Compositions for redirecting immunoglobulins to immune cells

Abstract

The present invention relates to heterologous polypeptide or multimeric proteins and nucleic acids encoding the same comprising at least one immunoglobulin binding domain and at least one immune cell surface polypeptide or protein binding domain. The molecules of the invention can be used as monotherapy or for enhancing standard, current or experimental antibody-based immunotherapy, such as cancer, autoimmunity or pathogenic infections.

Description

Composition for redirecting immunoglobulins to immune cells

[0001] Cross-references to related applications

[0002] This application claims the benefit of U.S. Provisional Application No. 63 / 516,632, filed July 31, 2023, which is incorporated herein by reference in its entirety for all purposes. Technical Field

[0003] The field of this invention relates generally to immunology, and more specifically to proteins used to regulate disease. Background Technology

[0004] While natural antibodies, therapeutic antibodies, recombinant therapeutic antibodies, and Fc receptor-binding recombinant therapeutic proteins can offer significant benefits in regulating diseases such as cancer, autoimmunity, organ rejection, and pathogenic infections such as viruses, bacteria, parasites, or fungi, the cell types they can bind to are limited by their affinity and specificity for Fc receptors.

[0005] Having a mechanism for binding antibodies, therapeutic antibodies, recombinant therapeutic antibodies, and Fc receptors to recombinant therapeutic proteins with additional cell surface proteins and cell types would be advantageous. Summary of the Invention

[0006] This invention relates to heterologous polypeptides or multimeric proteins and nucleic acids encoding them, comprising at least one immunoglobulin-binding domain and at least one immune cell surface protein-binding domain, thereby creating immunoglobulin redirection (IgR) molecules capable of binding to immune cells and modulating cellular activities such as effector function or inhibition. The IgRs of this invention can be used as a monotherapy or in combination with one or more standard, current, or experimental therapeutic agents to treat conditions such as cancer, immune disorders, or pathogenic infections.

[0007] In some implementations, a heterologous polypeptide or multimeric protein and the nucleic acid encoding it comprise:

[0008] (a) at least one immunoglobulin-binding domain having affinity and specificity for one or more immunoglobulin isotypes, subtypes, allotypes and their variants, derivatives and analogs; and

[0009] (b) At least one immune cell surface protein binding domain;

[0010] (c) Optionally, at least one immunoglobulin-binding domain having affinity and specificity for one or more immunoglobulin isotypes, subtypes, allotypes and their variants, derivatives and analogs;

[0011] (d) Optionally, it is capable of binding at least one immunoglobulin-binding domain of wild-type immunoglobulin, but not limited to.

[0012] In some implementations, a heterologous polypeptide or multimeric protein and the nucleic acid encoding it comprise:

[0013] (a) at least two immunoglobulin-binding domains; and

[0014] (b) At least one immune cell surface protein binding domain;

[0015] (c) Optionally, at least two immunoglobulin-binding domains having affinity and specificity for one or more immunoglobulin isotypes, subtypes, allotypes and their variants, derivatives and analogs;

[0016] (d) Optionally, it is capable of binding at least two immunoglobulin-binding domains of wild-type immunoglobulins.

[0017] In some implementations, a heterologous polypeptide or multimeric protein and the nucleic acid encoding it comprise:

[0018] (a) at least one immunoglobulin-binding domain; and

[0019] (b) at least one immune cell surface protein-binding domain; and

[0020] (c) It can mediate immune cell activity in the presence of target-specific antibodies, which is significantly greater than that in the presence of non-specific antibodies that it can bind;

[0021] (d) Optionally, at least one immunoglobulin-binding domain having affinity and specificity for one or more immunoglobulin isotypes, subtypes, allotypes and their variants, derivatives and analogs;

[0022] (e) Optionally, it is capable of binding at least one immunoglobulin-binding domain of wild-type immunoglobulin;

[0023] (f) Optionally, it is able to mediate immune cell activity in the presence of target-specific and non-specific antibodies that it can bind, which is significantly higher than immune cell activity in the presence of non-specific antibodies.

[0024] (g) Optionally, it is capable of mediating immune cell activity in the presence of target-specific and non-specific antibodies to which it can bind, and such immune cell activity is not significantly lower than that in the presence of target-specific antibodies.

[0025] In some implementations, a heterologous polypeptide or multimeric protein and the nucleic acid encoding it comprise:

[0026] (a) at least two immunoglobulin-binding domains; and

[0027] (b) at least one immune cell surface protein-binding domain; and

[0028] (c) It can mediate immune cell activity in the presence of target-specific antibodies, which is significantly greater than that in the presence of non-specific antibodies that it can bind;

[0029] (d) Optionally, at least two immunoglobulin-binding domains having affinity and specificity for one or more immunoglobulin isotypes, subtypes, allotypes and their variants, derivatives and analogs.

[0030] (e) Optionally, it is capable of binding at least two immunoglobulin-binding domains of wild-type immunoglobulins;

[0031] (f) Optionally, it is able to mediate immune cell activity in the presence of target-specific and non-specific antibodies that it can bind, which is significantly higher than immune cell activity in the presence of non-specific antibodies.

[0032] (g) Optionally, it is capable of mediating immune cell activity in the presence of target-specific and non-specific antibodies to which it can bind, and such immune cell activity is not significantly lower than that in the presence of target-specific antibodies.

[0033] In some embodiments or in a combination of any of the preceding claims, a heterologous polypeptide and a nucleic acid encoding therethe, wherein:

[0034] (a) A heteropeptide is a single chain having at least three domains and an optional linker between the three domains, as shown in the figure below:

[0035] D1-D2-D3; or

[0036] (b) A heteropeptide is a single chain having at least four domains and optional linkers between the four domains, as shown in the figure below:

[0037] D1-D2-D3-D4

[0038] In some embodiments or in a combination of any of the preceding claims, a heterologous polypeptide or multimeric protein and a nucleic acid encoding thereas, wherein:

[0039] (a) At least one immunoglobulin-binding domain is derived from an antigen-binding domain, an antibody or antigen-binding fragment and its variants, derivatives or analogs;

[0040] (b) Optionally, at least one immunoglobulin binding domain is derived from an antigen-binding domain, an antibody or antigen-binding fragment and its variants, derivatives or analogs, including but not limited to VH and VL pairs, ScFv, Fab, IgG, sdAb-VL, sdAb-VH, VHH or avimer and their derivatives or analogs.

[0041] (c) Optionally, at least one immunoglobulin-binding domain is derived from an antigen-binding domain, antibody, or antigen-binding fragment and its variants, derivatives, or analogs that have affinity and specificity for one or more immunoglobulin isotypes, subtypes, allotypes, and their variants, derivatives, or analogs, including but not limited to: IgG, IgG1, IgG2, IgG3, IgG4, IgA, IgA1, IgA2, IgM, IgE, and IgD.

[0042] In some embodiments or in a combination of any of the preceding claims, a heterologous polypeptide or multimeric protein and a nucleic acid encoding thereas, wherein:

[0043] (a) At least one immunoglobulin-binding domain is derived from an Fc receptor or Fc binding domain, including but not limited to FcγRIII, mFcγRIV, FcγRIIa, FcγRIIb, FcγRIIc, FcγRI, mFcγRIII, mFcγRIIa, mFcγRIIb, mFcγRI, FcαRI, C1q, FcRL, FcRL5, pIgR, Fcα / μR, FcμR, FcεRI, FcεRII, FcRn, TRIM21, including its allotypes, derivatives and analogs;

[0044] (b) Optionally, the Fc receptor can bind to one or more immunoglobulin subclasses, their allotypes, derivatives and analogs, including but not limited to: IgG, IgG1, IgG2, IgG3, IgG4, IgA, IgA1, IgA2, IgM, IgE, IgD;

[0045] (c) Optionally, according to the residue number in SEQ ID NO: 9, FcγRIIa contains one or more mutations, including but not limited to R56H, K118N, T120V, L160Q and V172E;

[0046] (d) Optionally, according to the residue number in SEQ ID NO: 1, FcγRIII contains one or more mutations, including but not limited to S181P, K122N, T124V, Q176E, I90R, T118K, A119L and Y134F;

[0047] (e) Optionally, FcγR includes a domain from the first FcγR and a domain from an FcγR different from the first FcγR.

[0048] In some embodiments or in a combination of any of the preceding claims, a heterologous polypeptide or multimeric protein and the nucleic acid encoding it comprise:

[0049] (a) At least one immune cell surface protein binding domain derived from a natural soluble protein or ligand, including its variants, derivatives and analogs;

[0050] (b) Optionally, at least one immune cell surface protein-binding domain derived from a natural soluble protein or ligand, including its variants, derivatives and analogs, including but not limited to: cytokines, chemokines, pentamers, galactolectin-9, HMGB1, TGF-β, growth factors, pattern recognition proteins, lectins, enzymes, peptides, polysaccharides, lipids or metabolites.

[0051] In some embodiments or in a combination of any of the preceding claims, a heterologous polypeptide or multimeric protein and the nucleic acid encoding it comprise:

[0052] (a) At least one immune cell surface protein binding domain derived from the extracellular portion of a natural receptor or ligand, including its derivatives and analogs;

[0053] (b) Optionally, at least one immune cell surface protein-binding domain derived from the extracellular portion of a natural receptor or ligand, including its variants, derivatives, and analogs.Including but not limited to: CD3, CD3ε, CD3δ, CD3γ, TCR, TCRα, TCRβ, TCRγ, TCRδ or combinations thereof, TCR complex, CD8, CD4, CD2, CCR8, TNFR2, CD39, CD103, Fas ligand, MHC-I, MHC-II, MHC-G, HLA-DR, CD226, CD27, CD209, CD206, galactoglobulin-3, LSECtin, FGL1, CD112, CD155, HVEM, CEACAM-1, CD40, CD40L, CD137, CD137L, CD28, CD56, NKG2D, NKp46, PD1, PDL1, PDL1, CTLA4, CLEC5A, CD79, BCR, OX40, OX40L, TIM3, TIM1, TIGIT, CD7, LAG3, CD11a, CD11b, CD18, CD80, CD86 or FcγRIIIa, FcγRIV, FcγRIII b, FcγRIIa, FcγRIIc, FcγRIIb, FcγRI, C1q, FcRL5, pIgR, FcαRI, Fcα / μR, FcμR, FcεRI, FcεRII, FcRn, DC-SIGN, CD47, SIRP1α, CD96, VISTA, BTLA, B7- H3, chemokine receptors, cytokine receptors, growth factor receptors, pattern recognition receptors, enzymes, glycans, lipids, MICA, ULBP-1, ULBP-2, CD121a, CD121b, IL-18Rα, IL-18Rβ, CD25, CD122, CD132, CD124, CD213a13, CD127, CD360, CD19, CD20, CD5, IL-9R, CD213a1, CD213a2, IL-15Ra, CD123, CDw131, CDw125, CD131, CD116, CDw131, CD126, CD130, IL-11Ra, CD130, CD1 14. CD212, LIFR, CD130, OSMR, CDw210, IL-20Rα, IL-20Rβ, IL-14R, CDw217, CD118, CDw119, LTβR, CD120a, CD120b, BCMA, TACI, CD30, CD95 (Fas), GIT R, GITRL, LTbR, TRAILR1-4, Apo3, RANK, OPG, TGF-βR1, TGF-βR2, TGF-βR3, EpoR, TpoR, Flt-3, CD117, CD115, CDw136, dectin-1, dectin-2 and dectin-3. ,

[0054] In some embodiments or in a combination of any of the preceding claims, a heterologous polypeptide or multimeric protein and a nucleic acid encoding thereas, wherein:

[0055] (a) At least one immune cell surface protein binding domain is an antigen-binding domain, antibody, or antigen-binding fragment, including its variants, derivatives, or analogs, that has affinity and specificity for the extracellular portion of a natural receptor or ligand.

[0056] (b) Optionally, at least one immune cell surface protein-binding domain is an antigen-binding domain, antibody, or antigen-binding fragment, including its variants, derivatives, or analogs, that has affinity and specificity for the extracellular portion of a natural receptor or ligand.Including but not limited to: CD3, CD3ε, CD3δ, CD3γ, TCR, TCRα, TCRβ, TCRγ, TCRδ or combinations thereof, TCR complex, CD8, CD4, CD2, CCR8, TNFR2, CD39, CD103, Fas ligand, MHC-I, MHC-II, MHC-G, HLA-DR, CD226, CD27, CD209, CD206, galactolectin-3, LSECtin, FGL1, CD112, CD155, HVEM, CEACAM-1, CD40, CD40L, CD137, CD137L, CD28, CD56, NKG2D , NKp46, PD1, PDL1, PDL1, CTLA4, CLEC5A, CD79, BCR, OX40, OX40L, TIM3, TIM1, TIGIT, CD7, LAG3, CD11a, CD11b, CD18, CD80, CD86 or FcγRIIIa, Fcγ RIV, FcγRIIIb, FcγRIIa, FcγRIIc, FcγRIIb, FcγRI, C1q, FcRL5, pIgR, FcαRI, Fcα / μR, FcμR, FcεRI, FcεRII, FcRn, DC-SIGN, CD47, SIRP1α, CD96, VISTA, BTLA, B7-H3, chemokine receptors, cytokine receptors, growth factor receptors, pattern recognition receptors, enzymes, glycans, lipids, MICA, ULBP-1, ULBP-2, CD121a, CD121b, IL-18Rα, IL-18Rβ, CD25, CD122, CD132, CD124, CD213a13, CD127, CD360, CD19, CD20, CD5, IL-9R, CD213a1, CD213a2, IL-15Ra, CD123, CDw131, CDw125, CD131, CD116, CDw131, CD126, CD 130. IL-11Ra, CD130, CD114, CD212, LIFR, CD130, OSMR, CDw210, IL-20Rα, IL-20Rβ, IL-14R, CDw217, CD118, CDw119, LTβR, CD120a, CD120b, BCMA , TACI, CD30, CD95 (Fas), GITR, GITRL, LTbR, TRAILR1-4, Apo3, RANK, OPG, TGF-βR1, TGF-βR2, TGF-βR3, EpoR, TpoR, Flt-3, CD117, CD115, CDw136 dectin-1, dectin-2 and dectin-3. ,

[0057] In some embodiments or in a combination of any of the preceding claims, a heterologous polypeptide or multimeric protein and a nucleic acid encoding thereas, wherein:

[0058] (a) At least one immune cell surface protein binding domain is an antigen-binding domain, antibody or antigen-binding fragment and its derivatives or analogues that have affinity and specificity to the extracellular portion of a natural receptor or ligand present on, but not limited to, cells containing: lymphocytes, myeloid cells, T cells, B cells, NK cells, macrophages, monocytes, NK-T cells, neutrophils, dendritic cells, basophils, eosinophils and mast cells;

[0059] (b) Optionally, at least one immune cell surface protein binding domain is an antigen-binding domain, antibody, or antigen-binding fragment with affinity and specificity for the extracellular portion of a natural receptor or ligand present on, but not limited to, cells containing, but not limited to, VH and VL pairs, ScFv, Fab, IgG or sdAb-VL, sdAb-VH, VHH and their variants, derivatives, or analogs: lymphocytes, myeloid cells, T cells, B cells, NK cells, macrophages, monocytes, NK-T cells, neutrophils, dendritic cells, basophils, eosinophils, and mast cells.

[0060] In some embodiments or in a combination of any of the preceding claims, a heterologous polypeptide or multimeric protein and a nucleic acid encoding thereas are included, wherein an additional region comprises:

[0061] (a) at least one half-life-extending domain, including but not limited to HSA, anti-HSA and its derivatives or analogs; and

[0062] (b) Optionally, the antiserum albumin domain includes, but is not limited to, one or more CDR or FR regions as defined in SEQ ID NO: 558, 559, 560, 561, 562, 563, 564, 565, 566, 567, 568, 569, 570, 571, 572, 573, 574, 575, 576, 577 and 578 provided in Table 16, including variants, derivatives and analogs thereof.

[0063] In some embodiments or in a combination of any of the preceding claims, a multimeric protein and a nucleic acid encoding the same, wherein an additional region of the molecule comprises:

[0064] (a) Two half-life extension domains comprising, but not limited to, Fc polypeptides derived from immunoglobulins, wherein the Fc polypeptides substantially do not bind to the immunoglobulin-binding region; and

[0065] (b) Wherein the first Fc polypeptide and the second Fc polypeptide contain heteropolymerization domains, wherein the heteropolymerization domains may be one or more knob into hole mutations, etc.

[0066] In some embodiments or in a combination of any of the preceding claims, a multimeric protein and a nucleic acid encoding the same, wherein an additional region of the molecule comprises:

[0067] (a) Two half-life extension domains comprising, but not limited to, Fc polypeptides derived from immunoglobulins, wherein by selecting IgG heavy chain Fc polypeptides, the Fc substantially does not bind to the immunoglobulin binding region, the IgG heavy chain Fc polypeptides including, but not limited to, mutants L234A, L235A and P329A in constant heavy chain domain 2 (EU number) or alternatively P329G;

[0068] (b) wherein the first Fc polypeptide and the second Fc polypeptide contain heteropolymerization domains, wherein the heteropolymerization domains are selected from one or more knob into hole mutations, etc.

[0069] In some embodiments or in a combination of any of the preceding claims, the multimeric protein of any of the claims and the nucleic acid encoding it, wherein an additional region of the molecule comprises:

[0070] (a) Two half-life extension domains comprising, but not limited to, Fc polypeptides derived from immunoglobulins, wherein, by selecting IgG heavy chain Fc polypeptides, the Fc substantially does not bind to the immunoglobulin-binding region, said IgG heavy chain Fc polypeptides including, but not limited to, mutants L234A, L235A, and P329A in constant heavy chain domain 2 (EU number) or alternatively P329G; and

[0071] (b) wherein the first Fc polypeptide includes, but is not limited to, the mutant T366W and optionally S354C, and wherein the second Fc polypeptide includes, but is not limited to, T366S, L368A and Y407V and optionally Y349C in constant heavy chain domain 3 (EU number).

[0072] In some embodiments or in a combination of any of the preceding claims, a multimeric protein and a nucleic acid encoding the same, wherein an additional region of the molecule comprises:

[0073] (a) One or more immunoglobulin κ or λ constant light chains and their variants, derivatives and analogs;

[0074] (b) One or more immunoglobulin constant heavy chain domains 1 and all, none or part of the immunoglobulin hinge region and its variants, derivatives and analogs;

[0075] (c) Optionally, at least two immunoglobulin κ constant light chains and their variants, derivatives and analogs;

[0076] (d) Optionally, at least two immunoglobulin λ constant light chains and their variants, derivatives or analogs;

[0077] (e) Optionally, at least one immunoglobulin κ constant light chain and one immunoglobulin λ constant light chain, and variants, derivatives and analogs thereof;

[0078] (f) Optionally, one or more constant heavy chain domains 1 and all, none or some of the hinges contain mutations such as C233S and constant light chains contain mutations such as C214S (Kabat number).

[0079] (g) Optionally, one or more constant heavy chain domains 1 and all, none or some of the hinges contain mutations such as C233S and F174C and the constant light chain contains mutations such as C214S and S176C (Kabat number).

[0080] (h) Optionally, at least the first pair of constant heavy chains and all, none or some of the hinges do not contain mutations at C233 etc. and the constant light chains do not contain mutations at C214 etc., and at least the second pair of constant heavy chain domain 1 and all, none or some of the hinges contain mutations C233S etc. and the constant light chains contain mutations C214S etc. (Kabat number).

[0081] (i) Optionally, at least the first pair of constant heavy chains and all, no or some hinges do not contain mutations at C233, etc. and the constant light chains do not contain mutations at C214, etc., and at least the second pair of constant heavy chain domain 1 and all, no or some hinges contain mutations C233S and F174C, etc. and the constant light chains contain mutations C214S and S176C, etc. (Kabat number).

[0082] (j) Optionally, at least the first pair of constant heavy chain domain 1 and all, none or some of the hinges contain mutations such as C233S and so on, and the constant light chain contains mutations such as C214S and so on, and at least the second pair of constant heavy chain domain 1 and all, none or some of the hinges contain mutations such as C233S and F174C and so on, and the constant light chain contains mutations such as C214S and S176C and so on (Kabat number).

[0083] In some embodiments or in a combination of any of the preceding claims, a heterologous polypeptide or multimeric protein and a nucleic acid encoding thereas, wherein one or more additional regions of the molecule comprise:

[0084] (a) at least a second immunoglobulin-binding domain, wherein the second immunoglobulin-binding domain is identical to the first immunoglobulin-binding domain; or

[0085] (b) at least a second immunoglobulin-binding domain, wherein the second immunoglobulin-binding domain is different from the first immunoglobulin-binding domain;

[0086] (c) Optionally, at least a second immunoglobulin-binding domain, wherein the second immunoglobulin-binding domain is separated from the first immunoglobulin-binding domain by a suitable linker, the linker may have different lengths, such as 1 amino acid (e.g., Gly) to 20 amino acids, 2 amino acids to 15 amino acids, 3 amino acids to 12 amino acids, including 4 amino acids to 10 amino acids, 5 amino acids to 9 amino acids, 6 amino acids to 8 amino acids, or 7 amino acids to 8 amino acids, and may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 4 4, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59 or 60 amino acids, including but not limited to regions of human constant heavy chain domain 1, κ or λ chain domain, polypeptides containing linkers of 13 or fewer amino acids, linkers of 6 or fewer amino acids, spacer regions derived from constant heavy chain domain 1 such as ASTKGPSVFPLAP, ASTKGP or ASTKGPSVFPLAS, spacer regions derived from constant κ chain such as RTVAAPSVFIFPP or RTVAAP, spacer regions derived from constant λ chain such as SQPKAAPSVTLFP, GQPKANPTVTLFP, GQPKAAPSVTLFP, SQPKAA, GQPKAN or GQPKAA, (GGGS)1, (GGGS)2, (GGGS)3, (GGGS)4;

[0087] (d) Optionally, at least a second immunoglobulin-binding domain, wherein the second immunoglobulin-binding domain is identical to the first immunoglobulin-binding domain, wherein if the domains are derived from FcγR type Fc receptors, they are not arranged in head-to-tail tandem.

[0088] (e) Optionally, at least a second immunoglobulin-binding domain, wherein the second immunoglobulin-binding domain is identical to the first immunoglobulin-binding domain, wherein if the domains are derived from FcγR type Fc receptors, they are arranged in head-to-tail tandem.

[0089] In some embodiments or in a combination of any of the preceding claims, a heterologous polypeptide or multimeric protein and a nucleic acid encoding thereas, wherein an additional region of the molecule comprises:

[0090] (a) at least a second immune cell surface protein binding domain, wherein the second immune cell binding domain is different from the first immune cell surface protein binding domain; or

[0091] (b) at least a second immune cell surface protein binding domain, wherein the second immune cell surface protein binding domain is identical to the first immune cell surface protein binding domain;

[0092] (c) Optionally, at least a second immunoglobulin-binding domain, wherein the second immunoglobulin-binding domain is separated from the first immunoglobulin-binding domain by a suitable linker, the linker may have different lengths, such as 1 amino acid (e.g., Gly) to 20 amino acids, 2 amino acids to 15 amino acids, 3 amino acids to 12 amino acids, including 4 amino acids to 10 amino acids, 5 amino acids to 9 amino acids, 6 amino acids to 8 amino acids, or 7 amino acids to 8 amino acids, and may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 4 4, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59 or 60 amino acids, including but not limited to regions of human constant heavy chain domain 1, κ or λ chain domain, polypeptides containing linkers of 13 or fewer amino acids, linkers of 6 or fewer amino acids, spacer regions derived from constant heavy chain domain 1 such as ASTKGPSVFPLAP, ASTKGP or ASTKGPSVFPLAS, spacer regions derived from constant κ chain such as RTVAAPSVFIFPP or RTVAAP, spacer regions derived from constant λ chain such as SQPKAAPSVTLFP, GQPKANPTVTLFP, GQPKAAPSVTLFP, SQPKAA, GQPKAN or GQPKAA, (GGGS)1, (GGGS)2, (GGGS)3, (GGGS)4;

[0093] In some embodiments or in a combination of any of the preceding claims, a heterologous polypeptide or multimeric protein and a nucleic acid encoding thereas, wherein one or more regions of the molecule comprise:

[0094] (a) A single amino acid mutation or multiple amino acid mutations in which the immunoglobulin binding domain substantially does not bind itself or another region or multiple regions of the molecule.

[0095] (b) Optionally, one or more amino acid mutations are made in one or more constant domains of human IgG1, IgG2, IgG3 or IgG4, their variants, derivatives and analogs;

[0096] (c) Optionally, one or more amino acid mutations in constant heavy chain domain 1, including but not limited to F122Y, P126S and K213E (Kabat number).

[0097] (d) Optionally, one or more amino acids in the hinge region are mutated;

[0098] (e) Optionally, one or more amino acid mutations in constant heavy chain domain 2, including but not limited to N276K, L309V, L234A, L235A and P329A or alternatively P329G (EU number).

[0099] (f) Optionally, one or more amino acids in the constant heavy chain domain 3 are mutated;

[0100] (g) Optionally, one or more amino acid mutations in the human κ or λ domain.

[0101] (h) Optionally, one or more amino acid mutations in one or more light chain or heavy chain FR domains.

[0102] In some embodiments or in a combination of any of the preceding claims, a heterologous polypeptide or multimeric protein and a nucleic acid encoding thereas, wherein one or more regions of the molecule comprise:

[0103] (a) At least one immunoglobulin-binding domain comprising, but not limited to, one or more CDR or FR regions as defined in SEQ ID NO:579 to SEQ ID NO:718 provided in Table 17, including variants, derivatives and analogs thereof; or

[0104] (b) At least one immunoglobulin-binding domain, which consists of anti-Fc AVIG, SEQ ID NO: 132, its variants, derivatives and analogs.

[0105] In some embodiments or in a combination of any of the preceding claims, a heterologous polypeptide or multimeric protein and a nucleic acid encoding thereas, wherein one or more regions of the molecule comprise:

[0106] (a) At least one immune cell surface protein binding domain comprising, but not limited to, one or more CDR or FR regions as defined in SEQ ID NO: 719 to SEQ ID NO: 1558 provided in Tables 18, 19, 20, 21, 22, 23, 24, 25, 26, 27 and 28, including variants, derivatives and analogs thereof.

[0107] In some embodiments or in a combination of any of the preceding claims, a heterologous polypeptide or multimeric protein and a nucleic acid encoding the same, wherein an additional region of the molecule contains a free cysteine ​​residue at or near the C-terminus.

[0108] In some embodiments or in a combination of any of the preceding claims, a heterologous polypeptide or multimeric protein and a nucleic acid encoding thereon, wherein an additional region of the molecule contains a covalently linked PEG-lipid.

[0109] In some embodiments or in a combination of any of the preceding claims, a heterologous polypeptide or multimeric protein and the nucleic acid encoding it comprise:

[0110] (a) At least one immunoglobulin-binding domain that blocks immunoglobulins from binding to one or more of their homologous receptors; or

[0111] (b) At least one immunoglobulin-binding domain that does not block the binding of immunoglobulins to one or more of their homologous receptors; or

[0112] (c) Optionally, at least two immunoglobulin-binding domains that block immunoglobulins from binding to one or more of their homologous receptors; or

[0113] (d) Optionally, at least two immunoglobulin-binding domains that do not block the binding of immunoglobulins to one or more of their homologous receptors; or

[0114] (e) Optionally, at least two immunoglobulin-binding domains, wherein a first immunoglobulin-binding domain blocks immunoglobulins from binding to one or more of their homologous receptors, and a second immunoglobulin-binding domain does not block immunoglobulins from binding to one or more of their homologous receptors.

[0115] In some implementations, a multimeric protein and a nucleic acid encoding it are included, wherein regions of the molecule contain:

[0116] (a) One or more immunoglobulin κ or λ constant light chains and their variants, derivatives and analogs;

[0117] (b) One or more immunoglobulin constant heavy chain domains 1 and all, none or part of the immunoglobulin hinge region and its variants, derivatives and analogs;

[0118] (c) Optionally, at least two immunoglobulin κ constant light chains and their variants, derivatives and analogs;

[0119] (d) Optionally, at least two immunoglobulin λ constant light chains and their variants, derivatives and analogs;

[0120] (e) Optionally, at least one immunoglobulin κ constant light chain and one immunoglobulin λ constant light chain, and variants, derivatives and analogs thereof;

[0121] (f) Optionally, one or more constant heavy chain domains 1 and all, none or some of the hinges contain mutations such as C233S and constant light chains contain mutations such as C214S (Kabat number).

[0122] (g) Optionally, one or more constant heavy chain domains 1 and all, none or some of the hinges contain mutations such as C233S and F174C and the constant light chain contains mutations such as C214S and S176C (Kabat number).

[0123] (h) Optionally, at least the first pair of constant heavy chains and all, none or some of the hinges do not contain mutations at C233 etc. and the constant light chains do not contain mutations at C214 etc., and at least the second pair of constant heavy chain domain 1 and all, none or some of the hinges contain mutations C233S etc. and the constant light chains contain mutations C214S etc. (Kabat number).

[0124] (i) Optionally, at least the first pair of constant heavy chains and all, no or some hinges do not contain mutations at C233, etc. and the constant light chains do not contain mutations at C214, etc., and at least the second pair of constant heavy chain domain 1 and all, no or some hinges contain mutations C233S and F174C, etc. and the constant light chains contain mutations C214S and S176C, etc. (Kabat number).

[0125] (j) Optionally, at least the first pair of constant heavy chain domain 1 and all, none or some of the hinges contain mutations such as C233S and so on, and the constant light chain contains mutations such as C214S and so on, and at least the second pair of constant heavy chain domain 1 and all, none or some of the hinges contain mutations such as C233S and F174C and so on, and the constant light chain contains mutations such as C214S and S176C and so on (Kabat number).

[0126] In some embodiments or in a combination of any of the preceding claims, a multimeric protein and a nucleic acid encoding the same, wherein an additional region of the molecule comprises:

[0127] (a) Two half-life extension domains comprising, but not limited to, Fc polypeptides derived from immunoglobulins, wherein the first and second Fc polypeptides comprise heteropolymerization domains selected from one or more knob-into-hole mutations, etc.; and

[0128] (b) Optionally, the first Fc polypeptide includes, but is not limited to, the mutant T366W and optionally S354C; and the second Fc polypeptide includes, but is not limited to, T366S, L368A and Y407V and optionally Y349C in constant heavy chain domain 3 (EU number);

[0129] (c) Optionally, wherein by selecting an IgG heavy chain Fc polypeptide, the Fc substantially does not bind to one or more of its homologous Fc receptors.

[0130] (d) Optionally, wherein by selecting an IgG heavy chain Fc polypeptide, the Fc substantially does not bind to one or more of its homologous Fc receptors, said IgG heavy chain Fc polypeptide including, but not limited to, mutations L234A, L235A and P329A in constant heavy chain domain 2 (EU number) or alternatively P329G.

[0131] In some embodiments or in a combination of any of the preceding claims, the method of treating a disease using the heterologous polypeptide or multimeric protein of any of claims 1-26 and the nucleic acid encoding thereus is as follows:

[0132] (a) Single therapy; or

[0133] (b) In combination with one or more standard, current or experimental therapeutic agents to treat cancer, immune disorders and pathogenic infections.

[0134] In some embodiments or in a combination of any of the preceding claims, the method of treating a disease using the heterologous polypeptide or multimeric protein of any of claims 1-27 and the nucleic acid encoding thereus is as follows:

[0135] (a) Monotherapy that can bind to endogenous immunoglobulins; or

[0136] (b) In combination with one or more standard, current or experimental therapeutic agents to treat cancer, immune disorders and pathogenic infections.

[0137] In some embodiments or in a combination of any of the preceding claims, a kit comprises a heterologous polypeptide or multimeric protein and a nucleic acid encoding thereon. Attached Figure Description

[0138] Figure 1 illustrates how immunoglobulins (Ig) mediate effector functions by bridging diseased cells (e.g., cancer, pathogen infection, self-binding autoimmune-driven Ig) to immune cells via binding to cell surface antigens (Ag) on ​​diseased cells and Fc receptors (FcR) expressed on immune cells.

[0139] Figure 2. (A) Depicts an IgR fusion protein comprising an immunoglobulin-binding domain (IgBD) and an immune cell surface protein-binding domain (ICBD), wherein the immune cell surface protein-binding domain comprises an immunoglobulin redirection molecule with an optional linker. (B) Depicts IgR binding to an immunoglobulin bound to a diseased cell and redirecting it to an immune cell surface protein. (C) Depicts an IgR comprising two immunoglobulin-binding domains and one immune cell-binding domain, along with an optional linker. (D) Depicts an IgR comprising one immunoglobulin-binding domain and two immune cell-binding domains, along with an optional linker.

[0140] Figure 3. (A) depicts an IgR containing a half-life extension domain fused to an immunoglobulin-binding domain and an immune cell-binding domain via an optional linker. (B) depicts an IgR containing a half-life extension domain fused to two immunoglobulin-binding domains and an immune cell-binding domain via an optional linker. (C) depicts an IgR containing a half-life extension domain fused to an immunoglobulin-binding domain and two immune cell-binding domains via an optional linker.

[0141] Figure 4. (A) depicts an IgR containing an immunoglobulin-binding domain fused to an Fc, which is attached to another Fc having a heavy / light chain containing an immunoglobulin-binding domain (VH / VL pairs attached to their corresponding constant heavy / light chains). (B) depicts an IgR containing an immunoglobulin-binding domain fused to an Fc, which is attached to another Fc having a heavy / light chain containing two immunoglobulin-binding domains (one VH / VL pair attached to its corresponding constant heavy and light chains, and one VH / VL pair fused as ScFv to the C-terminus of the constant light chain). (C) describes an IgR comprising an immunoglobulin-binding domain fused to an Fc, which is attached to another Fc having a heavy chain comprising an immunoglobulin-binding domain (the VH / VL pair is attached as ScFv to the N-terminus of a constant heavy chain). (D) depicts an IgR comprising an immunoglobulin-binding domain fused to an Fc, which is attached to another Fc having a heavy chain comprising an immunoglobulin-binding domain and a second immunoglobulin-binding domain (the immunoglobulin-binding domain fused to the VH / VL pair is attached as ScFv to the N-terminus of a constant heavy chain). (E) depicts an IgR comprising an immunoglobulin-binding domain fused to the N-terminus of a constant heavy chain, a second immunoglobulin-binding domain fused to the N-terminus of a constant light chain on the same Fc, the same Fc being attached to another Fc having a heavy chain / light chain (VH / VL pairs attached to their respective constant heavy chain / light chain) comprising an immunoglobulin-binding domain. (F) depicts an IgR comprising an immunoglobulin-binding domain fused to the N-terminus of a constant heavy chain via an optional linker, a second immunoglobulin-binding domain fused to the N-terminus of a constant light chain via an optional linker on the same Fc being attached to another Fc having a heavy chain / light chain (VH / VL pairs attached to their respective constant heavy chain / light chain) comprising an immunoglobulin-binding domain; and a second immunoglobulin-binding domain fused to the N-terminus of a constant light chain via an optional linker. (G) describes an IgR comprising an immunoglobulin-binding domain fused to an Fc via an optional linker and attached to another Fc having a heavy / light chain (VH / VL pairs attached to their corresponding constant heavy / light chains) containing an immune cell-binding domain; and a second immunoglobulin-binding domain fused to the C-terminus of the constant light chain via an optional linker.(H) depicts an IgR comprising an immunoglobulin-binding domain (VH / VL pairs attached to their corresponding full-length constant heavy / light chains), the immunoglobulin-binding domain being attached to another Fc having a heavy / light chain containing an immunoglobulin-binding domain (VH / VL pairs attached to their corresponding full-length constant heavy / light chains). (I) depicts an IgR comprising two immunoglobulin-binding domains, wherein a first VH / VL pair is attached to its corresponding full-length constant heavy / light chain, and a second VH / VL is attached to the N-terminus of the first VH / VL pair via an optional linker, wherein its Fc is attached to another Fc having a heavy / light chain containing an immunoglobulin-binding domain (VH / VL pairs attached to their corresponding full-length constant heavy / light chains). (J) describes an IgR comprising two immunoglobulin-binding domains, wherein a first VH / VL pair is attached to its corresponding full-length constant heavy / light chain, the two immunoglobulin-binding domains being attached to another Fc having a heavy / light chain comprising a second immunoglobulin-binding domain (VH / VL pair being attached to its corresponding full-length constant heavy / light chain), wherein the immune cell-binding domain is attached to the C-terminus of one of the light chains of the immunoglobulin-binding domain via an optional linker.

[0142] Figure 5. (A) depicts an IgR comprising a Fab fusion body, wherein an immune cell binding domain VH / VL pair is attached to its corresponding constant heavy chain 1 and constant light chain, and an immunoglobulin binding domain is fused to the C-terminus of the constant light chain via an optional linker. (B) depicts an IgR comprising a Fab fusion body, wherein an immune cell binding domain VH / VL pair is attached to its corresponding constant heavy chain 1 and constant light chain, and an immunoglobulin binding domain is fused to the C-terminus of the constant heavy chain, and an immunoglobulin domain is fused to the C-terminus of the constant light chain via an optional linker. (C) depicts an IgR comprising a Fab fusion body, wherein an immune globulin binding domain VH / VL pair is attached to its corresponding constant heavy chain 1 and constant light chain, and an immune cell binding domain is fused to the C-terminus of the constant light chain via an optional linker. (D) depicts an IgR comprising a Fab fusion body, wherein an immunoglobulin-binding domain is attached to the N-terminus of a constant heavy chain 1, and an immune cell-binding domain is fused to the C-terminus of a constant light chain via an optional linker. (E) depicts an IgR comprising a Fab fusion body, wherein an immunoglobulin-binding domain is attached to the N-terminus of a constant heavy chain 1, a second immunoglobulin-binding domain is attached to the N-terminus of a constant light chain, and an immune cell-binding domain is fused to the C-terminus of a constant light chain via an optional linker. (F) depicts an IgR comprising a Fab fusion body, wherein an immunoglobulin-binding domain is attached to the N-terminus of a constant heavy chain 1, an immunocell-binding domain is fused to the C-terminus of the constant heavy chain via an optional linker, and a second immunocell-binding domain is fused to the C-terminus of the constant light chain via an optional linker. (G) depicts an IgR comprising a Fab fusion body, wherein an immunoglobulin-binding domain is attached to the N-terminus of a constant heavy chain 1, a second immunoglobulin-binding domain is attached to the N-terminus of a constant light chain, an immunocell-binding domain is fused to the C-terminus of the constant heavy chain via an optional linker, and a second immunocell-binding domain is fused to the C-terminus of the constant light chain via an optional linker. (H) depicts an IgR comprising a Fab fusion body, wherein immunoglobulin-binding domains VH / VL are attached to their corresponding constant heavy chain 1 and constant light chain, and an immunocell-binding domain is fused to the C-terminus of the constant light chain via an optional linker, and a second immunocell-binding domain is fused to the C-terminus of the constant heavy chain via an optional linker.(I) Describes an IgR comprising a Fab fusion having two immunoglobulin-binding domains, wherein a first VH / VL pair is attached to its corresponding constant heavy chain 1 and constant light chain, and a second VH / VL pair is attached to the N-terminus of the first VH / VL pair via an optional linker; and an immunocellular binding domain fused to the C-terminus of the constant light chain via an optional linker; and a half-life extension domain fused to the C-terminus of the constant heavy chain via an optional linker. (J) Describes an IgR comprising a Fab fusion having immunocellular binding domains VH / VL pairs attached to their corresponding constant heavy chain 1 and constant light chain, and an immunoglobulin-binding domain fused to the C-terminus of the constant heavy chain, and an immunoglobulin domain fused to the C-terminus of the constant light chain via an optional linker; and wherein the half-life extension domain is attached to the C-terminus of one of the immunoglobulin domains via an optional linker. (K) describes an IgR comprising a Fab fusion body in which an immune cell binding domain VH / VL pair is attached to its corresponding constant heavy chain 1 and constant light chain, and an immunoglobulin binding domain is fused to the N-terminus of a variable heavy chain, and an immunoglobulin domain is fused to the N-terminus of a variable light chain via an optional linker. (L) describes an IgR comprising a Fab fusion body in which an immune cell binding domain VH / VL pair is attached to its corresponding constant heavy chain 1 and constant light chain, and an immunoglobulin binding domain is fused to the N-terminus of a variable heavy chain via an optional linker; an immunoglobulin domain is fused to the N-terminus of a variable light chain via an optional linker; and an immunoglobulin domain is fused to the C-terminus of one of the constant chains via an optional linker.

