Receptor signaling that is conditionally activated by using scaffold proteins.

Abstract

The present invention relates to one or more binding molecules capable of inducing receptor signaling of a receptor complex, subject to non-competitive and / or biparatopic binding to a scaffold protein. The present invention also relates to the therapeutic use of one or more binding molecules in cancer and autoimmune diseases. TIFF2026510624000048.tif78170

Description

[Technical Field]

[0001] The present invention relates to one or more target-binding molecules capable of inducing receptor signaling of a receptor complex, subject to binding to a scaffold protein. [Background technology]

[0002] Most agonist ligands, including but not limited to cytokines, are limited in their therapeutic use due to the risk of toxicity. Localizing the agonist activity of ligands is a promising approach to improve the efficacy and safety of therapeutic molecules. Several approaches are currently under development, including protease-activating ligands (Nature Communications 2021; 12: 2768), antibody-ligand conjugations (Nature 2022; 610: 161-172), and split cytokines (WO2020 / 106708A1). However, the selectivity of these approaches remains limited. In addition, many receptors, including but not limited to cytokine receptors, induce signaling by forming homodimers, heterodimers, or oligomers on the cell surface for effective signaling. This disclosure relates to target-binding molecules that induce effective signaling of receptor complexes in a manner dependent on binding to scaffold proteins, addressing these needs. [Prior art documents] [Patent Documents]

[0003] [Patent Document 1] WO2020 / 106708A1 [Non-patent literature]

[0004] [Non-Patent Document 1] Hsu, E.J., Cao, X., Moon, B. et al. A cytokine receptor-masked IL2 prodrug selectively activates tumor-infiltrating lymphocytes for potent antitumor therapy. Nat Commun 12, 2768 (2021) [Non-Patent Document 2] Deak, L.C., Nicolini, V., Hashimoto, M., et al. PD1-cis-IL-2R agonism yields better effectors from stem-like CD8+T cells. Nature 610, 161-172 (2022) [Non-Patent Document 3] Wang, X., Rickert, M., Garcia, K.C. Structure of the quaternary complex of interleukin-2 with its alpha, beta, and gammac receptors. Science 310, 1159-1163 (2005) [Non-Patent Document 4] Mitra, S., Ring, A.M., Amarnath, S., et al. Interleukin-2 activity can be fine tuned with engineered receptor signaling clamps. Immunity 42, 826-838 (2015) [Non-Patent Document 5] Song, D., Liu, X., Dong, C., et al. Two novel human anti-CD25 antibodies with antitumor activity inversely related to their affinity and in vitro activity. Scientific Reports 11, 22966 (2021). [Non-Patent Document 6] Yen, M., Ren, J., Liu, Q., et al. Facile discovery of surrogate cytokine agonists. Cell 185, 1414-1430.e19 (2022)

Non-Patent Document 7

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Non-Patent Document 10

[0005] Effective receptor signaling is a key requirement for the efficacy of agonist ligands. A well-known example is cytokines, which are key immune mediators present in many lesion sites, and their effects, when utilized, can significantly improve the immune response. This is also true for interleukin-2 (IL-2). While many therapies with agonist ligands have been developed, concerns remain regarding high toxicity and low efficacy. [Means for solving the problem]

[0006] The inventors considered that the ability to deliver agonists capable of site - specifically activating receptor signaling would overcome the problems of systemic toxicity and low efficacy. To achieve such site - specific activation of receptor signaling, the inventors developed, inter alia, combinations of target - binding molecules or protein complexes capable of inducing receptor signaling of a receptor complex in the presence of a scaffold protein. The combination of target - binding molecules or protein complex of the present invention comprises a first binding domain and a third binding domain, each of which can bind non - competitively to the scaffold protein. The combination of target - binding molecules or protein complex of the present invention comprises a second binding domain capable of binding to a first receptor protein and a fourth binding domain capable of binding to a second receptor protein. The first receptor protein and the second receptor protein are each receptor subunits capable of associating to form a receptor complex. When all four binding domains are bound, the receptor complex can be activated to induce signaling. By making the presence of the scaffold protein a condition for the activation of the receptor complex and the specific binding of the present invention, receptor signaling can be conditionally induced, thus significantly increasing specificity and reducing toxicity.

