D.
  • constructs with costimulatory activity binding to CD3 and CD28
  • Ml, and Tl showed slightly improved T cell counts and T cell activation over the constructs binding just to CD3, M3 and T3, while the constructs binding just to the tumor target and CD28, M2 and T2, showed activity comparable to the no antibody and isotype control constructs.
  • T cell Dependent Cytotoxicity Antibody-mediated redirected T cell killing of HEK293/TROP-2 cells (generated in-house) was measured in a TDCC assay in which human PBMCs as effector cells (E) and TROP2 transduced HEK293 cells as target cells (T) at an E:T ratio 3: 1 were incubated with serial dilutions of indicated antibodies in 96- well plates for 72 hours at 37°C in a humidified 5% CO2 incubator. After incubation, the impedance was used to evaluate the cell viability by RTCA as described above. The percent of cytotoxicity following antibody treatment was calculated by normalization of cell index from no antibody treatment group; results are provided in FIG. 44. The IC50 values determined for antibodies Tl and T3 were 0.04792 and 0.3422, respectively. Data are representative of 2 independent donors.
  • Antibody-mediated redirected T cell killing of A431-Luc cells was measured in a TDCC assay in which human PBMCs as effector cells (E) and A431- Luc cells as target cells (T) at an E:T ratio 1:1 were incubated with serial dilutions of indicated antibodies in 96-well plates for 96 hours at 37°C in a humidified 5% CO2 incubator.
  • a luminescence-based viability assay was used to determine the viable cells as described below.
  • the % of cell viability following antibody treatment with T3, Tl, or D16 was calculated by normalization of viable cells from no antibody treatment group. Data are representative of 3 independent donors. Results are provided in FIG. 45.
  • the IC50 values determined for antibodies T3, Tl, and D16 were 0.04448, 0.01560, and 0.001432, respectively.
  • Luciferase tagged adherent target tumor cells were added to the 96-well cell culture plates at the specified seeding densities and cultured in a humidified cell incubator with 5% CO2 at 37 °C for 24 hours to allow target tumor cells to settle and attach to the bottom. Effector cells (PBMC or T cells) and test articles were then added to the wells before resuming the culture. After incubation, the viability of the luciferase tagged tumor cells was determined by luminescent readout using the BIO-GLOTM luciferase assay system from Promega (catalogue number G7940), and the data was analyzed using GraphPad Prism.
  • T cell dependent cytotoxicity (TDCC) activity of various solid tumor targeting constructs as shown in Table 23 on the ovarian adenocarcinoma cell line SKOV-3 (BPS Bioscience, catalogue No 7842) was assessed by luminescence as follows.
  • Antibody- mediated redirected T cell killing of SKOV3-Luc cells was measured in a TDCC assay in which human PBMCs as effector cells (E) and SKOV3-Luc cells as target cells (T) at an E:T ratio 3 : 1 were incubated with serial dilutions of indicated antibodies in 96-well plates for 120 hours at 37°C in a humidified 5% CO2 incubator.
  • Antibody-mediated redirected T cell killing of OVCAR3 cells was measured in a TDCC assay in which human PBMCs as effector cells (E) and OVCAR3-Luc cells as target cells (T) at an E:T ratio of 1 :3 were incubated with serial dilutions of antibodies D16, T3, Tl, D17, M3 and Ml in 96-well plates for 120 hours (5 days) at 37°C in a humidified 5% CO2 incubator.
  • Cytokine Release Cytokine release mediated by the test articles was determined as described above using a Human Thl/Th2 Cytokine Cytometric Bead Array (CBA) Kit II from BD Biosciences using supernatant samples from the 72-hour (3 -day) time point. Levels of IL-2, IL-4, IL-6, IL-10, TNFa (also referred to herein as TNF) and IFN-y mediated by the test articles are shown for in FIGs. 48A-48F, respectively.