[0143] Figure 6. (A) depicts an IgR comprising a Fab fusion body, wherein an immunoglobulin-binding domain VH / VL pair is attached to its corresponding constant heavy chain 1 and constant light chain, and an immunoglobulin-binding domain is fused to the C-terminus of the constant light chain via an optional linker, and a PEG-lipid is covalently attached to the C-terminus of the constant heavy chain 1. (B) depicts an IgR comprising a Fab fusion body, wherein an immunoglobulin-binding domain VH / VL pair is attached to its corresponding constant heavy chain 1 and constant light chain, and an immunoglobulin-binding domain is fused to the C-terminus of the constant light chain via an optional linker, and a PEG-lipid is covalently attached to the C-terminus of the constant heavy chain 1. (C) depicts an IgR comprising a Fab fusion body, wherein an immunoglobulin-binding domain is attached to the N-terminus of the constant heavy chain 1 via an optional linker, an immunoglobulin-binding domain VH / VL pair ScFv is fused to the C-terminus of the constant light chain via an optional linker, and a PEG-lipid is covalently attached to the C-terminus of the constant heavy chain 1. (D) Depicts an IgR comprising a Fab fusion compound, wherein an immunoglobulin-binding domain is attached to the N-terminus of a constant heavy chain 1 via an optional linker, a second immunoglobulin-binding domain is attached to the N-terminus of a constant light chain via an optional linker, an immune cell-binding domain is fused to the C-terminus of the constant light chain via an optional linker, and a PEG-lipid is covalently attached to the C-terminus of the constant heavy chain 1. (E) Depicts an IgR comprising an immunoglobulin-binding domain fused to the N-terminus of the immune cell-binding domain VH / VL to ScFv and a PEG-lipid covalently attached to the C-terminus of ScFv. (F) depicts an IgR comprising a Fab fusion body, wherein an immune cell binding domain VH / VL ScFv is attached to the N-terminus of a constant heavy chain 1 via an optional linker, a second immune cell binding domain VH / VL ScFv is attached to the N-terminus of a constant light chain, an immunoglobulin binding domain is fused to the C-terminus of the constant light chain via an optional linker, and a PEG-lipid is covalently attached to the C-terminus of the constant heavy chain 1. (G) depicts an IgR comprising a Fab fusion body, wherein an immunoglobulin binding domain is attached to the N-terminus of a constant heavy chain 1, an immune cell binding domain VH / VL ScFv is attached to the N-terminus of the constant light chain, a second immune cell binding domain VH / VL ScFv is fused to the C-terminus of the constant light chain via an optional linker, and a PEG-lipid is covalently attached to the C-terminus of the constant heavy chain 1.

[0144] Figure 7. (A) depicts an IgR containing an Fc-binding domain and a T-cell receptor complex-binding domain, which is capable of redirecting immunoglobulins to T cells. (B) depicts how the FcB / αTCR IgR molecule enhances Ig-mediated effector function against cancer cells by recruiting and driving T-cell-mediated effector function.

[0145] Figure 8. A, C, E, G, I, and K show SDS-PAGE gel maps of IgR under reducing (R) and non-reducing conditions. B, D, F, H, J, and L show chromatograms of IgR obtained by high-performance liquid chromatography-size exclusion chromatography (HPLC-SEC).

[0146] Figure 9. A, C, E, G, I, and K show SDS-PAGE gel chromatograms of IgR under reducing (R) and non-reducing conditions. B, D, F, H, J, and L show chromatograms of IgR obtained by high-performance liquid chromatography-size exclusion chromatography (HPLC-SEC).

[0147] Figure 10. Depicts T-cell activation assays using a co-culture of a target cancer cell line and an engineered T-cell line, Jurkat NFAT-luc, which expresses luciferase in its T-cell receptor when the target cell binding antibody is combined with an Fc-binding / T-cell receptor binding fusion protein.

[0148] Figure 11. Validation of T cell activation assay using co-cultures of CD20+ Raji cells and Jurkat NFAT-Luc cells in all wells, with individual cells and anti-CD20 rituximab as negative controls and anti-CD20 / anti-CD3 motuzumab as positive controls.

[0149] Figure 12. Validation of T cell activation assay using co-cultures of CD20+ Raji cells and Jurkat NFAT-Luc cells in all wells, with anti-CD20 / anti-CD3 motuzumab as a positive control.

[0150] Figure 13. Evaluation of T cell activation mediated by FcγRIIIA / antiCD3 or FcγRIIIA / antiTCR fusion proteins in combination with and without rituximab (Rtx) when co-cultured with CD20+ Raji B cells and Jurkat-NFAT-Luc T cells.

[0151] Figure 14. Evaluation of T cell activation mediated by FcγRIIIA / anti-CD3 fusion with and without half-life extension strategies when co-cultured with CD20+ Raji B cells and Jurkat-NFAT-Luc T cells.

[0152] Figure 15. Evaluation of T cell activation mediated by FcγRIIIA / anti-CD3 or anti-Fc / anti-CD3 fusions in combination with rituximab and without such combinations when co-cultured with CD20+ Raji B cells and Jurkat-NFAT-Luc T cells.

[0153] Figure 16. Evaluation of T cell activation mediated by FcγRIIIA / antiCD3 or FcγRIIIA / antiTCR fusion protein in combination with trastuzumab (Tras) or rituximab (Rtx) when co-cultured with HER2+ SKBR3 breast cancer cells and Jurkat-NFAT-Luc T cells.

[0154] Figure 17. Evaluation of T cell activation mediated by FcγRIIIA / anti-CD3 fusion in combination with trastuzumab (Tras) or rituximab (Rtx) with or without half-life extension strategies when co-cultured with HER2+SKBR3 breast cancer cells and Jurkat-NFAT-Luc T cells.

[0155] Figure 18. Evaluation of T cell activation mediated by FcγRIIIA / anti-CD3 or anti-Fc / anti-CD3 fusions in combination with trastuzumab (Tras) or rituximab (Rtx) when co-cultured with HER2+SKBR3 breast cancer cells and Jurkat-NFAT-Luc T cells.

[0156] Figure 19 shows SDS-PAGE gel images of IgR under reduced (R) and non-reduced (NR) conditions in Example 18 and markers (M) in kDa.

[0157] Figure 20 shows the chromatogram of IgR in Example 18 by high performance liquid chromatography-size exclusion chromatography (HPLC-SEC).

[0158] Figure 21. Evaluation of T cell activation mediated by combination of anti-CD3 IgR with rituximab (Rtx) or trastuzumab (Trz) when co-cultured with CD20+ Raji B cells and Jurkat-NFAT-Luc T cells in Example 18.

[0159] Figure 22 shows SDS-PAGE gel images of IgR under reduced (R) and non-reduced (NR) conditions in Example 19 and markers (M) in kDa.

[0160] Figure 23 shows the chromatogram of IgR in Example 19 by high performance liquid chromatography-size exclusion chromatography (HPLC-SEC).

[0161] Figure 24. Evaluation of T cell activation mediated by combination of anti-CD3 IgR with rituximab (Rtx) or trastuzumab (Trz) when co-cultured with CD20+ Raji B cells and Jurkat-NFAT-Luc T cells in Example 19.

[0162] Figure 25. Evaluation of T cell activation mediated by anti-CD3 IgR with rituximab (Rtx) or trastuzumab (Trz) or a combination of rituximab and trastuzumab when co-cultured with CD20+ Raji B cells and Jurkat-NFAT-Luc T cells in Example 20.

[0163] Figure 26 shows SDS-PAGE gel images of IgR under reduced (R) and non-reduced (NR) conditions in Example 21 and markers (M) in kDa.

[0164] Figure 27 shows the chromatogram of IgR obtained by high performance liquid chromatography-size exclusion chromatography (HPLC-SEC) in Example 21.

[0165] Figure 28. Evaluation of T cell activation mediated by anti-CD3 IgR with rituximab (Rtx) or trastuzumab (Trz) or a combination of rituximab and trastuzumab when co-cultured with CD20+ Raji B cells and Jurkat-NFAT-Luc T cells in Example 21.

[0166] Figures 29.A and B show SDS-PAGE gel images of IgR under reduced (R) and non-reduced (NR) conditions and markers (M) in kDa in Example 22.

[0167] Figure 30 shows the chromatogram of IgR obtained by high performance liquid chromatography-size exclusion chromatography (HPLC-SEC) in Example 22.

[0168] Figure 31. Evaluation of T cell activation mediated by combination of anti-CD3 IgR with rituximab (Rtx) or trastuzumab (Trz) when co-cultured with CD20+ Raji B cells and Jurkat-NFAT-Luc T cells in Example 22.

[0169] Figure 32. Evaluation of T cell activation mediated by combination of anti-CD3 IgR with rituximab (Rtx) or trastuzumab (Trz) when co-cultured with CD20+ Raji B cells and Jurkat-NFAT-Luc T cells in Example 22.

[0170] Figure 33. Evaluation of T cell activation mediated by anti-CD3 IgR with rituximab (Rtx) or trastuzumab (Trz) or a combination of rituximab and trastuzumab when co-cultured with CD20+ Raji B cells and Jurkat-NFAT-Luc T cells in Example 22.

[0171] Figure 34. Evaluation of T cell activation mediated by anti-CD3 IgR alone or in combination with anti-CD3 OKT3 mIgG2a when co-cultured with CD20+ Raji B cells and Jurkat-NFAT-Luc T cells in Example 22; or rituximab alone (Rtx); or anti-CD3 OKT3 mIgG2a alone.

[0172] Figure 35. Evaluation of T cell co-stimulation mediated by combination of anti-CD28 IgR with rituximab (Rtx) or trastuzumab (Trz) when co-cultured with CD20+ Raji B cells and Jurkat-NFAT-Luc T cells in wells pre-coated with 1 μg / mL OKT3 in Example 22.

[0173] Figure 36. A and B show SDS-PAGE gel images of IgR under reduced (R) and non-reduced (NR) conditions and markers (M) in kDa in Example 23.

[0174] Figures 37.A and B show chromatograms of IgR high performance liquid chromatography-size exclusion chromatography (HPLC-SEC) in Example 23.

[0175] Figure 38. Evaluation of T cell activation mediated by anti-CD3 IgR with rituximab (Rtx) or trastuzumab (Trz) or a combination of rituximab and trastuzumab when co-cultured with CD20+ Raji B cells and Jurkat-NFAT-Luc T cells in Example 23.

[0176] Figure 39. Evaluation of T cell activation mediated by anti-CD3 IgR with rituximab (Rtx) or trastuzumab (Trz) or a combination of rituximab and trastuzumab when co-cultured with CD20+ Raji B cells and Jurkat-NFAT-Luc T cells in Example 23.

[0177] Figure 40. Evaluation of T cell activation mediated by anti-CD3 IgR with rituximab (Rtx) or trastuzumab (Trz) or a combination of rituximab and trastuzumab when co-cultured with CD20+ Raji B cells and Jurkat-NFAT-Luc T cells in Example 23.

[0178] Figure 41. A and B show SDS-PAGE gel images of IgR under reduced (R) and non-reduced (NR) conditions in Example 24 and markers (M) in kDa.

[0179] Figure 42. A and B show chromatograms of IgR obtained by high performance liquid chromatography-size exclusion chromatography (HPLC-SEC) in Example 24.

[0180] Figure 43. Evaluation of T cell activation mediated by anti-CD3 IgR with rituximab (Rtx) or trastuzumab (Trz) or a combination of rituximab and trastuzumab when co-cultured with CD20+ Raji B cells and Jurkat-NFAT-Luc T cells in Example 24.

[0181] Figure 44. Evaluation of T cell co-stimulation mediated by anti-CD28 IgR in combination with rituximab (Rtx) or trastuzumab (Trz) when co-cultured with CD20+ Raji B cells and Jurkat-NFAT-Luc T cells in wells pre-coated with 1 μg / mL OKT3 in Example 24.

[0182] Figure 45. Evaluation of T cell co-stimulation mediated by anti-CD137 IgR with rituximab (Rtx) or trastuzumab (Trz) or a combination of rituximab and trastuzumab when co-cultured with CD20+ Raji B cells and HEK-Luc-CD137 cells in Example 24.

[0183] Figure 46. Evaluation of T cell co-stimulation mediated by anti-CD89 IgR with OKT3 or trastuzumab (Trz) or a combination of OKT3 and trastuzumab when co-cultured with Jurkat-NFAT-Luc T cells and CHO-K1-CD89 cells in Example 24.

[0184] Figure 47 shows SDS-PAGE gel images of IgR under reduced (R) and non-reduced (NR) conditions in Example 25 and markers (M) in kDa.

[0185] Figure 48. A and B show chromatograms of IgR high performance liquid chromatography-size exclusion chromatography (HPLC-SEC) in Example 25. Detailed Implementation

[0186] The field of this invention relates generally to immunology, and more specifically to heterologous polypeptides or multimeric proteins and nucleic acids encoding them, comprising at least one immunoglobulin-binding domain and at least one polypeptide or protein-binding domain on the surface of immune cells, for the regulation of diseases such as cancer, autoimmunity, organ rejection, and pathogenic infections (such as viruses, bacteria, parasites, or fungi).

[0187] This invention relates to, but is not limited to, heterologous polypeptides or multimeric proteins comprising an immunoglobulin-binding domain having affinity and specificity for a portion of an immunoglobulin molecule (Ig); and at least one domain having affinity and specificity for at least one extracellular polypeptide present on immune cells.

[0188] This invention relates to heterologous polypeptides or multimeric proteins and nucleic acids encoding them, comprising at least one immunoglobulin-binding domain and at least one immune cell surface protein-binding domain, thereby creating immunoglobulin redirection (IgR) molecules capable of binding to immune cells and modulating cellular activities such as effector function or inhibition. The IgRs of this invention can be used as a monotherapy or in combination with one or more standard, current, or experimental therapeutic agents to treat conditions such as cancer, immune disorders, or pathogenic infections.

[0189] Antibody-based or antibody fragment-based immunotherapies, such as plasma-derived intravenous immunoglobulin (IVIG), polyclonal antibodies, monoclonal antibodies, antibody fusion proteins, single-chain variable fragments (ScFv), domain antibodies (e.g., VHH or nanobodies), and antibody-drug conjugates, can treat a variety of diseases, particularly cancer, inflammatory diseases, and infectious diseases. These therapies may rely on eliminating diseased cells by binding to Fc receptors on effector cells and recognizing cell surface molecules that are differentially present relative to normal cells (e.g., proteins overexpressed on cancer cells or viruses or viral components budding from infected cells). The binding of antibody-based immunotherapies to diseased cells can induce cell death via various mechanisms, such as antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP), antibody-dependent neutrophil extracellular trapping (NETosis), complement-dependent cytotoxicity (CDC), or direct cytotoxic activity from the payload of antibody-drug conjugates (ADCs). Combining IgR with antibody-based therapies can induce novel cell types and cell functions. In addition, IgR can be delivered as a monotherapy and relies on the patient's own antibodies to regulate the disease.

[0190] Immunotherapy includes, but is not limited to, cell-based therapies, antibody therapies, cytokines, chemokines, growth factors, and receptor or ligand fusions used to elicit an immune response against diseased cells, tissues, or organs while preserving healthy cells, tissues, or organs. Disease areas where immunotherapy is used include, but are not limited to, cancer, autoimmune diseases, inflammation, allogeneic diseases, and infectious diseases.

[0191] definition

[0192] Unless otherwise defined, all industry terms, symbols, and other technical and scientific terms or words used herein are intended to have the same meaning as commonly understood by one of ordinary skill in the art to which the claimed subject matter pertains. In some instances, terms with commonly understood meanings are defined herein for clarity and / or for ease of reference, and the inclusion of such definitions herein should not necessarily be construed as indicating a material difference from the meanings commonly understood in the art.

[0193] As used herein, unless the context clearly specifies otherwise, the singular forms “a,” “an,” and “the / said” include plural referents. For example, “a” or “an” means “at least one / at least one” or “one or more / one or more types.” It should be understood that the aspects and variations described herein include aspects and variations that are “composed of” and / or “substantially composed of”.

[0194] Throughout this disclosure, all aspects of the claimed subject matter are presented in scope. It should be understood that this scope-based description is merely for convenience and brevity and should not be construed as an immutable limitation on the scope of the claimed subject matter. Therefore, the scope description should be considered as having specifically disclosed all possible subscopes and individual numerical values ​​within that scope. For example, in the case of providing a range of values, it should be understood that every intermediate value between the upper and lower limits of that range, as well as any other stated value or intermediate value within the stated range, is covered within the claimed subject matter. The upper and lower limits of these smaller ranges may be independently included within the smaller range and also covered within the claimed subject matter, subject to any express exclusions within the stated range. Where the stated range includes one or two limits, the range excluding any one or both of these included limits is also included within the claimed subject matter. This applies regardless of the width of the scope.

[0195] As used herein, the term "about" refers to a typical range of error for a corresponding value that is readily known to those skilled in the art. References to a "about" value or parameter include (and describe) embodiments for that value or parameter itself. For example, a description referring to "about X" includes a description of "X". The term "about" may also cover variations that can be as high as ±5%, but can also be ±4%, 3%, 2%, 1%, etc. Whether or not modified by the term "about", the claims include equivalents of the quantity.

[0196] As used herein, a “domain” (typically three or more, usually five or seven or more amino acids, such as a sequence of 10 to 200 amino acid residues) refers to a portion of a molecule (such as a protein or encoding a nucleic acid) that is structurally and / or functionally distinct from other parts of the molecule and is identifiable. For example, a domain includes those portions of a polypeptide chain that can form independently folded structures within a protein composed of one or more structural motifs and / or are identifiable by functional activity (such as binding activity). Proteins may have one or more distinct domains. For example, domains can be identified, defined, or distinguished by the homology of their primary sequence or structure with members of a related family (such as homology with motifs). In another example, domains can be distinguished by their function (such as their ability to interact with biomolecules). A domain can independently exhibit a biological function or activity such that the domain, either independently or fused to another molecule, can exercise activity, such as, for example, binding. A domain can be a linear sequence of amino acids or a non-linear sequence of amino acids. Many polypeptides contain multiple domains. Definitions are provided for illustrative purposes, but in some respects, specific structural domains can be identified by their names. If necessary, appropriate software can be used to identify structural domains.

[0197] As used herein, "immunoglobulin heavy chain" is a polypeptide comprising at least a portion of the antigen-binding domain of an immunoglobulin and at least a portion of the variable region or the constant region of an immunoglobulin heavy chain. Therefore, immunoglobulin-derived heavy chains contain important regions of the amino acid sequence of members of the immunoglobulin gene superfamily. For example, the heavy chain in the Fab fragment is an immunoglobulin-derived heavy chain.

[0198] As used herein, an "immunoglobulin light chain" is a polypeptide comprising at least a portion of the antigen-binding domain of an immunoglobulin and at least a portion of either the variable region or the constant region of an immunoglobulin light chain. Therefore, immunoglobulin-derived light chains contain important regions of the amino acid sequence of members of the immunoglobulin gene superfamily.

[0199] As used in this article, "Fc region" or "Fc domain" refers to a crystallizable fragment, which is the region where the antibody interacts with the cell surface receptor (Fc receptor).

[0200] As used herein, an "immunoglobulin molecule" is a protein that comprises immunologically active portions of an immunoglobulin heavy chain and an immunoglobulin light chain covalently coupled together, and is capable of specifically binding to an antigen. For example, an immunoglobulin molecule can be IgG, IgE, IgD, IgA, IgM, and IgY. For example, subclasses of immunoglobulin molecules can be IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2.

[0201] In some respects, the specific dose level for any particular subject or patient may depend on a variety of factors, including the activity of the specific compound used, age, weight, general health condition, route of administration, severity of illness, and rate of excretion. The therapeutically effective amount for a given situation can be readily determined through routine laboratory testing and is within the skill and judgment of an average clinician. The terms “therapeutically effective amount” and “therapeuticly effective dose” are used interchangeably herein and refer to the amount of a compound that results in the prevention or improvement of a patient’s systemic or desired biological outcome. The terms “subject” and “patient” are used interchangeably herein and refer to mammals, such as humans, non-human primates (e.g., baboons, orangutans, monkeys, gorillas), or non-primate mammals (e.g., mice, rats, dogs, pigs).

[0202] The protein-binding domain on the surface of immune cells can be any suitable molecule that can bind to a receptor, such as small organic molecules, antigen-binding parts of antibodies (e.g., Fab fragments, Fab' fragments, F(ab')2 fragments, scFv fragments, Fv fragments, dsFv biantibodies, dAb fragments, Fd' fragments, Fd fragments, or isolated complementarity-determining regions (CDRs), antibody mimics (e.g., aptamers, affibody, affilin, affimer, anticalin, avimer, DARPin, etc.), nucleic acids, lipids, etc.

[0203] IgRs can contain any portion that inhibits the ability of immunoglobulins to bind to and / or activate their receptors. The blocking portion can inhibit the ability of immunoglobulins to bind to and / or activate their receptors by spatially blocking and / or by non-covalently binding to the immunoglobulin. Examples of suitable blocking portions include full-length or immunoglobulin-binding fragments of the immunoglobulin's homologous receptor or mutant proteins. Antibodies and fragments thereof that bind to immunoglobulins can also be used, including polyclonal antibodies, recombinant antibodies, human antibodies, humanized antibodies, single-chain variable fragments (scFv), single-domain antibodies such as heavy chain variable domains (VH), light chain variable domains (VL), and variable domains (VHH) of camel-type nanobodies, dAbs, etc. Other suitable antigen-binding domains that bind immunoglobulins can also be used, including non-immunoglobulin proteins that mimic antibody binding and / or structures, such as anticalin, affilin, affibody molecules, affimer, affitin, alphabody, avimer, DARPin, fynomer, kunitz domain peptides, monomeric antibodies, and binding domains based on other engineered scaffolds such as SpA, GroEL, fibronectin, lipid transport proteins, and CTLA4 scaffolds. Further examples of suitable blocking peptides include peptides that spatially inhibit or block the binding of immunoglobulins to their homologous receptors.

[0204] Advantageously, such moieties can also be used as half-life extension elements. For example, peptides modified by conjugation with water-soluble polymers such as PEG can spatially inhibit or prevent the binding of immunoglobulins to their receptors. Peptides or fragments thereof with long serum half-lives, such as serum albumin (human serum albumin), immunoglobulin Fc, transferrin, etc., as well as fragments and mutant proteins of such peptides, can also be used.

[0205] Antibodies and antigen-binding domains that bind to proteins with long serum half-lives (such as HSA, immunoglobulins, or transferrin) or to receptors that recycle to the plasma membrane (such as FcRn or transferrin receptors) can also inhibit immunoglobulins, especially when bound to their antigens. Examples of such antigen-binding peptides include single-chain variable fragments (scFv), single-domain antibodies such as heavy chain variable domains (VH), light chain variable domains (VL), and variable domains (VHH) of camel-type nanobodies, dAbs, etc. Other suitable antigen-binding domains that bind immunoglobulins can also be used, including non-immunoglobulin proteins that mimic antibody binding and / or structures, such as anticalin, affilin, affibody molecules, affimer, affitin, alphabody, avimer, DARPin, fynomer, Kunitz domain peptides, monomeric antibodies, and binding domains based on other engineered scaffolds such as SpA, GroEL, fibronectin, lipid transport proteins, and CTLA4 scaffolds.

[0206] Engineered scaffolds can include sdAb, scFv, Fab, VHH, fibronectin type III domain, immunoglobulin-like scaffolds, DARPin, cystine binding peptides, lipid transport proteins, triple helix bundle scaffolds, protein G-associated albumin binding modules, or DNA or RNA aptamer scaffolds.

[0207] Serum half-life extension elements can also be antigen-binding peptides that bind to proteins with long serum half-lives, such as serum albumin, transferrin, etc. Examples of such peptides include antibodies and fragments thereof, including polyclonal antibodies, recombinant antibodies, human antibodies, humanized antibodies, single-chain variable fragments (scFv), single-domain antibodies such as heavy chain variable domain (VH), light chain variable domain (VL), and variable domains (VHH) of camel-type nanobodies, dAb, etc. Other suitable antigen-binding domains include non-immunoglobulin proteins that mimic antibody binding and / or structures, such as anticalin, affilin, affinity protein molecules, affimer, affitin, alphabody, avimer, DARPin, fynomer, Kunitz domain peptides, monomeric antibodies, and binding domains based on other engineered scaffolds such as SpA, GroEL, fibronectin, lipid transport proteins, and CTLA4 scaffolds. Other examples of antigen-binding peptides include ligands for desired receptors, ligand-binding moieties of receptors, lectins, and peptides that bind to or associate with one or more target antigens.

[0208] The binding moiety can be any type of peptide. For example, in some cases, the binding moiety is a native peptide, a synthetic peptide, or a fibronectin scaffold, or an engineered bulk serum protein. Bulk serum proteins include, for example, albumin, fibrinogen, or globulin. In some embodiments, the binding moiety is an engineered scaffold. Engineered scaffolds include, for example, sdAb, scFv, Fab, VHH, fibronectin type III domains, immunoglobulin-like scaffolds (Halaby et al., 1999), DARPin, cystine knot peptides, lipid carrier proteins, triple-helix bundle scaffolds, protein G-associated albumin binding modules, or DNA or RNA aptamer scaffolds.

[0209] In some implementations, the heterologous polypeptide or multimeric protein and the nucleic acid encoding it comprise:

[0210] (a) At least one immunoglobulin-binding domain,

[0211] (b) and at least one immune cell surface protein binding domain.

[0212] In some implementations, the heterologous polypeptide or multimeric protein and the nucleic acid encoding it comprise:

[0213] (a) At least one immunoglobulin-binding domain, wherein the immunoglobulin-binding domain is an antigen-binding domain, antibody, or antigen-binding fragment with affinity and specificity for one or more immunoglobulin isotypes and their derivatives (including but not limited to IgG, IgA, IgM, IgE, IgD), including but not limited to ScFv, Fab, single-domain antibodies, VHH, nanobodies, avimer, Fab, (Fab)2, Kunitz domain, small modular immunopharmaceuticals (SMIPs), adnectin, affibody, DARPin, anticalin, or synthetic peptides (such as Fc-III), and their derivatives or analogs, wherein,

[0214] (b) and at least one immune cell surface protein binding domain.

[0215] In various implementations, the immunoglobulin-binding domain is a fully human or humanized antigen-binding domain, antibody, or antigen-binding fragment, including its derivatives and analogs.

[0216] In some embodiments, at least one immunoglobulin-binding domain is an antigen-binding domain, an antibody, or an antigen-binding fragment, including its derivatives and analogs, which has affinity and specificity for one or more immunoglobulin isotypes (including IgG, IgA, IgM, IgE, and IgD) of Fc, Fc-glycan, hinge, constant heavy chain (including CH1, CH2, CH3, CH4), constant light chain κ, constant light chain λ, variable heavy chain framework region, variable light chain framework region, Fab, or (Fab)2.

[0217] Immunoglobulin-binding antibodies have been previously reported (Hamilton & Morrison, 1993), (Hermans et al., 2015), (Hermans et al., 2017), (Bonvin et al., 2015), (Belin et al., 2004; Carrier et al., 1995), (Leonard et al., 2022), and (Ji Yi HK, 2021).

[0218] In some embodiments, the immunoglobulin-binding domain is anti-Fc VHH (SEQ ID NO: 48), anti-IgG containing VH / VL against 17F12 (SEQ ID NO: 134, SEQ ID NO: 135), anti-CH1 VHH (SEQ ID NO: 130), or a CDR thereof. In other embodiments, the immunoglobulin-binding domain is derived from anti-IgG clones HG2-25, 8E11, 8F1, NH3 / 130.5.2, NH3 / 15.8, HP6045, MS-278, anti-IgG Fc MK1A6, JDC-10, H2, 6F11C8, AbD27686, R10Z8E9, M1310G05, 97924, PABZ-080, RF-AN, A4, NA6, HP6017 (SEQ ID NO: 288, SEC ID: 48). 290), HP6070, GG-7, Fc-III peptide (Univ, 2010), clone EM-07; anti-IgA clones in (Chang et al., 2017), H15A43, B35064B, A9604D2; anti-IgM clones in (Frey et al., 2018), SA-DA4, M15 / 8, B481; anti-IgE clone HuMaE11 / Omalizumab, TES-C21; anti-IgD clone IGD26, IADB6; anti-λ clone N10 / 2, JDC-12; anti-κ clone NH3 / 41.34, SB81A; anti-IgG Fab clone 4A11; anti-IgG CH2 clone 8A4.

[0219] In some embodiments, at least one immunoglobulin-binding domain is an antigen-binding domain, an antibody, or an antigen-binding fragment, including its derivatives and analogs, which has affinity and specificity for the Fc region of one or more immunoglobulin isotypes (including IgG, IgA, IgM, IgE, and IgD) and blocks the binding of the Fc receptor to the Fc region of one or more immunoglobulin isotypes (including IgG, IgA, IgM, IgE, and IgD).

[0220] In some embodiments, at least one immunoglobulin-binding domain is an antigen-binding domain, an antibody, or an antigen-binding fragment, including its derivatives and analogs, which has affinity and specificity for the Fc region of one or more immunoglobulin isotypes (including IgG, IgA, IgM, IgE, and IgD) and does not block the binding of the Fc receptor to the Fc region of one or more immunoglobulin isotypes (including IgG, IgA, IgM, IgE, and IgD).

[0221] Fc receptors are classified based on the isotypes of antibodies they can bind. For example, Fcγ receptors (FcγR) typically bind to IgG antibodies, such as one or more of their subtypes (i.e., IgG1, IgG2, IgG3, IgG4); Fcα receptors (FcαR) typically bind to IgA antibodies; Fcα / μ (Fcα / μR) receptors typically bind to IgA and IgM; Fcε receptors (FcεR) typically bind to IgE antibodies; and IgDR receptors typically bind to IgD. In addition, various other Fc receptors of various isotypes have been reported, such as the Fc neonatal receptor (FcRn) that binds IgG (Raghavan et al., 1994); FcRL4 and FcRL5 that bind IgG and IgA (Wilson et al., 2012); DC-SIGN that binds IgG (Anthony et al., 2008); TRIM21 that binds IgG (Keeble et al., 2008); MMR that binds IgG glycans (Dong et al., 1999); Dectin-1 and Dectin-2 that bind glycans on IgG (Boesch et al., 2014; Karsten et al., 2012); mannose-binding lectin 2 (MBL2) that binds glycans on Ig (Arnold et al., 2006); C1q that binds IgG, IgM, and IgA; and pIgR that binds IgA and IgM. In some implementations, the Fc binding domain is an Fc receptor, including Fcγ receptor, Fcα receptor, Fcε receptor, and Fcα / μ receptor. Examples of Fcγ receptors include, but are not limited to, CD64A (FcγRI), CD64B (FcγRI), CD64C (FcγRI), CD32A (FcγRIIA, including H131 and R131 allotypes), CD32B (FcγRIIB), CD32C (FcγRIIC), CD16A (FcγRIIIA, including V176 and F158 allotypes, also referring to V158 and F158 allotypes, excluding leader sequences or L66H mutations (de Vries et al., 1996), and CD16B (FcγRIIIB, including SH, NA1, and NA2 allotypes). An example of an Fcα receptor is FcaR1 (CD89). Examples of Fcε receptors include, but are not limited to, FcεRI and FcεRII / CD23.

[0222] The binding of Fc receptors on innate and adaptive immune cells and tissue-specific cells mediates a variety of cellular functions via binding to the Fc regions of immunoglobulins (Ig) such as IgG, IgA, IgE, IgM, and IgD. Ig-Fc can bind to Fc receptors to varying degrees depending on allotype, subclass, allotype, glycosylation, and via point mutation. IgG can bind to activated FcγRs, such as CD16 (FcγRIIIa or FcγRIIIb), CD32A or CD32C (FcγRIIa or FcγRIIc), CD64 (FcγRI), and C1q, and mediate cellular functions such as antigen presentation, internalization, superoxide production, ADCC, phagocytosis, cytokine secretion, NETosis, adhesion induction, respiratory burst, degranulation, complement fixation, and apoptosis. IgG can bind inhibitory FcγRs such as CD32B (FcγRIIb) and can mediate internalization and downregulation in B cells, mast cells, macrophages, and NK cells (Boesch et al., 2015). IgG binds to FcRn in a pH-dependent manner and can mediate internalization, IgG transport, and phagocytosis. Additional IgG Fc receptors, such as complement C1q, FcRL5, pIgR, and DC-SIGN, have been shown to bind Fc domains to varying degrees (Boesch et al., 2014). IgA can bind pIgR, FcαRI, Fcα / μR, and C1q and can mediate functions such as transcellularity, phagocytosis, ADCC, oxidative burst, cytokine production, TGFβ downregulation, antigen presentation, complement fixation, and immune complex capture (PMID 25700208). IgM can bind to pIgR, Fcα / μR, and C1q, and can mediate functions such as transcellularity, antigen presentation, immune complex capture, and complement fixation. IgE can bind to FcεRI and FcεRII, and can mediate functions such as immune cell regulation, internalization, antigen capture, antigen presentation, histamine, cytokine production, and cytotoxicity (Delespesse et al., 1989; Shin & Greer, 2015). IgD can bind to IgDR and can mediate basophil stimulation, immune activation, pro-inflammatory effects, and the release of antimicrobial mediators (Chen & Cerutti, 2011).Selecting FcγR types and variants through allotypes, amino acid mutants, domain exchange fusions, modified glycosylation sites, and glycoforms can alter affinity and IgG subclass specificity, as previously illustrated (“The Second and Third Extracellular Domains of FcγRI (CD64) Confer the Unique High Affinity Binding of IgG2a,” 1998) (Hulett & Hogarth, 1998; “The Second and Third Extracellular Domains of FcγRI (CD64) Confer the Unique High Affinity Binding of IgG2a,” 1998) (Oganesyan et al., 2015) (website, not attributed) (Shibata-Koyama et al., 2009). Possible FcγR variants with enhanced affinity or modified IgG subclass specificity are shown in Example 10.

[0223] The immunoglobulin-binding domains of heterologous peptides and multimeric proteins described herein include Fc-binding domains or peptides capable of binding the Fc portion of immunoglobulin (Ig) molecules (e.g., IgG, IgA, IgM, IgE, or IgD). Suitable Fc-binding domains can be derived from natural proteins, such as mammalian Fc receptors, certain bacterial proteins such as protein A or protein G, and viral proteins such as TspB (Müller et al., 2013), gE (Para et al., 1980), gI (Dubin et al., 1990), gpI (Litwin et al., 1992), FcγR-like HCV core proteins (Namboodiri et al., 2007), gp34, gp68, gpRL13, gp95, and fcr-1 (Corrales-Aguilar et al., 2014). Furthermore, the Fc-binding domain can be a synthetic peptide specifically engineered to bind the Fc portion of any Ig molecule described herein with high affinity and specificity.

[0224] In some implementations, the Fc binding domain is the extracellular ligand-binding domain of the mammalian Fc receptor. As used herein, an "Fc receptor" is a cell surface-binding receptor expressed on the surface of many tissues and immune cells, including endothelial cells, epithelial cells, Langerhans cells, B cells, dendritic cells, natural killer (NK) cells, macrophages, monocytes, myeloid progenitor cells, platelets, neutrophils, mast cells, polymorphonuclear leukocytes, syncytiotrophoblasts, basophils, and eosinophils, and exhibits binding specificity to the Fc domain of an antibody. An Fc receptor typically consists of an immunoglobulin (Ig)-like domain that has binding specificity to the Fc (crystallizable) portion of an antibody. In some cases, the binding of the Fc receptor to the Fc portion of an antibody can mediate effector functions such as antibody-dependent cell-mediated cytotoxicity (ADCC).

[0225] The Fc receptor used to construct the heteropeptide or multimeric protein as described herein can be a naturally occurring polymorphic variant (e.g., CD16A V158 and F158 variants or CD16B NA1, NA2, SH variants) that may have increased or decreased affinity for Fc compared to the wild-type counterpart. Alternatively, the Fc receptor can be a functional variant of the wild-type counterpart carrying one or more mutations (e.g., substitutions of up to 10 amino acid residues) that alter the binding affinity to the Fc moiety of an Ig molecule. In some cases, mutations can alter the glycosylation pattern of the Fc receptor and thus change the binding affinity to Fc. In some embodiments, one or more point mutations in the Fc receptor eliminate enzyme cleavage sites, such as the ADAM17 cleavage site on FcγR3A and FcγR3B, as in FcγR3AV-S197P, SEQ ID NO: 23, FcγR3AF-S197PSEQ ID NO: 271, FcγR3B-NA1-S197P SEQ ID NO: 272, FcγR3B-NA2-S197P SEQ ID NO: 273, and FcγR3B-SH-S197P SEQ ID NO: 274. Optionally, some or all of the enzyme cleavage sites (such as the ADAM17 cleavage site) may be removed, but the Fc receptor sequence may be shortened, as in FcγR3AV-short SEQ ID NO:3, FcγR3AF-short SEQ ID NO:4, FcγR3B-NA1-short, SEQ ID NO:275, FcγR3B-NA2-short, SEQ ID NO:276, FcγR3B-SH-short, and SEQ ID NO:277.