[0007] Exemplary aspects Based on such findings, the present invention specifically includes the following exemplary aspects and embodiments.

[0008] [A - 1] The present invention particularly relates to a first target - binding molecule comprising a first binding domain capable of binding to a scaffold protein and a second binding domain capable of binding to a first receptor protein, and a second target - binding molecule comprising a third binding domain capable of binding to the scaffold protein and a fourth binding domain capable of binding to a second receptor protein comprising a combination of target - binding molecules, The first target-binding molecule and the second target-binding molecule are capable of binding to the scaffold protein non-competitively, and the first receptor protein and the second receptor protein are each receptor subunits capable of associating to form a receptor complex, and the combination of the target-binding molecules is capable of inducing receptor signaling of the receptor complex, relating to the combination of the target-binding molecules. [A-1a] The present invention relates to a first target-binding molecule comprising a first binding domain capable of binding to a scaffold protein and a second binding domain capable of binding to a first receptor protein, and a second target-binding molecule comprising a third binding domain capable of binding to the scaffold protein and a fourth binding domain capable of binding to a second receptor protein comprising a combination of target-binding molecules, wherein the first target-binding molecule and the second target-binding molecule are capable of binding to the scaffold protein non-competitively, and the first receptor protein and the second receptor protein are each receptor subunits capable of associating to form a receptor complex, relating to the combination of the target-binding molecules. When the four binding domains as defined above are bound, the receptor complex induces signaling. [A-2] In a preferred embodiment of the combination of the target-binding molecules of [A-1] or [A-1a], the first binding domain and the third binding domain are capable of binding to the scaffold protein bivalently. [A-3] In a preferred embodiment of any one of the combinations of target-binding molecules from [A-1] to [A-2], the combination of target-binding molecules induces receptor signaling at a first concentration or amount of the scaffold protein, and does not induce receptor signaling at a second concentration or amount of the scaffold protein, wherein the first concentration or amount of the scaffold protein is compared to the second concentration or amount of the scaffold protein. If receptor signaling is increased by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, or 500%, then receptor signaling is increased by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, or 500%. In a preferred embodiment of any one of the target-binding molecule combinations from [A-1] to [A-3], the first binding domain is optionally connected to the second binding domain via a linker, and / or the third binding domain is optionally connected to the fourth binding domain via a linker. In a preferred embodiment, the linker is 20, 15, 10 amino acids, or less. In a more preferred embodiment, the linker is 10 amino acids or less. In a preferred embodiment of any one of the target-binding molecule combinations [A-5] [A-1] to [A-4], the first binding domain and the third binding domain are capable of biparatopically binding to the scaffold protein within the domain of the scaffold protein. In a preferred embodiment of one of the target-binding molecule combinations [A-5a] [A-1] to [A-4], the first binding domain and the third binding domain are each selected from the group consisting of a neutralizing binding domain and a non-neutralizing binding domain. Preferably, the first binding domain is a neutralizing binding domain and the third binding domain is a non-neutralizing binding domain, or vice versa. In a preferred embodiment of the combination of target-binding molecules from any one of [A-6] [A-1] to [A-5a], the scaffold protein comprises two, three, four, or more subunits. In a preferred embodiment of any one of the target binding molecule combinations [A-7] [A-1] to [A-6], the first binding domain and the third binding domain can each bind to a subunit of a scaffold protein that can associate to form a scaffold protein. In a preferred embodiment of any one of the target-binding molecule combinations