  • CBA Human Thl/Th2 Cytokine Cytometric Bead Array
  • Cytokine Release Cytokine release mediated by the test articles was determined as described above using supernatant samples from the 72-hour (3-day) time point. Levels of IL-2, IL-4, IL-6, IL- 10, TNF, and IFN-y mediated by the test articles are shown for in FIGs. 50A-50F, respectively.
  • Example 25 T-Cell Dependent Cellular Toxicity Assay Using High, Medium and Low-TROP-2 Expressing Cell Lines
  • T1 anti-TROP-2 x anti-CD3 x anti-CD28 IgM antibody
  • T3 anti-TROP-2 x anti-CD3 IgM
  • TROP-2 transduced HEK293T cells with TROP-2 high expression TROP-2 high expression
  • A431 melanoma cells with TROP-2 high expression and SKOV-3 ovarian cancer cells with TROP-2 medium/low expression, as target cells (T)
  • T target cells
  • E:T ratios of 3: 1, 1 :1, and 3:1 respectively, in the presence of serial dilutions of T1 or T3 antibody.
  • Incubation was carried out for 3-4 days at 37°C in a humidified 5% CO2 incubator. After incubation, the impedance was used to evaluate the cell viability by RTCA.
  • mice were engrafted with 10 7 human PBMCs on day -14. On day 0, mice were implanted subcutaneously with 10 7 TROP-2 transduced HEK293T cells.
  • Animals were dosed intravenously biweekly with antibody T1 or antibody T3, at doses of 0.3 mg/kg, 1 mg/kg, or 3 mg/kg, or vehicle (lx PBS) (see Table 26), starting on day 3 for a total of 12 doses (biwx!2).
  • antibody dosing was stopped after 7 doses to compare the prolonged effect of treatment with the illustrative anti-TROP-2 x anti-CD3 x anti-CD28 IgM antibody versus the illustrative anti-TROP-2 x anti-CD3 IgM antibody (that lacks a CD28 co-stimulatory signal).
  • FIGs. 53A Graphs of tumor volume over time for the various treatment groups is provided in FIGs. 53A (0.3 mg/kg groups), 53B (1 mg/kg groups) and 53C (3 mg/kg groups).
  • treatment with an IgM antibody such as T1 resulted in enhanced in vivo antitumor activity over exemplary IgM antibody T3 (lacking CD28 co-stimulation), e.g., when evaluated in a humanized HEK293/TROP- 2 xenograft model, particularly at low doses.
  • T1 IgM antibody treatment groups i.e., following treatment with an exemplary TROP-2 x CD3 x CD28 IgM antibody (i.e., with CD28 co-stimulation).
  • Mouse peripheral blood samples were harvested on day 12 (24 hours pre-4 th dose). Blood samples were processed and stained for flow cytometry analysis with an antibody cocktail containing ZOMBIE AQUATM BV510 viability dye, anti-human CD45 (clone 2D1, APC/Cy7), anti-mouse CD45 (clone 30F-11, AF700), anti-CD4 (clone OKT4, BV785), anti-CD8 (clone SKI, PerCp/Cy5.5), anti-Bcl_xL (clone 54H6, APC), purchased from either Biolegend or Cell Signaling Technology. Samples were then analyzed by flow cytometry analysis.
  • ZOMBIE AQUATM BV510 viability dye anti-human CD45 (clone 2D1, APC/Cy7)
  • anti-mouse CD45 clone 30F-11, AF700
  • anti-CD4 clone OKT4, BV785
  • anti-CD8 clone SKI, PerCp
  • Antibody-mediated, redirected T-cell killing of MKN45-Luc cells was measured in a TDCC assay in which human PBMCs as effector cells (E) and MKN45-Luc cells as target cells (T) at an E:T ratio of 3: 1 were incubated with serial dilutions of exemplary antibodies Ml and M3 in 96-well plates for 4 days at 37°C in a humidified 5% CO2 incubator. Following incubation, a luminescence-based viability assay as previously described was used to determine the viable cells. The percent of cell viability following antibody treatment was calculated by normalization of viable cells from the “no antibody” treatment group. Data are representative of 3 independent donors. Results are shown graphically in FIG. 56.