[0226] In some implementations, the heterologous polypeptide or multimeric protein and the nucleic acid encoding it comprise:

[0227] (a) At least one immunoglobulin-binding domain derived from an Fc receptor, said Fc receptor including, but not limited to, FcγRIIIa, FcγRIV, FcγRIIIb with LVGSKNV in domain 2 replaced by MGKHRY, FcγRIIa, FcγRIIc, FcγRIIb, FcγRI, FcγRI, FcγRI with domain 3 removed, FcγRI, FcRL5, pIgR, FcαRI, Fcα / μR, FcμR, FcεRI, FcεRII, FcRn, or TRIM21, including its allotypes, derivatives, and analogs.

[0228] (b) and at least one immune cell surface protein binding domain.

[0229] In some implementations, the heterologous polypeptide or multimeric protein and the nucleic acid encoding it comprise:

[0230] (a) At least one immunoglobulin-binding domain derived from the Fc receptor, wherein one or more point mutations in the Fc receptor can eliminate enzyme cleavage sites, such as the ADAM17 cleavage site.

[0231] (b) Optionally, in the Fc receptor, some or all enzyme cleavage sites, such as the ADAM17 cleavage site, have been removed.

[0232] (c) and at least one immune cell surface protein binding domain.

[0233] In some implementations, the heterologous polypeptide or multimeric protein and the nucleic acid encoding it comprise:

[0234] (a) At least one immunoglobulin-binding domain derived from an Fc receptor comprising FcγR3AV-S197P, SEQ ID NO: 23, FcγR3AF-S197P SEQ ID NO: 271, FcγR3B-NA1-S197P SEQ ID NO: 272, FcγR3B-NA2-S197P SEQ ID NO: 273, FcγR3B-SH-S197P SEQ ID NO: 274, including any allotypes, derivatives and analogs thereof.

[0235] (b) Optionally, derived from an Fc receptor comprising FcγR3AV-short SEQ ID NO: 3, FcγR3AF-short SEQ ID NO: 4, FcγR3B-NA1-short, SEQ ID NO: 275, FcγR3B-NA2-short, SEQ ID NO: 276, FcγR3B-SH-short, SEQ ID NO: 277, including any allotypes, derivatives and analogs thereof.

[0236] (c) and at least one immune cell surface protein binding domain.

[0237] In some implementations, the heterologous polypeptide or multimeric protein and the nucleic acid encoding it comprise:

[0238] (a) At least one immunoglobulin-binding domain derived from an Fc receptor, including but not limited to FcγRIIIa V158 SEQ ID NO: 1, FcγRIIIa-short V158 SEQ ID NO: 3, FcγRIIIa F158 SEQ ID NO: 2, FcγRIIIa-short F158 SEQ ID NO: 4, FcγRIV SEQ ID NO: 8, FcγRIIIbNA1 SEQ ID NO: 6, FcγRIIIb NA2 SEQ ID NO: 7, FcγRIIIb SH SEQ ID NO: 5, FcγRIIa H131 SEQ ID NO: 9, FcγRIIa R131 SEQ ID NO: 10, FcγRIIb / c SEQ ID NO: 11, FcγRI SEQ ID NO: 12, FcRL5 SEQ ID NO: 13, pIgR SEQ ID NO: 14, FcαRI SEQ ID NO: 15, Fcα / μR SEQ ID NO: 16, FcμR SEQ ID NO: 17, FcεRI SEQ ID NO: 18, FcεRII SEQ ID NO: 19, FcRn SEQ ID NO: 20 or c1q, including any allotypes, derivatives and analogs thereof.

[0239] (b) and at least one immune cell surface protein binding domain.

[0240] In some embodiments, at least one immunoglobulin-binding domain is derived from a bacterial or viral Fc receptor, including but not limited to protein A, protein G or protein A / G, TspB, gE, gI, gpI, FcγR-like HCV core protein, gp34, gp68, gpRL13, gp95, fcr-1, including their derivatives and analogs.

[0241] Antibody-based immune checkpoint pathways and co-stimulatory modulators have begun to offer novel immunotherapies for cancer treatment (e.g., checkpoint inhibition by binding PD1, PDL1, CTLA4, OX40, TIM3, LAG3, TIGIT, and CD47; co-stimulation by binding CD137, CD28, CD2, and CD7; and treatment of inflammatory diseases by inhibiting immune cell activation via binding SIRP1α, CD11a, CD18, CD80, CD86, and PD1).

[0242] Multispecific agents bind to tumor-associated antigens or tumor-associated antigen peptide-MHC complexes and can be redirected to surface proteins on lymphocytes such as T cells, NK cells, or myeloid cells such as macrophages, monocytes, or neutrophils. They have gained clinical attention by targeting effector-mediated receptors, including but not limited to T-cell receptor (TCR) complexes, including CD3, CD3ε, CD3δ, CD3γ, TCR, TCRα, TCRβ, TCRγ, TCRδ, or combinations thereof, CD8, CD4, CD2, Fas ligand, CD40, CD40L, CD137, CD28, CD56, NKG2D, NKp46, PD1, PDL1, CTLA4, CLEC5A, CD79, BCR, OX40, TIM3, TIGIT, CD7, LAG3, CD11a, CD18, CD80, and CD86. dectin-1, dectin-2, dectin-3, FcγRIIIa, FcγRIV, FcγRIIIb, FcγRIIa, FcγRIIc, FcγRIIb, FcγRI, C1q, FcRL5, pIgR, FcαRI, Fcα / μR, FcμR, FcεRI, or FcεRII.

[0243] In some embodiments or in combination with previous embodiments, the heterologous polypeptide or multimeric protein and the nucleic acid encoding it comprise:

[0244] (a) At least one immunoglobulin-binding domain,

[0245] (b) and at least one immune cell surface protein binding domain, which is derived from a natural soluble ligand derivative or analogue thereof or the extracellular portion of a natural receptor or ligand found on immune cells, including but not limited to lymphocytes, myeloid cells, T cells, B cells, NK cells, macrophages, monocytes, NK-T cells, neutrophils, dendritic cells, basophils, eosinophils and mast cells.

[0246] In some embodiments or in combination with previous embodiments, the heterologous polypeptide or multimeric protein comprises:

[0247] (a) At least one immunoglobulin-binding domain,

[0248] (b) At least one immune cell surface protein-binding domain derived from a natural soluble ligand, including but not limited to cytokines, chemokines, pentameric proteins, galactoglobin-9, HMGB1, TGF-β, growth factors, pattern recognition proteins, lectins, and enzymes, including their derivatives or analogs or the extracellular portion of natural receptors or ligands, including but not limited to PD1, PDL1, PDL2, CTLA4, OX40, OX40L, TIM3, TIM1, LAG3, TIGIT, CD137, CD137L, CD28, CD2, CD7, CD11a, CD11b, CD18, CD80, CD86, MHC-I, MHC-II, MHC-G, HLA-DR, CD209, CD206, galactoglobin-3, LSECtin, FGL1, CD112, CD155, HVEM, CEACAM-1, Fas ligand, TCR complex, TCRα, TCRβ, TCRγ, TCRδ, CD3, CD3ε, CD3γ, CD3δ, CD226, CD27, CD47, SIRP1α, CCR8, TNFR2, CD103, CD39, TIGIT, CD96, VISTA, BTLA, B7-H3, CD8, CD4, dectin-1, dectin-2, dectin-3, chemokine receptors, cytokine receptors, growth factor receptors, pattern recognition receptors, NKG2D, NKp46, MICA, ULBP-1, ULBP-2, BCR, CD79, CD40, CD40L, and enzymes, including their derivatives or analogues.

[0249] In some embodiments or in combination with previous embodiments, the heterologous polypeptide or multimeric protein comprises:

[0250] (a) At least one immunoglobulin-binding domain,

[0251] (b) At least one immune cell surface protein binding domain, which is an antigen-binding domain, antibody, or antigen-binding fragment with affinity and specificity for the extracellular portion of a natural receptor or ligand, including its derivatives or analogs, wherein the natural receptor or ligand includes, but is not limited to, PD1, PDL1, PDL2, CTLA4, OX40, OX40L, TIM3, TIM1, LAG3, TIGIT, CD137, CD137L, CD28, CD2, CD7, CD11a, CD11b, CD18, CD80, CD86, MHC-I, MHC-II, MHC-G, HLA-DR, CD209, CD206, galactolectin-3, LSECtin, FGL1, CD112, CD155, HVEM, CEACAM-1, Fas ligand, TCR complex, TCRα, TCR β, TCRγ, TCRδ, CD3, CD3ε, CD3γ, CD3δ, CD226, CD27, CD47, SIRP1α, CCR8, TNFR2, CD103, CD39, TIGIT, CD96, VISTA, BTLA, B7-H3, CD8, CD4, chemokine receptors, cytokine receptors, growth factor receptors, pattern recognition receptors, NKG2D, NKp46, MICA, ULBP-1, ULBP-2, BCR, CD79, CD40, CD40L, FcγRIIIa, FcγRIV, FcγRIIIb, FcγRIIa, FcγRIIc, FcγRIIb, FcγRI, FcRL5, pIgR, FcαRI, Fcα / μR, FcμR, FcεRI, FcεRII, DC-SIGN, CLEC5A dectin-1, dectin-2, dectin-3.

[0252] In some embodiments or in combination with previous embodiments, the heterologous polypeptide or multimeric protein comprises:

[0253] (a) At least one immunoglobulin-binding domain,

[0254] (b) At least one immune cell surface protein binding domain, which is an antigen-binding domain, antibody, or antigen-binding fragment, including derivatives or analogs thereof, having affinity and specificity for the extracellular portion of a natural receptor or ligand, including but not limited to lymphocytes, myeloid cells, T cells, B cells, NK cells, macrophages, monocytes, NK-T cells, neutrophils, dendritic cells, basophils, eosinophils, and mast cells.

[0255] In some respects, there is a desire for heterologous peptides or multimeric proteins with long half-lives in vivo. Various half-life extension strategies have been previously described (Strohl, 2015), including but not limited to proteins, peptides, and protein fusions, such as: human serum albumin (HSA), anti-HSA antibodies, transferrin, anti-transferrin antibodies, Fc, PEGylated, XTENated, PAS-modified, HAP-modified, ELP-modified, GLP-modified, and CTP-modified. Monomeric Fc fusion proteins have been previously described (Wang et al., 2017), which can achieve longer half-lives and binding to some Fc receptors while maintaining a smaller molecular weight.

[0256] In some areas, it is desirable to increase the half-life of heteropeptides or multimeric proteins containing Fc peptides, where the Fc region has one or more amino acid mutations that can further enhance the molecule's half-life in vivo. Several Fc mutation strategies have previously been described (Saunders, 2019) that can achieve half-life enhancement, including but not limited to one or more amino acid mutations: R435H, N434A, M252Y / S254T / T256E, M428L / N434S, T252L / T253S / T254F, E294 (deleted) / T307P / N434Y, T256N / A378V / S383N / N434Y, or glycosylation engineering, such as E294 (deleted), which induces increased sialylation of Fc-glycans, which in turn increases the half-life.

[0257] In some respects, it is desirable to have an Fc domain that is substantially non-binding to immunoglobulin-binding domains such as Fc receptors or antibodies. Several strategies capable of significantly reducing Fc receptor binding in the Fc region have been previously described (Saunders, 2019; Strohl, 2015), including but not limited to one or more amino acid mutations: L235E, L234A / L235A, S228P / L235E in IgG4; L234A / L235A / P239G, P331S / L234E / L235F, N265A, G237A, E318A, E233P, G236R / E328R, and IgG2 / IgG4 crosslinking. Glycosylation engineering, modification, or removal of IgG2 / IgG3 cross subclasses, H268Q / V209L / A330S / P331S, V234A / G237A / P238S / H268A / A330S / P331S, A330L, N270A, K322A, P329A, P331A, IgG2 / IgG3 cross subclasses, V264A, F241A, N297A, N297G, N297Q, S228P / F234A / L235A, or N297, including but not limited to high-mannose or enzymatic deglycosylation or trimming.

[0258] In some respects, IgR is a multimeric protein containing an immunoglobulin-binding domain and a human IgG CH1 domain, which is an anti-CH1 antibody fragment, and the human IgG CH1 domain has a mutation F122Y (Kabat number) to eliminate the ability of IgR to bind to itself, "self-bind," or "self-associate."

[0259] In some respects, IgR is a multimeric protein comprising some or all of an immunoglobulin-binding domain and a human constant heavy chain domain having one or more amino acid mutations. The immunoglobulin-binding domain is an anti-IgG antibody fragment containing clone 17F12 CDR. These amino acid mutations include, but are not limited to, CH1-P13S; CH1-K87E; hinge IgG1-EPKSCDKTH, hinge IgG2-ERKCCV, hinge IgG3-ELKTPLGDTTH, IgG4-ESKYGP to IgG-EFTP; CH2-E64R; CH3-T10I; CH3-S14P; CH3-K30T; CH3-T71I; CH3-V92T to eliminate the "self-binding" ability of IgR.

[0260] In some respects or in combination with previous embodiments, the heterologous polypeptide or multimeric protein comprises:

[0261] (a) At least one immunoglobulin-binding domain,

[0262] (b) and at least one immune cell surface protein binding domain,

[0263] (c) and at least one half-life extension domain, including but not limited to human serum albumin (HSA), anti-HSA, transferrin, anti-transferrin, PEGylated, XTENized, PAS-ized, HAP-ized, ELP-ized, GLP-fused, CTP-fused, or Fc polypeptides derived from immunoglobulins, wherein the Fc substantially does not bind to the Fc binding region.

[0264] In some embodiments or in combination with previous embodiments, the heterologous polypeptide or multimeric protein comprises:

[0265] (a) At least one immunoglobulin-binding domain,

[0266] (b) and at least one immune cell surface protein binding domain,

[0267] (c) and at least one half-life extension domain, including but not limited to Fc peptides derived from immunoglobulins, wherein by selecting IgG heavy chain Fc peptides including mutant L234A, L235A and P329A or alternatively P329G, the Fc substantially does not bind to the immunoglobulin binding domain.

[0268] In some embodiments or in combination with previous embodiments, the heterologous polypeptide or multimeric protein comprises:

[0269] (a) At least one immunoglobulin-binding domain,

[0270] (b) and at least one immune cell surface protein binding domain,

[0271] (c) and at least one half-life extension domain, including but not limited to Fc peptides derived from immunoglobulins, wherein the Fc peptides substantially do not bind to the immunoglobulin binding domains, and the Fc peptides include mutant M252Y / S254T / T256E.

[0272] In some aspects or in combination with the foregoing aspects or embodiments, it is desirable to use mutations in one or more amino acids in the constant region of an antibody to generate multispecific heteropeptides or heteropolymeric peptides having at least one immunoglobulin-binding domain and at least one immune cell surface protein-binding domain. Various protein engineering strategies have previously been described (H. Liu et al., 2017) to generate multispecific proteins, including but not limited to Quadromas, Knobs-in-holes homologous light chains, CrossMab Fab, CrossMab VH-VL, CrossMab CH1-CL, TriMab, OAscFab-IgG, dsFv-IgG, DuetMab, cFae-IgG1, charge-paired-ScFv-Fc, SEEDbody, biarm LUZ-Y, κ-λ body, bispecific T cell connector (bite), biantibodies, Tamdab, DART, BiKE, TriKE, mFc-VH, or Fcab. The previously described protein engineering strategies involve introducing one or more amino acid mutations in the Fc region to generate heteropolymeric proteins, including but not limited to mutations in Fc CH3-1 and CH3-2, which respectively include: T366Y and Y407T; S354C / T366W and Y349C, T366S, L368A, Y407V; S364H / F405A and Y349T / T394F; T350V / L351Y / F405A / Y407V and T350V / T366L / K392L / T394W; K392D / K409D and E356K / D399K; IgG1 D221E / P228E / L368E and IgG1 D221R / P228R / K409R; IgG2 C223E / P228E / L368E and IgG2 C223R / E225R / P228R / K409R; K360E / K409W and Q347R / D399V / F405T; K360E / K409W / Y349C and Q347R / D399V / F405T / S354C; 366K (+351K) and 351D or E or D at 349, 368, 349 or 349 + 355; Duobody F405L and K409R, SEEDbody IgG / A chimera, BEAT residues from the TCRα interface and residues from the TCRβ interface; K360D / D399M / Y407A and E345R / Q347R / T366V / K409V; or Y349S / K370Y / T366M / K409V and E356G / E357D / S364Q / Y407A.(Brinkmann & Kontermann, 2017; H. Liu et al., 2017).

[0273] In some embodiments or in combination with previous embodiments, the multimeric protein comprises:

[0274] (a) At least one immunoglobulin-binding domain,

[0275] (b) and at least one immune cell surface protein binding domain,

[0276] (c) and two half-life extension domains, comprising, but not limited to, Fc polypeptides derived from immunoglobulins, wherein the Fc substantially does not bind to the Fc binding region.

[0277] (d) The first Fc polypeptide and the second Fc polypeptide contain heteropolymerization domains, wherein the heteropolymerization domains may be one or more knob into hole mutations, leucine zippers, electrostatics, etc.

[0278] In some embodiments or in combination with previous embodiments, the multimeric protein comprises:

[0279] (a) At least one immunoglobulin-binding domain,

[0280] (b) and at least one immune cell surface protein binding domain,

[0281] (c) and two half-life extension domains comprising, but not limited to, Fc polypeptides derived from immunoglobulins, wherein, by selecting IgG heavy chain Fc polypeptides comprising mutant L234A, L235A, and P329A or alternatively P329G, the Fc substantially does not bind to the Fc binding region.

[0282] (d) The first Fc polypeptide and the second Fc polypeptide contain heteropolymerization domains, which may be knob into hole mutations, leucine zippers, electrostatics, etc.

[0283] In some embodiments or in combination with previous embodiments, the multimeric protein comprises:

[0284] (a) At least one immunoglobulin-binding domain,

[0285] (b) and at least one immune cell surface protein binding domain,

[0286] (c) and two half-life extension domains comprising, but not limited to, Fc polypeptides derived from immunoglobulins, wherein, by selecting IgG heavy chain Fc polypeptides comprising mutant L234A, L235A, and P329A or alternatively P329G, the Fc substantially does not bind to the Fc binding region.

[0287] (d) wherein the first Fc polypeptide comprises the mutant T366W and optionally S354C, and the second Fc polypeptide comprises T366S, L368A and Y407V and optionally Y349C.

[0288] In some embodiments or in combination with previous embodiments, the multimeric protein comprises:

[0289] (a) At least one immunoglobulin-binding domain,

[0290] (b) and at least one immune cell surface protein binding domain,

[0291] (c) and two half-life extension domains comprising, but not limited to, Fc polypeptides derived from immunoglobulins, wherein, by selecting IgG heavy chain Fc polypeptides comprising mutants L234A, L235A, and P329A or alternatively P329G, the Fc substantially does not bind to the Fc binding region, and the Fc polypeptide comprises mutants M252Y / S254T / T256E.

[0292] (d) wherein the first Fc polypeptide comprises the mutant T366W and optionally S354C, and the second Fc polypeptide comprises T366S, L368A and Y407V and optionally Y349C.

[0293] In some embodiments or in combination with previous embodiments, it is desirable to generate a multimeric protein with an introduced amino acid mutation that removes the natural interchain disulfide bond formation between the constant heavy chain and the constant light chain, and in some cases, as previously described (Brinkmann et al., 1993; Geddie et al., 2022) (Nakamura et al., 2018), in one of the chains (Hagihara & Saerens, 2014; Nakamura et al., 2018) or as a solution to improve light chain pairing within a multispecific antibody (Vaks et al., 2018) (Geddie et al., 2022), to introduce buried interchain disulfide between the constant heavy chain and the constant light chain.

[0294] In some embodiments or in combination with previous embodiments, the multimeric protein comprises:

[0295] (a) At least one immunoglobulin-binding domain,

[0296] (b) and at least one immune cell surface protein binding domain,

[0297] (c) and at least one immunoglobulin κ or λ light chain, its derivatives and analogs,

[0298] (d) and at least one CH1 domain derivative and its analogue,

[0299] (e) Optionally, the constant heavy chain domain 1 contains mutations such as C233S and the constant light chain contains mutations such as C214S to prevent the formation of interchain disulfide bonds.

[0300] (f) Optionally, the constant heavy chain domain 1 contains mutations such as C233S and F174C and the constant light chain contains mutations such as C214S and S176C to prevent the formation of interchain disulfide bonds and to create buried interchain disulfide bonds.

[0301] In some aspects or in combination with other embodiments or aspects, it is desirable to have multiple immunoglobulin-binding domains to alter the biological activity of heterologous peptides or multimeric proteins containing at least one immune cell surface protein-binding domain or to enhance their overall affinity and cohesion. Strategies for generating immunoglobulin-binding domains comprising multiple FcγR or multimeric Fc fusion bodies arranged in a head-to-tail tandem have previously been described or anticipated (Hogarth & Wines, 2014; Johnson et al., 2010; Rueger et al., 2018).

[0302] In some embodiments or in combination with previous embodiments, the heterologous polypeptide or multimeric protein comprises:

[0303] (a) First immunoglobulin-binding domain,

[0304] (b) and at least one immune cell surface protein binding domain,

[0305] (c) and the second immunoglobulin-binding domain, wherein the second immunoglobulin-binding domain differs from the first immunoglobulin domain.

[0306] (d) Optionally, the second immunoglobulin-binding domain is identical to the first immunoglobulin-binding domain, wherein if the domains are derived from FcγR type Fc receptors, they are not arranged in head-to-tail tandem.

[0307] In some aspects or in combination with other embodiments or aspects, it is desirable to have multiple immune cell surface protein binding domains to alter the biological activity of heterologous peptides or multimeric proteins containing at least one immunoglobulin binding domain or to enhance their overall affinity and co-occurrence. For example, it has been previously described that conjugation of CD3 and co-stimulatory or immune checkpoint targets can provide benefits for T cell conjugates such as CD3 and CD28 (Promsote et al., 2023), CD3 and CD137 (L. Liu et al., 2019), and CD3 and PD1 (Herrmann et al., 2018).

[0308] In some embodiments or in combination with previous embodiments, the heterologous polypeptide or multimeric protein comprises:

[0309] (a) At least one immunoglobulin-binding domain,

[0310] (b) and the binding domain of the first immune cell surface protein,

[0311] (c) and the binding domain of the second immune cell surface protein.

[0312] (d) Optionally, a second immune cell surface protein binding domain, wherein the second immune cell surface protein binding domain is the same as the first immune cell surface protein binding domain.

[0313] In some respects, cysteine ​​is used in the antibody fragments described herein to enable site-specific conjugation to lipids or polymers. Antibodies modified by engineering cysteine ​​into specific sites for conjugation have been previously described (Pillow et al., 2014) (Junutula et al., 2008) (Beck et al., 2017) (Jeffrey et al., 2013) (Tumey et al., 2017). Optionally, non-natural amino acids can be incorporated into specific sites for conjugation (Hallam et al., 2015).

[0314] In some embodiments or in combination with previous embodiments, the heterologous polypeptide or multimeric protein comprises:

[0315] (a) At least one immunoglobulin-binding domain,

[0316] (b) and at least one immune cell surface protein binding domain,

[0317] (c) and free cysteine, which is located at or near the C-terminus, or optionally at or near the N-terminus.

[0318] In some embodiments or in combination with previous embodiments, the heterologous polypeptide or multimeric protein comprises:

[0319] (a) At least one immunoglobulin-binding domain,

[0320] (b) and at least one immune cell surface protein binding domain,

[0321] (c) and covalently linked lipid components.

[0322] In some embodiments or in combination with previous embodiments, the heterologous polypeptide or multimeric protein comprises:

[0323] (a) At least one immunoglobulin-binding domain,

[0324] (b) and at least one immune cell surface protein binding domain,

[0325] (c) and covalently linked lipids, PEG-lipids or optionally cholesterol components.

[0326] In some embodiments or combinations of previous embodiments, the present invention contemplates the use of heterologous peptides or multimeric proteins as a monotherapy or in combination with one or more standard therapeutic agents, current therapeutic agents, or experimental therapeutic agents to treat diseases such as cancer, immune disorders, and pathogenic infections. These therapeutic agents include, but are not limited to: rituximab, trastuzumab, cetuximab, bevacizumab, oflamuzumab, pertuzumab, oxotuzumab, ramucirumab, ramucirumab, alemtuzumab, necitumumab, dartuximab, daratumumab, erlotuzumab, olatumumab, atezolizumab, and ionotuzumab-oxozamicin. ozogamicin), averumab, durvalumab, gemtuzumab (orzomicin), moglizamab (moglizamab-kpkc), cimiprimab (cimiprimab-rwlc), epavamab (epavamab-lzsg), ​​mosetumumab (mosetumumab-tdfk), polotuzumab (polotuzumab-piiq), veentumumab ( Verentuximab-ejfv), [fam-]detrastuzumab (fam-detrastuzumab-nxki), ixartuximab (ixartuximab-irfc), goxatocinumab (goxatocinumab-hziy), tancituzumab (tancituzumab-cxix), mabelantuzumab (mabelantuzumab-blmf), nacituzumab-gqgk, maglucituzumab-cmkb, Loncastuximab tesirine), dostarlimab, amivantamab, tisotumab vedotin, tisotumab-tftv, renalalimab, texilimab, nivolumab, pembrolizumab, ipilimumab, sotopimab, casirivimab, imdevimab, renalalimab, tesaxagvir, cigavir, nisecvir, and soravtansine.

[0327] In some embodiments or in combinations of previous embodiments, the present invention contemplates a kit comprising a heterologous polypeptide or multimeric protein.

[0328] As described herein, pharmaceutical compositions comprise one or more linker sequences. The linker sequences provide flexibility between peptides, enabling, for example, immunoglobulin-binding domains and immune cell surface protein-binding domains to simultaneously bind to their targets. The linker sequences may be located between any or all of the immunoglobulin-binding domain, immune cell surface binding domain, serum half-life extension element, and / or blocking moiety. Suitable linkers may have different lengths, such as 1 amino acid (e.g., Gly) to 20 amino acids, 2 amino acids to 15 amino acids, 3 amino acids to 12 amino acids, including 4 amino acids to 10 amino acids, 1 amino acid to 9 amino acids, 6 amino acids to 8 amino acids, or 7 amino acids to 8 amino acids, and may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 1 3, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 amino acids. The blocking portion can prevent one or more immunoglobulin-binding domains or one or more immune cell surface protein-binding domains, or both, from significantly binding to their targets. The linker may contain cleavage sites specific to environmental conditions, including but not limited to pH, redox potential, electrical potential, and enzymes such as metalloproteinases. The connector can be designed to conditionally break or change conformation to release the blocking portion that prevents immunoglobulin binding domains or immune cell surface protein binding domains from significantly binding to their targets.

[0329] This method may further involve administering one or more adjunctive agents to treat cancer, such as chemotherapeutic agents (e.g., doxorubicin, daunorubicin, bleomycin, melphalan, vincristine, fluorouracil, thiotepa, methotrexate, bismuth subcitrate, mitoxantrone, thioguanine, cytarabine, procarbazine), immuno-oncology agents (e.g., anti-PD-L1, anti-CTLA4, anti-PD-1, anti-CD47, anti-GD2, anti-SIRP1α), cell therapy (e.g., CAR-T, T-cell therapy), and oncolytic viruses, etc.

[0330] In some embodiments, the heterologous polypeptide comprises: (a) one or more Fc-binding domains having affinity and specificity for the Fc moiety of an immunoglobulin molecule (Ig); and (b) at least one domain having affinity and specificity for at least one extracellular polypeptide present on immune cells, including derivatives and analogs thereof, wherein the Fc-binding domain comprises an extracellular domain of FcγRI, FcγRIIA, FcγRIIB / C, FcγRIIIA, FcγRIIIB, FcαRI, Fcα / μR, FcεRI, FcεRII, pIgR, or FcRn, including allotypes, derivatives, and analogs thereof. The choice of the ligand-binding domain of the Fc receptor used for the heterologous polypeptide described herein will be apparent to those skilled in the art. For example, it may depend on factors such as the isotype of the antibody required to bind to the Fc receptor and the desired affinity of the binding interaction.

[0331] In some embodiments, the heterologous polypeptide comprises: (a) one or more Fc-binding domains having affinity and specificity for the Fc moiety of an immunoglobulin molecule (Ig); and (b) at least one domain having affinity and specificity for at least one extracellular polypeptide present on immune cells, including derivatives and analogs thereof, wherein the Fc-binding domain may be a synthetic polypeptide specifically engineered to bind with high affinity and specificity to the Fc moiety of any Ig molecule described herein. For example, such an Fc-binding domain may be an antibody or an antigen-binding fragment thereof that specifically binds to the Fc moiety of an immunoglobulin. Examples include, but are not limited to, fragment antigen-binding regions (Fab) or (Fab)2, single-chain variable fragments (scFv), domain antibodies, or nanobodies. Alternatively, the Fc-binding domain can be a synthetic peptide that specifically binds to the Fc moiety, such as, but not limited to, the Kunitz domain, small modular immunopharmaceuticals (SMIPs), adnectin, avimer, affibody, DARPin or anticalin, or Fc-III peptides (multi-resolution JaLC, undated), which can be identified by screening a library of peptide combinatorial libraries with Fc-binding activity.

[0332] In some embodiments, the heterologous polypeptide comprises: (a) one or more Fc-binding domains having affinity and specificity for the Fc portion of an immunoglobulin molecule (Ig); and (b) at least one domain having affinity and specificity for at least one extracellular polypeptide present on immune cells, including derivatives and analogs thereof, wherein the Fc-binding domain may be derived from natural proteins, such as mammalian Fc receptors, certain bacterial proteins, such as protein A or protein G, and viral proteins, such as TspB, gE, gI, gpI, FcγR-like HCV core protein, gp34, gp68, gpRL13, gp95, and fcr-1.

[0333] In some embodiments, the heterologous polypeptide comprises: (a) one or more Fc-binding domains having affinity and specificity for the Fc moiety of an immunoglobulin molecule (Ig); and (b) at least one domain having affinity and specificity for at least one extracellular polypeptide present on immune cells, including derivatives and analogs thereof, wherein the extracellular polypeptide (b) comprises the following extracellular domains: FcγRI, FcγRII, FcγRIII, FcαRI, Fcα / μR, FcεRI, FcεRII, pIgR, FcRn, and T-cell receptor (TCR) complexes (including CD3ε, CD3δ, CD3γ). TCRα, TCRβ, TCRγ, TCRδ or combinations thereof), CD8, CD2, Fas ligand, CD40L, CD16, CD32, CD64, NKG2D, NKp46, PD1, PDL1, CTLA4, OX40, TIM3, LAG3, TIGIT, CD137, CD28, CD28, CD7, CD11a, CD18, CD80, CD86, CD121a, CD121b, IL-18Rα, IL-18Rβ, CD25, CD122, CD132, CD124, CD213a13, CD12 7. CD360, CD19, CD20, CD5, IL-9R, CD213a1, CD213a2, IL-15Ra, CD123, CDw131, CDw125, CD131, CD116, CDw131, CD126, CD130, IL-11 Ra, CD130, CD114, CD212, LIFR, CD130, OSMR, CDw210, IL-20Rα, IL-20Rβ, IL-14R, CD4, CDw217, CD118, CDw119, LTβR, CD120a, CD120 b, CD137L, BCMA, TACI, CD27, CD30, CD95 (Fas), GITR, GITRL, CLEC5A, LTbR, HVEM, TRAILR1-4, Apo3, RANK, OPG, TGF-βR1, TGF-βR2, T GF-βR3, EpoR, TpoR, Flt-3, CD117, CD115 or CDw136, CD47, SIPR1α, CCR8, TNFR2, CD103, CD39, CD79, dectin-1, dectin-2, dectin-3.

[0334] In some embodiments, the heterologous polypeptide comprises: (a) one or more immunoglobulin-binding domains having affinity and specificity for one or more regions of an immunoglobulin molecule (Ig); and (b) at least one domain having affinity and specificity for at least one extracellular polypeptide present on an immune cell, including derivatives and analogs thereof; and (c) wherein the heterologous polypeptide is additionally fused to a transmembrane domain and optionally one or more intracellular domains.

[0335] In some embodiments, the heterologous polypeptide comprises: (a) one or more immunoglobulin-binding domains having affinity and specificity for one or more regions of an immunoglobulin molecule (Ig); and (b) at least one domain having affinity and specificity for at least one extracellular polypeptide present on immune cells, including derivatives and analogs thereof; and (c) wherein the heterologous polypeptide is conjugated to one or more chemical compounds such as lipids, PEG, cytotoxic drug agents.

[0336] The various implementation plans are as follows.

[0337] 1. A heterologous polypeptide, said heterologous polypeptide comprising:

[0338] At least one immunoglobulin-binding domain; and

[0339] At least one immune cell surface protein binding domain;

[0340] The at least one immunoglobulin-binding domain is derived from an Fc receptor or Fc binding domain, including but not limited to FcγRIII, mFcγRIV, FcγRIIa, FcγRIIb, FcγRIIc, FcγRI, mFcγRIII, mFcγRIIa, mFcγRIIb, mFcγRI, FcαRI, C1q, FcRL, FcRL5, pIgR, Fcα / μR, FcμR, FcεRI, FcεRII, FcRn, TRIM21, its allotypes, derivatives and analogs;

[0341] The at least one immunoglobulin-binding domain is derived from an antigen-binding domain, an antibody or antigen-binding fragment and its variants, derivatives or analogs, the variants, derivatives or analogs comprising VH and VL pairs, ScFv, Fab, IgG, sdAb-VL, sdAb-VH, VHH or avimer and their derivatives or analogs.

[0342] The heteropeptide comprises one or more half-life extension domains, the half-life extension domains comprising an anti-HSA antigen-binding domain, an antibody or antigen-binding fragment, and variants, derivatives or analogs thereof including VHH or single-domain antibodies, as well as immunoglobulin IgG Fc domains and variants, derivatives or analogs thereof.

[0343] The heterologous polypeptide comprises at least two immunoglobulin-binding domains; and

[0344] The heterologous polypeptide contains at least two immune cell surface protein binding domains.

[0345] 2. A heterologous polypeptide, said heterologous polypeptide comprising:

[0346] At least one immunoglobulin-binding domain; and

[0347] At least one immune cell surface protein binding domain;

[0348] The at least one immunoglobulin-binding domain comprises all or a portion of the Fc receptor of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 23, 77, 80, 271, 272, 273, 274, 275, 276, 277, 294, 296, 298, 300, 324, and 325;

[0349] The at least one immunoglobulin-binding domain is derived from FcγRIIa containing one or more mutations as described in SEQ ID NO: 9, including R56H, K118N, T120V, L160Q, and V172E; or

[0350] The at least one immunoglobulin-binding domain is derived from FcγRIII containing one or more mutations as described in SEQ ID NO: 1, including S181P, K122N, T124V, Q176E, I90R, T118K, A119L, and Y134F.

[0351] The heterologous polypeptide comprises at least two immunoglobulin-binding domains; and

[0352] The heterologous polypeptide contains at least two immune cell surface protein binding domains.

[0353] 3. A heterologous polypeptide, said heterologous polypeptide comprising:

[0354] At least one immunoglobulin-binding domain; and

[0355] At least one immune cell surface protein binding domain;

[0356] The at least one immunoglobulin binding domain is derived from an antigen-binding domain, an antibody or antigen-binding fragment and its variants, derivatives or analogs, including but not limited to VH and VL pairs, ScFv, Fab, IgG, sdAb-VL, sdAb-VH, VHH or avimer and their derivatives or analogs.

[0357] The at least one immunoglobulin-binding domain comprises SEQ ID NO: 569, 570, 571, 572, 573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584, 585, 586, 587, 588, 589, 590, 591, 592, 593, 594, 595, 596, 597, 598, 599, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611, 612, 613, 614, 61 5, 616, 617, 618, 619, 620, 621, 622, 623, 624, 625, 626, 627, 628, 629, 6230, 631, 632, 633, 634, 635, 636, 637, 638, 639, 640, 641, 642, 643, 644, 645, 646, 647, 648, 649, 650, 651, 652, 653, 654, 655, 656, 657, or at least one of the CDR or FR regions in SEQ ID NO: 132;

[0358] The heterologous polypeptide comprises at least two immunoglobulin-binding domains; and

[0359] The heterologous polypeptide contains at least two immune cell surface protein binding domains.

[0360] 4. A heterologous polypeptide, said heterologous polypeptide comprising:

[0361] At least one immunoglobulin-binding domain;

[0362] At least one immune cell surface protein binding domain;

[0363] At least one of the immune cell surface protein binding domains is derived from an antigen-binding domain, an antibody or antigen-binding fragment and its variants, derivatives or analogs, including but not limited to VH and VL pairs, ScFv, Fab, IgG, sdAb-VL, sdAb-VH, VHH and its derivatives or analogs.

[0364] The immune cell surface protein binding domain described herein includes SEQ ID NO: 580, 606, 608, 609, 610, 611, 612, 615, 626, 635, 636, 637, 638, 639, 658, 659, 660, 661, 662, 663, 664, 665, 666, 667, 668, 669, 670, 671, 672, 673, 674, 675, 676, 677, 678, 679, 680, 681, 682, 683, 684, 685, 686, 687, 688, 689, 690, 691, 692, 693, 694, 695, 696, 697, 698, 699, 700, 701, 702, 703, 70 4, 705, 706, 707, 708, 709, 710, 711, 712, 713, 714, 715, 716, 717, 718, 719, 720, 721, 722, 723, 724, 725, 726, 727, 728, 729, 730, 731, 732, 733, 734, 735, 736, 737, 738, 739, 740, 741, 742, 743, 744, 745, 746, 747, 748, 749, 750, 751, 752, 753, 754, 755, 756, 757, 758, 759, 760, 761, 762, 763, 764, 765, 766, 767, 768, 769, 770, 771, 772, 773, 774, 775, 776, 777, 778, 779, 780, 781, 782, 783, 784, 785, 786, 787, 788, 789, 790, 791, 792, 793, 794, 795, 796, 797, 798, 799, 800, 801, 802, 803, 804, 805, 806, 807, 808, 809, 810, 811, 812, 813, 814, 815, 816, 817, 818, 819, 820, 821, 822, 823, 824, 82 5, 826, 827, 828, 829, 840, 841, 842, 843, 844, 845, 846, 847, 848, 849, 840, 841, 842, 843, 844, 845, 846, 847, 848, 849, 850, 851, 852, 853, 854, 855, 856, 857, 858, 859, 860, 861, 862, 863, 864, 865, 866, 867, 868, 869, 870, 871, 872, 873, 874, 875, 876, 877, 878, 879, 880, 881, 882, 883, 884, 885,886, 887, 888, 889, 890, 891, 892, 893, 894, 895, 896, 897, 898, 899, 900, 901, 902, 903, 904, 905, 906, 907, 908, 909, 910, 911, 912, 913, 914, 915, 916, 917, 918, 919, 920, 921, 922, 923, 924, 925, 926, 927, 928, 929, 9 40, 941, 942, 943, 944, 945, 946, 947, 948, 949, 940, 941, 942, 943, 944, 945, 946, 947, 948, 949, 950, 951, 952, 953, 954, 955, 956, 957, 958, 959, 960, 961, 962, 963, 964, 965, 966, 967, 968, 969, 970, 971, 972, 973, 974 975, 976, 977, 978, 979, 980, 981, 982, 983, 984, 985, 986, 987, 988, 989, 990, 991, 992, 993, 994, 995, 996, 997, 998, 999, 1000, 1001, 1002, 1003, 1004, 1005, 1006, 1007, 1008, 1009, 1010, 1011, 1012, 1013, 1014, 10 One or more CDR or FR zones from the following: 15, 1016, 1017, 1018, 1019, 1020, 1021, 1022, 1023, 1024, 1025, 1026, 1027, 1028, 1029, 1030, 1031, 1032, 1033, 1034, 1035, 1036, 1037, 1038, 1039, 1040, 1041, 1042, 1043, 1044, 1045, 1046, and 1047;

[0365] The heterologous polypeptide comprises at least two immunoglobulin-binding domains; and

[0366] The heterologous polypeptide contains at least two immune cell surface protein binding domains.