from [A-8] [A-1] to [A-7], the scaffold protein is a membrane-bound protein, a soluble protein, or an insoluble protein deposit. In a preferred embodiment of any one of the target-binding molecule combinations from [A-9] [A-1] to [A-8], the scaffold protein is a cell surface marker, an immune cell surface marker, a T cell exhaustion marker, a tumor-specific marker, a tumor-associated marker, or a protein whose expression is characteristic of a disease or disorder. In a preferred embodiment of the combination of target-binding molecules from any one of [A-10] [A-1] to [A-9], the scaffold protein is PD1, PDL1, CSF2RB, CD4, CD5, CD6, CD7, CD8, CD9, CCR4, CD8, CD25, CD27, CD38, CD39, CD45, CD62L, CCR6, CD69, CD103, CD73, CD127, GITR, LRRC32, ICOS, TIGIT, RBD, MUC1, CEA, CTLA4, IL2Rα, CXCR5, neuropilin-1, TIM3, L The group is selected from AG3, TNFα, CD19, CD20, CD22, CD30, CD33, glycoprotein NMB, CD56, CD70, CD79A, CD79B, CD138, PSCA, PSMA, BCMA, FcRH5, GPRC5D, FAP, LRRC15, E-selectin, EphB2, melanotransferrin, HER2, TROP2, nectin 4, EGFRvIII, IL13RA2, TMEFF2, Muc16, EpCAM, FcRH2, AFP, PSA, amyloid β, MBP, and ASGPR. In a more preferred embodiment, the scaffold protein is selected from the group consisting of PD1, PDL1, TNFα, CD25, MUC1, CEA, and CD8. In a preferred embodiment of the combination of target-binding molecules, one of [A-11] [A-1]~[A-10], the first receptor protein and the second receptor protein are, independently, IL2Rβ, IL2Rγ, IL4R, IL13RA1, IL7R, IL9R, IL21R, TSLPR, IL3RA, CSF2RB, IL5RA, CSF2RA, IL6R, gp130, IL11RA, IL12RB1, IL12RB2, IL27RA, IL31RA, OSMR, CNTFR, LIFR, IL10RA, IL10RB, IL20RA, IL20RB, IL22RA1, IL28RA, IFNAR1, IFNAR2, IFNGR1, IFNGR2, IL1R1, IL1RAP, IL18R1, IL18RAP, ST2, IL17RA, IL1 7RC, TLR3, TLR4, TLR7, TLR9, CSF1R, TNFR1, TNFR2, LTBR, ​​HVEM, FAS, CD28, cMET, DR3, DR4, DR5, NGFR , RANK, FN14, CD40, 4-1BB, OX40, GITR, TGFBR1, TGFBR2, ACVRL1, ACVR2A, BMPR2, ACVR2B, ACVR1B, ACV In a more preferred embodiment, the first receptor protein and the second receptor protein are each independently selected from the group consisting of IL2Rβ, IL2Rγ, Fzd1, Fzd2, Fzd3, Fzd4, Fzd5, Fzd6, Fzd7, Fzd8, Fzd9, Fzd10, LRP5, LRP6, and LGR5. In a preferred embodiment of one of the target-binding molecule combinations from [A-11a] [A-1] to [A-10], the scaffold protein is PD1, the first receptor protein is IL2Rβ, and the second receptor protein is IL2Rγ. [A-11b] In a preferred embodiment of one of the target-binding molecule combinations from [A-1] to [A-10], the scaffold protein is IL2Rα, the first receptor protein is IL2Rβ, and the second receptor protein is IL2Rγ. In a preferred embodiment of any one of the target-binding molecule combinations from [A-11c] [A-1] to [A-10], the target-binding molecule competes for binding with any one of the target-binding molecules or protein complexes from Tables 1 to 17, or binds to the same epitope. In a preferred embodiment of the target-binding molecule combination from any one of [A-1] to [A-10], the scaffold protein, the first receptor protein, and the second receptor protein are combinations selected from any of the following (i) to (vi): (i) The scaffold protein is PDL1, the first receptor protein is IL2Rβ, and the second receptor protein is IL2Rγ; (ii) The scaffold protein is TNFa, the first receptor protein is IL2Rβ, and the second receptor protein is IL2Rγ; (iii) The scaffold protein is CD25, the first receptor protein is IL2Rβ, and the second receptor protein is IL2Rγ; (iv) The scaffold protein is PDL1, the first receptor protein is CSF2RA, and the second receptor protein is CSF2RB; (v) The scaffold protein is MUC1 or CEA, the first receptor protein is the Fzd receptor, and the second receptor protein is Lrp; or (vi) The scaffold protein is CD8, the first receptor protein is IL2Rβ, and the second receptor protein is IL2Rγ. In a preferred embodiment of the combination of target-binding molecules from any one of [A-1] to [A-11d], the receptor complex is IL-2R. In a more preferred embodiment, inducing IL-2 receptor complex signaling involves evaluating the activation of the IL-2 receptor complex using a colorimetric enzyme assay that determines alkaline phosphatase activity. In an even more preferred embodiment, alkaline phosphatase activity is determined by measuring optical density at optionally 620 nm. In the most preferred embodiment, receptor signaling of the receptor complex is at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 1% in the presence of the combination of target-binding molecules from [A-1] to [A-11c] compared to the absence of the combination of target-binding molecules from [A-1] to [A-11c]. This is induced when there is an increase in optical density of 80%, 190%, 200%, 210%, 220%, 230%, 240%, 250%, 260%, 270%, 280%, 290%, 300%, 310%, 320%, 330%, 340%, 350%, 360%, 370%, 380%, 390%, 400%, 410%, 420%, 430%, 440%, 450%, 460%, 470%, 480%, 490%, or 500%. In a preferred embodiment of any one of the combinations of target-binding molecules from [A-12] [A-1] to [A-11e], each binding domain is an antigen-binding domain containing VH and VL, sdAb, VHH, scFv, Fab, scFab, Fab', Fab'-SH, F(ab')2, diabody, triabody, Fv, aptamer, aphibody, cytokine, ligand, or split cytokine. In a preferred embodiment of any one of the combinations of target-binding molecules from [A-13] [A-1] to [A-12], the first target-binding molecule and the second target-binding molecule are optionally linked via a linker. In this embodiment, the combination is a single protein or a protein complex. In a preferred embodiment of any one of the target-binding molecule combinations from [A-14] [A-1] to [A-13], the first target-binding molecule and the second target-binding molecule are each optionally connected to a half-life extension domain via a linker. In a preferred embodiment of the combination of target-binding molecules [A-15] and [A-14], the first target-binding molecule is connected to the half-life extension domain in the first binding domain, and the second target-binding molecule is connected to the half-life extension domain in the third binding domain. In a preferred embodiment of the target-binding molecule combination of [A-16] and [A-14], the half-life extension domain comprises an Fc domain, an FcRn-binding domain, an albumin-binding domain, albumin or a variant thereof, or polyethylene glycol. In preferred embodiments of the target-binding molecule combinations of [A-17] and [A-14], the half-life extension domain comprises an Fc domain, which optionally includes modifications that reduce antibody-dependent cell-mediated cytotoxicity (ADCC), reduce complement-dependent cytotoxicity (CDC), increase FcRn binding, increase pI, or promote heterodimerization. [A-18] The present invention also relates to a nucleic acid molecule encoding one of the target-binding molecule combinations from [A-1] to [A-17]. [A-19] The present invention also relates to a plurality of nucleic acid molecules encoding a combination of target-binding molecules, one of [A-1] to [A-17], wherein the plurality comprises at least one first nucleic acid molecule and at least one second nucleic acid molecule, the first nucleic acid molecule encoding a first target-binding molecule as defined in one of [A-1] to [A-17], and the second nucleic acid molecule encoding a second target-binding molecule as defined in one of [A-1] to [A-17]. [A-20] The present invention also relates to a vector comprising a nucleic acid molecule of [A-18] or a plurality of nucleic acid molecules of [A-19]. [A-20a] The present invention also relates to a first vector comprising a first nucleic acid molecule of [A-19] and a second vector comprising a second nucleic acid molecule of [A-19]. [A-21] The present invention also relates to a host cell comprising a nucleic acid molecule of [A-18], or a plurality of nucleic acid molecules of [A-19], or a vector of [A-20]. [A-21a] The present invention also relates to a first host cell comprising a first nucleic acid molecule of [A-19] or a first vector of [A-20a], and a second host cell comprising a second nucleic acid molecule of [A-19] or a second vector of [A-20a]. [A-22] The present invention also relates to a method for producing a combination of target-binding molecules, (i) A step of culturing host cells of [A-21] or host cells of [A-21a] under conditions suitable for protein expression; (ii) optionally a step of lysing the host cells; and (iii) Step of isolating the combination of target binding molecules. The method, including the method described herein. [A-23] The present invention also relates to a method for producing a combination of target-binding molecules, (i) A step of culturing host cells of [A-21] or first host cells of [A-21a] under conditions suitable for protein expression in order to obtain a first target binding molecule; (ii) A step of culturing host cells of [A-21] or second host cells of [A-21a] under conditions suitable for protein expression in order to obtain a second target binding molecule; (iii) the step of isolating the first target-binding molecule and the second