  • mice were dosed intravenously biweekly with antibody Ml, antibody M3, or vehicle (lx PBS) at doses of antibody of 3 mg/kg or 10 mg/kg, starting on day 3 for a total of 12 doses (biwxl2).
  • Results are shown graphically as average tumor volume over time (days post MKN45 cell implant) in FIGs. 57A (3 mg/kg dose) and 57B (10 mg/kg dose). As shown in FIGs.
  • IgM antibody such as Ml (anti-mesothelin (MSLN) x anti-CD3 x anti-CD28, i.e., with CD28 co-stimulation)
  • Ml anti-mesothelin
  • MSLN anti-mesothelin
  • CD28 co-stimulation treatment with an IgM antibody such as Ml (anti-mesothelin (MSLN) x anti-CD3 x anti-CD28, i.e., with CD28 co-stimulation)
  • MSLN anti-mesothelin
  • MSLN anti-mesothelin
  • Ml IgM antibody treatment groups i.e., following treatment with an exemplary MSLN xCD3xCD28 IgM antibody (i.e., with an IgM antibody with CD28 co-stimulation).
  • Intra-tumoral T cell counts Mouse tumor samples were harvested on day 43 (end point). Tumor samples were processed and stained with an antibody cocktail containing viability dye (ZOMBIE AQUATM BV510), anti-human CD45 (clone 2D1, APC/Cy7), anti-mouse CD45 (clone 30F-11, AF700), anti-CD4 (clone OKT4, BV785), and anti-CD8 (clone SKI, PerCp/Cy5.5), all reagents purchased from Biolegend. Samples were then subject to flow cytometry and gated for absolute CD4+ T cell counts (FIG. 60A) and absolute CD8+ T cell counts (FIG. 60B). An increase in the absolute cell counts for both CD4 and CD8 T cells were observed in tumor following CD28 co-stimulation at the higher dose.
  • Example 29 Comparison of various IgG and IgM anti-CD28 anti-CD3 antibody combinations in non-targeted T cell activation and cytokine release
  • constructs listed in Table 27 either alone or in combination were bound to the solid phase in a plate-based in vitro assay and were contacted with human PBMCs from two different donors.
  • the PBMCs were assayed for T cell activation and the resulting culture supernatants were assayed for cytokine release.
  • Frozen human PBMCs from two different donors were thawed in a 37°C water bath, washed with culture media, and then resuspended in culture media at 0.25 x 10 6 cells/mL.
  • the resuspended human PBMCs (200 pL, containing 5 x 10 4 cells) were dispensed into each well of the washed plates coated with antibodies. The plates were then incubated for 72 hours at 37°C with 5% CO2 and 95% humidity. The cell pellets and culture supernatants were recovered.
  • IL-2 levels in the cell culture supernatant samples were analyzed using the Human Thl/Th2 Cytokine Cytometric Bead Array (CBA) Kit II (BD, cat# 551809).
  • CBA Human Thl/Th2 Cytokine Cytometric Bead Array
  • the IL-2 levels measured for one exemplary PBMC donor are shown in FIG. 59C.
  • the cultured PBMC pellets were stained with fluorescent labeled antibodies against CD3, CD4, CD8 and CD25 for flow analysis to evaluate T cell activation status (CD25 positive percentage) of CD4 and CD8 T cell populations.
  • T cell activation of PBMCs of the same exemplary PBMC donor are shown in FIG. 59A (CD4+ T cells) and FIG. 59B (CD8+ T cells).
  • Anti-CD28 W as an IgG4 antibody (D28), showed strong T cell activation and strong IL-2 release even in the absence of CD3 stimulation, while anti-CD28 Z as an IgG4 antibody (D27) showed only weak T cell activation and no significant IL-2 release in the absence of CD3 stimulation.