[0367] 5. A heterologous polypeptide, said heterologous polypeptide comprising:

[0368] At least one immunoglobulin-binding domain;

[0369] At least one immune cell surface protein binding domain;

[0370] At least one half-life extension domain;

[0371] The at least one half-life extension domain comprises an anti-HSA antigen-binding domain, an antibody or antigen-binding fragment, and variants, derivatives or analogs thereof including VHH or single-domain antibodies, wherein the VHH or single-domain antibody comprises one or more CDR or FR regions from SEQ ID NO: 558, 559, 560, 561, 562, 563, 564, 565, 566, 567 and 568;

[0372] The heterologous polypeptide comprises at least two immunoglobulin-binding domains; and

[0373] The heterologous polypeptide contains at least two immune cell surface protein binding domains.

[0374] 6. A heterologous polypeptide, said heterologous polypeptide comprising:

[0375] At least one immunoglobulin-binding domain;

[0376] At least one immune cell surface protein binding domain;

[0377] At least one half-life extension domain;

[0378] The heterologous polypeptide is a single chain with the following structure:

[0379] D1-D2-D3

[0380] and

[0381] The at least one half-life extension domain comprises an anti-HSA antigen-binding domain, an antibody or antigen-binding fragment, and variants, derivatives or analogs thereof including VHH or single-domain antibodies, wherein the VHH or single-domain antibody comprises one or more CDR or FR regions from SEQ ID NO: 558, 559, 560, 561, 562, 563, 564, 565, 566, 567 and 568.

[0382] 7. The heteropeptide of claim 5, wherein the heteropeptide further comprises one or more linkers between the domains.

[0383] 8. A heterologous polypeptide, said heterologous polypeptide comprising:

[0384] At least two immunoglobulin-binding domains;

[0385] At least one immune cell surface protein binding domain;

[0386] At least one half-life extension domain;

[0387] The heterologous polypeptide is a single chain having at least four domains with the following structure:

[0388] D1-D2-D3-D4

[0389] and

[0390] The at least one half-life extension domain comprises an anti-HSA antigen-binding domain, an antibody or antigen-binding fragment, and variants, derivatives or analogs thereof including VHH or single-domain antibodies, wherein the VHH or single-domain antibody comprises one or more CDR or FR regions from SEQ ID NO: 558, 559, 560, 561, 562, 563, 564, 565, 566, 567 and 568.

[0391] 9. The heteropeptide of claim 6, wherein the heteropeptide further comprises one or more linkers between the domains.

[0392] 10. A multimeric protein, wherein a region of the molecule comprises:

[0393] At least one immunoglobulin-binding domain;

[0394] At least one immune cell surface protein binding domain; and

[0395] Two half-life extension domains, comprising a first Fc polypeptide and a second Fc polypeptide derived from immunoglobulins, wherein the Fc polypeptides are substantially non-binding to the immunoglobulin binding domains.

[0396] The first Fc polypeptide and the second Fc polypeptide contain heteropolymerization domains, wherein the heteropolymerization domains are at least one knob into hole mutation.

[0397] The multimeric protein comprises at least two immunoglobulin-binding domains; and

[0398] The multimeric protein contains at least two immune cell surface protein-binding domains.

[0399] 11. A multimeric protein, wherein the regions of the molecule include:

[0400] At least one immunoglobulin-binding domain;

[0401] At least one immune cell surface protein binding domain;

[0402] Two half-life extension domains, comprising a first Fc polypeptide and a second Fc polypeptide derived from immunoglobulins, wherein by selecting an IgG heavy chain Fc polypeptide containing mutant L234A, L235A and P329A or P329G in constant heavy chain domain 2 (EU number), the Fc polypeptide substantially does not bind to the immunoglobulin binding domain.

[0403] The first Fc polypeptide and the second Fc polypeptide contain heteropolymerization domains, wherein the heteropolymerization domains are at least one knob into hole mutation.

[0404] The multimeric protein comprises at least two immunoglobulin-binding domains; and

[0405] The multimeric protein contains at least two immune cell surface protein-binding domains.

[0406] 12. A multimeric protein, wherein the regions of the molecule include:

[0407] At least one immunoglobulin-binding domain;

[0408] At least one immune cell surface protein binding domain;

[0409] Two half-life extension domains, comprising, but not limited to, a first Fc polypeptide and a second Fc polypeptide derived from immunoglobulins, wherein by selecting an IgG heavy chain Fc polypeptide containing mutant L234A, L235A and P329A or P329G in constant heavy chain domain 2 (EU number), the Fc substantially does not bind to the immunoglobulin binding domain.

[0410] The first Fc polypeptide contains the mutant T366W; and

[0411] The second Fc polypeptide contains T366S, L368A and Y407V in constant heavy chain domain 3 (EU number);

[0412] The first Fc polypeptide further comprises S354C, and the second Fc polypeptide further comprises Y349C in the constant heavy chain domain 3 (EU number);

[0413] The multimeric protein comprises at least two immunoglobulin-binding domains; and

[0414] The multimeric protein contains at least two immune cell surface protein-binding domains.

[0415] 13. A multimeric protein, wherein the regions of the molecule include:

[0416] At least one immunoglobulin-binding domain;

[0417] At least one immune cell surface protein binding domain;

[0418] At least one constant light chain of immunoglobulin κ or λ, its variants, derivatives and analogs;

[0419] At least one portion of an immunoglobulin constant heavy chain domain 1 and an immunoglobulin hinge region, and variants, derivatives and analogs thereof;

[0420] The one or more constant heavy chain domains 1 and at least a portion of the hinge contain the mutation C233S and the constant light chain contains the mutation C214S (Kabat number).

[0421] The one or more constant heavy chain domains 1 and at least a portion of the hinge contain mutations C233S and F174C and the constant light chain contains mutations C214S and S176C (Kabat number).

[0422] Wherein at least the first pair of constant heavy chains and at least a portion of the hinge do not contain a mutation at C233 and do not contain a mutation at C214 in the constant light chain, and at least the second pair of constant heavy chain domain 1 and at least a portion of the hinge contain mutations such as C233S and the constant light chain contains mutations such as C214S (Kabat number).

[0423] Wherein at least the first pair of constant heavy chains and at least a portion of the hinge do not contain a mutation at C233 and do not contain a mutation at C214 in the constant light chain, and at least the second pair of constant heavy chain domain 1 and at least a portion of the hinge contain mutations C233S and F174C and the constant light chain contains mutations C214S and S176C (Kabat number).

[0424] At least the first pair of constant heavy chain structural domains 1 and at least a portion of the hinge contain mutation C233S and the constant light chain contains mutation C214S, and at least the second pair of constant heavy chain structural domains 1 and at least a portion of the hinge contain mutations C233S and F174C and the constant light chain contains mutations C214S and S176C (Kabat number).

[0425] The heteropeptide comprises one or more half-life extension domains, the half-life extension domains comprising an anti-HSA antigen-binding domain, an antibody or antigen-binding fragment, and variants, derivatives or analogs thereof including VHH or single-domain antibodies, as well as immunoglobulin IgG Fc domains and variants, derivatives or analogs thereof.

[0426] The multimeric protein comprises at least two immunoglobulin-binding domains; and

[0427] The multimeric protein contains at least two immune cell surface protein-binding domains.

[0428] 14. A multimeric protein, wherein one or more regions of the molecule comprise:

[0429] At least two immunoglobulin-binding domains;

[0430] At least one immune cell surface protein binding domain;

[0431] At least a second immunoglobulin-binding domain, wherein the second immunoglobulin-binding domain is separated from the first immunoglobulin-binding domain by a linker of 1 to 20 amino acids, the linker comprising regions of a human constant heavy chain domain 1, a κ chain domain, and a λ chain domain, a linker containing 13 or fewer amino acids, a polypeptide containing a linker containing 6 or fewer amino acids, a spacer region ASTKGPSVFPLAP, ASTKGP, or ASTKGPSVFPLAS derived from the constant heavy chain domain 1, a spacer region RTVAAPSVFIFPP or RTVAAP derived from the constant κ chain, and a spacer region SQPKAAPSVTLFP, GQPKANPTVTLFP, GQPKAAPSVTLFP, SQPKAA, GQPKAN, or GQPKAA, (GGGS)1, (GGGS)2, (GGGS)3, or (GGGS)4 derived from the constant λ chain.

[0432] At least one of them contains an immunoglobulin-binding domain comprising SEQ ID NO: 569, 570, 571, 572, 573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584, 585, 586, 587, 588, 589, 590, 591, 592, 593, 594, 595, 596, 597, 598, 599, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611, 612, 613, 614, 615, 6 One or more CDR or FR regions from 16, 617, 618, 619, 620, 621, 622, 623, 624, 625, 626, 627, 628, 629, 6230, 631, 632, 633, 634, 635, 636, 637, 638, 639, 640, 641, 642, 643, 644, 645, 646, 647, 648, 649, 650, 651, 652, 653, 654, 655, 656, 657, or the aviser defined in SEQ ID NO: 132; and

[0433] At least a second immunoglobulin-binding domain is therein, wherein the second immunoglobulin-binding domain is identical to the first immunoglobulin-binding domain; or

[0434] It includes at least a second immunoglobulin-binding domain, wherein the second immunoglobulin-binding domain is different from the first immunoglobulin-binding domain.

[0435] 15. A multimeric protein, wherein at least one region comprises:

[0436] At least one immunoglobulin-binding domain;

[0437] At least one immune cell surface protein binding domain;

[0438] One or more amino acid mutations in one or more constant or framework domains of human IgG1, IgG2, IgG3 or IgG4, their variants, derivatives and analogs, wherein the immunoglobulin binding domain substantially does not bind itself or another region or regions of the molecule.

[0439] One or more amino acid mutations in constant heavy chain domain 1 include F122Y, P126S and K213E (Kabat number).

[0440] One or more amino acid mutations in constant heavy chain domain 2 include N276K, L309V, L234A, L235A, and P329A or P329G (EU number).

[0441] The heterologous polypeptide or multimeric protein and the nucleic acid encoding it contain at least two immunoglobulin-binding domains; and

[0442] The heterologous polypeptide or multimeric protein and the nucleic acid encoding it contain at least two immune cell surface protein-binding domains.

[0443] 16. A multimeric protein, wherein the regions of the molecule include:

[0444] At least one constant light chain of immunoglobulin κ or λ, its variants, derivatives, and analogs; and

[0445] At least one immunoglobulin constant heavy chain domain 1 and at least a portion thereof, and variants, derivatives and analogues thereof; and

[0446] Two half-life extension domains comprising a first Fc polypeptide and a second Fc polypeptide derived from immunoglobulins, wherein the first Fc polypeptide and the second Fc polypeptide comprise heteropolymerization domains selected from at least one knob into hole mutation.

[0447] The first Fc polypeptide contains T366W, and the second Fc polypeptide contains T366S, L368A and Y407V in constant heavy chain domain 3 (EU number).

[0448] This involves selecting IgG heavy chain Fc polypeptides that contain L234A, L235A, and P329A in constant heavy chain domain 2 (EU number), wherein the Fc substantially does not bind to one or more of its homologous Fc receptors.

[0449] One or more of the constant heavy chain domains 1 and all, none or part of the hinges contain the mutation C233S and the constant light chain contains the mutation C214S (Kabat number).

[0450] One or more of the constant heavy chain domain 1 and at least a portion of the hinge contain mutations C233S and F174C and the constant light chain contains mutations C214S and S176C (Kabat number).

[0451] Wherein at least the first pair of constant heavy chains and at least a portion of the hinge do not contain a mutation at C233 and do not contain a mutation at C214 in the constant light chain, and at least the second pair of constant heavy chain domain 1 and at least a portion of the hinge contain a mutation at C233S and the constant light chain contains a mutation C214S (Kabat number).

[0452] Wherein at least the first pair of constant heavy chains and at least a portion of the hinge do not contain a mutation at C233 and do not contain a mutation at C214 in the constant light chain, and at least the second pair of constant heavy chain domain 1 and at least a portion of the hinge contain mutations at C233S and F174C, and the constant light chain contains mutations C214S and S176C (Kabat number).

[0453] At least the first pair of constant heavy chain structural domains 1 and at least a portion of the hinge contain a mutation at C233S and the constant light chain contains a mutation at C214S, and at least the second pair of constant heavy chain structural domains 1 and at least a portion of the hinge contain mutations C233S and F174C and the constant light chain contains mutations C214S and S176C (Kabat number).

[0454] 17. The multimeric protein according to claim 16,

[0455] The first Fc polypeptide further comprises S354C, and the second Fc polypeptide further comprises Y349C in the constant heavy chain domain 3 (EU number); and

[0456] The IgG heavy chain Fc polypeptide further includes P329G in the constant heavy chain domain 2 (EU number).

[0457] 18. The heteropeptide or multimeric protein according to claim 1, 2, 3, 4, 13, 14 or 16, wherein an additional region of the molecule contains a free cysteine ​​residue at or near the C-terminus.

[0458] 19. The heteropeptide or multimeric protein according to claim 1, 2, 3, 4, 13, 14 or 16, wherein an additional region of the molecule comprises a covalently linked PEG-lipid.

[0459] 20. A treatment method comprising administering a heterologous polypeptide or multimeric protein according to claim 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 16 or 17 as a monotherapy to a patient in need of treating a disease.

[0460] 21. A treatment method comprising administering a heterologous polypeptide or multimeric protein according to claim 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 16 or 17 in combination with at least one selected from standard therapeutic agents, current therapeutic agents or experimental therapeutic agents to a subject in need of treating cancer, immune disorders or pathogenic infections.

[0461] 22. A nucleotide encoding a heteropeptide or multimeric protein according to any one of claims 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 16 or 17.

[0462] 23. A kit comprising a heterologous polypeptide or multimeric protein according to any one of claims 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 16 or 17.

[0463] Example

[0464] Example 1. Expression and purification of FcγR3A / αCD3 and FcγR3A / αTCR fusions

[0465] The Fc-binding heterologous peptide was encoded into a plasmid DNA expression vector and transiently expressed as a recombinant protein in Chinese hamster ovary (CHO) cells. The protein was purified using immobilized metal chromatography by incorporating a GGHHHHHH (SEQ ID NO:21) tag at the C-terminus or by protein A (if the protein contains the Fc region).

[0466] The extracellular domain of the CD16A Fc receptor (FcγR3A-V158, SEQ ID NO: 1) with the S197P mutation (FcγR3AV-S197P, SEQ ID NO: 23) to eliminate susceptibility to ADAM17 cleavage was fused via a (G4S)3 linker (SEQ ID NO: 22) to: a humanized version of the αCD3 ScFv clone HzUCHT1 with a VL-VH orientation using a (G4S)3 linker (VHSEQ ID NO: 24; VL SEQ ID NO: 25, ScFv-HzUCHT1 SEQ ID NO: 26); to form an FcγR3A / ScFv-HzUCHT1 (SEQ ID NO: 27; JIB1, SEQ ID NO: 28) fusion, as depicted in Figure 2A; and an αCD3 ScFv clone TR66 with a VH-VL orientation using a (G4S)3 linker (TR66-VH). SEQ ID NO: 29; TR66-VL SEQ ID NO: 30, ScFv-TR66 SEQ ID NO: 31), to form the FcγR3A / ScFv-TR66 fusion (SEQ ID NO: 32; JIB2, SEQ ID NO: 33), as depicted in Figure 2A; and αTCR VHH clone V700 (VHH-V700 SEQ ID NO: 34), to form the FcγR3A / VHH-V700 fusion (SEQ ID NO: 35; JIB3, SEQ ID NO: 36), as depicted in Figure 2A. Figure 8 and Table 1 show that the protein was well expressed in high purity (by the amount recovered using A280 after single-step purification) as determined by both SDS-PAGE and SEC.

[0467] Table 1. Protein expression and monomers of IgR generated in Example 1 as determined by SEC.

[0468] Protein CHO (mg / L) Monomer content as determined by SEC: JIB1 23 185.4%; JIB2 25 66 8.6%; JIB3 37 084.1%. surface

[0469] Example 2. Expression and purification of αHSA / FcγR3A / αCD3 fusion

[0470] The Fc-binding heterologous polypeptide was expressed and purified as described in Example 1.

[0471] Humanized VHH targeting HSA (SEQ ID NO: 37) was fused to the extracellular domain of a human CD16A Fc receptor with an S197P mutation via a (G4S)3 linker. This S197P mutation was fused to a humanized version of the αCD3 ScFv clone HzUCHT1 with a VL-VH orientation using a (G4S)3 linker, forming the αHSA-VHH / FcγR3A / αCD3-ScFv fusion (SEQ ID NO: 38; JIB5, SEQ ID NO: 39), as depicted in Figure 3A. For purification purposes, a 6his tag with a GG spacer region was fused to the C-terminus of the ScFv. Figure 8 and Table 2 show that the protein was well expressed in high purity as determined by both SDS-PAGE and SEC. Comparing Figures 8A-B with 8I-J shows that adding anti-HSA VHH to the N-terminus of FcγRIIIA did not promote significant aggregation, indicating that FcγRIIIA is resistant to binding regions at both its N-terminus and C-terminus.

[0472] Table 2. Protein expression and monomers of IgR generated in Example 2 as determined by SEC.

[0473] Protein CHO (mg / L) % monomer content as determined by SEC: JIB 543378.8% surface

[0474] Example 3. Expression and purification of FcγR3A-Fc / αCD3-Fc heterodimer protein

[0475] The Fc-binding heterodimer protein was expressed and purified as described in Example 1. The FcγR3A-Fc / αCD3-Fc fusion was expressed as three polypeptide chains to assemble into a soluble protein comprising two heterodimers assembled using a knock-in-hole mutation to form a heavy chain and a light chain assembled with one of the two heterodimer heavy chains, as depicted in Figure 4A. A heavy chain consisting of the extracellular domain of the human CD16A Fc receptor with the S197P mutation is fused via a (G4S)3 linker to a partial human IgG1 heavy chain with the following mutations: C233S to prevent disulfide bond formation; T366S, L368A, Y407V to generate a “hole” Fc heavy chain; and L234A, L235A, and P329A (C233S / Fc-LALAPA-hole, SEQ ID NO: 40) to eliminate Fc receptor binding; forming the FcγR3A-Fc polypeptide (FcγR3A-S197P / Fc-LALAPA-hole SEQ ID NO: 41). Another heavy chain consists of a variable heavy chain (VH-SP34, SEQ ID NO: 42) of αCD3 clone SP34 and a constant human IgG1 heavy chain with the following mutations: T366W, to produce a “knob” Fc heavy chain; and L234A, L235A, and P329A (hIgG1-HC / LALAPA-knob, SEQ ID NO: 43), to eliminate Fc receptor binding; forming the SP34 human IgG1 heavy chain (SP34-Fc-LALAPA-knob, SEQ ID NO: 44). A light chain consists of a variable light chain (SP34-VL, SEQ ID NO: 45) of αCD3 clone SP34 and a constant human λ light chain (hLC7, SEQ ID NO: 46), forming the SP34 human light chain (SP34-hLC7, SEQ ID NO: 47). Figure 8 and Table 3 show that the protein was well expressed in high purity by both SDS-PAGE and SEC assays. Figure 8K-L indicates that the knock-in-hole mutation promotes a near 1:1 ratio of FcγR3A-Fc and SP34-Fc arms, and the FcRKO mutation prevents self-binding between FcγRIIIA and the heterodimer Fc, which is corroborated by the high purity results of the protein.

[0476] Table 3. Protein expression and monomers of IgR generated in Example 3 as determined by SEC.

[0477] Protein CHO (mg / L) % monomer content as determined by SEC: JIB610790.8% surface

[0478] Example 4. Expression and purification of αFc / αCD3 fusion

[0479] The Fc-binding heterologous polypeptide was expressed and purified as described in Example 1.

[0480] The αFc VHH (Fc-10-VHH, SEQ ID NO: 48) capable of binding to human Fc was fused via a (G4S)3 linker to a humanized version of αCD3 ScFv cloned with VL-VH orientation using a (G4S)3 linker to form the αFc-VHH / ScFv-HzUCHT1 fusion (SEQ ID NO: 49, SEQ ID NO: 50), as depicted in Figure 2A. Figure 8 and Table 4 show that the protein was well expressed in high purity as determined by both SDS-PAGE and SEC.

[0481] Table 4. Protein expression of IgR generated in Example 4 and monomers as determined by SEC.

[0482] Protein CHO (mg / L) % monomer content as determined by SEC: JIB 4315 97.7% surface

[0483] Example 5. Expression and purification of FcγRIV / αCD3 fusion

[0484] The Fc-binding heterologous polypeptide was expressed and purified as described in Example 1.

[0485] The extracellular domain of the mouse FcγRIV Fc receptor (mFcγRIV ECD, SEQ ID 8) was fused via a (G4S)3 linker to form FcγRIV / ScFv-2C11 fusions (SEQ ID NO: 54, SEQ ID 55) using a (G4S)4 linker with a VH-VL orientation. 58) FcγRIV / ScFv-KT3 fusion complexes (SEQ ID NO: 59, SEQ 60) were formed, as depicted in Figure 2A; αCD3 ScFv clones 500A2 (500A2-VH SEQ ID NO: 61; 500A2-VL SEQ ID NO: 62, ScFv-500A2 SEQ ID NO: 63) with a (G4S)4 linker were used to form FcγRIV / ScFv-500A2 fusion complexes (SEQ ID NO: 64, SEQ ID 65), as depicted in Figure 2A. Figure 9 and Table 5 show that the protein was well expressed in high purity as determined by both SDS-PAGE and SEC.

[0486] Table 5. Protein expression and monomers of IgR generated in Example 5 as determined by SEC.

[0487] Protein CHO (mg / L) Monomer % as determined by SEC: FcγRIV / ScFv-2C11-his 2481.4; FcγRIV / ScFv-KT3-his 5692.6; FcγRIV / ScFv-500A2-his 7575.4 surface

[0488] Example 6. Expression and purification of αMSA / FcγRIV / αCD3 fusion

[0489] The Fc-binding heterologous polypeptide was expressed and purified as described in Example 1.

[0490] VHH (SEQ ID NO: 66) targeting mouse serum albumin (MSA) was fused via a (G4S)3 linker to the extracellular domain of the mouse FcγRIV Fc receptor (FcγRIV, SEQ ID NO: 24), which was then fused via a (G4S)3 linker to αCD3 ScFv clone 500A2 with a VH-VL orientation using a (G4S)4 linker to form αMSA-VHH / FcγRIV / αCD3-ScFv fusions (SEQ ID NO: 67, SEQ ID NO: 68), as depicted in Figure 3A. For purification purposes, a 6his tag with a GG spacer region was fused to the C-terminus of ScFv. Figure 9 shows that the protein was well expressed with reasonable purity from single-step purification as determined by both SDS-PAGE and SEC assays.

[0491] Table 6. Protein expression and monomers of IgR generated in Example 6 as determined by SEC.

[0492] Protein CHO (mg / L) SEC determination of monomer % αMSA-VHH / FcγRIV / αCD3-ScFv-his 4854.0 surface

[0493] Example 7. Expression and purification of FcγRIV-Fc / αCD3-Fc heterodimer protein

[0494] The Fc-binding heterodimeric protein was expressed and purified as described in Example 1.

[0495] The FcγRIV-Fc / αCD3-Fc fusion was expressed as three polypeptide chains to assemble into a soluble protein comprising two heterodimers assembled using a knob-in-hole mutation to form a heavy chain and a light chain assembled with one of the two heterodimer heavy chains, as depicted in Figure 4A. A heavy chain is formed by the extracellular domain of the mouse FcγRIV Fc receptor, which is fused via a (G4S)3 linker to a portion of the mouse IgG2a heavy chain with the following mutations: E356K, T364S, M368L, T370K, D399K, and R411T for generating the Knob-in-holes A (KiH-A) Fc heavy chain; and L234A, L235A, and P329G for eliminating Fc receptor binding (KiH-A / LALAPG / mIgG2a-Fc, SEQ ID NO: 69); forming the FcγRIV-Fc polypeptide (FcγRIV / mIgG2a-Fc SEQ ID NO: 70). Another heavy chain consists of a variable heavy chain of αCD3 clone 500A2 and a constant heavy chain of human IgG2a with the following mutations: T364S, M368L, T370K, K409D, R411T, and K439D for generating the Knob-in-holes B (KiH-B) Fc heavy chain; and L234A, L235A, and P329G for eliminating Fc receptor binding (mIgG2a-HC / KiH-B / LALAPG, SEQ ID NO: 71); forming the 500A2 human IgG2a heavy chain (500A2-HC-Fc, SEQ ID 72). The light chain is composed of a variable light chain (500A2-VL, SEQ ID NO: 32) of αCD3 clone 500A2 and a mouse constant κ light chain (mLC, SEQ ID NO: 73), forming the 500A2 mouse light chain (500A2-mLC, SEQ ID NO: 74). Figure 9 and Table 6 show that the protein was well expressed in high purity by both SDS-PAGE and SEC assays. The knock-in-hole mutation induced an approximately 1:1 ratio between FcγRIV-Fc and 500A2-Fc, and the FcR-KO Fc mutation prevented any self-binding between FcγRIV and the heterodimeric fusion protein Fc.

[0496] Table 7. Protein expression and monomers of IgR generated in Example 7 as determined by SEC.

[0497] Protein CHO (mg / L) monomer % determined by SEC SEQ IDs 70, 72, 7423596.8 surface

[0498] Example 8. By combining anti-CD20 IgG with IgRs targeting CD3 or TCR, T cells were redirected to hematological malignancies.

[0499] To evaluate the ability of Fc-binding T-cell conjugation (FcB-TCE) IgR to bridge hematologic cancer cells to T cells, CD20+ Raji cells and a co-culture reporter system of CD3 / TCR+ Jurkat-CD28-NFAT-Luc were used, as shown in Figure 10. Commercially available anti-CD20 rituximab IgG1 (Biointron) is used to modulate CD20+ Raji cells and is bridged to T cells via various IgR proteins, including FcγR3A / ScFv-HzUCHT1 fusion (SEQ ID NO: 28); FcγR3A / ScFv-TR66 fusion (SEQ ID NO: 33); FcγR3A / VHH-V700 fusion (SEQ ID NO: 36); αHSA-VHH / FcγR3A / αCD3-ScFv fusion (SEQ ID NO: 39); FcγR3A-Fc / SP34-Fc heterodimer (SEQ ID NO: 41, SEQ ID NO: 44, SEQ ID NO: 47); and αFc-VHH / ScFv-HzUCHT1 fusion (SEQ ID NO: 50).

[0500] Bioassays were performed as follows: (1) Raji and Jurkat-CD28-NFAT1-luc were collected from cell culture flasks and centrifuged at 300 x gravity for 5 minutes; (2) the density of Raji was adjusted to 5E5 cells / mL and the density of Jurkat-CD28-NFAT-luc was adjusted to 2.5E6 cells / mL; (3) 40 μL of Raji and Jurkat-CD28-NFAT-luc were added to 96-well U-bottom culture plates (NEST); (4) 20 μL of rituximab, motuzumab, or culture medium, i.e., RPMI 1640 (Corning) + 1% fetal bovine serum (BI), were added to the wells. The working concentration of rituximab or motuzumab was 10 times the final target concentration, or if combined with IgR, 10 μL of rituximab and 10 μL of motuzumab were used at 10 times the final target concentration. (5) Gently mix the cells and proteins and incubate them in a plate at 37°C for 6 hours; (6) After incubation, add 100 μl of Bio-Lite luciferase assay reagent (Vazyme) and transfer it to a TC-treated white transparent bottom ViewPlate-96 (Perkin Elmer) and measure the luminescence using an ELISA reader.

[0501] To validate the functionality of the assay system, commercially available anti-CD20 / anti-CD3 bispecific motuzumab (Biointron) was used as a positive control to directly bridge Raji B cells to Jurkat T cells, while cells alone or cells plus rituximab served as negative controls. Figure 11 shows that there was no statistically significant difference between cells alone and cells plus rituximab, indicating that rituximab alone cannot bridge B cells to T cells. In contrast, motuzumab at each concentration evaluated was statistically significantly different from cells alone, as expected, since it is specifically designed to bridge CD20+ cells to CD3+ T cells.

[0502] Next, the ability of 0.1 nM, 1 nM, and 10 nM IgR to bridge CD20+ Raji B cells with T cells and mediate T cell activation was evaluated in the presence and absence of 1 nM rituximab, and in a run with motuzumab as a positive control (Figure 12). As shown in Figures 13, 14, and 15, all six designs were able to drive T cell activation when combined with rituximab compared to the absence of rituximab. Figure 13 shows that the anti-TCR V700 fusion exhibited lower potency than the anti-CD3 TR66 or anti-CD3 HzUCHT1 fusion, and the TR66 fusion exhibited slightly higher potency than HzUCHT1, highlighting the importance of optimizing around T cell targets, epitopes within T cell targets, and affinity for those T cell targets. Figure 14 shows that IgR designs with half-life extension strategies, such as those fused to anti-HSA VHH or fused to IgG Fc with the FcR KO mutation, exhibit similar potency, indicating that adding VHH to the N-terminus of the FcγRIIIA region does not interfere with rituximab IgG-Fc binding, and that Fc fusion to the C-terminus of the FcγRIIIA region does not interfere with rituximab IgG-Fc binding. Figure 15 shows that using anti-Fc VHH instead of Fc receptors such as FcγRIIIA can mediate higher potency, indicating that the epitope, affinity, and stoichiometry of the Fc binding domain to IgG-Fc are important parameters to optimize (e.g., the FcγR:IgG stoichiometry is 1:1, while the anti-Fc VHH:IgG stoichiometry can be 2:1 or 1:1 depending on the epitope). Furthermore, most of the evaluated IgRs exhibited maximum luminescence values ​​similar to those of motuzumab, indicating that the combination of IgR with target cell-specific immunoglobulins can mediate T cell activity similar to that of conventional T cell conjugates (TCEs).

[0503] Example 9. By combining anti-HER2 IgG with IgR targeting CD3 or TCR, T cells were redirected to solid cancer cells.

[0504] To evaluate the ability of Fc-binding T-cell conjugation (FcB-TCE) IgR to bridge hematologic cancer cells to T cells, HER2+ SKBR3 cells and a co-culture reporter system of CD3 / TCR+ Jurkat-CD28-NFAT-Luc were used, as shown in Figure 10. Commercially available anti-HER2 trastuzumab IgG1 (Biointron) is used to modulate HER2+ SKBR3 cells and is bridged to T cells via various IgR proteins, including FcγR3A / ScFv-HzUCHT1 fusion (SEQ ID NO: 28); FcγR3A / ScFv-TR66 fusion (SEQ ID NO: 33); FcγR3A / VHH-V700 fusion (SEQ ID NO: 36); αHSA-VHH / FcγR3A / αCD3-ScFv fusion (SEQ ID NO: 39); FcγR3A-Fc / SP34-Fc heterodimer (SEQ ID NO: 41, SEQ ID NO: 44, SEQ ID NO: 47); and αFc-VHH / ScFv-HzUCHT1 fusion (SEQ ID NO: 50).

[0505] Bioassays were performed as follows: (1) SKBR3 cells were collected and cultured overnight at 37°C in 100 μL / well in complete medium at 1E5 cells / mL; (2) the cells were resuspended the next day in 50 μL of Jurkat-CD28-NFAT-luc at 1E6 cells / mL; (3) 25 μL of rituximab (negative control), trastuzumab, or medium, i.e., RPMI 1640 (Corning) + 1% fetal bovine serum (BI), were added to the wells. The working concentration of rituximab or trastuzumab was 4 times the final target concentration of 1 nM. 25 μL of IgR was used at 4 times the final target concentrations of 0.1 nM, 1 nM, and 10 nM; (5) the cells and proteins were gently mixed and incubated in plates at 37°C for 6 hours; (6) after incubation, 100 μL of traceuticals were added to the wells. I dispensed μl of Bio-Lite luciferase assay reagent (Vazyme) and transferred it to a TC-treated white transparent bottom ViewPlate-96 (Perkin Elmer), and measured the luminescence using a microplate reader.

[0506] In the presence of 1 nM trastuzumab, the ability of 0.1 nM, 1 nM, and 10 nM IgR to bridge HER2+ SKBR3 breast cancer cells with T cells and mediate T cell activation was evaluated, while its ability to prevent T cell activation in the presence of anti-CD20 rituximab was also verified. As shown in Figures 17, 18, and 19, in the absence of CD20 in SKBR3 cells, all six designs drove more T cell activation when combined with trastuzumab compared to IgG and rituximab unrelated to the SKBR3 cancer cell line. Figure 16 shows that the anti-TCR V700 fusion exhibited lower potency than the anti-CD3 TR66 or anti-CD3 HzUCHT1 fusion, and the TR66 fusion exhibited higher potency than HzUCHT1, highlighting the importance of optimizing around T cell targets, epitopes within T cell targets, and affinity for those T cell targets. Figure 17 shows that IgR designs with half-life extension strategies, such as those fused to anti-HSA VHH or fused to IgG Fc with an FcR KO mutation, exhibit similar potency, indicating that adding VHH to the N-terminus of the FcγRIIIA region does not interfere with trastuzumab IgG-Fc binding, and that Fc fusion to the C-terminus of the FcγRIIIA region does not interfere with trastuzumab IgG-Fc binding. Figure 18 shows that using anti-FcVHH instead of Fc receptors such as FcγRIIIA can mediate higher potency, indicating that the epitope, affinity, and chemometry of the Fc binding (FcB) region for IgG-Fc are important parameters to optimize (e.g., the FcγR:IgG chemometry is 1:1, while the anti-Fc VHH:IgG chemometry can be 2:1 or 1:1 depending on the epitope). The ability of IgR combined with rituximab in Example 8 and trastuzumab in Example 9 to activate T cells across both blood and solid tumor cancer cells and across two different antigen targets demonstrates that IgR can exert a broad range of effects, independent of immunoglobulin target antigens and target cell types.

[0507] Example 10. Expression, purification, and evaluation of FcγR variants fused to αCD3

[0508] Fc-binding heteropeptides can be expressed and purified as described in Example 1.

[0509] The extracellular domain of the human CD16A Fc receptor (FcγR3A-V158, SEQ ID NO: 1) can be shortened by removing 15 amino acids from the C-terminus (FcγR3AV-short, SEQ 3), which can then be fused via a (G4S)3 linker to a humanized version of αCD3 ScFv cloned with VL-VH orientation using a (G4S)3 linker to form the FcγR3AV-short / ScFv-HzUCHT1 fusion (SEQ ID NO: 75, SEQ ID NO: 76), as depicted in Figure 2A. The extracellular domain of a human CD16A Fc receptor with an S197P mutation (FcγR3AV-S197P) and LVGSKNV in domain 2 of FcγR3A replaced by MGKHRY from FcγR1 (SEQ 77) (FcγR3A-V158, SEQ ID NO: 1) can be fused via a (G4S)3 linker to: a humanized version of HzUCHT1 cloned with αCD3 ScFv in VL-VH orientation using a (G4S)3 linker to form an FcγR3A-MGKHRY / ScFv-HzUCHT1 fusion (SEQ ID NO: 78, SEQ ID 79), as depicted in Figure 2A. The extracellular domain of the human CD64 Fc receptor (FcγR1, SEQ ID NO: 12) can be truncated to remove domain 3 (FcγR1-D1-D2, SEQ 80) and fused via a (G4S)3 linker to: a humanized version of HzUCHT1 cloned from αCD3 ScFv with VL-VH orientation using a (G4S)3 linker to form the FcγR1-D1-D2 / ScFv-HzUCHT1 fusion (SEQ ID NO: 81, SEQ ID NO: 82), as depicted in Figure 2A. The extracellular domain of the human CD32A Fc receptor (FcγR2A-H131, SEQ ID NO: 9) can be fused via a (G4S)3 linker to form a humanized version of HzUCHT1 cloned from αCD3 ScFv with a VL-VH orientation using a (G4S)3 linker, to form FcγR2AH / ScFv-HzUCHT1 fusions (SEQ ID NO: 83, SEQ ID NO: 84), as depicted in Figure 2A. The extracellular domain of the human CD32B / C Fc receptor (FcγR2BC, SEQ ID NO: 11) can be fused via a (G4S)3 linker to form a humanized version of HzUCHT1 cloned from αCD3 ScFv with a VL-VH orientation using a (G4S)3 linker, to form FcγR2BC / ScFv-HzUCHT1 fusions (SEQ ID NO: 85, SEQ ID NO: 86), as depicted in Figure 2A.The extracellular domain of the mouse Fc receptor RIV (FcγRIV, SEQ ID NO: 8) can be fused via a (G4S)3 linker to a humanized version of HzUCHT1 cloned from αCD3 ScFv with a VL-VH orientation using a (G4S)3 linker to form FcγR2BC / ScFv-HzUCHT1 fusions (SEQ ID NO: 87, SEQ ID NO: 88), as depicted in Figure 2A.

[0510] Optionally, other anti-CD3, anti-TCR, or other immune cell surface peptide-binding domains can be used and fused into the FcγR variants described herein. Optionally, the FcγR described herein can be fused in a Fab form with or without lipid conjugation or in an Fc fusion form. The functionality of the IgR with or without tumor-targeting IgG can be assessed using the assays reported as described in Examples 8 and 9 or using a cytotoxicity assay utilizing primary immune cells.

[0511] Example 11. Expression and purification of FcγR / αCD3 multimeric proteins in various Fab forms

[0512] Fc-binding multimer proteins can be expressed and purified as described in Example 1, or optionally using protein A (for VH3 family heavy chains), protein L (for VL1, VL3, VL4 family light chains), or anti-CH1 (for any family) affinity chromatography.