target-binding molecule from host cells; and (iv) The step of combining the first target binding molecule and the second target binding molecule in order to obtain a combination of target binding molecules. The method, including the method described herein. [A-24] The present invention also relates to a pharmaceutical composition comprising any one of the target-binding molecules from [A-1] to [A-17]. [A-25] The present invention also relates to a method for treating a disease in a subject, comprising the step of administering the pharmaceutical composition of [A-24] to a subject in need of such treatment. In a preferred embodiment of the method for treating a disease in the subjects of [A-26] and [A-25], the disease is cancer or an autoimmune disease. [A-26a] In a preferred embodiment, the cancer or autoimmune disease of [A-25] is a cancer or autoimmune disease that is sensitive to improvement or prevention by increased or enhanced IL-2R signaling. [A-27] The present invention also relates to a pharmaceutical composition of [A-24] for use in therapy. In preferred embodiments of the pharmaceutical compositions for the use of [A-28] and [A-27], the therapy is cancer immunotherapy or autoimmune disease immunotherapy. [A-28a] In a preferred embodiment, the cancer or autoimmune disease of [A-28] is a cancer or autoimmune disease that is sensitive to improvement or prevention by increased or enhanced IL-2R signaling. [A-29] The present invention also relates to the use of the pharmaceutical composition of [A-24] in the manufacture of pharmaceuticals for the treatment of diseases. [A-30] In a preferred embodiment of the use of claim [A-29], the disease is cancer or an autoimmune disease. [A-30a] In a preferred embodiment, the cancer or autoimmune disease of [A-30] is a cancer or autoimmune disease that is sensitive to improvement or prevention by increased or enhanced IL-2R signaling. In a preferred embodiment of the use of any one of [A-1] to [A-17], a combination of target-binding molecules, any one method of [A-25] or [A-26], any one of [A-27] or [A-28], or any one of [A-29], [A-30], or [A-30a], the scaffold protein is PD-1, the first receptor protein is IL-2Rβ, and the second receptor protein is IL-2Rγ. In a preferred embodiment of the use of any one of [A-1] to [A-17], a combination of target-binding molecules, any one method of [A-25] or [A-26], any one of [A-27] or [A-28], or any one of [A-29], [A-30], or [A-30a], the scaffold protein is IL-2Rα, the first receptor protein is IL-2Rβ, and the second receptor protein is IL-2Rγ. In a combination of any one of the target-binding molecules from [A-1] to [A-17], the method of claim [A-26], the pharmaceutical composition for use of [A-28], or a preferred embodiment of the use of [A-30] or [A-30a], cancer or autoimmune disease is characterized by the expression of PD1, IL-2Rα, IL-2Rβ, or IL-2Rγ. [A-33] The present invention also relates to a first target-binding molecule as defined in any one of [A-1] to [A-17] for use in therapy, wherein the therapy comprises administering the first target-binding molecule in combination with a second target-binding molecule as defined in any one of [A-1] to [A-17], and optionally the first target-binding molecule and the second target-binding molecule are administered simultaneously, sequentially, or separately. [A-34] The present invention also relates to a second target-binding molecule as defined in any one of [A-1] to [A-17] for use in therapy, wherein the therapy comprises administering the second target-binding molecule in combination with a first target-binding molecule as defined in any one of [A-1] to [A-17], and optionally the first target-binding molecule and the second target-binding molecule are administered simultaneously, sequentially, or separately. In preferred embodiments of the first target-binding molecule for use with [A-35] [A-33] or the second target-binding molecule for use with [A-34], the disease is cancer or an autoimmune disease. [A-35a] In a preferred embodiment, the cancer or autoimmune disease of [A-35] is a cancer or autoimmune disease that is sensitive to improvement or prevention by increased or enhanced IL-2R signaling. In preferred embodiments of the first or second target-binding molecule for use in [A-36] [A-35], cancer or autoimmune disease is characterized by the expression of PD1, IL2Rα, IL-2Rβ, or IL-2Rγ. In preferred embodiments of the first or second target-binding molecule for use in any one of [A-37] [A-33] to [A-36], the scaffold protein