  • Neither anti-CD28 W nor anti-CD28 Z, expressed on the J chain of an anti-virus isotype control IgM (D22 and D21, respectively) showed significant T cell activation of cytokine release in the absence of CD3 stimulation.
  • cytokine release from D22 and D21 remained low even in the presence of separate anti-CD3 IgG4 molecules (D25 + D21 or D25 + D22) or separate anti-viral IgM isotype control molecules expressing anti-CD3 on the J chain (D20 + D21 or D20 + D22).
  • separate anti-viral IgM isotype control molecules expressing anti-CD3 on the J chain D20 + D21 or D20 + D22.
  • efficient T cell activation and moderate IL-2 release was observed for the trispecific anti-viral IgM isotype control molecules in which both anti-CD28 Z or anti- CD28 W and anti-CD3 AA were co-expressed on the J chain (D15 and D23, respectively).

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Abstract

La présente divulgation propose des molécules de liaison multimères impliquant des lymphocytes T comprenant cinq, quatre ou deux unités de liaison bivalentes qui se lient à un antigène cible et une chaîne J modifiée, la chaîne J modifiée comprenant une chaîne J ou un fragment fonctionnel ou un variant de celle-ci, un domaine de liaison à l'antigène anti-CD3, et un polypeptide hétérologue comprenant un agoniste polypeptidique d'une molécule costimulatrice de lymphocytes T. La molécule costimulatrice de lymphocytes T peut être, par exemple, CD28 ou 4-1BB. La présente divulgation propose en outre des molécules de liaison multimères comprenant cinq, quatre ou deux unités de liaison bivalentes qui se lient à un antigène cible et une chaîne J modifiée, la chaîne J modifiée comprenant une chaîne J ou un fragment fonctionnel ou un variant de celle-ci et un trimère de ligand 4-1BB (4-1BBL) qui peut engager 4-1BB et l'activer sur une cellule cible.
PCT/US2024/010591 2023-01-06 2024-01-05 Molécules de liaison multimères costimulatrices de lymphocytes t et utilisations associées Ceased WO2024148336A1 (fr)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US12486336B2 (en) 2015-09-30 2025-12-02 Igm Biosciences, Inc. Binding molecules with modified J-chain

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WO2002047721A1 (fr) * 2000-12-14 2002-06-20 Fujisawa Pharmaceutical Co., Ltd. Anticorps anti-cd28 silencieux et leur utilisation
WO2017059387A1 (fr) * 2015-09-30 2017-04-06 Igm Biosciences, Inc. Molécules de liaison à chaîne j modifiée
US20190100597A1 (en) * 2015-03-04 2019-04-04 Igm Biosciences, Inc. Cd20 binding molecules and uses thereof
WO2022109023A1 (fr) * 2020-11-17 2022-05-27 Igm Biosciences, Inc. Utilisations de molécules recruteuses de cellules effectrices avec des fractions de puissances différentes

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Publication number Priority date Publication date Assignee Title
WO2002047721A1 (fr) * 2000-12-14 2002-06-20 Fujisawa Pharmaceutical Co., Ltd. Anticorps anti-cd28 silencieux et leur utilisation
US20190100597A1 (en) * 2015-03-04 2019-04-04 Igm Biosciences, Inc. Cd20 binding molecules and uses thereof
WO2017059387A1 (fr) * 2015-09-30 2017-04-06 Igm Biosciences, Inc. Molécules de liaison à chaîne j modifiée
WO2022109023A1 (fr) * 2020-11-17 2022-05-27 Igm Biosciences, Inc. Utilisations de molécules recruteuses de cellules effectrices avec des fractions de puissances différentes

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US12486336B2 (en) 2015-09-30 2025-12-02 Igm Biosciences, Inc. Binding molecules with modified J-chain

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