[0513] The constant heavy chain CH1 region and optionally some hinges (CH1 SEQ ID 89) and constant κ light chain (κ SEQ ID NO: 90) and optionally λ (LC1 SEQ ID NO: 91, LC2 SEQ ID NO: 92, LC3 SEQ ID NO: 93, LC7 SEQ ID NO: 46) of human IgG can be fused to the VH and VL regions of the antigen-binding domain on the surface of immune cells, respectively, where appropriate. Optionally, cysteine ​​residues formed on the interchain disulfide bonds of the constant heavy chain (C233S, CH1-NoDS SEQ ID NO: 94) and constant light chain (C214S, κNoDS SEQ ID NO: 95, hLC1NoDS SEQ ID NO: 96, hLC2NoDS SEQ ID NO: 97, hLC3NoDS SEQ ID NO: 98, hLC7NoDS SEQ ID NO: 99) can be mutated to serine residues, etc. Optionally, in addition to mutating the interchain disulfide bond forming cysteine ​​to serine, additional buried disulfide bonds can be introduced into both the constant heavy chain (F174C and C233S, CH1-bDS SEQ ID NO: 100) and the constant light chain (S176C and C214S, κbDS SEQ ID NO: 101, hLC1bDS SEQ ID NO: 102, hLC2bDS SEQ ID NO: 103, hLC3bDS SEQ ID NO: 104, hLC7bDS SEQ ID NO: 105) to form cysteine.

[0514] The VH / VL pair of HzUCHT1 can fuse to the CH1 heavy chain and the κ light chain, wherein the extracellular domains of the Fc receptor, such as FcγR3AV, fuse to the C-terminus of the constant light chain, as depicted in Figure 5A, or optionally or additionally fuse to the constant heavy chain via a (G4S)3 linker, as depicted in Figure 5B (CH1NoDS-HzUCHT1, SEQ ID NO: 106, CH1NoDS-HzUCHT1 / FcγR3AV-short, SEQ ID NO: 107, κNoDS-HzUCHT1 / FcγR3AV-short, SEQ ID NO: 108), or optionally fuse to the N-terminus of the variable heavy chain or the variable light chain via a (G4S)3 linker. FcγR3AV can be fused to the N-terminus of the CH1 domain, and optionally HzUCHT1 ScFv is fused to the C-terminus and co-expressed with a constant light chain, wherein HzUCHT1ScFv is fused to the C-terminus and optionally FcγR3AV is fused to the N-terminus, as depicted in Figures 5D, 5E, 5F, and 5G (FcγR3AV-short / CH1bDS SEQ ID NO: 109, FcγR3AV-short / CH1bDS / ScFv-HzUCHT1 SEQ ID NO: 110, κbDS / ScFv-HzUCHT1 SEQ ID NO: 111, FcγR3AV-short / κbDS / ScFv-HzUCHT1: 112). The presence or absence of IgR functionality with tumor-targeting IgG can be assessed using the assays reported as described in Examples 8 and 9 or using a cytotoxicity assay utilizing primary immune cells.

[0515] Example 12. Expression, purification, and lipid conjugation of FcγR / αCD3 multimeric peptides in various Fab forms

[0516] Fc-binding multimer proteins can be expressed and purified as described in Example 11.

[0517] As described in Example 11, a constant heavy chain CH1 domain (C233S, CH1-NoDS-Cys SEQ ID NO: 113) having a native hinge region (up to the first cysteine) and an interchain disulfide bond mutated to serine, etc., can be paired with the NoDS light chain sequence and the VH / VL pair of anti-immune cell surface proteins. Optionally, the constant heavy CH1 domain having a native hinge region (up to the first cysteine) and an interchain disulfide bond mutated to serine can introduce additional mutations in the heavy chain (F174C and C233S, CH1-bDS-Cys SEQ ID NO: 114) and the light chain sequence to produce a Fab with embedded disulfide bonds, as described in Example 11. PEG-lipids can be covalently attached to the C-terminal cysteine ​​of a constant heavy chain, as depicted in Figures 6A, 6B, 6C, 6D, 6F, and 6G; or covalently attached to the C-terminal cysteine ​​of ScFv, as depicted in Figure 6E.

[0518] The VH / VL pair of HzUCHT1 can attach to its corresponding CH1 heavy chain and κ light chain, and is fused via (G4S)3 at the C-terminus of the constant heavy chain to an immunoglobulin-binding domain such as FcγR3AV (HzUCHT1-CH1NoDS-Cys, SEQ ID NO: 115, HzUCHT1-CH1bDS-Cys, SEQ ID NO: 116, FcγR3AV-short / κNoDS-HzUCHT1, SEQ ID 108, FcγR3AV-short / κbDS-HzUCHT1, SEQ ID NO: 117), as depicted in Figure 6A. The presence or absence of IgR functionality for tumor-targeting IgG can be assessed using the assays reported as described in Examples 8 and 9, or using a cytotoxicity assay utilizing primary immune cells.

[0519] Following the expression and purification of the Fab fusion protein with a C-terminal cysteine ​​residue, lipid conjugation can be performed. First, stock solutions of maleimide-PEG2K-DSPE / MeOH-PEG2K-DSPE are prepared by dissolving maleimide-PEG2K-DSPE in 1 mM citrate at pH 6.7 to 3.4 mM (10 mg / L), and dissolving MeOH-PEG2K-DSPE in 1 mM citrate at pH 6.7 to 3.4 mM (9.54 mg / mL). These stock solutions can be stored separately or after mixing at the desired molar ratio at -80°C. For a 2:3 Mal:MeOH ratio, assuming both stock solutions are 3.4 mM, volumetric mixing is possible; for example, 200 µl of Mal stock solution + 300 µl of MeOH stock solution will yield a 500 µl mixture with a 2:3 molar ratio. If the Fab concentration has not yet reached approximately 3 mg / mL, the protein is concentrated using a 10 kD ultracel regenerated cellulose membrane, etc., and then recovered using fresh PBS. The protein is spiked with 0.5 M EDTA pH 7.5 for the final PBS + 5 mM EDTA pH 7.5.

[0520] For reduction, TCEP was dissolved in PBS + 5 mM EDTA to a final concentration of 100 mM. TCEP concentrate was spiked into the protein solution to a final concentration of approximately 0.2 mM, and reduction was carried out at room temperature for approximately 1.5 hours. After reduction, free cysteine ​​or other small molecules were removed using a 40K Zeba desalting column, and Fab was replaced with PBS + 5 mM EDTA. Following the Zeba desalting, A280 was obtained via nanodroplets, and the protein concentration was calculated. Therefore, it was possible to calculate how much Mal-PEG-DSPE / MeOH-PEG-DSPE was added for an approximately 2:1 MAL:protein ratio, which corresponds to an approximately 5:1 PEG-DSPE to protein ratio.

[0521] For the reaction, the protein was added to the MAL / MeOH mixture, mixed with a pipette, and incubated at 37°C for approximately 3 hours. After the reaction, 15 mM cysteine ​​was spiked at a 1:10 v / v ratio (for final dilution with 1.5 mM cysteine) and incubated at 37°C for approximately 15 min to quench unreacted Mal-PEG-DSPE.

[0522] For purification, dilute with 25 mM HEPES, 150 mM NaCl, pH 7.5 (HBS), concentrate with a 100 kD UF membrane, dilute > 25x, and repeat the concentration / dilution three times consecutively. This allows unreacted protein to pass through the filter while retaining the lipid-conjugated protein micelles. Carefully recover the protein micelles from the membrane, washing 1-2 times with 10-15 μL. Ideally, the final volume should be only 35-50 μL. Measure the volume using a pipette and A280 using a nanodrop or cuvette to recover the material. Run SDS-PAGE with the unreduced and reduced original material and the reduced final conjugate; you should be able to see the movement of the heavy chain bands between the conjugated Fab and the PEG-lipid-conjugated Fab. Optionally, a similar PEG-lipid conjugation and purification procedure can be performed when the Fc-binding domain / ScFv fusion protein contains a C-terminal cysteine ​​residue.

[0523] Example 13. Expression and purification of multiple FcR or Fc binding domains fused to αCD3

[0524] Fc-binding heteropeptides and heteropolymers can be expressed and purified as described in Examples 1 and 11.

[0525] Multiple immunoglobulin-binding domains of the same or different types can be fused to ScFv or Fab targeting immune cell surface proteins (as depicted in 4D, 4E, 4F, 4G), optionally having or not having a half-life extension domain, such as anti-HSA (as depicted in Figure 3B) or in the form of an Fc-Fc heterodimer protein. FcγR3A can be fused to HzUCHT1ScFv, followed by ligation of another FcγR3A (FcγR3AV-short / ScFv-HzUCHT1 / FcγR3AV-short, SEQ ID NO: 118), as depicted in Figure 2C, with an optional domain order. FcγR2A can be fused to HzUCHT1ScFv, followed by ligation of FcγR3A (FcγR2AH131 / ScFv-HzUCHT1 / FcγR3AV-short, SEQ ID NO: 119), as depicted in Figure 2C, with an optional domain order. FcαRI can be fused to FcγR3A, followed by connection to HzUCHT1 ScFv (FcαRI / FcγR3AV-short / ScFv-HzUCHT1, SEQ ID NO: 120), as depicted in Figure 2C. The two tandem domains 1 of FcαRI can be fused to HzUCHT1 ScFv (FcαRI-D1 / FcαRI-D1 / ScFv-HzUCHT1, SEQ ID NO: 121), as depicted in Figure 2C, and optionally have FcγR3A. Anti-Fc VHH can be fused to another anti-Fc, followed by connection to HzUCHT1 ScFv (anti-Fc / anti-Fc / ScFv-HzUCHT1, SEQ ID NO: 122), as depicted in Figure 2C. Anti-Fc VHH can fuse to FcγR3A, followed by ligation to HzUCHT1 ScFv (anti-Fc / FcγR3AV-short / ScFv-HzUCHT1, SEQ ID NO: 123), as depicted in Figure 2C. FcγR3A can fuse to the C-terminus of the CH1 / HzUCHT1 heavy chain (CH1-HzUCHT1 / FcγR3AV-short, SEQ ID NO: 124) and the C-terminus of the κ / HzUCHT1 light chain (κ-HzUCHT1 / FcγR3AV-short, SEQ ID NO: 125), as depicted in Figure 5B.FcγR3A can be fused to the N-terminus of IgG1-LALAPA-knobFc to form FcγR3AV-short / Fc-LALAPA-knob SEQ ID NO: 126, and optionally fused to the N-terminus of ScFv-HzUCHT1-Fc-LALAPA-hole SEQ ID 128 to form FcγR3AV-short / κ-HzUCHT1-Fc-LALAPA-hole SEQ ID NO: 129, to produce a heteropolymeric protein having one or two immunoglobulin-binding domains, as depicted in Figures 4C and 4D. Where appropriate, the presence or absence of IgR functionality targeting tumor-targeting IgG and / or IgA can be assessed using the assays reported as described in Examples 8 and 9 or using a cytotoxicity assay utilizing primary immune cells.

[0526] FcγR3A can fuse via a (G4S)3 linker to the C-terminus of a partial human IgG1 heavy chain with the following mutations: C233S for preventing disulfide bond formation; T366S, L368A, and Y407V for generating a "hole" Fc heavy chain; and L234A, L235A, and P329A for eliminating Fc receptor binding; forming the FcγR3A-Fc polypeptide (FcγR3A-short / hIgG1-Fc-LALAPA-hole SEQ ID NO: 41). Another heavy chain can be composed of a variable heavy chain from the αCD3 clone HzUCHT1 and a constant human IgG1 heavy chain with the following mutations: T366W for generating the “knob” Fc heavy chain; and L234A, L235A, and P329A for eliminating Fc receptor binding; forming the HzUCHT1 human IgG1 heavy chain (HzUCHT1-HC-Fc-LALAPA-knob, SEQ ID NO: 127). The light chain is composed of a variable light chain from the αCD3 clone HzUCHT1 and a constant human κ light chain, wherein FcγR3AV is fused to the C-terminus via a (G4S)3 linker, forming the HzUCHT1 human light chain FcγR3AV fusion (κ-HzUCHT1 / FcγR3AV-short, SEQ ID NO: 125), as depicted in Figure 4G. Expression of these three chains will produce a heteropolymer protein with two immunoglobulin-binding domains.

[0527] Using the Fab form, FcγR3A can be fused to the C-terminus of CH1 (CH1-HzUCHT1 / FcγR3AV-short, SEQ ID NO: 124) and the C-terminus of the constant light chain (κ-HzUCHT1 / FcγR3AV-short, SEQ ID NO: 125) to form a heteropolymeric protein with two immunoglobulin domains, as depicted in Figure 5B.

[0528] Example 14. Expression and purification of the immunoglobulin-binding domain fused to αCD3

[0529] Immunoglobulin-binding heteropeptides and heteropolymers can be expressed and purified as described in Examples 1 and 11.

[0530] Immunoglobulin-binding domains such as anti-CH1 VHH (VHH-CH128 SEQ ID 130) can be fused to various immunocell-binding domains specific to CD3, TCR, or other immune cell surface peptides using a (G4S)3 linker to form VHH-CH128 / ScFv fusions. Anti-CD3 HzUCHT1 ScFv is fused to CH128 VHH via a (G4S)3 linker to form CH128 / ScFv-HzUCHT1 SEQ ID NO: 131. Aviders targeting the Fc region of IgG (AVIG SEQ ID 132) can be fused to HzUCHT1 ScFv to form AVIG / ScFv-HzUCHT1 SEQ ID NO: 133. Anti-IgG VH / VL pairs (17F12 VH SEQ ID NO: 134, 17F12 VL SEQ ID NO: 135) can be fused to the immune cell targeting domain in the form of ScFv (ScFV-17F12 SEQ ID 136) to form ScFv-17F12 / ScFv-HzUCHT1: 137, or optionally as Fab, where one of the immune cell targeting domains is attached to a light constant light chain, a heavy chain, or both (CH1-NoDS-17F12 SEQ ID NO: 138, CH1-NoDS-17F12 / ScFv-HzUCHT1 SEQ ID NO: 140, κNoDS-17F12 / ScFv-HzUCHT1 SEQ ID NO: 137). The C-terminus of 141), as depicted in Figures 5C and 5H, or the Fc fusion having one or more point mutations in the constant heavy chain to significantly reduce 17F12 binding, as depicted in Figure 4A, wherein IgBD is the complete Fab containing 17F12 VH / VL pairs attached to its corresponding constant heavy and light chains. Optionally, an anti-IgG Fab having an immune cell-targeting domain attached to the C-terminus of the light constant light chain (CH1-NoDS-17F12-Cys SEQ ID NO: 139, κNoDS-17F12 / ScFv-HzUCHT1 SEQ ID NO: 141) can be conjugated to lipids via a free cysteine ​​residue at or near the C-terminus, as depicted in Figure 6B. The function of IgR with or without tumor-targeting IgG can be assessed using the assays reported as described in Examples 8 and 9 or using a cytotoxicity assay utilizing primary immune cells.

[0531] Example 15. Expression and purification of FcγR fused to αCD3 and αTCR

[0532] Fc-binding heteropeptides can be expressed and purified as described in Example 1.

[0533] Immunoglobulin-binding domains, such as the extracellular domains of human CD16A Fc receptors with the S197P mutation (FcγR3AV-S197P SEQ ID 23) or optionally with 15 amino acids removed from the C-terminus, can be fused using a (G4S)3 linker to various VH / VL pairs specific for CD3 and TCR to form FcγR3AV-ScFv fusions: anti-CD3 Hu291 (Hu291-VH SEQ: 142, Hu291-VL SEQ ID NO: 143, ScFv-Hu291 SEQ ID NO: 144, FcγR3AV-short / ScFv-Hu291 SEQ ID NO: 145), anti-CD3 huCLB-T3 / 4.A (huCLB-VH SEQ ID NO: 146, huCLB-VL SEQ ID NO: 147, ScFv-huCLB SEQ ID NO: 148). 148, FcγR3AV-short / ScFv-huCLB SEQ ID NO: 149), anti-CD3 HuYTH 12.5 (12.5-VH SEQ ID NO: 150, 12.5-VL SEQ ID NO: 151, ScFv-12.5 SEQ ID NO: 152, FcγR3AV-short / ScFv-12.5 SEQ ID NO: 153), anti-CD3 TR66 - high affinity (TR66HF-VH SEQ ID NO: 154, TR66HF-VL SEQ ID NO: 155, ScFv-TR66HF SEQ ID NO: 156, FcγR3AV-short / ScFv-TR66HF SEQ ID NO: 157), anti-CD3 G19-4 (G19-4-VH SEQ ID NO: 158, G19-4-VL SEQ ID NO: 159, ScFv-G19-4 SEQ ID NO: 160, FcγR3AV-short / ScFv-G19-4 SEQ ID NO: 161), anti-CD3 BC3 (BC3-VH SEQ ID NO: 162, BC3-VL SEQ ID NO: 163, ScFv-BC3 SEQ ID NO: 164, FcγR3AV-short / ScFv-BC3 SEQ ID NO:165), anti-CD3 Hz06 (Hz06-VH SEQ ID NO: 166, Hz06-VL SEQ ID NO: 167, ScFv-Hz06 SEQID NO: 168, FcγR3AV-short / ScFv-Hz06 SEQ ID NO: 169), anti-CD3 Ly17.2G3 (Ly17-VHSEQ ID NO: 170, Ly17-VL SEQ ID NO: 171, ScFv-Ly17 SEQ ID NO: 172, FcγR3AV-short / ScFv-Ly17 SEQ ID NO: 173), anti-TCR TOL-101 (TOL-VH SEQ ID NO: 174, TOL-VLSEQ ID NO: 175, ScFv-TOL SEQ ID NO: 176, FcγR3AV-short / ScFv-TOL SEQ ID NO: 177), anti-TCR hJOV1 (hJOV1-VH SEQ ID NO: 178, hJOV1-VL SEQ ID NO: 179, ScFv-hJOV1SEQ ID NO: 180. FcγR3AVshort / ScFv-hJOV1 SEQ ID NO: 181), anti-TCR BMA-031 (BMA-VH SEQ ID NO: 182, BMA-VL SEQ ID NO: 183, FcγR3AV-short / ScFv-BMA SEQ ID NO: 184). Optionally, the VH / VL pairs in this embodiment or other embodiments may be incorporated in the form of an Fc fusion as described in Example 3, or in the form of a Fab with or without a C-terminus and with or without lipid conjugation as described in Examples 11 and 12. The function of IgR with or without tumor-targeting IgG can be assessed using the reported assays as described in Examples 8 and 9 or using a cytotoxicity assay.

[0534] Example 15. Expression and purification of FcγR, a co-stimulatory, immune checkpoint, and immunosuppressive target fused to T cells.

[0535] Fc-binding heteropeptides and heteropolymers can be expressed and purified as described in Examples 1 and 11.

[0536] Immunoglobulin-binding domain (IGN) Fc fusion proteins can be co-expressed with immune cell-binding domain (ICB) Fc proteins to generate multiple heterodimer proteins, in which one or two IGN domains are combined with one or two ICB domains, as depicted in Figure 4. Three polypeptide chains can be assembled into a soluble protein comprising two heterodimers assembled using a knock-in-hole mutation to form a heavy chain and a light chain assembled with one of the two heterodimer heavy chains. One heavy chain can consist of IGN domains such as those fused via a (G4S)3 linker to a human CD16A Fc receptor with the following mutations in the partial human IgG1 heavy chain: C233S for preventing disulfide bond formation; T366S, L368A, and Y407V for generating a "hole" Fc heavy chain; and L234A, L235A, and P329A for eliminating Fc receptor binding; forming the FcγR3A-Fc polypeptide. Another heavy chain can consist of variable heavy chains from various T-cell co-stimulatory and immune checkpoint-targeting clones (anti-CD137 5B9, uroselumab, P566 (uroselumab), Hz4B4-2; anti-CD28 2E12, TGN1412, 9.3; anti-PD1 nivolumab, pembrolizumab; anti-CTLA4 ipilimumab; anti-CD11a hMHM24; anti-CD18 antibody rhuMab) and constant human IgG1 heavy chains with the following mutations: T366W for generating the "knob" Fc heavy chain; and L234A, L235A, and P329A for eliminating Fc receptor binding. Light chains can consist of corresponding variable light chains from various T-cell co-stimulatory and immune checkpoint-targeting clones, and appropriate human constant κ or λ light chains. Optionally, these T cell co-stimulatory or immune checkpoint-targeting clones can be generated as ScFv and fused to the C-terminus or N-terminus of the immunoglobulin-binding domain. Another T cell-targeting Fab or Fc-fusion or anti-T cell-targeting ScFv light chain, such as anti-CD3 or TCR, can be fused to the C-terminal light chain of the anti-T cell immune checkpoint or co-stimulatory light chain, for example, CH1NoDS-17F12 / ScFv-9.3 SEQ ID NO: 220, κNoDS-17F12 / ScFv-HzUCHT1 SEQ ID NO: 141, Fig. 5H; FcγR3AV-short / CH1bDS / ScFv-9.3 SEQ ID NO: 221, κbDS / ScFv-HzUCHT1 SEQ ID NO: 111, FcγR3AV-short / κbDS / ScFv-HzUCHT1 SEQ ID 112, Fig. 5F, 5G). The presence or absence of IgR with tumor-targeting IgG function can be assessed using the assays reported as described in Examples 8 and 9 or using cytotoxicity or activation assays utilizing primary immune cells.

[0537] Example 16. Expression and purification of FcγR, an effector function, co-stimulatory agent, and immune checkpoint target fused to myeloid cells, B cells, and NK cells.

[0538] Fc-binding heteropeptides and heteropolymers can be expressed and purified as described in Examples 1 and 11.

[0539] Immunoglobulin-binding domains (such as Fc receptor, anti-Fc, anti-CH1, and anti-IgG) can be fused to immune cell surface polypeptide-binding domains on myeloid cells, B cells, and NK cells in the form of ScFv fusion, or optionally incorporated in the form of Fc fusion (with mutations to appropriately knock out anti-CH1 or anti-IgG binding) or Fab (with mutations to appropriately knock out anti-CH1 or anti-IgG binding) as described in Example 3. These constructs may or may not have a C-terminus and may or may not undergo lipid conjugation, as described in Examples 11 and 12. The functionality of IgRs with or without tumor-targeting IgG can be assessed using reporter assays, primary cell activation assays, cytotoxicity assays, or phagocytosis assays similar to those in Examples 8 and 9. FcγR3a can be fused to anti-CD89 VH / VL pairs (14A8, A77, 8D2) to target myeloid cells. FcγR3a can fuse to anti-CD40 VH / VL pairs (40.2.220), CD40L, and anti-CD79 (SN8, 2F2) to target B cells. FcγR3a can also fuse to anti-NKp46 VH / VL pairs (NKp46-1, NKp46-4) to target NK cells.

[0540] Example 17. Expression and purification of the half-life extension domain fused to the immunoglobulin binding domain and the immune cell binding domain.

[0541] Fc-binding heteropeptides and heteropolymers can be expressed and purified as described in Examples 1 and 11.

[0542] Anti-HSA or HSA can be fused to the N-terminus or C-terminus, or optionally fused between the immunoglobulin-binding domain and the immune cell-binding domain, as depicted in Figure 3. Anti-HSA VHH or HSA (SEQ ID NO: 266) can be fused to the C-terminus of FcγR3A, which is fused to anti-CD3 ScFv UCHT1, which is fused to another FcγR3A to form anti-HSA / FcγR3A / ScFv-HzUCHT1 / FcγR3A SEQ ID NO: 267 and HSA / FcγR3A-short / ScFv-HzUCHT1 / FcγR3A SEQ ID NO: 268. HSA can also be fused to FcγR3A-short / ScFv-HzUCHT1 to form HSA / FcγR3A-short / ScFv-HzUCHT1 SEQ ID 269. Optionally, anti-HSA can be fused to an immunoglobulin-binding domain and two immune cell-binding domains (anti-HSA / FcγR3A / ScFv-HzUCHT1 / ScFv-9.3 SEQ ID NO: 270).

[0543] Example 18. Expression, purification, and evaluation of FcγR variants, IgG-binding variants, and multimeric proteins fused to αCD3.

[0544] IgG-binding heteropeptides and heteropolymers can be expressed and purified as described in Examples 1 and 11. IgG-binding polymers can be expressed and purified as described in Example 1, or optionally using protein A (for VH3 family heavy chains), protein L (for VL1, VL3, VL4 family light chains), or anti-CH1 (for any family) affinity chromatography.

[0545] The following fusion compounds from Example 10 have been generated: FcγR3AV-short / ScFv-HzUCHT1-his (JIB15, SEQ ID NO: 76), FcγR3A-MGKHRY / ScFv-HzUCHT1-his (JIB16, SEQ ID 79), FcγR1-D1-D2 / ScFv-HzUCHT1 (JIB17, SEQ ID NO: 82), FcγR2AH / ScFv-HzUCHT1-his (JIB18, SEQ ID 84), and FcγR2BC / ScFv-HzUCHT1 (JIB23, SEQ ID NO: 86). Meanwhile, VHH-CH128 / ScFv-HzUCHT1-his was generated using SEQ ID NO: 131, and product JIB21 (SEQ ID NO: 86) was generated using SEQ ID NO: 21 from Example 14. 278), as depicted in Figure 2A.

[0546] The VH of HzUCHT1 fuses to the CH1 heavy chain and the his tag (CH1NoDS-HzUCHT1 SEQ ID NO: 106; his SEQ ID NO: 21) to form CH1NoDS-HzUCHT1-his (SEQ ID NO: 279), and the VL of HzUCHT1 fuses to the κ light chain (κNoDS, SEQ ID NO: 108), wherein the extracellular domain of the Fc receptor FcγR3AV-S197P (SEQ ID NO: 23) fuses via (G4S)3 to the C-terminus of the constant light chain (κNoDS-HzUCHT1-FcγR3AV-S197P, SEQ ID NO: 280), as depicted in Figure 5A, to form JIB19 (SEQ ID NO: 279 and SEQ ID NO: 280). 280), or optionally or additionally, fused to the C-end of a constant heavy chain via a (G4S)3 joint to form CH1NoDS-HzUCHT1-FcγR3AV-S197P-his, SEQ ID NO: 281 (CH1NoDS-HzUCHT1 SEQ ID NO: 106; FcγR3AV-S197P SEQ ID NO: 23, his SEQ ID NO: 21), as depicted in FIG5B, to form JIB20 (SEQ ID 280, SEQ ID 281).

[0547] Anti-IgG clone 17F12 (a known human IgG1, IgG2, IgG3, and IgG4 binding domain that cross-reacts with non-human primate IgG) was fused to a human CH1 heavy chain (SEQ ID 138) with a his tag (SEQ ID 21) to form CH1NoDS-17F12-his (SEQ ID 282) and a κ light chain (κNoDS-17F12, SEQ ID NO: 112). A humanized version of the αCD3 ScFv clone HzUCHT1 oriented with a VL-VH orientation was fused to the C-terminus of a constant light chain via a (G4S)3 linker to form κNoDS-17F12 / ScFv-HzUCHT1 (SEQ ID NO: 141), thereby generating the heterodimeric polypeptide JIB22 (SEQ ID NO: 282, SEQ ID NO: 141), as depicted in Figure 5C.

[0548] Figures 19 and 20 and Table 8 show that the proteins were well expressed and had high to reasonable purity as determined by SDS-PAGE and SEC, except for JIB16 and JIB22, whose monomer percentages as determined by SEC were 4.3% and 13.3%, respectively. Interestingly, when comparing their SDS-PAGE patterns, laddering was observed in anti-IgG 17F12 molecule JIB22 even when the sample was reduced and denatured, suggesting that the binding region of the VH / VL pair may bind a portion of the constant region.

[0549] Table 8. Protein expression and monomers of IgR produced in Example 18 as determined by SEC.

[0550] Protein CHO (mg / L) % of monomers determined by SEC: JIB152 156 9.6%; JIB16 9 14.3%; JIB17 11 96.0%; JIB18 8 87 4.6%; JIB19 17 0 89.4%; JIB20 18 4 84.9%; JIB21 5 86 7.0%; JIB22 7 0 13.3%; JIB23 10 16 3.8% surface

[0551] Next, using the methods described in Example 8, the ability of 0.1 nM and 1 nM IgR to bridge CD20+ Raji B cells with T cells and mediate T cell activation in the presence of 1 nM rituximab was evaluated, along with its ability to prevent T cell activation in the presence of 1 nM trastuzumab or when run with motuzumab as a positive control (Figure 21). The use of FcγR3A-short in JIB15 did not reduce its potency compared to the untruncated FcγR3A form in JIB1, indicating that the FcγR3A amino acids near the C-terminus of the extracellular domain (including the absence of the ADAM17 cleavage site) are not essential for IgG binding. Fusions containing FcγR2A (JIB18), FcγR2BC (JIB23), and anti-CH1 VHH (JIB21) mediated T cell activation in the presence of rituximab, with significantly greater activation than in the presence of trastuzumab. This indicates that IgR can utilize multiple Fc receptor types that block binding to homologous immunoglobulin Fc receptors in vivo, and that IgR can utilize IgG binding domains that do not block binding to homologous immunoglobulin Fc receptors in vivo. Compared to FcγR2A-based JIB18 and FcγR2BC-based JIB23, a similarly designed IgR (JIB17) containing FcγR1 lacking domain 3 failed to mediate significant rituximab signaling compared to trastuzumab-based activation, suggesting that the removal of domain 3 in FcγR1 significantly reduces its function. Compared to fusions containing FcγR3A and HzUCHT1 (such as JIB1 and JIB15), a reduced potency was observed in the HzUCHT1 Fab fusion, where one FcγR3A is fused to the C-terminus of the constant light chain (JIB19). Given this result, we surprisingly observed that an IgR identical to JIB19 but with a second FcγR3A fused to the C-terminal constant heavy chain, totaling two FcγR3As per IgR molecule (JIB20), exhibited significant activation (greater than JIB1, JIB15, or JIB19) with 1 nM and 0.1 nM rituximab, and significantly greater than activation in the presence of non-target-specific trastuzumab, with no elevated levels of non-specific activation from trastuzumab relative to cells alone. These results suggest that IgR molecules with two or more IgG-binding domains can have enhanced potency compared to those with only one IgG-binding domain.

[0552] Example 19. Expression and purification of variant FcR, IgG binding domain or Fc binding domain fused to αCD3

[0553] IgG and Fc-binding heterologous peptides and heterologous multimers can be expressed and purified as described in Examples 1 and 11.

[0554] Wild-type VH (SEQ ID NO: 134) of anti-IgG clone 17F12 and a mutant form of VL for improved expression and stability (17F12QUAD-VL, SEQ ID NO: 283) were fused into a mutant form of the CH1 constant heavy chain containing P126S and K213E (Kabat number) to eliminate the self-binding of 17F12 (C4CH1-bDS, SEQ ID NO: 284) to form (C4CH1-bDS-17F12-his, SEQ ID NO: 285) and a κ light chain (κbDS-17F12QUAD, SEQ ID NO: 286). A humanized version of the αCD3 ScFv clone HzUCHT1 oriented with a (G4S)3 linker in a VL-VH orientation was fused into the C-terminus of the constant light chain to form (κ-bDS-17F12QUAD-ScFv-HzUCHT1, SEQ ID NO: 286). 287), thereby generating the heterodimeric polypeptide JIB33 (SEQ ID NO: 285 and 287), as depicted in Figure 5C.

[0555] Wild-type anti-Fc clone HP6017 is known to bind to human IgG1, IgG2, IgG3, IgG4 and non-human primate IgG. HP6017-VH, SEQ ID NO: 288, is fused to the embedded disulfide CH1 heavy chain (CH1-bDS, SEQ ID NO: 100) to form CH1-bDS-HP6017-his (SEQ ID NO: 289), and the wild-type VL (HP6017-VL, SEQ ID NO: 290) is fused to the embedded disulfide κ light chain (κbDS, SEQ ID NO: 101) to form κbDS-HP6017 (SEQ ID NO: 291). Furthermore, a humanized version of the αCD3 ScFv clone HzUCHT1 oriented with a (G4S)3 linker in a VL-VH configuration is fused to the C-terminus of the constant light chain to form (κ-bDS-HP6017 / ScFv-HzUCHT1, SEQ ID NO: 292), thereby generating the heterodimeric polypeptide JIB34 (SEQ ID NO: 292). 289 and 292), as depicted in Figure 5C.

[0556] The anti-Fc avimer from Example 14, namely AVIG SEQ ID NO: 133, is fused to the his tag to form the AVIG / ScFv-HzUCHT1-his fusion JIB35 (SEQ ID NO: 293), as depicted in Figure 2A.

[0557] According to the residue numbering in SEQ ID NO: 9, the extracellular domain of the human CD32A Fc receptor contains point mutations R56H, K118N, T120V, L160Q, and V172E (FcγR2A_mut, SEQ ID NO: 294) to increase Fc affinity and fuse via (G4S)3 linker to a humanized version of the αCD3 ScFv clone HzUCHT1 with a VL-VH orientation using the (G4S)3 linker to form the FcγR2A_mut / ScFv-HzUCHT1-his fusion JIB36 (SEQ ID 295), as depicted in Figure 2A.

[0558] According to the residue number in SEQ ID NO: 1, the extracellular domain of the human CD16A Fc receptor contains point mutations K122N, T124V, Q176E (FcγR3A_mut1, SEQ ID NO: 296) to increase Fc affinity and fuse via (G4S)3 linker to a humanized version of αCD3 ScFv clone HzUCHT1 with VL-VH orientation using (G4S)3 linker to form FcγR3A_mut1 / ScFv-HzUCHT1-his fusion JIB37 (SEQ ID 297), as depicted in Figure 2A.

[0559] According to the residue numbering in SEQ ID NO: 1, the extracellular domain of the human CD16A Fc receptor contains point mutations I90R, T118K, A119L, and Y134F (FcγR3A_mut2, SEQ ID NO: 298) to alter Fc affinity and fuse via (G4S)3 linker to a humanized version of αCD3 ScFv clone HzUCHT1 with VL-VH orientation using (G4S)3 linker to form FcγR3A_mut2 / ScFv-HzUCHT1-his fusion JIB38 (SEQ ID 299), as depicted in Figure 2A.

[0560] Domain 2 of the extracellular domain of the human CD64 Fc receptor is fused with domain 1 of the CD16A Fc receptor to form FcγR1-D1-FcγR3A-D2 (SEQ ID NO: 300), and then fused via a (G4S)3 linker to a humanized version of αCD3 ScFv clone HzUCHT1 oriented with a (G4S)3 linker in a VL-VH orientation to form the FcγR1-D1-FcγR3A-D2 / ScFv-HzUCHT1-his fusion JIB39 (SEQ ID 301), as depicted in Figure 2A.

[0561] The VH / VL pair of HzUCHT1 was fused to the CH1 heavy chain and κ light chain, wherein the extracellular domain of the Fc receptor FcγR3AV was fused to the N-terminus of the variable heavy chain (FcγR3AV-S197P / HzUCHT1-CH1-bDS-his, SEQ ID NO:302) and the variable light chain (FcγR3AV-S197P-HzUCHT-κ-bDS SEQ ID NO:303) via a (G4S)3 linker, and the chains of SEQ ID 302 and 303 were co-expressed to generate JIB40, as shown in Figure 5K.

[0562] Figures 22 and 23 and Table 9 show that the proteins were well expressed and had high purity as determined by SDS-PAGE and SEC, except for JIB34 and JIB39, as shown in Table 2 below, where their monomer percentages as determined by SEC were 51.8% and 21.1%, respectively. Interestingly, compared with the wild-type counterpart JIB22, the 17F12 variant JIB33 contains a mutation in its CH1 constant domain to prevent self-binding and a mutation in its VL to improve expression, and did not show ladder-like banding as determined by SDS-PAGE in the reduced lane (Figure 22). Furthermore, JIB33 has a significantly improved monomer percentage as determined by SEC compared with JIB22, 86.8% vs. 13.3%, respectively. These results indicate that the point mutations P126S and K213E in the constant CH1 domain eliminate self-binding in JIB33, and that the binding epitopes of the 17F12 clone are located in the CH1 domain. Although the purity determined by SEC was moderate, unlike wild-type 17F12JIB22, the SDS-PAGE of HP6017 protein JIB34 did not show ladder-like bands in the reduced lane (Fig. 22), indicating that it does not self-bind when it only contains the CH1 and κ constant regions and lacks the Fc region, which is consistent with its known human IgG1, IgG2, IgG3, and IgG4 Fc binding domains. The SDS-PAGE profile of JIB39, the FcγR1-D1-FcγR3A-D2 / ScFv-HzUCHT1-his fusion, showed ladder-like bands in the non-reduced lane, indicating disulfide bond mismatch.

[0563] Table 9. Protein expression and monomers of IgR produced in Example 19 as determined by SEC.

[0564] Protein CHO (mg / L) Monomer content determined by SEC: JIB338786.8% JIB347451.8% JIB356878.4% JIB3614085.5% JIB3714077.8% JIB3810380.8% JIB395221.1% JIB409981.9% surface

[0565] Next, using the methods described in Example 8, the ability of 0.1 nM and 1 nM IgR to bridge CD20+ Raji B cells and mediate T cell activation in the presence of 1 nM rituximab was evaluated, along with their ability to prevent T cell activation in the presence of 1 nM trastuzumab or when run with motuzumab as a positive control (Figure 24). Compared to 1 nM trastuzumab, the 17F12 variant HzUCHT1 IgR, JIB33, exhibited some T cell activation in the presence of 1 nM rituximab. Compared to trastuzumab, the anti-Fc HP6017 HzUCHT1 IgR, JIB34, exhibited significant T cell activation under rituximab conditions at both 1 nM and 0.1 nM IgR levels. This suggests that using a Fab-like form of human pan-subclass (binding human IgG1, IgG2, IgG3, and IgG4) anti-Fc antibody fused to immune cell surface proteins is a feasible architecture to bridge target cell-bound antibodies to immune cell receptors and avoid the immune cell activity of nonspecific (unbound) antibodies. Compared to trastuzumab, the AVIG HzUCHT1 fusion, JIB35, showed some T cell activation under both 1 nM and 0.1 nM rituximab conditions. Compared to the wild-type FcγR3A-HzUCHT1 fusion JIB1, both FcγR3A_mut1-HzUCHT1 JIB36 and FcγR2A_mut-HzUCHT1 JIB37 IgRs exhibited increased T cell activation potency at both 1 nM and 0.1 nM IgR levels in the presence of rituximab, without increasing the signal from trastuzumab alone, suggesting that Fc receptors containing one or more mutations can be used to enhance IgR potency. In contrast, FcγR3A_mut2-HzUCHT1 JIB37 IgR showed reduced levels of T cell activation at both 1 nM and 0.1 nM levels in the presence of rituximab. In the presence of rituximab, at both concentrations, HzUCHT1 Fab-like molecules (one FcγR3A fused to the N-terminus of its VH and one FcγR3A fused to its VL, each JIB40 IgR containing a total of two FcγR3A) showed lower T cell activation than a single FcγR3A containing FcγR3A-ScFv-HzUCHT1 JIB1 IgR.

[0566] Example 20. Evaluation of the ability of IgR to bridge cancer cells to immune cells in the presence of tumor-specific IgG and high concentrations of non-specific IgG.