is PD-1, the first receptor protein is IL-2Rβ, and the second receptor protein is IL-2Rγ. In preferred embodiments of the first or second target-binding molecule for use in any one of [A-37a] [A-33] to [A-36], the scaffold protein is IL-2Rα, the first receptor protein is IL-2Rβ, and the second receptor protein is IL-2Rγ. [A-38] The present invention also relates to a first target-binding molecule comprising a first binding domain capable of binding to a scaffold protein and a second binding domain capable of binding to a first receptor protein, and A second target-binding molecule comprising a third binding domain capable of binding to the scaffold protein and a fourth binding domain capable of binding to a second receptor protein. A protein complex comprising, The first target-binding molecule and the second target-binding molecule are capable of binding non-competitively to the scaffold protein. The first receptor protein and the second receptor protein are receptor subunits that can each associate to form a receptor complex. The protein complex is capable of inducing receptor signaling of the receptor complex. This relates to the protein complex. [A-38a] The present invention also, A first target-binding molecule comprising a first binding domain capable of binding to a scaffold protein and a second binding domain capable of binding to a first receptor protein, and A second target-binding molecule comprising a third binding domain capable of binding to the scaffold protein and a fourth binding domain capable of binding to a second receptor protein. A protein complex comprising, The first target-binding molecule and the second target-binding molecule are capable of binding non-competitively to the scaffold protein. The first receptor protein and the second receptor protein are receptor subunits that can associate to form a receptor complex. This relates to the protein complex. When the four binding domains defined above bind, the receptor complex induces signal transduction. [A-39] The present invention also relates to a protein complex comprising a first binding domain, a second binding domain, a third binding domain, and a fourth binding domain, The first binding domain is capable of binding to the scaffold protein, the second binding domain is capable of binding to the first receptor protein, the third binding domain is capable of binding to the scaffold protein, and the fourth binding domain is capable of binding to the second receptor protein. The first binding domain and the third binding domain are capable of binding non-competitively to the scaffold protein. The first receptor protein and the second receptor protein are receptor subunits that can each associate to form a receptor complex. The protein complex is capable of inducing receptor signaling of the receptor complex. This relates to the protein complex. [A-39a] The present invention also relates to a protein complex comprising a first binding domain, a second binding domain, a third binding domain, and a fourth binding domain, The first binding domain is capable of binding to the scaffold protein, the second binding domain is capable of binding to the first receptor protein, the third binding domain is capable of binding to the scaffold protein, and the fourth binding domain is capable of binding to the second receptor protein. The first binding domain and the third binding domain are capable of binding non-competitively to the scaffold protein. The first receptor protein and the second receptor protein are receptor subunits that can associate to form a receptor complex. This relates to the protein complex. When the four binding domains defined above bind, the receptor complex induces signal transduction. In a preferred embodiment of any one of the protein complexes [A-40], [A-38], to [A-39a], the first binding domain and the third binding domain are capable of biparatopic binding to the scaffold protein. In a preferred embodiment of any one of the protein complexes [A-41] [A-38]~[A-40], the protein complex induces receptor signaling at a first concentration or amount of the scaffold protein, and does not induce receptor signaling at a second concentration or amount of the scaffold protein, wherein the first concentration or amount of the scaffold protein is less than the second concentration or amount of the scaffold protein. If receptor signaling is increased by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, or 500%, receptor signaling will increase by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, or 500%. In a preferred embodiment of any one of the protein complexes [A-42] [A-38] to [A-41], the first binding domain is optionally linked to the second binding domain via a linker, and / or the third binding domain is optionally linked to the fourth binding domain via a linker. In a preferred embodiment, the linker is 20, 15, 10 amino acids, or less. In a more preferred embodiment, the linker is 10 amino acids or less. In a preferred embodiment of any one of the protein complexes [A-43] [A-38] to [A-42], the first binding domain and the third binding domain are capable of biparatopically binding to the scaffold protein within the domain of the scaffold protein. In a preferred embodiment of any one of the protein complexes [A-43a] [A-38] to [A-42], the first binding domain and the third binding domain are each selected from the group consisting of a neutralizing binding domain and a non-neutralizing binding domain. Preferably, the first binding domain is a neutralizing binding domain and the third binding domain is a non-neutralizing binding domain, or vice versa. In a preferred embodiment of any one of the protein complexes [A-44] [A-38] to [A-43a], the scaffold protein comprises two, three, four, or more subunits. In a preferred embodiment of any one of the protein complexes [A-45] [A-38] to [A-44], the first binding domain and the third binding domain can each bind to a subunit of a scaffold protein that can associate to form a scaffold protein. In a preferred embodiment of any one of the protein complexes [A-46] [A-38] to [A-45], the scaffold protein is a membrane-bound protein, a soluble protein, or an insoluble protein deposit. In a preferred embodiment of any one of the protein complexes [A-47] [A-38] to [A-46], the scaffold protein is a cell surface marker, an immune cell surface marker, a T cell exhaustion marker, a tumor-specific marker, a tumor-associated marker, or a protein whose expression is characteristic of a disease or disorder. In a preferred embodiment of any one protein complex of [A-48] [A-38]~[A-47], the scaffold protein is PD1, PDL1, CSF2RB, CD4, CD5, CD6, CD7, CD8, CD9, CCR4, CD8, CD25, CD27, CD38, CD39, CD45, CD62L, CCR6, CD69, CD103, CD73, CD127, GITR, LRRC32, ICOS, TIGIT, RBD, MUC1, CEA, CTLA4, IL2Rα, CXCR5, neuropilin-1, TIM3, LA The group is selected from G3, TNFα, CD19, CD20, CD22, CD30, CD33, glycoprotein NMB, CD56, CD70, CD79A, CD79B, CD138, PSCA, PSMA, BCMA, FcRH5, GPRC5D, FAP, LRRC15, E-selectin, EphB2, melanotransferrin, HER2, TROP2, nectin 4, EGFRvIII, IL13RA2, TMEFF2, Muc16, EpCAM, FcRH2, AFP, PSA, amyloid β, MBP, and ASGPR. In a more preferred embodiment, the scaffold protein is selected from the group consisting of PD1, PDL1, TNFα, CD25, MUC1, CEA, and CD8. In a preferred embodiment of any one protein complex of [A-49] [A-38]~[A-48], the first receptor protein and the second receptor protein are, independently, IL2Rβ, IL2Rγ, IL4R, IL13RA1, IL7R, IL9R, IL21R, TSLPR, IL3RA, CSF2RB, IL5RA, CSF2RA, IL6R, gp130, IL11RA, IL12RB1, IL12RB2, IL27RA, IL31RA, OSMR, CNTFR, LIFR, IL10RA, IL10RB, IL20RA, IL20RB, IL22RA1, IL28RA, IFNAR1, IFNAR2, IFNGR1, IFNGR2, IL1R1, IL1RAP, IL18R1, IL18RAP, ST2, IL17RA, IL17R C. In a more preferred embodiment, the first receptor protein and the second receptor protein are each independently selected from the group consisting of IL2Rβ, IL2Rγ, Fzd1, Fzd2, Fzd3, Fzd4, Fzd5, Fzd6, Fzd7, Fzd8, Fzd9, Fzd10, LRP5, LRP6, and LGR5. In a preferred embodiment of any one of the protein complexes [A-49a] [A-38] to [A-49], the scaffold protein is PD1, the first receptor protein is IL2Rβ, and the second receptor protein is IL2Rγ. [A-49b] In a preferred embodiment of any one of the protein complexes [A-38] to [A-49], the scaffold protein is IL2Rα, the first receptor protein is IL2Rβ, and the second receptor protein is IL2Rγ. In a preferred embodiment of any one of the protein complexes in [A-49c] [A-38] to [A-49], the protein complex competes for binding with any one of the target-binding molecules or protein complexes in Tables 1 to 17, or binds to the same epitope. In a preferred embodiment of any one of the protein complexes [A-49d] [A-38]~[A-49], the scaffold protein, the first receptor protein, and the second receptor protein are combinations selected from any of the following (i)~(vi): (i) The scaffold protein is PDL1, the first receptor protein is IL2Rβ, and the second receptor protein is IL2Rγ; (ii) The scaffold protein is TNFa, the first receptor protein is IL2Rβ, and the second receptor protein