[0567] Rituximab specifically binds to Raji cells but not to T cells' anti-CD20 IgG1. Trastuzumab does not specifically bind to Raji cells or T cells' anti-HER2 IgG1. When IgRs do not bind to their targets, regardless of their target specificity, IgRs can bind different immunoglobulins to the same IgR-binding epitopes with the same affinity in the liquid phase. Therefore, when not binding to their targets, rituximab and trastuzumab can compete for the same IgR immunoglobulin-binding domains. The ability of 100 nM, 10 nM, and 1 nM JIB4, JIB20, JIB34, and JIB37 IgR to bridge CD20+ Raji B cells and mediate T cell activation was evaluated in the presence of 100 nM, 10 nM, and 1 nM rituximab, with or without 1 mg / mL trastuzumab (anti-HER2, does not specifically bind to Raji cells or T cells), to mimic high concentrations of potentially IgR-competitive non-target-specific immunoglobulins present in vivo, or in the presence of 1 mg / mL trastuzumab, without rituximab, to evaluate non-specific T cell activation due to IgR in the presence of only high concentrations of non-specific immunoglobulins. Using the bioassays described in Example 8, 100 nM, 10 nM, and 1 nM rituximab alone, 1 mg / mL trastuzumab alone were run as negative controls, and motuzumab alone was run as a positive control.

[0568] Figure 25 shows that αFc-VHH / ScFv-HzUCHT1 JIB4 and anti-Fc HP6017-HzUCHT1 JIB34 IgRs, in the absence of 1 mg / mL trastuzumab, exhibited the greatest potency with rituzumab by maintaining high signal intensity under both 10 nM and 1 nM IgR and rituzumab conditions. However, all IgRs showed significantly greater signal intensity under both IgR and rituzumab conditions compared to the IgR and trastuzumab conditions. Surprisingly, despite potential competition for the IgG binding domain of IgR between rituzumab and trastuzumab, all IgRs were able to mediate T cell activation in the presence of both rituzumab and 1 mg / mL trastuzumab, which was significantly greater than the activation observed in the presence of IgR alone in the presence of trastuzumab. This suggests that IgRs can utilize affinity when binding to targets present on cells or in immune complexes due to the multivalent availability of immunoglobulins. Furthermore, at 100 nM, all IgRs mediated T cell activation in the presence of rituximab and 1 mg / mL trastuzumab, which was not significantly lower than T cell activation in the presence of rituximab alone. We also surprisingly observed that at 10 nM, JIB20, with each IgR molecule containing two immunoglobulin-binding domains, mediated T cell activation in the presence of rituximab and 1 mg / mL trastuzumab, which was not significantly lower than T cell activation in the presence of rituximab alone, and greater than T cell activation under the same conditions with one immunoglobulin-binding domain, JIB4, JIB34, and JIB37, although JIB20 had lower potency than JIB4 and JIB34, and similar potency to high-affinity JIB37 in the absence of 1 mg / mL trastuzumab. These results demonstrate that even in the presence of high concentrations of non-target-specific immunoglobulins (similar to in vivo conditions) that bind the same immunoglobulin-binding domains to IgR, IgR can mediate biologically functional synapse formation and immune cell activity by bridging target-bound immunoglobulins to immune cell surface proteins. These results also indicate that, in the presence of high concentrations of non-specific immunoglobulins that bind the same immunoglobulin-binding domains to IgR, having more than one IgG-binding domain per IgR molecule can enhance the potency of IgR and its ability to bridge target-bound immunoglobulins to immune cell surface proteins.

[0569] Example 21. Expression, purification, and evaluation of αCD3 IgR that binds to IgG multivalently via multiple anti-IgG, anti-Fc, or FcγR domains.

[0570] IgG and Fc-binding heterologous peptides and heterologous multimers were expressed and purified as described in Examples 1 and 11.

[0571] The VH (SEQ ID 29) of TR66 fuses to the embedded disulfide CH1 heavy chain (SEQ ID 100), wherein the extracellular domain (SEQ ID NO: 23) of the Fc receptor FcγR3AV-S197P fuses to the C-terminus of the constant heavy chain via (G4S)3 and a his tag (SEQ ID NO: 21), forming CH1bDS-TR66-FcγR3AV-S197P-his (SEQ ID NO: 304), and the VL (SEQ ID NO: 30) of TR66 fuses to the κ light chain (κbDS, SEQ ID NO: 101), wherein the extracellular domain (SEQ ID NO: 23) of the Fc receptor FcγR3AV-S197P fuses to the constant light chain (κbDS-HzUCHT1-FcγR3AV-S197P, SEQ ID NO: 101) via (G4S)3. The FcγR3AV-S197P is fused to the C-terminus of both TR66 VH and VL (JIB46: SEQ ID NO: 306, SEQ ID NO: 307) to form JIB44 (SEQ ID NO: 304 and SEQ ID NO: 305). Alternatively, as shown in FIG5K, FcγR3AV-S197P is fused to the N-terminus of both TR66 VH and VL (JIB46: SEQ ID NO: 306, SEQ ID NO: 307) to obtain two Fc binding domains per molecule, and as shown in FIG5L, it is further fused to the C-terminus of the constant heavy chain (JIB45: SEQ ID NO: 308, SEQ ID NO: 307) to obtain three Fc binding domains per molecule.

[0572] CH1bDS was fused to C233S-Fc-LALAPA-hole (SEQ ID NO: 40), and the redundant “EPKSSDKTHT” on SEQ 40 was removed to form CH1bDS-Fc-LALAPA-hole (SEQ 309). FcγR3AV-S197P (SEQ 23) was fused to the N-terminus of SEQ 309 and κbDS via a (G4S)3 linker to form FcγR3AV-S197P-CH1bDS-Fc-LALAPA-hole (SEQ ID NO: 310) and FcγR3AV-S197P-κbDS (SEQ ID NO: 311). Each molecule containing two FcγR IgRs, i.e., JIB47, was generated by expressing all four chains: SEQ 310, SEQ 311, SEQ 44, and SEQ 47, as shown in Figure 4E.

[0573] The VH of HP6017 is fused to the N-terminus and C-terminus of the spacer region derived from the CH1 domain (SEQ ID NO: 312) to form HP6017-2xVH (SEQ 313). The VH of TR66 is fused to the C-terminus of HP6017-2xVH via the SEQ 311 connector and attached to the N-terminus of CH1bDS to form HP6017-2xVH-TR66-VH-CH1bDS-his (SEQ ID NO: 314). The VL of HP6017 is fused to the N-terminus and C-terminus of the spacer region derived from the κ domain (SEQ ID NO: 315) to form HP6017-2xVL (SEQ 316). The VL of TR66 was fused to the C-terminus of HP6017-2xVL via the SEQ 314 linker and attached to the N-terminus of κbDS, forming HP6017-2xVL-TR66-VL-κbDS (SEQ ID NO: 317). As shown in Figure 5M, expressing both strands (SEQ ID NO: 314, SEQ ID NO: 317) generates an IgR, JIB48, containing two Fc-binding domains in each molecule. Furthermore, HP6017-2xVH was fused to the N-terminus of CH1bDS-his to form HP6017-2xVH-CH1bDS-his (SEQ ID NO: 318), and HP6017-2xVL was fused to the N-terminus of κbDS, with TR66-ScFv fused to the C-terminus via a (G4S)3 linker to form HP6017-2xVL-κbDS-TR66-ScFv (SEQ ID NO: 319). Expression of SEQ 318 and SEQ ID NO: 319 yielded IgR JIB49, each molecule containing two Fc-binding domains, as shown in Figure 5I (without the half-life extension domain). Furthermore, TR66-VH is fused to CH1bDS and TR66-VL is fused to κbDS, both having (G4S)3 connectors at their C-termini, followed by HP6017-ScFv oriented with VL-VH (with (G4S)3 connectors between them), having his tag at the C-terminus of the heavy chain, forming SEQ ID NO: 320 and SEQ ID NO: 321, which are expressed to form JIB50, as shown in Figure 5B.

[0574] Tables 10, 26, and 27 show that, except for JIB48, JIB49, and JIB50, which have monomer content of 8.8%, 54.7%, and 43.6% as determined by SEC, all IgRs were well expressed with high purity. JIB44, which is similar to the high-performance JIB20 Fab-like design (two FcRs per IgR), but uses a buried inter-chain disulfide bond strategy and an inter-chain disulfide bond knockout strategy, showed similar high purity, at 84.7% and 84.9% monomer content, respectively, indicating that high purity can be obtained for this Fab-like architecture regardless of whether a buried or knockout strategy is used. The consistently low purity of HP6017 in different protein architectures and in different CD3-binding clones suggests that VH and VL sequence optimization is successful in attempting to improve its biophysical properties. Although JIB49 and JIB50 exhibit moderate purity, the following structures are represented: a double VH-VH and VL-VL Fab-like structure with ScFv linked to constant regions (JIB49) or a Fab-like structure with ScFv linked to the C-terminus of each constant domain, both employing a buried interchain disulfide bond strategy, indicating that these are viable architectures for IgR. The high purity of JIB47, with two Fc-binding FcγR domains and a full-length Fc, suggests that the LALAPA mutation is sufficient to knock out self-binding and aggregation, consistent with the results for JIB6. Furthermore, the use of a CH1 and constant κ chain pair employing a buried interchain disulfide bond strategy combined with a second CH1 and constant λ chain pair employing a natural interchain disulfide bond strategy does not affect the purity of the IgR molecule, indicating correct heavy and light chain pairing.

[0575] Table 10. Protein expression and monomers of IgR produced in Example 21 as determined by SEC.

[0576] Protein CHO (mg / L) Monomer content determined by SEC: JIB4490 84.7%; JIB451 528 9.5%; JIB461 809 2.0%; JIB471 678 7.2%; JIB488 8.8%; JIB491 154.7%; JIB502 343.6%. surface

[0577] Next, the ability of IgRs to bridge CD20+ Raji B cells and mediate T cell activation was evaluated at 10 nM and 1 nM in the presence of 10 nM rituximab with or without 1 mg / mL trastuzumab, or at 1 mg / mL trastuzumab without rituximab. Using the bioassays described in Examples 8 and 20, 10 nM rituximab alone, 1 mg / mL trastuzumab alone, and cells alone were run as negative controls, and motuzumab alone was run as a positive control. Figure 28 shows that all IgRs were able to mediate T cell activation in the presence of rituximab alone, with significantly greater activation than IgRs in the presence of 1 mg / mL trastuzumab alone. Furthermore, under 10 nM conditions, JIB20, JIB44, JIB47, and JIB49 were able to mediate T cell activation in the presence of rituximab and 1 mg / mL trastuzumab, with activation significantly greater than T cell activation in the presence of 1 mg / mL trastuzumab alone, but not significantly less than T cell activation in the presence of rituximab alone. In the presence of rituximab, with or without 1 mg / mL trastuzumab, the TR66 Fab (JIB44) with FcRs linked to the C-terminus of both its light and heavy chains was more potent than a similar design using HzUCHT1 (JIB20) at 0.1 nM IgR. Furthermore, at both 1 nM and 0.1 nM IgR, it was more potent than TR66 (JIB46) with FcRs linked to the N-terminus of both its light and heavy chains, and additionally TR66 (JIB45) with an FcR linked to the C-terminus of its heavy chain. While this result indicates that the selection of immune cell surface receptor-binding clones can influence IgR potency, it also demonstrates that IgR Fab-like architectures with immunoglobulin-binding domains linked to the C-terminus of both the heavy and light chains consistently exhibit high potency, even in the presence of competitive nonspecific immunoglobulins. It also showed that FcRs attached to the C-termini of both the heavy and light chains of Fab-like IgRs may be superior to those attached to the N-termini of the VH and VL regions, even when the third Fc-binding domain (binder) of each molecule is fused to the C-terminus. The dual Fc-binding Fc fusion with SP34 (JIB47) and the dual HP6017-VH-VH / VL-VL Fab with TR66-ScFv (JIB49) both exhibited potent T-cell activation in the presence or absence of 1 mg / mL trastuzumab, suggesting that two Fc-binding FcγRs or two Fc-binding antibody domains are viable compositions for generating target-cell-specific activation of immune cells while overcoming potential competition from high concentrations of nonspecific IgG.Furthermore, JIB47 contains a light chain / heavy chain pair using CH1bDS and κbDS and a second light chain / heavy chain pair containing WT CH1 and λ sequences. This suggests that the difference in interchain disulfide bond states between the two constant light chain and heavy chain pairs is a feasible means to achieve the desired constant light chain and heavy chain pairing in order to maintain the specificity of multispecific proteins and the intended target binding.

[0578] Example 22. Expression, purification, and evaluation of αCD3 and αCD28 IgRs that bind multivalently to IgG via multiple anti-IgG, anti-Fc, or FcγR domains.

[0579] IgG and Fc-binding heterologous peptides and heterologous multimers were expressed and purified as described in Examples 1 and 11.

[0580] TR66 Fab with embedded disulfide bonds has two different anti-Fc VHH (anti-Fc VHH2: SEQ ID NO: 322; anti-Fc VHH3, SEQ ID NO: 323), which fuse the extracellular domains (SEC ID 324, SEQ ID NO: 325) of mouse FcγR3 and FcγR2b to the C-terminus of its heavy chain (with a C-terminal his tag) and light chain, and co-express them to form JIB51 (SEQ ID NO: 326, SEQ ID NO: 327), JIB52 (SEQ ID NO: 328, SEQ ID NO: 329), JIB53 (SEQ ID NO: 330, SEQ ID NO: 331), and JIB54 (SEQ ID NO: 332, SEQ ID NO: 333), as depicted in Figure 5B. Furthermore, anti-Fc VHH2 is fused with VHH2, or VHH3 is fused with VHH3, and both are fused with TR66ScFv to form JIB55 (SEQ ID NO: 334) and JIB56 (SEQ ID NO: 335), as depicted in Figure 2C.

[0581] To improve the biophysical properties of HP6017, such as increasing the monomer percentage as determined by SEC after single-step purification, three different humanization strategies were employed. HzHP6017A (VH SEQ ID NO: 336, VL SEQ ID NO: 337) was designed by transplanting the CDR of HP6017 (according to Kabat (Kabat, 1992)) into the most closely matched human lines (IGHV1-46*01 and IGKV1-33*01) in the IMGT database (https: / / www.imgt.org / 3Dstructure-DB / cgi / DomainGapAlign.cgi) and back-mutating the mouse HP6017 wild-type sequence at key positions for IgG assembly (Chothia et al., 1985)—positions with known risk factors (Studnicka et al., 1994) (Footé & Winter, 1992). HzHP6017B (VH SEQ ID NO: 338, VL SEQ ID NO: 339) was designed by transplanting a CDR (according to Kabat) into the IGHV1-2*02 and IGKV1-12*01 human germlines in the IMGT database, and introducing the VH frame mutations R93S and D97E and the VL frame mutation V11L according to the IMGT numbers. HzHP6017C (VH SEQ ID NO: 340, VL SEQ ID NO: 341) was designed using the previously described BioPhi method (Prihoda et al., 2022). Table 11 shows the percentage of genetic and allele identity with the highest-matching human germline sequences in the IMGT database, and highlights that the humanization method of the HzHP6017B-VH / VL sequence produced the highest identity with human germline sequences. Then, the humanized VH and VL pairs are fused to the CH1bDS and κbDS sequences via (G4S)3 connectors such as JIB34 (where HzUCHT1 ScFv is attached to the C-terminus of the light chain), thereby generating JIB57 (SEQ ID NO: 342, SEQ ID NO: 343), JIB58 (SEQ ID NO: 344, SEQ ID NO: 345) and JIB59 (SEQ ID NO: 346, SEQ ID NO: 347), as shown in Figure 5C.

[0582] Table 11. Identity of the highest-matching human germline sequences from the IMGT database.

[0583] Variable-strand IMGT gene and allele identity %HP6017 VHIGHV1-46*0.169.4%HP6017 VLIGKV1-33*0.167.4%HzHP6017A VHIGHV1-46*0.175.5%HzHP6017A VLIGKV1-33*0.172.6%HzHP6017B VHIGHV1-46*0.188.8%HzHP6017B VLIGKV1-39*0.184.2%HzHP6017C VHIGHV1-46*0.179.6%HzHP6017C VLIGKV1-39*0.181.1% surface

[0584] A humanized version of VHH-CH128, HzVHH-CH128 (SEQ ID NO: 348), was designed by transplanting the CDR (according to Kabat) into a human germline sequence and introducing the IGHV3-64*04 mutations S24A, V42F, K48Q, Y52A, and S54A according to the IMGT number. VHH-CH128 was fused to another VHH-CH128 via a (G4S)3 linker, and then fused to HzUCHT1-ScFv, thereby generating JIB60, namely VHH-CH128-VHH-CH128-HzUCHT1-ScFv-his (SEQ ID NO: 349), as depicted in Figure 2C.

[0585] Two tandem extracellular domains of mouse FcγRIV were fused to the HzUCHT1-ScFv (G4S)3 linker to generate JIB61, namely mFcγRIV-mFcγRIV-HzUCHT1-ScFv-his (SEQ ID NO: 350), as depicted in Figure 2C. αFc-10-VHH was fused to the N-terminus of HzUCHT1-ScFv via the (G4S)3 linker, followed by anti-HSA-VHH1 (SEQ ID NO: 351) or αMSA-VHH, forming JIB62, namely αFc-10-VHH-HzUCHT1-ScFv-anti-HSA-VHH1 (SEQ ID NO: 352), and JIB63, namely αFc-10-VHH-HzUCHT1-ScFv-αMSA-VHH (SEQ ID NO: 353), as depicted in Figure 3A.

[0586] To explore different architectures and anti-CD3 clones using anti-IgG 17F12 clones, TR66 Fab of C4CH1-bDS and κbDS was fused with 17F12QUAD-ScFv at the C-terminus of the light and heavy chains via (G4S)3 linkers to generate JIB64 (SEQ ID NO: 354, SEQ ID NO: 355), as depicted in Figure 5B.

[0587] The anti-CD28 IgR protein was designed by fusing the extracellular domain of human FcγR2A (SEQ ID NO: 9) to the C-terminus of CH1bDS and κbDS, forming proteins containing 2E12 (SEQ ID NO: 205, SEQ ID NO: 207), TGN1412 (SEQ ID NO: 210, SEQ ID NO: 212), 9.3 (SEQ ID NO: 215, SEQ ID NO: 217), 8GA8 (8GA8-VH SEQ ID NO: 358, 8GA8-VL SEQ ID NO: 359), 9D7 (9D7-VH SEQ ID NO: 360, 9D7-VL SEQ ID NO: 361), and TN228 (TN228-VH SEQ ID NO: 362, TN228-VL SEQ ID NO: 362). The VH and VL pairs of CH1bDS-FcγR2A-his (SEQ ID NO: 356) and κbDS-FcγR2A (SEQ ID NO: 357) of NO: 363 are used to generate JIB67 (SEQ ID NO: 364, SEQ ID NO: 365), JIB68 (SEQ ID NO: 366, SEQ ID NO: 367), JIB69 (SEQ ID NO: 368, SEQ ID NO: 369), JIB70 (SEQ ID NO: 370, SEQ ID NO: 371), JIB71 (SEQ ID NO: 372, SEQ ID NO: 373) and JIB72 (SEQ ID NO: 374, SEQ ID NO: 375), as depicted in Figure 5B.

[0588] Tables 12, 29, and 30 show that, except for JIB57, JIB59, JIB64, and JIB70, which had monomers of 46.9%, 57.5%, 43.4%, and 14.7% as determined by SEC, respectively, all IgRs were well expressed in high purity after single-step purification. Compared to wild-type HP6017-VH / VL for JIB34, humanized HP6017A-VH / VL for JIB57, and humanized HP6017C-VH / VL for JIB59, the HP6017B-VH / VL humanization strategy for JIB58 produced the highest purity IgR among the four, with 79.4% monomer content as determined by SEC, compared to 51.8% for JIB34, 46.9% for JIB57, and 57.5% for JIB59, indicating that the HP6017B humanization strategy has the added benefit of improving the biophysical properties of IgR. JIB51, JIB52, JIB53, and JIB54 IgRs with Fab-like structures and two immunoglobulin domains (extended from the heavy and light chains, respectively) showed that the fused VHH domain did not mismatch with the VL of the Fab, and there were no expression or purity issues when using the mouse Fc receptor. Similarly, JIB55, JIB56, JIB60, and JIB61, which have two tandem anti-immunoglobulin-binding domains, including anti-Fc and anti-CH1 VHH or mouse Fc receptors fused in a head-to-tail orientation, and a C-terminal anti-CD3 domain, showed no problems in terms of expression or purity. Anti-CD28 JIB67, JIB68, JIB69, JIB70, JIB71, and JIB72 all possess the same Fab-like structure as the anti-CD3 JIB20, JIB44, JIB51, JIB52, JIB53, and JIB52, but have two FcγR2As fused to both the heavy and light chains, compared to immunoglobulin-binding VHH, FcγR3A, or mouse FcγR. The high expression and purity of all these constructs (except JIB70) suggest that this particular IgR architecture can be produced with typically high expression and purity from a single-step purification process, provided the starting components are suitable. In the case of JIB70, the low purity and expression are caused by the VH / VL pair used in the Fab-like scaffold rather than by the incorporated immunoglobulin-binding domain.

[0589] Table 12. Protein expression and monomers of IgR produced in Example 22 as determined by SEC.

[0590] Protein CHO (mg / L) % of monomers determined by SEC JIB512 93 98.0% JIB522 24 95.0% JIB538 897.1% JIB541 27 99.4% JIB551 25 77.0% JIB56 94 92.0% JIB571 50 46.9% JIB582 25 79.4% JIB592 15 57.5% IB6035896.6%JIB6113083.3%JIB6211999.0%JIB6330798.6%JIB643543.4%JIB67 6799.4%JIB6813298.9%JIB6917698.7%JIB70814.7%JIB718496.6%JIB7221795.4% surface

[0591] Next, the ability of anti-CD3 IgR JIB34, JIB57, JIB58, JIB59, JIB64, JIB4, JIB62, JIB63, JIB44, JIB53, JIB54, and JIB61 to bridge CD20+ Raji B cells with Jurkat T cells and mediate T cell activation was evaluated in the presence of 1 nM rituximab for assessing target cell-specific activation or in the presence of 1 nM trastuzumab for assessing non-specific activation. Using the bioassays described in Example 8, 1 nM rituximab alone, 1 nM trastuzumab alone, and cells alone were run as negative controls, and motuzumab alone was run as a positive control. Figure 31 shows that the humanized anti-Fc HP6017 IgRs (JIB57, JIB58, and JIB59) exhibited T cell activation comparable to that of wild-type mouse HP6017 IgR in both 1 nM and 0.1 nM rituximab, and the activation was significantly greater than that in the presence of trastuzumab, indicating that the humanization method minimally reduced antibody potency without increasing non-specific activation. Furthermore, JIB58 exhibited the highest potency among the three humanized HP6017 IgRs evaluated, except for having the highest percentage of identity with its most closely matched human germline sequence and the highest percentage of monomers as determined by SEC. JIB64, specifically TR66 Fab containing two IgG binding domains per molecule with 17F12QUAD ScFv linked to both the light and heavy chains, exhibited potency comparable to WT and humanized HP6017 IgRs (JIB20, JIB57, JIB58, and JIB59) at both 1 nM and 0.1 nM rituximab. This contrasts with the JIB33 17F12QUAD Fab-like IgR in Example 19, which produced almost no T cell activation relative to background. This suggests that eliminating the self-binding C4CH1 P126S and K213E mutations and improving expression and stability with the 17F12QUAD VL mutation can achieve high purity of 17F12 Fab while maintaining the target binding of the 17F12-VH and 17F12QUAD-VL pairs. Furthermore, Fab-like structures should be further explored using alternative immune cell surface protein binding clones. JIB62 and JIB63 are similar to JIB4, in which anti-Fc-10-VHH is fused to HzUCHT1, but anti-HSA or anti-MSA is fused to the C-terminus.All of them exhibited potency comparable to 1 nM rituximab, and caused only a slight decrease in T cell activation at 0.1 nM rituximab, without increasing activation in the presence of trastuzumab. This indicates the presence of a serum albumin-binding domain at the C-terminus of the IgR, which causes minimal interference with its IgG- and immune cell target binding and function, and does not increase non-specific immune cell activity. Figure 32 compares TR66 Fabs containing two FcγRs, each containing human FcγR3A (JIB44), mFcγR3 (JIB53), or mFcγR2b (JIB54), respectively. The results show that the IgRs containing human FcγR3A have the highest potency at both 1 nM and 0.1 nM rituximab, and the potency of the three IgRs is comparable to the known monovalent affinity (K) of human IgG1. D The ordering is consistent, with FcγR3A (68 nM) > mFcγR2b (1100 nM) > mFcγR3 (9300 nM) (Dekkers et al., 2017; Patel et al., 2019). This data suggests that even when included in approximately 10 μM unit valence K... D IgRs can also function within the range of low-affinity IgG binding domains. IgR JIB61, with two mFcγRIVs tandemly linked to HzUCHT1-ScFv, exhibits potency between JIB53 and JIB54.

[0592] The ability of anti-CD3 IgR JIB51, JIB52, JIB55, JIB56, JIB60, JIB65, and JIB50 to bridge CD20+ Raji B cells with Jurkat T cells and mediate T cell activation was evaluated in the presence of 10 nM rituximab with or without 1 mg / mL trastuzumab, or in the presence of 1 mg / mL trastuzumab without rituximab. Using the bioassays described in Examples 8 and 20, 10 nM rituximab alone, 1 mg / mL trastuzumab alone, and cells alone were run as negative controls, and motuzumab alone was run as a positive control. Figure 33 compares two formulations: one is a TR66 Fab-like design (JIB51, JIB52), where each IgR is linked to an anti-Fc VHH at the C-terminus of both the heavy and light chains; the other is two identical anti-Fc VHHs tandemly linked to TR66-ScFv (JIB55, JIB56). Both formulations mediate T cell activation in the presence of 10 nM or 1 nM rituximab, with significantly greater activation than in the presence of 1 mg / mL trastuzumab. Interestingly, in the presence of 1 mg / mL trastuzumab alone, the TR66 Fab-like formulation appears to produce less nonspecific activation than the tandem design, and the signal ratio between IgR in the presence of rituximab and 1 mg / mL trastuzumab is greater than that in the presence of trastuzumab alone. Under 1 nM rituximab, JIB60, an anti-CH1 IgR with two VHHs tandemly linked to HzUCHT1, appeared to have lower potency than the tandem anti-Fc IgRs JIB55 and JIB56. JIB50 TR66 Fab, each IgR containing two anti-Fc HP6017-ScFv (one linked to HC and one to LC), showed comparable T cell activation to JIB51 and JIB52 in the absence or presence of 1 mg / mL of 1 nM rituximab.

[0593] The ability of anti-CD3 IgR JIB51, JIB52, and JIB53 combined with 1 nM anti-CD3 OKT3 mIgG2a (Biolegend, 317325) to bind mouse IgG and mediate T cell activation was evaluated relative to 1 nM of IgR alone and 1 nM of OKT3 alone. 1 nM of rituximab alone (Rtx) was used as a negative control. Test items were co-cultured with CD20+ Raji B cells and Jurkat-NFAT-Luc T cells, and luminescence was read out using a procedure from Example 8. Figure 34 shows that JIB51 and JIB53 mediated significantly greater activation in the presence of OKT3 relative to OKT3 alone, and JIB51 did not show significantly greater activation when used alone relative to rituximab alone or cells alone. JIB52 did not show significantly greater activation in OKT3 alone relative to OKT3 alone. These results indicate that JIB51 cross-reacts with mouse IgG and cross-reacts more extensively with mouse FcγR3, which contains JIB53 IgR.

[0594] A modified version of the co-culture assay described in Example 8 was used to evaluate anti-CD28 IgR JIB67, JIB68, JIB69, JIB71, and JIB72, wherein 96-well U-shaped plates were coated overnight at 4°C with 100 μl of 1 μg / mL anti-CD3 OKT3 (Biointron, B6928, mouse IgG2a (D134G) CH1+ human IgG1 CH2+ CH3- mouse κ) and washed once with PBS the next day before the cell co-culture assay to remove any unbound OKT3 from the wells. The ability of IgR to bridge CD20+ Raji B cells with Jurkat T cells and mediate T cell co-stimulation (OKT3 IgG on the plate provides signal 1) at 10 nM and 1 nM was assessed in the presence of 10 nM rituximab for assessing target-cell-specific T cell co-stimulatory activity or in the presence of 10 nM trastuzumab for assessing non-specific T cell co-stimulatory activity. 10 nM rituximab alone, 10 nM trastuzumab alone, and cells alone were run as negative controls, and 10 nM and 1 nM anti-CD28 mIgG1 clone CD28.2 (Thermo, 16-0289-85) alone were used as positive T cell co-stimulation controls. Figure 35 shows that when combined with rituximab, all anti-CD28 IgRs had similar potency to anti-CD28 CD28.2 IgG, and in some cases, greater potency than anti-CD28 CD28.2 IgG. All IgRs combined with rituximab showed significantly greater T cell co-stimulatory activity relative to their activity when combined with non-target cell-specific trastuzumab. JIB67, JIB69, and JIB72 did not show significantly higher activity than trastuzumab alone when combined with trastuzumab, indicating that these IgRs have minimal non-specific T cell co-stimulatory activity. In contrast, when JIB68 and JIB71 were combined with trastuzumab, compared with trastuzumab alone, JIB68 and JIB71 exhibited some non-specific T cell co-stimulatory activity.

[0595] Example 23. Expression, purification, and evaluation of αCD3 and αTCRα / βIgR that bind multivalently to IgG via multiple anti-IgG, anti-Fc, or FcγR domains.

[0596] IgG and Fc-binding heterologous peptides and heterologous multimers were expressed and purified as described in Examples 1 and 11.

[0597] Using SP34 Fab with CH1NoDS and hLC7NoDS, FcγR2A is fused to the C-terminus of both the constant heavy chain and the light chain via a (G4S)3 connector, followed by attaching an αHSA-VHH with a C-terminal his tag to the heavy chain to form JIB73 (SEQ ID NO: 376, SEQ ID NO: 377), as depicted in Figure 5J. Similarly, using HzUCHT1 with CH1bDS and κbDS, FcγR2A is fused to the C-terminus of both the constant heavy chain and the light chain via a (G4S)3 connector, followed by attaching an αHSA-VHH with a C-terminal his tag to the heavy chain to form HzUCHT1-based JIB74 (SEQ ID NO: 378, SEQ ID NO: 379), as depicted in Figure 5J. Using CH1bDS and κbDS (or hLC7bDS for HuYTH 12.5 and Hz06), TR66, HzTR66, Hu291, huCLB-T3 / 4.A, HuYTH 12.5, BC3, Hz06, and αTCR BMA-031 Fab, FcγR2A is fused to the C-terminus of both the constant heavy chain and the light chain via a (G4S)3 connector. A C-terminal his tag is then attached to the heavy chain to form TR66-based JIB75 (SEQ ID NO: 380, SEQ ID NO: 381), HzTR66-based JIB76 (SEQ ID NO: 382, ​​SEQ ID NO: 383), Hu291-based JIB77 (SEQ ID NO: 384, SEQ ID NO: 385), and huCLB-T3 / 4.A-based JIB78 (SEQ ID NO: 386, SEQ ID NO: 387). 387), JIB79 based on HuYTH 12.5 (SEQ ID NO: 388, SEQ ID NO: 389), JIB80 based on BC3 (SEQ ID NO: 390, SEQ ID NO: 391), JIB81 based on Hz06 (SEQ ID NO: 392, SEQ ID NO: 393), and JIB82 based on BMA-031 (SEQ ID NO: 394, SEQ ID NO: 395), as depicted in Figure 5B. HzTR66 was generated using a method similar to that of HzHP6017B, wherein HzTR66 VH SEQ ID NO: 396 and VL SEQ ID NO: 397 were designed by transplanting CDRs (according to Kabat) into the human germlines IGHV1-2*02 and IGKV1-12*01 in the IMGT database and introducing VH framework mutations R93S and D97E and VL framework mutation V11L according to the IMGT numbers.

[0598] FcγR2A is fused to the N-terminus of CH1bDS-Fc-LALAPA-hole and κbDS via a (G4S)3 linker, forming FcγR2A-CH1bDS-Fc-LALAPA-hole (SEQ 398) and FcγR2A-κbDS (SEQ 399). SP34-Fc-LALAPA-knob is mutated with C233S and SP34 VL is fused with hLC7NoDS to knock out interchain disulfide bonds, forming SP34-NoDS-Fc-LALAPA-knob (SEQ ID NO: 400) and SP34-hLC7NoDS (SEQ ID NO: 401). HzUCHT1-VH is exchanged with SP34 in SP34-Fc-LALAPA-knob to form HzUCHT1-Fc-LALAPA-knob (SEQ ID NO: 402). HzUCHT1 VL is fused with κNoDS to form HzUCHT1-κNoDS (SEQ ID NO: 403). SP34-based JIB85 and HzUCHT1-based JIB84 are generated by co-expression of FcγR2A-CH1bDS-Fc-LALAPA-hole and FcγR2A-κbDS, as well as SP34-NoDS-Fc-LALAPA-knob and SP34-hLC7NoDS or HzUCHT1-Fc-LALAPA-knob and HzUCHT1-κNoDS, as depicted in Figure 4E. JIB86 was generated by co-expressing SP34-NoDS-Fc-LALAPA-knob and SP34-hLC7NoDS-FcγR2A (SEQ ID NO: 377), wherein FcγR2A was fused to the N-terminus of C233S / Fc-LALAPA-hole via a (G4S)3 linker, thereby generating FcγR2A-C233S / Fc-LALAPA-hole (SEQ ID NO: 404), as depicted in Figure 4G. JIB87 and JIB88 are formed by fusing a second FcγR2A to the N-terminus of FcγR2A-C233S / Fc-LALAPA-hole via a (G4S)3 connector (SEQ ID NO: 405), and co-expressed via a (G4S)3 connector with C233S / Fc-LALAPA-knob (SEQ ID NO: 406) fused to HzUCHT1-ScFv (SEQ ID NO: 407) or SP34-ScFv (SEQ ID NO: 408), as depicted in Figure 4C, but with an additional IgBD at the N-terminus of the first IgBD.

[0599] JIB89 is produced by fusing FcγR2A via a (G4S)3 connector, followed by connecting SP34-ScFv, FcγR2A, and αHSA-VHH to form FcγR2A-SP34-ScFv-FcγR2A-αHSA-VHH (SEQ ID NO: 409), as depicted in Figure 3B (in rearranged order). Similarly, JIB90 is produced, but using HzUCHT1-ScFv to form FcγR2A-HzUCHT1-ScFv-FcγR2A-αHSA-VHH (SEQ ID NO: 410). JIB91 and JIB92 are IgRs containing two Fc binding domains in each molecule, and have heavy chain HzHP6017B-2xVH-CH1bDS-αHSA-VHH (SEQ ID NO: 411) and light chain HzHP6017B-2xVL-κbDS-HzUCHT1-ScFv (SEQ ID NO: 412) or HzHP6017B-2xVL-κbDS-SP34-ScFv (SEQ ID NO: 413), as depicted in Figure 5I. JIB93 is an IgR containing two Fc-binding domains and a heterodimeric Fc per molecule, as depicted in Figure 4I, and is generated using four chains. The first light chain contains HzHP6017B-2xVL-κbDS (SEQ ID NO: 414), and the first heavy chain contains HzHP6017B-2xVH-CH1bDS-Fc_mut-LALAPA-hole (SEQ ID NO: 415), which has additional Fc mutations N276K and L309V (EU number) to eliminate the self-Fc binding of HzHP6017B. The second light chain contains SP34-hLC7NoDS (SEQ ID NO: 401), and the second heavy chain contains SP34-VL-CH1NoDS-Fc_mut-LALAPA-knob (SEQ ID NO: 416), which has additional Fc mutations N276K and L309V. V, to eliminate the Fc binding of HzHP6017B itself. JIB94 is an IgR containing two Fc binding domains per molecule, having a heavy chain containing SP34-VH-CH1NoDS-HzHP6017B-ScFv-his (SEQ ID NO: 417) and a light chain containing SP34-VL-hLC7NoDS-HzHP6017B-ScFv (SEQ ID NO: 418), as depicted in Figure 5B.

[0600] JIB95 is an IgR containing two anti-IgG binding domains per molecule. It has a heavy chain Hz17F12-2xVH-C4CH1bDS-αHSA-VHH-his (SEQ ID NO: 419), wherein CH1 is contained in JIB33 for eliminating the P126S and K213E mutations that self-bind Hz17F12, and the VH-VH spacer contains the P126S mutation (C4CH1 spacer, SEQ ID NO: 557); and a light chain Hz17F12-2xVL-κbDS-SP34-ScFv (SEQ ID NO: 420), as depicted in Figure 5I (without the half-life extension domain). Hz17F12 was generated using a method similar to that of HzHP6017B, wherein Hz17F12 VHSEQ ID NO: 421 and VL SEQ ID NO: 422 were designed by transplanting CDRs (according to Kabat) into the human germline IGHV1-2*02 and IGKV4-1*01 in the IMGT database and introducing VH framework mutations R93S and D97E and VL framework mutation K24R according to the IMGT numbers. JIB96 is an IgR containing two IgG binding domains and a heterodimer Fc per molecule, as depicted in Figure 4I, and is produced using four chains. The first light chain contains Hz17F12-2xVL-κbDS (SEQ ID NO: 423), and the first heavy chain contains Hz17F12-2xVH-C4CH1bDS-Fc-LALAPA-hole (SEQ ID NO: 424), which has P126S and K213E mutations in CH1 and a P126S mutation in the C4CH1 spacer region to eliminate the self-binding of Hz17F12. The second light chain contains SP34-hLC7NoDS (SEQ ID NO: 401), and the second heavy chain contains SP34-VL-C4CH1NoDS-Fc-LALAPA-knob (SEQ ID NO: 401). 425), which has P126S and K213E mutations in CH1 to eliminate the self-binding of Hz17F12. JIB97 is similar to JIB95, using the heavy chain Hz17F12-2xVH-C4CH1bDS-αHSA-VHH-his, but in which TR66 is linked to LC in Hz17F12-2xVL-κbDS-TR66-ScFv (SEQ ID NO: 426), as depicted in Figure 5I (without the half-life extension domain).

[0601] Tables 13, 36, and 37 show that, except for JIB91 and JIB92 which showed no detectable protein expression and JIB93 which had 44.7% monomer content as determined by SEC, all IgRs were well expressed at high or moderate purity after single-step purification. All three constructs contained a dual HzHP6017B VH-VH / VL-VL design with constant domain spacers. In contrast, JIB49 contained the same dual HP6017 VH-VH / VL-VL design using the same spacers as JIB91 and JIB92, but was able to express it, indicating structural differences between the humanized sequence and the wild-type mouse sequence, resulting in the loss of expression in the dual variable chain form. Although JIB93 contained a dual HzHP6017B VH-VH / VL-VL design, the molecular weight of the chains on the reduced SDS-PAGE gel indicated that the HzHP6017B heavy and light chains were not recovered, as the molecular weights were more consistent with those of the SP34 heavy and light chains. The SP34 heavy chain containing Fc mutations N276K and L309V to eliminate HzHP6017B self-binding was recovered, indicating that these mutations do not interfere with expression and protein recovery by protein A affinity chromatography. Furthermore, the Hz17F12 dual VH-VH / VL-VL construct, with the same VL-VL spacer region and a similar VH-VH spacer region as JIB91 and JIB92, showed no expression problems regardless of whether Fc was present (JIB96) or absent (JIB95, JIB97), indicating that the expression deletion of the dual HzHP6017B VH-VH / VL-VL design is specific to the HzHP6017B clone.

[0602] Notably from SEC and SDS-PAGE, JIB84 and IB85, containing four distinct polypeptide chains (including the heterodimer Fc region), were well expressed and exhibited high purity, with monomer content of 84.2% and 84.6%, respectively. Importantly, JIB84 contains a first light / heavy chain pair with CH1bDS / κbDS embedded interchain disulfide bonds and a second light / heavy chain pair with CH1NoDS / κNoDS but without interchain disulfide bonds; and JIB85 contains a first light / heavy chain pair with CH1bDS / κbDS and a second light / heavy chain pair with CH1NoDS / λNoDS but without interchain disulfide bonds. The high-purity results demonstrate that using different interchain disulfide bonding strategies between two light / heavy chain pairs in a single molecule enables the production of high-purity multimeric proteins, where constant light and heavy chains pair with their intended counterparts. This reinforces the findings for JIB47 in Example 21, which used CH1bDS / κbDS embedded sulfides and the CH1-WT / λ-WT native disulfide bonding strategy. This also shows that differential interchain disulfide bonding strategies can utilize constant light / heavy chain pairs with the same light chain type (such as CH1 / κ and CH1 / κ or CH1 / λ and CH1 / λ) or different light chain types (such as CH1 / κ and CH1 / λ). Purity data also show that triple-stranded, double-stranded, and single-stranded IgR designs for JIB86; JIB87 and JIB88; and JIB89 and JIB90 are feasible strategies, enabling the creation of two immunoglobulin-binding domains, one immune cell surface protein-binding domain, and extended half-life within a single IgR molecule.

[0603] Table 13. Protein expression and monomers of IgR produced in Example 23 as determined by SEC.

[0604] Protein CHO (mg / L) Monomer content determined by SEC: JIB73 182 94.9% JIB74 132 92.8% JIB75 106 97.5% JIB76 122 97.7% JIB77 76 97.4% JIB78 35 99.2% JIB79 95 96.7% JIB80 84 96.8% JIB81 47 100.0% JIB82 76 97.0% JIB84 56 84.2 %JIB854 684.6%JIB861 1494.0%JIB8781 89.4%JIB881 1691.8%JIB898 793.8%JIB904 884.4%JIB91 Not detected, not applicable JIB92 Not detected, not applicable JIB93 2344.70%JIB94 266.1%JIB951 184.9%JIB961 169.5%JIB973 62.7% surface

[0605] Using the bioassays described in Examples 8 and 20, the ability of anti-CD3 IgRs JIB73, JIB74, JIB84, JIB85, JIB87, JIB88, JIB89, and JIB90 to bridge CD20+ Raji B cells with Jurkat T cells and mediate T cell activation at 10 nM and 1 nM was evaluated with or without 1 mg / mL trastuzumab, or with 1 mg / mL trastuzumab without rituximab. Figure 38 compares the SP34 and HzUCHT1 clones in the same four unique IgR constructs. All IgRs showed potent T cell activation in the presence of 10 nM and 1 nM rituximab, with significantly greater activation than in the presence of trastuzumab. In all four designs, the IgRs using SP34 (JIB73, JIB85, JIB88, JIB89) exhibited significantly higher activity in the presence of rituximab and 1 mg / mL trastuzumab than in the presence of trastuzumab alone at 1 mg / mL. However, the activity in the presence of rituximab and 1 mg / mL trastuzumab was not significantly lower than that in the presence of rituximab alone. Compared to the presence of trastuzumab alone at 1 mg / mL or rituximab alone, the relative activity of the four IgRs using HzUCHT1 (JIB74, JIB84, JIB87, JIB90) in the presence of rituximab and 1 mg / mL trastuzumab was less significant in the SP34 design. These data suggest that incorporating the anti-HSA domain into the Fab-like structures in JIB73 and JIB74 does not interfere with function. Positive data from JIB84 and JIB85 demonstrate that a differential interchain disulfide bond strategy between two light and heavy chain pairs can create functional pairing structures that maintain highly potent, highly expressed, and highly pure specific target binding to multispecific proteins of the CH1 / κ and CH1 / κ pairs or CH1 / κ and CH1 / λ pairs. Also noteworthy are single-chain designs employing the D1-D2-D3-D4 strategy (JIB89 and JIB90), where D1 and D3 are immunoglobulin-binding domains, D2 is an immune cell surface protein-binding domain, and D4 is a serum albumin-binding domain, all exhibiting potent activity in the presence of rituximab alone. Furthermore, the SP34 design showed a high activity ratio between the combination of IgR and rituximab and 1 mg / mL trastuzumab compared to the combination of IgR and 1 mg / mL trastuzumab alone, in which case the original signal RLU ratio was approximately 3.

[0606] Using the bioassays described in Examples 8 and 20, the ability of anti-CD3 IgRs JIB75, JIB76, JIB77, JIB78, JIB79, JIB80, JIB81 and anti-TCRIgR JIB82, which used the Fab-like structure shown in Figure 5B and had two FcγR2A fused to the C-terminus of constant light and heavy chains, to bridge CD20+ Raji B cells with Jurkat T cells and mediate T cell activation was evaluated at both 10 nM and 1 nM, with the presence or absence of 1 mg / mL trastuzumab, or with 1 mg / mL trastuzumab and without rituzumab. Figure 39 shows that, except for JIB76, which is based on HzTR66, all IgRs exhibited significantly higher activity in the presence of rituzumab or rituzumab and 1 mg / mL trastuzumab than in the presence of 1 mg / mL trastuzumab alone. In the presence of rituximab and 1 mg / mL trastuzumab, anti-CD3 IgRs JIB78, JIB79, JIB80, JIB81 and anti-TCR IgR all exhibited T cell activity, which was not significantly lower than that in the presence of rituximab alone, indicating that IgR can exert its effects across multiple TCR complex epitopes, including different CD3 co-receptors and TCR chains.

[0607] Using the bioassays described in Examples 8 and 20, the ability of anti-CD3 IgRs JIB86, JIB93, JIB94, JIB95, JIB96, and JIB97, which used the Fab-like structure shown in Figure 5B and had two FcγR2A fused to the C-terminus of constant light and heavy chains, to bridge CD20+ RajiB cells with Jurkat T cells and mediate T cell activation was evaluated at both 10 nM and 1 nM, with the two FcγR2A fused to the C-terminus of constant light and heavy chains. Figure 40 shows that, except for JIB93, all evaluated IgRs showed significantly higher T cell activation in the presence of rituximab than in the presence of trastuzumab alone. For JIB93, the lack of specificity and purity results between rituximab and trastuzumab was consistent, with the HP6017B VH-VH / VL-VL strategy appearing to eliminate HP6017B heavy and light chain expression, while SP34 light and heavy chains were still expressed by SDS-PAGE assays, potentially causing them to be unpaired with their HP6017B counterparts, resulting in highly nonspecific binding and activation in T cell activation assays. In the presence of rituximab and 1 mg / mL trastuzumab, T cell activity in JIB86 was significantly higher than in the presence of 1 mg / mL trastuzumab alone, but not significantly lower than in the presence of rituximab alone. Although JIB94 SP34-Fab-like IgR with two HzHP6017B-ScFv (one linked on each constant strand) and 17F12-based JIB95, JIB96, and JIB96 did not exhibit a high rate of T cell activation relative to trastuzumab alone in the presence of rituximab and 1 mg / mL trastuzumab, it is important to note that four strands, JIB96, containing dual Hz17F12 VH-VH / VL-VL using C4CH1 spacer and κ spacer regions, as well as embedded disulfide bonds and self-binding C4CH1 knockout mutations, successfully paired with SP34 heavy and light chain pairs using C4CH1 knockout mutations and lacking native disulfide bonds. This further demonstrates that the differential inter-strand disulfide bond strategy is a viable means of ensuring the expected light and heavy chain pairing to maintain target binding and function.

[0608] Example 24. Expression, purification, and evaluation of αCD3, αCD28, αCD137, and αCD89 IgRs that bind multivalently to IgG via multiple anti-Fc or FcγR domains.

[0609] IgG and Fc-binding heterologous peptides and heterologous multimers were expressed and purified as described in Examples 1 and 11.

[0610] Anti-CD28 IgR is generated by fusing VH and VL pairs into CH1bDS and κbDS, wherein human FcγR2A is fused to the C-terminus of both the heavy chain (with his tag) and the light chain via a (G4S)3 linker to form JIB100 (SEQ ID NO: 427, SEQ ID NO: 428) based on Hz2E12, JIB101 (SEQ ID NO: 429, SEQ ID NO: 430) based on Hz9.3, JIB102 (SEQ ID NO: 431, SEQ ID NO: 432) based on Hz8GA8, JIB103 (SEQ ID NO: 433, SEQ ID NO: 434) based on Hz9D7, JIB104 (SEQ ID NO: 435, SEQ ID NO: 436) based on HzTN228, and JIB105 (SEQ ID NO: 437, SEQ ID NO: 436) based on Hz28.3. 438), JIB106 (SEQ ID NO: 439, SEQ ID NO: 440) based on Hz5.11A1, JIB107 (SEQ ID NO: 441, SEQ ID NO: 440) based on Hz5.11A1-C55S, and JIB108 (SEQ ID NO: 442, SEQ ID NO: 443) based on TY24876, as depicted in Figure 5B. Hz2E12 VH SEQ ID NO: 444 and VL SEQ ID NO: 445, and Hz9.3 VH SEQ ID NO: 446 and VL SEQ ID NO: 447 were designed by transplanting CDRs (according to Kabat) into the IGHV4-59*01 and IGKV4-1*01 human germlines in the IMGT database and introducing the VL frame mutation K24R according to the IMGT number. Hz8GA8 VH SEQ ID NO: 448 and VL SEQ ID NO: 449 were designed by transplanting a CDR (according to Kabat) into the IGHV1-2*02 and IGKV1-12*01 human germlines in the IMGT database, and introducing the VH frame mutations R93S and D97E and the VL frame mutation V11L according to the IMGT numbers. Hz9D7 VH SEQ ID NO: 450 and VL SEQ ID NO: 451 and HzTN228 VH SEQ ID NO: 452 and VL SEQ ID NO: 453 were designed by transplanting a CDR (according to Kabat) into the IGHV4-59*01 and IGKV1-12*01 human germlines in the IMGT database, and introducing the VL frame mutation V11L according to the IMGT numbers.Hz28.3 VH SEQ ID NO: 454 and VL SEQ ID NO: 455 were designed by transplanting a CDR (according to Kabat) into the IGHV1-2*02 and IGKV1-12*01 human lines in the IMGT database, and introducing the VH frame mutations R93S and D97E and the VL frame mutation V11L according to the IMGT numbers. 5.11A1 WT VH SEQ ID NO: 456 and VL SEQ ID NO: 457 were humanized into Hz5.11A1 VH SEQ ID NO: 458 and VL SEQ ID NO: 459 by transplanting a CDR (according to Kabat) into the IGHV1-2*02 and IGKV1-12*01 human lines in the IMGT database, and introducing the VH frame mutations R93S and D97E and the VL frame mutation V11L according to the IMGT numbers. Hz5.11A1-C55S VH SEQ ID NO: 460 was generated by introducing the framework mutation C55S according to the IMGT number in SEQ ID NO: 458. Furthermore, JIB109 (SEQ ID NO: 461) was generated by fusing human FcγR2A to the N-terminus of 1h-79-807-sdAb-VL (SEQ ID NO: 462), followed by linking a second FcγR2A and his tag via a (G4S)3 linker, as depicted in Figure 2C, in a rearranged order.

[0611] Anti-CD3 IgR JIB110 is generated via SP34-VH-CH1NoDS-HzFc-10-VHH-his (SEQ ID NO: 463) and SP34-VL-hLC7NoDS-HzFc-10-VHH (SEQ ID NO: 464), as depicted in Figure 5B. Similarly, JIB111 is generated via SP34-VH-CH1NoDS-anti-Fc-HzVHH2-his (SEQ ID NO: 465) and SP34-VL-hLC7NoDS-anti-Fc-HzVHH2 (SEQ ID NO: 466), as depicted in Figure 5B. JIB112 was prepared by using 20G6 VH (SEQ ID NO: 467) and VL (SEQ ID NO: 468) to prepare the heavy chain 20G6-VH-CH1bDS-FcγR2A-his (SEQ ID NO: 469) and the light chain 20G6-VL-κbDS-FcγR2A (SEQ ID NO: 470), as depicted in Figure 5B. JIB113 (SEQ ID NO: 471), JIB114 (SEQ ID NO: 472), and JIB115 (SEQ ID NO: 473) are produced by fusing human FcγR2A to the N-terminus of SP34-sdAb-VH (SEQ ID NO: 474), 20G6-sdAb-VH (SEQ ID NO: 475), and Hz06-sdAb-VH (SEQ ID NO: 476), respectively, followed by connecting a second FcγR2A and his tag via (G4S)3, as depicted in FIG2C, in a rearranged order. HzFc-10-VHH (SEQ ID NO: 477), anti-Fc-HzVHH2 (SEQ ID NO: 478), SP34-sdAb-VH, 20G6-sdAb-VH, Hz06-sdAb-VH, and Hz6017-sdAb-VH (SEQ ID NO: 479) were generated by transplanting the heavy chain CDR (according to Kabat) into the IGHV3-64*04 human germline sequence and introducing frame mutations S24A, V42F, K48Q, Y52A, and S54A according to the IMGT number. JIB126 was generated by SP34-VH-CH1NoDS-HzHP6017-sdAb-VH-his (SEQ ID NO: 480) and SP34-VL-hLC7NoDS-HzHP6017-sdAb-VH (SEQ ID NO: 481), as depicted in Figure 5B.JIB127 is an IgR containing two Fc-binding domains per molecule, wherein the heterodimer Fc and the immune cell-binding ScFv are fused to a light chain, as depicted in Figure 4J, and is produced using four chains. The first light chain contains HzHP6017B-VL-κbDS (SEQ ID NO: 482), the first heavy chain contains HzHP6017B-VH-CH1bD-Fc_mut-LALAPA-hole (SEQ ID NO: 483), which has additional Fc mutations N276K and L309V to eliminate the self-Fc binding of HzHP6017B, the second light chain contains HzHP6017B-VL-κNoDS-SP34-ScFv (SEQ ID NO: 484), and the second heavy chain contains HzHP6017B-VH-CH1bD-Fc_mut-LALAPA-knob (SEQ ID NO: 484). 485), which has additional Fc mutations N276K and L309V to eliminate Fc binding of HzHP6017B itself. To address the expression problem of the dual HzHP6017 VH-VH / VL-VL design in Example 23, three different spacer strategies were evaluated between VH-VH and VL-VL pairs. JIB128, JIB129, and JIB130 are identical to JIB92 as depicted in Figure 5I, except that: for JIB128, the HzHP6017B VL-VL spacer region derived from the constant κ domain is replaced by a shorter spacer region from the constant κ light chain (SEQ ID NO: 486), forming HzHP6017-2xVLshort-κbDS-SP34-ScFv (SEQ ID NO: 487) and co-expressed with JIB92 HC (SEQ ID NO: 411); for JIB129, the HzHP6017B VH-VH spacer region derived from the constant CH1 domain is replaced by a shorter spacer region derived from the constant CH1 chain (SEQ ID NO: 488), forming HzHP6017B-2xVHshort-CH1bDS-αHSA-VHH-his (SEQ ID NO: 489) and co-expressed with JIB92 LC (SEQ ID NO: 411). NO: 413) co-expression; for JIB130, the VH-VH and VL-VL spacer regions of HzHP6017B derived from the constant region of IgG are replaced by the (G4S)3 spacer region, forming HzHP6017-2xVLG4S3-κbDS-SP34-ScFv (SEQ ID NO: 490) and HzHP6017B-2xVHG4S3-CH1bDS-αHSA-VHH-his (SEQ ID NO: 491).

[0612] Anti-CD137 IgR is generated via VH and VL pairs fused to CH1bDS and κbDS or λbDS (for P566), wherein human FcγR2A is fused to the C-terminus of both the heavy chain (with his tag) and the light chain via a (G4S)3 linker to form JIB116 (SEQ ID NO: 492, SEQ ID NO: 493) based on 5B9, JIB117 (SEQ ID NO: 494, SEQ ID NO: 495) based on Hz5B9, JIB118 (SEQ ID NO: 496, SEQ ID NO: 497) based on uroselumab, JIB119 (SEQ ID NO: 498, SEQ ID NO: 499) based on P566, JIB120 (SEQ ID NO: 500, SEQ ID NO: 501) based on Hz4B4-1, and JIB121 (SEQ ID NO: 500, SEQ ID NO: 501) based on Hz4B4-2. 502, SEQ ID NO: 503), as depicted in Figure 5B. Hz4B4-1 is a variant of Hz4B4-2 with a seven-fold reduced affinity (Hz4B4-1-VH SEQ ID NO: 504, Hz4B4-1-VL SEQ ID NO: 505). Hz5B9 VH SEQ ID NO: 506 and VL SEQ ID NO: 507 were designed by transplanting the CDR (according to Kabat) into the IGHV4-59*01 and IGKV2-28*01 human lineages in the IMGT database.

[0613] Anti-CD89 IgR is generated by fusing VH and VL pairs to CH1bDS and κbDS, wherein human FcγR2A is fused to the C-terminus of both the heavy chain (with his tag) and the light chain via (G4S)3 linkers to form 14A8-based JIB122 (SEQ ID NO: 508, SEQ ID NO: 509), 8D2-based JIB123 (SEQ ID NO: 510, SEQ ID NO: 511), A77-based JIB124 (SEQ ID NO: 512, SEQ ID NO: 513), and HzA77-based JIB125 (SEQ ID NO: 514, SEQ ID NO: 515), as depicted in Figure 5B. HzA77 VH (SEQ ID NO: 516) and VL (SEQ ID NO: 517) were designed by transplanting CDR (according to Kabat) into the human germline IGHV1-2*02 and IGKV2-28*01 in the IMGT database and introducing the VH framework mutations R93S and D97E according to the IMGT number.

[0614] Tables 14, 41, and 42 show that, except for JIB113, JIB126, JIB127, and JIB128, all IgRs were well expressed in high purity after single-step purification. JIB113 was used to convert the SP34 VH / VL pair antibody into a single domain of variable heavy chain only (sdAb-VH). Although the protein was recovered and relatively pure as determined by SDS-PAGE, the monomer percentage as determined by SEC was very low, only 12.3%, indicating non-specific interactions with the matrix or itself. Similarly, JIB126, converted to sdAb-VH and fused to each constant chain of the SP34 Fab using the HzHP6017B VH / VL pair, was not expressed, indicating that the conversion to sdAb-VH was unsuccessful. In contrast, the Hz06 and 20G6 VH / VL pairs were successful in converting to the sdAb-VH construct from both expression and purity perspectives. The JIB102 IgR using the humanized Hz8GA8 VH / VL sequence had a monomer content of 98.8%, which contrasts sharply with the wild-type 8GA8 VH / VL construct JIB70 from Example 22, where the monomer content was 14.7%. This suggests that the humanization strategy for Hz8GA8 has the added benefit of improving its biophysical properties.

[0615] JIB127 evaluated Fc-Fc heterodimers with HzHP6017B VH / VL pairs on each Fc arm and SP34 ScFv linked to one of the HzHP6017B light chains using a differential interchain disulfide pairing strategy. However, although SDS-PAGE gels of the supernatant (Fig. 41B) showed that some proteins were expressed in the 25 kDa and 50 kDa regions in the reduced lanes, no proteins were recovered after a single purification. These results are consistent with previous findings, suggesting that the N276K and L309V mutations may only partially reduce the self-binding of HP6017 or HzHP6017B to the Fc region. JIB128, JIB129, and JIB130 evaluated alternative spacer regions between HzHP6017B dual VH-VH / VL-VL designs to address challenges previously observed in HzHP6017B VH-VH / VL-VL designs JIB91, JIB92, and JIB93, all of which used CH1 / κ spacer regions. Alternative spacer strategies included CH1 / short κ spacer regions (JIB128), short CH1 / κ spacer regions (JIB129), and (G4S)3 / (G4S)3. The SEC results in Table 14 show that short κ spacer regions between variable light chains did not solve the expression problem, while short CH1 spacer regions or flexible (G4S)3 spacer regions between the two variable domain pairs addressed the expression and purity challenges of dual VH-VH / VL-VL designs when using HzHP6017B cloning.

[0616] Table 14. Protein expression and monomers of IgR produced in Example 24 as determined by SEC.

[0617] Protein CHO (mg / L) SEC determination of monomer % JIB100 41100.0% JIB10152 95.9% JIB102160 98.8% JIB103113 94.6% JIB10486 98.0% JIB10527 99.0% JIB10657 96.8% JIB107118 98.5% JIB10837 100.0% JIB10925 87.4% JIB110123 95.4% JIB11161 92.1% JIB11233 97.6% JIB1136 12.3% JIB11453 9 1.8% JIB115 1979 2.9% JIB116 3298.9% JIB117 4596.2% JIB118V 1599 0.3% JIB119 13399.8% JIB120 4098.7% JIB121 2997.7% JIB122 3999.6% JIB123 111 100.0% JIB124 55 100.0% JIB125 3778.5% JIB126 Not detected Not applicable JIB127 Low detection Not applicable JIB128 0.3 Not applicable JIB129 4390.0% JIB130 1378.5% surface

[0618] Using the bioassays described in Examples 8 and 20, the ability of anti-CD3 IgR JIB110, JIB111, JIB112, JIB114, JIB115, JIB129, JIB130, and JIB49 to bridge CD20+ Raji B cells with Jurkat T cells and mediate T cell activation at 10 nM and 1 nM rituximab, with or without 1 mg / mL trastuzumab, or with 1 mg / mL trastuzumab, was evaluated. The results in Figure 43 show that JIB110, JIB111, JIB112, JIB129, JIB130, and JIB49 exhibited significantly greater T cell activation at one or both concentrations of rituximab than in the presence of 1 mg / mL trastuzumab. In the presence of 10 nM rituximab and 1 mg / mL trastuzumab, both JIB110 and JIB111 showed T cell activation, which was not significantly lower than that of 10 nM rituximab alone. These results indicate that the humanization of Fc-10-VHH against Fc-HzVHH2 was successful. In contrast, while JIB112, i.e., a 20G6-based Fab-like IgR with two FcγR2A ligations at each LC and HC C terminus, was functional, the 20G6 and Hz06 FcγR2A-sdAb-VH-FcγR2A fusions (JIB114 and JIB115, respectively) did not retain the function of their constructs that were converted from VH / VL pairs to a single-domain VH. In the presence of rituximab alone, HzHP6017B dual VH-VH / VL-VL Fab-like IgRs with FcγR2A linked to the C-terminus of each LC and HC are potent T-cell activators, and similar to WT HzHP6017 dual VH-VH / VL-VL IgR JIB49, indicating that the short CH1 / κ spacer region between VH-VH / VL-VL pairs (JIB129) or the G4 / S3 spacer region between VL-VL and VH-VH pairs (JIB130) can not only achieve expression and high purity of HzHP6017 clones, but also retain potent function.

[0619] Using the method described in Example 22, the ability of anti-CD28 IgR JIB68, JIB100, JIB101, JIB102, JIB03, JIB04, JIB105, JIB106, JIB107, JIB108, and JIB109 to bridge CD20+ Raji B cells with Jurkat T cells and mediate T cell co-stimulation was evaluated at 10 nM and 1 nM, in the presence of 10 nM rituximab alone, 10 nM trastuzumab alone, and cells alone as negative controls, and 10 nM and 1 nM anti-CD28 mIgG1 clone CD28.2 alone as positive T cell co-stimulatory activity controls. The results in Figure 44 show that, in the presence of 10 nM rituximab, all evaluated IgRs were able to mediate T cell co-stimulation at 10 nM IgR, 1 nM IgR, or both, with co-stimulation being greater than or significantly greater than that in the presence of 10 nM trastuzumab. Many evaluated IgRs, including JIB100 (Hz2E12), JIB101 (Hz9.3), JIB103 (Hz9D7), and JIB104 (HzTN228), are humanized versions of the anti-CD28 IgR evaluated in Example 22 (JIB67, JIB69, JIB71, and JIB72, respectively), indicating that these humanized IgRs retained their co-stimulatory function. JIB102 (Hz8GA8) was not evaluated as a WT clone (JIB70) in Example 22 due to poor purity, although it exhibited some function as a highly pure humanized clone under 1 nM IgR conditions. JIB105 (Hz28.3), JIB108 (TY24876), and JIB109 (1h-79-807), which were not evaluated in Example 22, can serve as humanized or wild-type IgR-mediated co-stimulation. JIB109 is a three-domain single-peptide chain in which FcγR2A is fused to the C-terminus and N-terminus of the anti-CD28 single-domain VL antibody (sdAb-VL), indicating that this is a feasible IgR structure for achieving functional cell-cell synapses and signal transduction. JIB106 (Hz5.11A1) and JIB107 (Hz5.11A1-C55S) are humanized versions of TGN1412 originally derived from its developed WT clone 5.11A1, with humanization and a cysteine ​​mutation at position 55 to a serine (according to IMGT number). Compared to the alternative humanized 5.11A1 clone TGN1412, they reduced the amount of nonspecific co-stimulatory signal in the presence of 10 nM trastuzumab.

[0620] In the presence of 10 nM rituximab, the ability of anti-CD137 IgR JIB116, JIB117, JIB118V1, JIB119, JIB120, and JIB121 to bridge CD20+ Raji B cells with HEK-Luc-CD137 cells (Biointron) (as alternative reporter cells for T cells and NK cells) was evaluated to assess their ability to mediate co-stimulatory activity. The bioassays described in Examples 8 and 20 were used, but with HEK-Luc-CD137 cells instead of Jurkat T cells. 10 nM of rituximab alone and titrated uroglümumab (Biointron, B315002) were used alone as positive controls, wherein uroglümumab is an IgG4-S228PP antibody capable of binding to FcγR2B on Raji cells to mediate CD137 crosslinking. The results in Figure 45 show that, in the presence of 10 nM rituximab, all IgRs were able to mediate co-stimulatory activity via targeting and cross-linking CD137, and this co-stimulatory activity was significantly higher than that in the presence of 10 nM rituximab alone. These results indicate that IgRs can mediate CD137 in the presence of antibodies bound to target cells. + Immune cell co-stimulatory function. Positive controls of uroselumab titrated at multiple concentrations provided signals consistent with the assay capabilities for detecting CD137 crosslinking and co-stimulatory activity.

[0621] Evaluation of anti-CD89 IgR JIB122, JIB123, JIB124, and JIB125 bridging Jurkat-NFAT-Luc T cells and CHO-K1-CD89 + The ability of the Biointron, specifically the Jurkat-NFAT-Luc T cells, to act as target cells, where a positive signal indicates cross-linking, and the CHO-K1-CD89, a surrogate reporter for cytotoxicity, is demonstrated. + Cells as representatives of CD89 +Myeloid cells were used as alternative effector cells. The ability of IgR to mediate functional crosslinking activity was evaluated at 100 nM, 10 nM, and 1 nM in the presence of 1 nM OKT3 (Biointron, B6928) with or without 1 mg / mL trastuzumab, or in the presence of 1 mg / mL trastuzumab without OKT3. Trastuzumab alone at 1 mg / mL and cells alone were run as negative controls, and a dose titration of OKT3 alone (Biointron, B6928) or 1 nM OKT3 with 1 mg / mL trastuzumab was run as a positive control, setting a background signal. The bioassays described in Examples 8 and 20 were used, but Jurkat T cells were used instead of Raji B cells (target cells), and CHO-K1-CD89+ were used instead of Jurkat T cells (effector cells). The results in Figure 46 show that, except for JIB125, all IgRs were able to mediate CD89 crosslinking and substitution effector activity via target cell luciferase production in the presence of 1 nM OKT3, which was significantly higher than that in the presence of 1 nM OKT3 alone or 1 mg / mL trastuzumab alone. Furthermore, both 14A8- and 8D2-based IgRs were able to mediate CD89 crosslinking and substitution effector activity in the presence of 1 nM OKT3 and 1 mg / mL trastuzumab, which was significantly higher than that in the presence of 1 mg / mL trastuzumab alone or 1 mM OKT3 and trastuzumab alone. These results indicate that, similar to in vivo conditions, IgRs can mediate CD89 crosslinking against antibody-conditioned target cells in the presence of high concentrations of nonspecific IgG. + Myeloid cell effector function.

[0622] Example 25. Expression, purification, and evaluation of mouse reactive αCD3, αCD28, αCD137, and αCD40 IgRs that bind multivalently to IgG via multiple anti-Fc or FcγR domains.

[0623] IgG and Fc-binding heterologous peptides and heterologous multimers were expressed and purified as described in Examples 1 and 11. To enable in vivo studies in known immunogenic mouse tumor models that generate endogenous antitumor antibodies (such as CT26 colorectal tumor, 4T1 triple-negative breast tumor, and RENCA renal cortical tumor (Zappala et al., 2022)), IgRs were generated having domains of mouse peptides on their surfaces capable of binding to mouse immune cells and domains capable of binding to naturally occurring endogenous mouse IgG in vivo, comprising the major subclasses mIgG1, mIgG2a, and mIgG2b, which constitute 90% of the mIgG present in serum / blood in vivo. These IgRs should be able to bridge endogenous antitumor antibodies binding to cancer cells to immune cell receptors to drive cell activity and efficacy by limiting tumor growth relative to a vector control. Furthermore, when combined with exogenously introduced mAbs targeting tumor cells, the IgRs should synergistically achieve enhanced antitumor efficacy.

[0624] IgR is generated by fusing VH and VL pairs into mCH1bDS (SEQ ID NO: 518) and mκbDS (SEQ ID NO: 519), wherein mouse mFcγR2b (capable of binding mIgG1, mIgG2a, and mIgG2b) or anti-Fc-VHH2 (cross-reactive with mIgG1, mIgG2a, and mIgG2b) is fused to the C-terminus of both the heavy and light chains, wherein αMSA-VHH is fused to the C-terminus of the mCH1bDS domain, followed by a his tag to form: JIB137 (SEQ ID NO: 520, SEQ ID NO: 521) based on anti-mCD3 KT3 / mFcγR2b, JIB138 (SEQ ID NO: 522, SEQ ID NO: 523) based on KT3 / anti-Fc-VHH2, and JIB139 (SEQ ID NO: 529) based on 2C11 / mFcγR2b. JIB140 (SEQ ID NO: 524, SEQ ID NO: 525) based on anti-mCD28TY24876 / mFcγR2b, JIB142 (SEQ ID NO: 528, SEQ ID NO: 529) based on anti-mCD137 Lob12.3 / mFcγR2b, JIB143 (SEQ ID NO: 530, SEQ ID NO: 531) based on Lob12.3 / anti-Fc-VHH2, JIB144 (SEQ ID NO: 532, SEQ ID NO: 533) based on 3H3 / mFcγR2b, and JIB145 (SEQ ID NO: 534, SEQ ID NO: 525) based on anti-mCD40 1C10 / mFcγR2b. JIB146 (SEQ ID NO: 536, SEQ ID NO: 537) based on 1C10 / anti-Fc-VHH2 and JIB147 (SEQ ID NO: 538, SEQ ID NO: 539) based on FGK45 / mFcγR2b, as depicted in Figure 5J. JIB141 is generated by fusing mFcγR2b to the N-terminus of 1h-79-807-sdAb-VL, followed by connecting mFcγR2b, αMSA-VHH and his tag via (G4S)3 connector to form mFcγR2b-SP1h-79-807-sdAb-VL-mFcγR2b-αMSA-VHH-his (SEQ ID NO: 540), as depicted in Figure 3B, in rearranged order.

[0625] To enable exogenously introduced IgG to be combined with in vivo IgR for the treatment of solid tumors, anti-EphA2 monoclonal antibodies capable of binding to mouse tumors such as CT26 were generated by fusing anti-EphA2 VH (SEQ ID NO: 549) and VL (SEQ ID NO: 550) into mouse mIgG1 heavy chain (SEQ ID NO: 551) and mouse λ1 light chain (SEQ ID NO: 552) to produce JIB148. Similarly, to enable exogenously introduced IgG to combine with in vivo IgR for the treatment of liquid tumors, an anti-CD19 monoclonal antibody capable of binding to mouse hematologic mIgG2a heavy chain (SEQ ID NO: 555) and preferentially binding to FcγRIV and human FcγR3A / B was generated by fusing 1D3 VH (SEQ ID NO: 553) and VL (SEQ ID NO: 554) into mouse mIgG2a heavy chain (SEQ ID NO: 555) and mouse κ light chain (SEQ ID NO: 556). This antibody was then cultured in the presence of 20 μM kifunensine to produce a non-fucosylated Fc glycoform that preferentially binds to FcγRIV and human FcγR3A / B, thereby generating anti-mCD19 mAb 1D3-IgG2a-kif.

[0626] Table 15. Protein expression and monomers of IgR produced in Example 25 as determined by SEC.

[0627] Protein CHO (mg / L) SEC assay of monomers % JIB1371 168 2.85% JIB1382 759 6.14% JIB1391 338 2.8% JIB140 38 3.4% JIB141 82 4.3% JIB142 220 91.6% JIB143 32 19 3.2% JIB144 488 2.6% JIB145 25 29 1.6% JIB146 33 79 3.7% JIB147 87 70.9% JIB148 37 88 9.3% 1D3-IgG2a-kif 47 98.6% surface

[0628] Tables 15, 47, and 48 show that all IgR and IgG were well expressed in high purity after single-step purification, except for JIB141, which, designed using mFcγR2b-SP1h-79-807-sdAb-VL-mFcγR2b-αMSA-VHH-his, had 4.3% monomer content as determined by SEC. This contrasts with the similar JIB109 design, which used the same anti-CD28 clone, FcγRA-SP1h-79-807-sdAb-VL-FcγR2A-his, which had 87.4% monomer content as determined by SEC after single-step purification.

[0629] The scope of this invention is not intended to be limited to the specific disclosed embodiments, which are provided, for example, to illustrate various aspects of the invention. Various modifications to the described compositions and methods will become apparent from the description and teachings herein. Such variations may be practiced without departing from the true scope and spirit of this disclosure and are intended to fall within its scope.

[0630] References

[0631] Anthony, RM, Wermeling, F., Karlsson, MCI, & Ravetch, JV(2008). Identification of a receptor required for the anti-inflammatory activity of IVIG. Proceedings of the National Academy of Sciences of the United States of America, 105(50), 19571–19578.

[0632] Arnold, JN, Dwek, RA, Rudd, PM, & Sim, RB (2006). Mannanbinding lectin and its interaction with immunoglobulins in health and disease. Immunology Letters, 106(2), 103–110.

[0633] Beck, A., Goetsch, L., Dumontet, C., & Corvaïa, N. (2017). Strategiesand challenges for the next generation of antibody-drug conjugates. NatureReviews. Drug Discovery, 16(5), 315–337.

[0634] Belin, P., Dassa, J., Drevet, P., Lajeunesse, E., Savatier, A.,Boulain, J.-C., & Ménez, A. (2004). Toxicity-based selection of Escherichiacoli mutants for functional recombinant protein production: application to anantibody fragment. Protein Engineering, Design & Selection: PEDS, 17(5), 491–500.

[0635] Boesch, A. W., Alter, G., & Ackerman, M. E. (2015). Prospects forengineering HIV-specific antibodies for enhanced effector function and half-life. Current Opinion in HIV and AIDS, 10(3), 160–169.

[0636] Boesch, A. W., Brown, E. P., Cheng, H. D., Ofori, M. O., Normandin,E., Nigrovic, P. A., Alter, G., & Ackerman, M. E. (2014). Highly parallelcharacterization of IgG Fc binding interactions. mAbs, 6(4), 915–927.

[0637] Bonvin, P., Venet, S., Kosco-Vilbois, M., & Fischer, N. (2015).Purpose-Oriented Antibody Libraries Incorporating Tailored CDR3 Sequences.Antibodies, 4(2), 103–122.

[0638] Brinkmann, U., & Kontermann, R. E. (2017). The making of bispecificantibodies. mAbs, 9(2), 182.

[0639] Brinkmann, U., Reiter, Y., Jung, S. H., Lee, B., & Pastan, I. (1993).A recombinant immunotoxin containing a disulfide-stabilized Fv fragment.Proceedings of the National Academy of Sciences of the United States ofAmerica, 90(16), 7538–7542.

[0640] Carrier, A., Ducancel, F., Settiawan, N. B., Cattolico, L., Maillère,B., Léonetti, M., Drevet, P., Ménez, A., & Boulain, J. C. (1995). Recombinantantibody-alkaline phosphatase conjugates for diagnosis of human IgGs:application to anti-HBsAg detection. Journal of Immunological Methods, 181(2). https: / / doi.org / 10.1016 / 0022-1759(94)00344-v

[0641] Chang, T.-W., Hung, A. F.-H., & Lu, D. C.-S. (2017). Anti-human mIgAantibodies capable of lysing mIgA-B lymphocytes and decreasing IgA production(USPTO Patent No. 9688776). In US Patent (No. 9688776). https: / / patentimages.storage.googleapis.com / b9 / 7c / f0 / df399842c072a7 / US9688776.pdf

[0642] Chen, K., & Cerutti, A. (2011). The function and regulation ofimmunoglobulin D. Current Opinion in Immunology, 23(3), 345–352.

[0643] Chothia, C., Novotný, J., Bruccoleri, R., & Karplus, M. (1985).Domain association in immunoglobulin molecules. The packing of variabledomains. Journal of Molecular Biology, 186(3). https: / / doi.org / 10.1016 / 0022-2836(85)90137-8

[0644] Corrales-Aguilar, E., Hoffmann, K., & Hengel, H. (2014). CMV-encodedFcγ receptors: modulators at the interface of innate and adaptive immunity.Seminars in Immunopathology, 36(6), 627–640.

[0645] Dekkers, G., Bentlage, A. E. H., Stegmann, T. C., Howie, H. L.,Lissenberg-Thunnissen, S., Zimring, J., Rispens, T., & Vidarsson, G. (2017).Affinity of human IgG subclasses to mouse Fc gamma receptors. mAbs, 9(5),767–773.

[0646] Delespesse, G., Hofstetter, H., & Sarfati, M. (1989). Low-affinityreceptor for IgE (FcERII, CD23) and its soluble fragments. InternationalArchives of Allergy and Applied Immunology, 90 Suppl 1, 41–44.

[0647] de Vries, E., Koene, H. R., Vossen, J. M., Gratama, J. W., von demBorne, A. E., Waaijer, J. L., Haraldsson, A., de Haas, M., & van Tol, M. J.(1996). Identification of an unusual Fc gamma receptor IIIa (CD16) on naturalkiller cells in a patient with recurrent infections. Blood, 88(8), 3022–3027.

[0648] Dong, X., Storkus, W. J., & Salter, R. D. (1999). Binding and uptakeof agalactosyl IgG by mannose receptor on macrophages and dendritic cells.Journal of Immunology , 163(10), 5427–5434.

[0649] Dubin, G., Frank, I., & Friedman, H. M. (1990). Herpes simplex virustype 1 encodes two Fc receptors which have different binding characteristicsfor monomeric immunoglobulin G (IgG) and IgG complexes. Journal of Virology,64(6), 2725–2731.

[0650] Foote, J., & Winter, G. (1992). Antibody framework residues affectingthe conformation of the hypervariable loops. Journal of Molecular Biology,224(2). https: / / doi.org / 10.1016 / 0022-2836(92)91010-m

[0651] Frey, S., Opazo, F., & Götzke, H. (2018). Target detection using amonovalent antibody (WIPO Patent No. 2018167141:A2). In World Patent (No.2018167141:A2). https: / / patentimages.storage.googleapis.com / 95 / b5 / e5 / 8702c689c5d90e / WO2018167141A2.pdf

[0652] Geddie, M. L., Kirpotin, D. B., Kohli, N., Kornaga, T., Boll, B.,Razlog, M., Drummond, D. C., & Lugovskoy, A. A. (2022). Development ofdisulfide-stabilized Fabs for targeting of antibody-directednanotherapeutics. mAbs, 14(1). https: / / doi.org / 10.1080 / 19420862.2022.2083466

[0653] Hagihara, Y., & Saerens, D. (2014). Engineering disulfide bondswithin an antibody. Biochimica et Biophysica Acta, 1844(11), 2016–2023.

[0654] Halaby, D. M., Poupon, A., & Mornon, J. (1999). The immunoglobulinfold family: sequence analysis and 3D structure comparisons. ProteinEngineering, 12(7). https: / / doi.org / 10.1093 / protein / 12.7.563

[0655] Hallam, T. J., Wold, E., Wahl, A., & Smider, V. V. (2015). Antibodyconjugates with unnatural amino acids. Molecular Pharmaceutics, 12(6), 1848–1862.

[0656] Hamilton, R. G., & Morrison, S. L. (1993). Epitope mapping of humanimmunoglobulin-specific murine monoclonal antibodies with domain-switched,deleted and point-mutated chimeric antibodies. Journal of ImmunologicalMethods, 158(1), 107–122.

[0657] Hermans, W. J. J., Bezemer, S., & Mijnsbergen, Y. M. (2015). Single-domain antigen-binding proteins that bind mammalian IgG (USPTO Patent No.9040666). In US Patent (No. 9040666). https: / / patentimages.storage.googleapis.com / a6 / 30 / 24 / 5c6331255c5ef5 / US9040666.pdf

[0658] Hermans, W. J. J., Blokland, S., van Kesteren, M. A., Horrevoets, J.M., & Detmers, F. J. M. (2017). Antigen-binding protein directed againstepitope in the CH1 domain of human IgG antibodies (USPTO Patent No. 9850315).In US Patent (No. 9850315). https: / / patentimages.storage.googleapis.com / 61 / e4 / 6a / 1f1b6a48d95d10 / US9850315.pdf

[0659] Herrmann, M., Krupka, C., Deiser, K., Brauchle, B., Marcinek, A.,Ogrinc Wagner, A., Rataj, F., Mocikat, R., Metzeler, K. H., Spiekermann, K.,Kobold, S., Fenn, N. C., Hopfner, K.-P., & Subklewe, M. (2018). BifunctionalPD-1 × αCD3 × αCD33 fusion protein reverses adaptive immune escape in acutemyeloid leukemia. Blood, 132(23), 2484–2494.

[0660] Hogarth, P. M., & Wines, B. D. (2014). Multimeric Fc receptorpolypeptides (USPTO Patent No. 8729247). In US Patent (No. 8729247). https: / / patentimages.storage.googleapis.com / 2a / a6 / 81 / a6474991b98b27 / US8729247.pdf

[0661] Hulett, & Hogarth, P. M. (1998). The second and third extracellulardomains of FcgammaRI (CD64) confer the unique high affinity binding of IgG2a.Molecular Immunology, 35(14-15). https: / / doi.org / 10.1016 / s0161-5890(98)00069-8

[0662] Jeffrey, S. C., Burke, P. J., Lyon, R. P., Meyer, D. W., Sussman, D.,Anderson, M., Hunter, J. H., Leiske, C. I., Miyamoto, J. B., Nicholas, N. D.,Okeley, N. M., Sanderson, R. J., Stone, I. J., Zeng, W., Gregson, S. J.,Masterson, L., Tiberghien, A. C., Howard, P. W., Thurston, D. E., … Senter,P. D. (2013). A potent anti-CD70 antibody-drug conjugate combining a dimericpyrrolobenzodiazepine drug with site-specific conjugation technology.Bioconjugate Chemistry, 24(7), 1256–1263.

[0663] Johnson, L. S., Li, H., & Tuaillon, N. (2010). FcgammaRIIB FusionProteins and Compositions Thereof (USPTO Patent No. 20100196372:A1). In USPatent (No. 20100196372:A1). https: / / patentimages.storage.googleapis.com / 3c / b3 / a3 / 3703e4a4dd5edb / US20100196372A1.pdf

[0664] Junutula, J. R., Raab, H., Clark, S., Bhakta, S., Leipold, D. D.,Weir, S., Chen, Y., Simpson, M., Tsai, S. P., Dennis, M. S., Lu, Y., Meng, Y.G., Ng, C., Yang, J., Lee, C. C., Duenas, E., Gorrell, J., Katta, V., Kim,A., … Mallet, W. (2008). Site-specific conjugation of a cytotoxic drug to anantibody improves the therapeutic index. Nature Biotechnology, 26(8), 925–932.

[0665] Kabat, E. A. (1992). Sequences of Proteins of Immunological Interest.DIANE Publishing.

[0666] Karsten, C. M., Pandey, M. K., Figge, J., Kilchenstein, R., Taylor,P. R., Rosas, M., McDonald, J. U., Orr, S. J., Berger, M., Petzold, D.,Blanchard, V., Winkler, A., Hess, C., Reid, D. M., Majoul, I. V., Strait, R.T., Harris, N. L., Köhl, G., Wex, E., … Köhl, J. (2012). Anti-inflammatoryactivity of IgG1 mediated by Fc galactosylation and association of FcγRIIBand dectin-1. Nature Medicine, 18(9), 1401–1406.

[0667] Keeble, A. H., Khan, Z., Forster, A., & James, L. C. (2008). TRIM21is an IgG receptor that is structurally, thermodynamically, and kineticallyconserved. Proceedings of the National Academy of Sciences of the UnitedStates of America, 105(16), 6045–6050.

[0668] Leonard, E. K., Pellitero, M. A., Juelg, B., Spangler, J. B., &Arroyo-Currás, N. (2022). Antibody–Invertase Fusion Protein EnablesQuantitative Detection of SARS-CoV-2 Antibodies Using Widely AvailableGlucometers. Journal of the American Chemical Society. https: / / doi.org / 10.1021 / jacs.2c02537

[0669] Litwin, V., Jackson, W., & Grose, C. (1992). Receptor properties oftwo varicella-zoster virus glycoproteins, gpI and gpIV, homologous to herpessimplex virus gE and gI. Journal of Virology, 66(6), 3643–3651.

[0670] Liu, H., Saxena, A., Sidhu, S. S., & Wu, D. (2017). Fc Engineeringfor Developing Therapeutic Bispecific Antibodies and Novel Scaffolds.Frontiers in Immunology, 8. https: / / doi.org / 10.3389 / fimmu.2017.00038

[0671] Liu, L., Lam, C.-Y. K., Alderson, R., Long, V., Yang, Y., Burns, R.,Widjaja, L., Li, J., Wolf, C., Ciccarone, V., Tamura, J., Diedrich, G.,Bonvini, E., Johnson, S., & Moore, P. A. (2019, July 1). Abstract 1560:Selection of a bispecific trivalent HER2 x CD137 TRIDENT format providingoptimal tumor-anchored immune co-stimulation. Immunology. Proceedings: AACRAnnual Meeting 2019; March 29-April 3, 2019; Atlanta, GA. https: / / doi.org / 10.1158 / 1538-7445.sabcs18-1560

[0672] Müller, M. G., Ing, J. Y., Cheng, M. K.-W., Flitter, B. A., & Moe, G.R. (2013). Identification of a phage-encoded Ig-binding protein from invasiveNeisseria meningitidis. Journal of Immunology , 191(6), 3287–3296.

[0673] Nakamura, H., Oda-Ueda, N., Ueda, T., & Ohkuri, T. (2018). A novelengineered interchain disulfide bond in the constant region enhances thethermostability of adalimumab Fab. Biochemical and Biophysical ResearchCommunications, 495(1), 7–11.

[0674] Namboodiri, A. M., Budkowska, A., Nietert, P. J., & Pandey, J. P.(2007). Fc gamma receptor-like hepatitis C virus core protein bindsdifferentially to IgG of discordant Fc (GM) genotypes. Molecular Immunology,44(15), 3805–3808.

[0675] Oganesyan, V., Mazor, Y., Yang, C., Cook, K. E., Woods, R. M.,Ferguson, A., Bowen, M. A., Martin, T., Zhu, J., Wu, H., & Dall’Acqua, W. F.(2015). Structural insights into the interaction of human IgG1 with FcγRI:no direct role of glycans in binding. Acta Crystallographica. Section D,Biological Crystallography, 71(Pt 11), 2354.

[0676] Para, M. F., Baucke, R. B., & Spear, P. G. (1980). Immunoglobulin G(Fc)-binding receptors on virions of herpes simplex virus type 1 and transferof these receptors to the cell surface by infection. Journal of Virology, 34(2), 512–520.

[0677] Patel, K. R., Roberts, J. T., & Barb, A. W. (2019). MultipleVariables at the Leukocyte Cell Surface Impact Fc γ Receptor-DependentMechanisms. Frontiers in Immunology, 10, 223.

[0678] Pillow, T. H., Tien, J., Parsons-Reponte, K. L., Bhakta, S., Li, H.,Staben, L. R., Li, G., Chuh, J., Fourie-O’Donohue, A., Darwish, M., Yip, V.,Liu, L., Leipold, D. D., Su, D., Wu, E., Spencer, S. D., Shen, B.-Q., Xu, K.,Kozak, K. R., … Junutula, J. R. (2014). Site-specific trastuzumabmaytansinoid antibody-drug conjugates with improved therapeutic activitythrough linker and antibody engineering. Journal of Medicinal Chemistry, 57(19), 7890–7899.

[0679] Prihoda, D., Maamary, J., Waight, A., Juan, V., Fayadat-Dilman, L.,Svozil, D., & Bitton, D. A. (2022). BioPhi: A platform for antibody design,humanization, and humanness evaluation based on natural antibody repertoiresand deep learning. mAbs, 14(1), 2020203.

[0680] Promsote, W., Xu, L., Hataye, J., Fabozzi, G., March, K., Almasri, C.G., DeMouth, M. E., Lovelace, S. E., Talana, C. A., Doria-Rose, N. A., McKee,K., Hait, S. H., Casazza, J. P., Ambrozak, D., Beninga, J., Rao, E.,Furtmann, N., Birkenfeld, J., McCarthy, E., … Koup, R. A. (2023).Trispecific antibody targeting HIV-1 and T cells activates and eliminateslatently-infected cells in HIV / SHIV infections. Nature Communications, 14(1),1–13.

[0681] Raghavan, M., Chen, M. Y., Gastinel, L. N., & Bjorkman, P. J. (1994).Investigation of the interaction between the class I MHC-related Fc receptorand its immunoglobulin G ligand. Immunity, 1(4), 303–315.

[0682] Rueger, P., Schlothauer, T., & Seeber, S. (2018). METHOD FORPRODUCING SOLUBLE FcR AS Fc-FUSION WITH INERT IMMUNOGLOBULIN Fc-REGION ANDUSES THEREOF (USPTO Patent No. 20180265564:A1). In US Patent (No.20180265564:A1). https: / / patentimages.storage.googleapis.com / 64 / a7 / 41 / defc1adfda2cd5 / US20180265564A1.pdf

[0683] Saunders, K. O. (2019). Conceptual Approaches to Modulating AntibodyEffector Functions and Circulation Half-Life. Frontiers in Immunology, 10,449325.

[0684] Shibata-Koyama, M., Iida, S., Okazaki, A., Mori, K., Kitajima-Miyama,K., Saitou, S., Kakita, S., Kanda, Y., Shitara, K., Kato, K., & Satoh, M.(2009). The N-linked oligosaccharide at FcγRIIIa Asn-45: an inhibitoryelement for high FcγRIIIa binding affinity to IgG glycoforms lacking corefucosylation. Glycobiology, 19(2), 126.

[0685] Shin, J.-S., & Greer, A. M. (2015). The role of FcεRI expressed indendritic cells and monocytes. Cellular and Molecular Life Sciences: CMLS, 72(12), 2349–2360.

[0686] Strohl, W. R. (2015). Fusion Proteins for Half-Life Extension ofBiologics as a Strategy to Make Biobetters. BioDrugs: ClinicalImmunotherapeutics, Biopharmaceuticals and Gene Therapy, 29(4), 215.

[0687] Studnicka, G. M., Soares, S., Better, M., Williams, R. E., Nadell,R., & Horwitz, A. H. (1994). Human-engineered monoclonal antibodies retainfull specific binding activity by preserving non-CDR complementarity-modulating residues. Protein Engineering, 7(6). https: / / doi.org / 10.1093 / protein / 7.6.805

[0688] The second and third extracellular domains of FcγRI (CD64) conferthe unique high affinity binding of IgG2a. (1998). Molecular Immunology, 35(14-15), 989–996.

[0689] Tumey, L. N., Li, F., Rago, B., Han, X., Loganzo, F., Musto, S.,Graziani, E. I., Puthenveetil, S., Casavant, J., Marquette, K., Clark, T.,Bikker, J., Bennett, E. M., Barletta, F., Piche-Nicholas, N., Tam, A., O’Donnell, C. J., Gerber, H. P., & Tchistiakova, L. (2017). Site Selection: aCase Study in the Identification of Optimal Cysteine Engineered Antibody DrugConjugates. The AAPS Journal, 19(4), 1123–1135.

[0690] UniProt. (n.d.-a). Retrieved June 27, 2024, from https: / / www.uniprot.org / uniprotkb / P08637 / entry

[0691] UniProt. (n.d.-b). Retrieved June 27, 2024, from https: / / www.uniprot.org / uniprotkb / O75015 / entry

[0692] UniProt. (n.d.-c). Retrieved June 27, 2024, from https: / / www.uniprot.org / uniprotkb / A0A0B4J1G0 / entry

[0693] UniProt. (n.d.-d). Retrieved June 27, 2024, from https: / / www.uniprot.org / uniprotkb / P12318 / entry

[0694] UniProt. (n.d.-e). Retrieved June 27, 2024, from https: / / www.uniprot.org / uniprotkb / P31994 / publications

[0695] UniProt. (n.d.-f). Retrieved June 27, 2024, from https: / / www.uniprot.org / uniprotkb / P12314 / entry

[0696] UniProt. (n.d.-g). Retrieved June 27, 2024, from https: / / www.uniprot.org / uniprotkb / P01833 / entry

[0697] UniProt. (n.d.-h). Retrieved June 27, 2024, from https: / / www.uniprot.org / uniprotkb / P24071 / entry

[0698] UniProt. (n.d.-i). Retrieved June 27, 2024, from https: / / www.uniprot.org / uniprotkb / Q8WWV6 / entry

[0699] UniProt. (n.d.-j). Retrieved June 27, 2024, from https: / / www.uniprot.org / uniprotkb / O60667 / entry

[0700] UniProt. (n.d.-k). Retrieved June 27, 2024, from https: / / www.uniprot.org / uniprotkb / P12319 / entry

[0701] UniProt. (n.d.-l). Retrieved June 27, 2024, from https: / / www.uniprot.org / uniprotkb / P06734 / entry

[0702] UniProt. (n.d.-m). Retrieved June 27, 2024, from https: / / www.uniprot.org / uniprotkb / P55899 / entry

[0703] Univ, K. (2010). IgG BINDING PEPTIDE (USPTO Patent No.20100297606A1). In US Patent (No. 20100297606A1). https: / / eureka.patsnap.com / patent-US20100297606A1

[0704] Vaks, L., Litvak-Greenfeld, D., Dror, S., Shefet-Carasso, L.,Matatov, G., Nahary, L., Shapira, S., Hakim, R., Alroy, I., & Benhar, I.(2018). Design Principles for Bispecific IgGs, Opportunities and Pitfalls ofArtificial Disulfide Bonds. Antibodies (Basel, Switzerland), 7(3). https: / / doi.org / 10.3390 / antib7030027

[0705] Wang, C., Wu, Y., Wang, L., Hong, B., Jin, Y., Hu, D., Chen, G.,Kong, Y., Huang, A., Hua, G., & Ying, T. (2017). Engineered Soluble MonomericIgG1 Fc with Significantly Decreased Non-Specific Binding. Frontiers inImmunology, 8, 302778.

[0706] Website. (n.d.-a). https: / / www.ncbi.nlm.nih.gov / pmc / articles / PMC3220521 / ,

[0707] Website. (n.d.-b). https: / / www.uniprot.org / uniprotkb / Q96RD9 / entry

[0708] Wilson, T. J., Fuchs, A., & Colonna, M. (2012). Cutting edge: humanFcRL4 and FcRL5 are receptors for IgA and IgG. Journal of Immunology , 188(10), 4741–4745.

[0709] Zappala, F., Higbee-Dempsey, E., Jang, B., Miller, J., Yan, L.,Minutolo, N. G., Rosado González, G. T., Tsourkas, A., & Ozdemir, B. A.(2022). Rapid, site-specific labeling of “off-the-shelf” and native serumautoantibodies with T cell–redirecting domains. Science Advances. https: / / doi.org / 10.1126 / sciadv.abn4613

[0710] Multiple resolution - JaLC. (n.d.). Retrieved June 27, 2024, fromhttps: / / doi.org / 10.15017 / 1931748

[0711] Ji Yi HK. (2021). Mouse anti-human IgG monoclonal antibody (PatentNo. CN:112010981:B). In Patent (No. CN:112010981:B). https: / / patentimages.storage.googleapis.com / 72 / 3d / 18 / 159b852f4b070e / CN112010981B.pdf

Claims

1. A heterologous polypeptide comprising: at least one immunoglobulin-binding domain; and at least one immune cell surface protein-binding domain; wherein the at least one immunoglobulin-binding domain is derived from an Fc receptor or an Fc binding domain, including but not limited to FcγRIII, mFcγRIV, FcγRIIa, FcγRIIb, FcγRIIc, FcγRI, mFcγRIII, mFcγRIIa, mFcγRIIb, mFcγRI, FcαRI, C1q, FcRL, FcRL5, pIgR, Fcα / μR, FcμR, FcεRI, FcεRII, FcRn, TRIM21 The heteropeptide comprises, and isoforms, derivatives, and analogs thereof; wherein at least one immunoglobulin-binding domain is derived from an antigen-binding domain, an antibody, or an antigen-binding fragment thereof, or its variants, derivatives, or analogs thereof, the variants, derivatives, or analogs comprising VH and VL pairs, ScFv, Fab, IgG, sdAb-VL, sdAb-VH, VHH, or avimer thereof, or its derivatives or analogs thereof; wherein the heteropeptide comprises one or more half-life-extending domains, the half-life-extending domains comprising an anti-HSA antigen-binding domain, an antibody, or an antigen-binding fragment thereof, and variants, derivatives, or analogs thereof including VHH or single-domain antibodies, and immunoglobulin IgG Fc domain thereof, and its variants, derivatives, or analogs thereof; wherein the heteropeptide comprises at least two immunoglobulin-binding domains; and wherein the heteropeptide comprises at least two immune cell surface protein binding domains.

2. A heterologous polypeptide comprising: at least one immunoglobulin-binding domain; and at least one immune cell surface protein-binding domain; wherein the at least one immunoglobulin-binding domain comprises all or a portion of the Fc receptor of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 23, 77, 80, 271, 272, 273, 274, 275, 276, 277, 294, 296, 298, 300, 324, and 325; wherein the at least one immunoglobulin-binding domain is derived from FcγRIIa containing one or more mutations of SEQ ID NO: 9, the mutations including R56H, K118N, T120V, L160Q, and V172E; or wherein the at least one immunoglobulin-binding domain is derived from FcγRIIa containing one or more mutations of SEQ ID NO:

9. One or more mutated FcγRIII in 1, the mutations including S181P, K122N, T124V, Q176E, I90R, T118K, A119L and Y134F; wherein the heteropeptide comprises at least two immunoglobulin-binding domains; and wherein the heteropeptide comprises at least two immune cell surface protein-binding domains.

3. A heterologous polypeptide comprising: at least one immunoglobulin-binding domain; and at least one immune cell surface protein-binding domain; wherein the at least one immunoglobulin-binding domain is derived from an antigen-binding domain, an antibody or antigen-binding fragment, or variants, derivatives, or analogs thereof, including but not limited to VH and VL pairs, ScFv, Fab, IgG, sdAb-VL, sdAb-VH, VHH, or avimer and derivatives or analogs thereof; wherein the at least one immunoglobulin-binding domain comprises SEQ ID NO: 569, 570, 571, 572, 573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584, 585, 586, 587, 588, 589, 590, 591, 592, 593, 594, 595, 596, 597, 598, 599, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611, 612, 613, 614, 61 The heteropeptide comprises at least one of the CDR or FR regions of 5, 616, 617, 618, 619, 620, 621, 622, 623, 624, 625, 626, 627, 628, 629, 6230, 631, 632, 633, 634, 635, 636, 637, 638, 639, 640, 641, 642, 643, 644, 645, 646, 647, 648, 649, 650, 651, 652, 653, 654, 655, 656, and 657, or at least one of the avimers defined in SEQ ID NO: 132; wherein the heteropeptide comprises at least two immunoglobulin-binding domains; and wherein the heteropeptide comprises at least two immune cell surface protein-binding domains.

4. A heterologous polypeptide comprising: at least one immunoglobulin-binding domain; at least one immune cell surface protein-binding domain; wherein the at least one immune cell surface protein-binding domain is derived from an antigen-binding domain, an antibody or antigen-binding fragment and its variants, derivatives or analogs, said variants, derivatives or analogs including but not limited to VH and VL pairs, ScFv, Fab, IgG, sdAb-VL, sdAb-VH, VHH and their derivatives or analogs; wherein the immune cell surface protein-binding domain comprises SEQ ID NO: 580, 606, 608, 609, 610, 611, 612, 615, 626, 635, 636, 637, 638, 639, 658, 659, 660, 661, 662, 663, 664, 665, 666, 667, 668, 669, 670, 671, 672, 673, 674, 675, 676, 677, 678, 679, 680, 681, 682, 683, 684, 685, 686, 687, 688, 689, 690, 691, 692, 693 694, 695, 696, 697, 698, 699, 700, 701, 702, 703, 704, 705, 706, 707, 708, 709, 710, 711, 712, 713, 714, 715, 716, 717, 718, 719, 720, 721, 722, 723, 724, 725, 726, 727, 728, 729, 730, 731, 732, 733, 734, 735, 736, 737, 738, 739, 740, 741, 742, 743 744, 745, 746, 747, 748, 749, 750, 751, 752, 753, 754, 755, 756, 757, 758, 759, 760, 761, 762, 763, 764, 765, 766, 767, 768, 769, 770, 771, 772, 773, 774, 775, 776, 777, 778, 779, 780, 781, 782, 783, 784, 785, 786, 787, 788, 789, 790, 791, 792, 793 794, 795, 796, 797, 798, 799, 800, 801, 802, 803, 804, 805, 806, 807, 808, 809, 810, 811, 812, 813, 814, 815, 816, 817, 818, 819, 820, 821, 822, 823, 824, 825, 826, 827, 828, 829, 840, 841, 842, 843, 844, 845, 846, 847, 848, 849, 840, 841, 842, 843,844, 845, 846, 847, 848, 849, 850, 851, 852, 853, 854, 855, 856, 857, 858, 859, 860, 861, 862, 863, 864, 865, 866, 867, 868, 869, 870, 871, 872, 873, 874, 875, 876, 877, 878, 879, 880, 881, 882, 883, 884, 885, 886, 887, 888, 889, 890, 891, 892, 893, 894, 895, 896, 897, 898, 899, 900, 901, 9 02, 903, 904, 905, 906, 907, 908, 909, 910, 911, 912, 913, 914, 915, 916, 917, 918, 919, 920, 921, 922, 923, 924, 925, 926, 927, 928, 929, 940, 941, 942, 943, 944, 945, 946, 947, 948, 949, 940, 941, 942, 943, 944, 945, 946, 947, 948, 949, 950, 951, 952, 953, 954, 955, 956, 957, 958, 959, 96 0, 961, 962, 963, 964, 965, 966, 967, 968, 969, 970, 971, 972, 973, 974, 975, 976, 977, 978, 979, 980, 981, 982, 983, 984, 985, 986, 987, 988, 989, 990, 991, 992, 993, 994, 995, 996, 997, 998, 999, 1000, 1001, 1002, 1003, 1004, 1005, 1006, 1007, 1008, 1009, 1010, 1011, 1012, 1013, 1014, One or more CDR or FR regions selected from 1015, 1016, 1017, 1018, 1019, 1020, 1021, 1022, 1023, 1024, 1025, 1026, 1027, 1028, 1029, 1030, 1031, 1032, 1033, 1034, 1035, 1036, 1037, 1038, 1039, 1040, 1041, 1042, 1043, 1044, 1045, 1046, and 1047; wherein the heteropolypeptide comprises at least two immunoglobulin-binding domains; and wherein the heteropolypeptide comprises at least two immune cell surface protein-binding domains.

5. A heterologous polypeptide comprising: at least one immunoglobulin-binding domain; at least one immune cell surface protein-binding domain; at least one half-life extension domain; wherein the at least one half-life extension domain comprises an anti-HSA antigen-binding domain, an antibody or antigen-binding fragment, and variants, derivatives, or analogs thereof including VHH or single-domain antibodies, wherein the VHH or single-domain antibody comprises one or more CDR or FR regions selected from SEQ ID NO: 558, 559, 560, 561, 562, 563, 564, 565, 566, 567, and 568; wherein the heterologous polypeptide comprises at least two immunoglobulin-binding domains; and wherein the heterologous polypeptide comprises at least two immune cell surface protein-binding domains.

6. A heterologous polypeptide comprising: at least one immunoglobulin-binding domain; at least one immune cell surface protein-binding domain; at least one half-life extension domain; wherein the heterologous polypeptide is a single chain with the structure D1-D2-D3 and wherein the at least one half-life extension domain comprises an anti-HSA antigen-binding domain, an antibody or antigen-binding fragment, and variants, derivatives or analogs thereof including VHH or single-domain antibodies, wherein the VHH or single-domain antibody comprises one or more CDR or FR regions selected from SEQ ID NO: 558, 559, 560, 561, 562, 563, 564, 565, 566, 567 and 568.

7. The heteropeptide of claim 5, wherein the heteropeptide further comprises one or more linkers between the domains.

8. A heterologous polypeptide comprising: at least two immunoglobulin-binding domains; at least one immune cell surface protein-binding domain; at least one half-life extension domain; wherein the heterologous polypeptide is a single chain having at least four domains having the following structure: D1-D2-D3-D4 and wherein the at least one half-life extension domain comprises an anti-HSA antigen-binding domain, an antibody or antigen-binding fragment, and variants, derivatives or analogs thereof including VHH or single-domain antibodies, wherein the VHH or single-domain antibody comprises one or more CDR or FR regions selected from SEQ ID NO: 558, 559, 560, 561, 562, 563, 564, 565, 566, 567 and 568.

9. The heteropeptide of claim 6, wherein the heteropeptide further comprises one or more linkers between the domains.

10. A multimeric protein, wherein the molecule comprises: at least one immunoglobulin-binding domain; at least one immune cell surface protein-binding domain; and two half-life-extending domains comprising a first Fc polypeptide and a second Fc polypeptide derived from immunoglobulins, wherein the Fc polypeptide substantially does not bind to the immunoglobulin-binding domain; wherein the first Fc polypeptide and the second Fc polypeptide comprise a heteropolymerization domain, wherein the heteropolymerization domain is at least one knob-into-hole mutation; wherein the multimeric protein comprises at least two immunoglobulin-binding domains; and wherein the multimeric protein comprises at least two immune cell surface protein-binding domains.

11. A multimeric protein, wherein the molecule comprises: at least one immunoglobulin-binding domain; at least one immune cell surface protein-binding domain; two half-life-extending domains comprising a first Fc polypeptide and a second Fc polypeptide derived from immunoglobulins, wherein the Fc polypeptide substantially does not bind to the immunoglobulin-binding domain by selecting an IgG heavy chain Fc polypeptide containing mutants L234A, L235A, and P329A or P329G in constant heavy chain domain 2 (EU number); wherein the first Fc polypeptide and the second Fc polypeptide comprise a heteropolymerization domain, wherein the heteropolymerization domain is at least one knob into hole mutation; wherein the multimeric protein comprises at least two immunoglobulin-binding domains; and wherein the multimeric protein comprises at least two immune cell surface protein-binding domains.

12. A multimeric protein, wherein the molecule comprises: at least one immunoglobulin-binding domain; at least one immune cell surface protein-binding domain; two half-life extension domains comprising, but not limited to, a first Fc polypeptide and a second Fc polypeptide derived from immunoglobulins, wherein the Fc polypeptide substantially does not bind to the immunoglobulin-binding domain by selecting an IgG heavy chain Fc polypeptide containing mutants L234A, L235A, and P329A or P329G in constant heavy chain domain 2 (EU number); wherein the first Fc polypeptide contains a mutant T366W; and wherein the second Fc polypeptide contains T366S, L368A, and Y407V in constant heavy chain domain 3 (EU number); wherein the first Fc polypeptide further contains S354C, and wherein the second Fc polypeptide further contains Y349C in constant heavy chain domain 3 (EU number); wherein the multimeric protein comprises at least two immunoglobulin-binding domains; and wherein the multimeric protein comprises at least two immune cell surface protein-binding domains.

13. A multimeric protein, wherein the molecule comprises: at least one immunoglobulin-binding domain; at least one immune cell surface protein-binding domain; at least one immunoglobulin κ or λ constant light chain and its variants, derivatives, and analogs; at least a portion of at least one immunoglobulin constant heavy chain domain 1 and an immunoglobulin hinge region and its variants, derivatives, and analogs; wherein the one or more constant heavy chain domains 1 and at least a portion of the hinge contain the mutation C233S and the constant light chain contains the mutation C214S (Kabat number); wherein the one or more constant heavy chain domains 1 and at least a portion of the hinge contain the mutations C233S and F174C and the constant light chain contains the mutations C214S and S176C (Kabat number); wherein at least a first pair of constant heavy chains and at least a portion of the hinge do not contain a mutation at C233 and do not contain a mutation at C214 in the constant light chain, and at least a second pair of constant heavy chain domains 1 and at least a portion of the hinge contain the mutation C233S, etc., and the constant light chain contains a mutation. C214S, etc. (Kabat designation); wherein at least the first pair of constant heavy chains and at least a portion of the hinge do not contain a mutation at C233 and the constant light chain does not contain a mutation at C214, and at least the second pair of constant heavy chain structural domain 1 and at least a portion of the hinge contain mutations C233S and F174C and the constant light chain contains mutations C214S and S176C (Kabat designation); wherein at least the first pair of constant heavy chain structural domain 1 and at least a portion of the hinge contain mutation C233S and the constant light chain contains mutations C214S and S176C (Kabat designation); wherein at least the first pair of constant heavy chain structural domain 1 and at least a portion of the hinge contain mutation C233S and the constant light chain contains mutations C214S and S176C (Kabat designation); The constant light chain contains the mutant C214S, and at least the second pair of constant heavy chain domains 1 and at least a portion of the hinge contains the mutants C233S and F174C, and the constant light chain contains the mutants C214S and S176C (Kabat number); wherein the heteropeptide contains one or more half-life extension domains, the half-life extension domains containing an anti-HSA antigen-binding domain, an antibody or antigen-binding fragment, and variants, derivatives or analogs thereof including VHH or single-domain antibodies, and immunoglobulin IgG Fc domains and variants, derivatives or analogs thereof; wherein the multimeric protein contains at least two immunoglobulin-binding domains; and wherein the multimeric protein contains at least two immune cell surface protein-binding domains.

14. A multimeric protein, wherein one or more regions of the molecule comprise: at least two immunoglobulin-binding domains; at least one immune cell surface protein-binding domain; at least a second immunoglobulin-binding domain, wherein the second immunoglobulin-binding domain is separated from the first immunoglobulin-binding domain by a linker of 1 to 20 amino acids, the linker comprising regions of a human constant heavy chain domain 1, a κ chain domain, and a λ chain domain, a linker comprising 13 or fewer amino acids, a polypeptide comprising a linker comprising 6 or fewer amino acids, and a derivative of the constant heavy chain domain 1. The spacer region ASTKGPSVFPLAP, ASTKGP, or ASTKGPSVFPLAS; the spacer region RTVAAPSVFIFPP or RTVAAP derived from the constant κ chain; the spacer region SQPKAAPSVTLFP, GQPKANPTVTLFP, GQPKAAPSVTLFP, SQPKAA, GQPKAN, or GQPKAA, (GGGS)1, (GGGS)2, (GGGS)3, (GGGS)4; wherein at least one immunoglobulin-binding domain contains SEQ ID NO: 569, 570, 571, 572, 573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584, 585, 586, 587, 588, 589, 590, 591, 592, 593, 594, 595, 596, 597, 598, 599, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611, 612, 613, 614, 615, 6 One or more CDR or FR regions from 16, 617, 618, 619, 620, 621, 622, 623, 624, 625, 626, 627, 628, 629, 6230, 631, 632, 633, 634, 635, 636, 637, 638, 639, 640, 641, 642, 643, 644, 645, 646, 647, 648, 649, 650, 651, 652, 653, 654, 655, 656, 657, or SEQ Aviver as defined in ID NO: 132; and wherein at least a second immunoglobulin binding domain is the same as the first immunoglobulin binding domain; or wherein at least a second immunoglobulin binding domain is different from the first immunoglobulin binding domain.

15. A multimeric protein, wherein at least one region comprises: at least one immunoglobulin-binding domain; at least one immune cell surface protein-binding domain; one or more amino acid mutations in one or more constant or framework domains of human IgG1, IgG2, IgG3 or IgG4, variants, derivatives and analogs thereof, wherein the immunoglobulin-binding domain substantially does not bind itself or another region or regions of the molecule; wherein one or more amino acid mutations in constant heavy chain domain 1 comprise F122Y, P126S and K213E (Kabat designation); wherein one or more amino acid mutations in constant heavy chain domain 2 comprise N276K, L309V, L234A, L235A and P329A or P329G (EU designation); wherein the heterologous polypeptide or multimeric protein and the nucleic acid encoding thereof comprise at least two immunoglobulin-binding domains; and wherein the heterologous polypeptide or multimeric protein and the nucleic acid encoding thereof comprise at least two immune cell surface protein-binding domains.

16. A multimeric protein, wherein the molecule comprises: at least one immunoglobulin κ or λ constant light chain and its variants, derivatives, and analogs; and at least a portion of an immunoglobulin constant heavy chain domain 1 and an immunoglobulin hinge region and its variants, derivatives, and analogs; and two half-life extension domains comprising a first Fc polypeptide and a second Fc polypeptide derived from immunoglobulin, wherein the first Fc polypeptide and the second Fc polypeptide comprise heteropolymerization domains, wherein the heteropolymerization domains are selected from at least one Knob into Hole mutation; wherein the first Fc polypeptide comprises T366W, and wherein the second Fc polypeptide comprises T366S, L368A, and Y407V in constant heavy chain domain 3 (EU number); wherein by selecting an IgG heavy chain Fc polypeptide comprising L234A, L235A, and P329A in constant heavy chain domain 2 (EU number), the Fc substantially does not bind to one or more of its homologous Fc receptors; wherein one or more constant heavy chain domains 1 and all, none, or a portion of the hinge comprises the mutation C233S and the constant light chain comprises the mutation C214S (Kabat number); wherein one or more constant heavy chain domains 1 and at least a portion of the hinge comprises the mutations C233S and F174C and the constant light chain comprises the mutations C214S and S176C (Kabat number); wherein at least the first pair of constant heavy chains and at least a portion of the hinge do not contain a mutation at C233 and in the constant light chain The constant light chain contains mutations at C214 and at least a portion of the second pair of constant heavy chain structural domains 1 and the hinge, respectively, at C233S and at least a portion of the hinge, respectively, and at least a portion of the constant light chain contains mutations at C214S (Kabat number); wherein at least a portion of the first pair of constant heavy chain structural domains and the hinge does not contain mutations at C233S and at least a portion of the constant light chain contains mutations at C214S and at least a portion of the second pair of constant heavy chain structural domains 1 and the hinge, respectively, and at least a portion of the constant light chain contains mutations at C233S and F174C, respectively, and at least a portion of the constant light chain contains mutations at C214S and at least a portion of the second pair of constant heavy chain structural domains 1 and the hinge, respectively, and at least a portion of the constant light chain contains mutations at C214S and at least a portion of the hinge, respectively, and at least a portion of the second pair of constant heavy chain structural domains 1 and the hinge, respectively, and at least a portion of the constant light chain contains mutations at C214S and F174C, respectively, and at least a portion of the constant light chain contains mutations at C214S and S176C (Kabat number).

17. The multimeric protein of claim 16, wherein the first Fc polypeptide further comprises S354C, and wherein the second Fc polypeptide further comprises Y349C in the constant heavy chain domain 3 (EU number); and wherein the IgG heavy chain Fc polypeptide further comprises P329G in the constant heavy chain domain 2 (EU number).

18. The heteropeptide or multimeric protein of claim 1, wherein an additional region of the molecule contains a free cysteine ​​residue at or near the C-terminus.

19. The heteropeptide or multimeric protein of claim 1, wherein an additional region of the molecule comprises covalently linked PEG-lipids.

20. A treatment method comprising administering the heterologous polypeptide or multimeric protein of claim 1 as a monotherapy to a patient in need of treating a disease.

21. A treatment method comprising administering a combination of the heterologous polypeptide or multimeric protein of claim 1 with at least one selected from standard therapeutic agents, current therapeutic agents or experimental therapeutic agents to a subject in need of treating cancer, immune disorders or pathogenic infections.

22. A nucleotide encoding a heteropeptide or multimeric protein according to claim 1.

23. A kit comprising the heterologous polypeptide or multimeric protein according to claim 1.

Images