is IL2Rγ; (iii) The scaffold protein is CD25, the first receptor protein is IL2Rβ, and the second receptor protein is IL2Rγ; (iv) The scaffold protein is PDL1, the first receptor protein is CSF2RA, and the second receptor protein is CSF2RB; (v) The scaffold protein is MUC1 or CEA, the first receptor protein is the Fzd receptor, and the second receptor protein is Lrp; or (vi) The scaffold protein is CD8, the first receptor protein is IL2Rβ, and the second receptor protein is IL2Rγ. In a preferred embodiment of the protein complexes [A-49e] [A-38]~[A-49d], the receptor complex is IL-2R. In a more preferred embodiment, inducing IL-2 receptor complex signaling involves evaluating the activation of the IL-2 receptor complex using a colorimetric enzyme assay that determines alkaline phosphatase activity. In an even more preferred embodiment, alkaline phosphatase activity is determined by measuring optical density at an optional 620 nm. In the most preferred embodiment, receptor signaling of the receptor complex is at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180% in the presence of the protein complexes [A-38]~[A-49c] compared to the absence of the protein complexes [A-38]~[A-49c]. It is induced when there is an increase in optical density of 190%, 200%, 210%, 220%, 230%, 240%, 250%, 260%, 270%, 280%, 290%, 300%, 310%, 320%, 330%, 340%, 350%, 360%, 370%, 380%, 390%, 400%, 410%, 420%, 430%, 440%, 450%, 460%, 470%, 480%, 490%, or 500%. In a preferred embodiment of any one protein complex of [A-50] [A-38]~[A-49e], each binding domain is an antigen-binding domain containing VH and VL, sdAb, VHH, scFv, Fab, scFab, Fab', Fab'-SH, F(ab')2, diabody, triabody, Fv, aptamer, aphibody, cytokine, ligand, or split cytokine. In a preferred embodiment of any one of the protein complexes [A-51] [A-38] to [A-50], the protein complex further comprises a half-life extension domain. In a preferred embodiment of any one of the protein complexes [A-52], [A-38], and [A-40] to [A-51], the first target-binding molecule is connected to the half-life extension domain in the first binding domain, and the second target-binding molecule is connected to the half-life extension domain in the third binding domain. In a preferred embodiment of any one of the protein complexes [A-53] [A-51] to [A-52], the half-life extension domain comprises an Fc domain, an FcRn-binding domain, an albumin-binding domain, albumin or a variant thereof, or polyethylene glycol. In preferred embodiments of the protein complexes of [A-54] and [A-53], the half-life extension domain comprises an Fc domain, which optionally includes modifications that reduce antibody-dependent cell-mediated cytotoxicity (ADCC), reduce complement-dependent cell-mediated cytotoxicity (CDC), increase FcRn binding, increase pI, or promote heterodimerization. [A-55] The present invention also relates to a nucleic acid molecule or a plurality of nucleic acid molecules encoding any one of the protein complexes described in [A-38] to [A-54]. [A-56] The present invention also relates to a vector comprising the nucleic acid molecule or a plurality of nucleic acid molecules of [A-55]. [A-57] The present invention also relates to a host cell comprising the nucleic acid molecule of [A-55] or a plurality of nucleic acid molecules, or the vector of [A-56]. [A-58] The present invention also relates to a method for producing a protein complex, (i) A step of culturing host cells of [A-57] under conditions suitable for protein expression; (ii) optionally a step of lysing host cells; and (iii) Steps to isolate combinations of target binding molecules The method, including the method described herein. [A-59] The present invention also relates to a pharmaceutical composition comprising any one of the protein complexes [A-38] to [A-54]. [A-60] The present invention also relates to a method for treating a disease in a subject, comprising the step of administering the pharmaceutical composition of [A-59] to a subject in need of such treatment. In a preferred embodiment of the method for treating a disease in the subjects of [A-61] and [A-60], the disease is cancer or an autoimmune disease. [A-61a] In a preferred embodiment, the cancer or autoimmune disease of [A-61] is a cancer or autoimmune disease that is sensitive to improvement or prevention by increased or enhanced IL-2R signaling. [A-62] The present invention also relates to a pharmaceutical composition of [A-59] for use in therapy. In preferred embodiments of the pharmaceutical compositions for use of [A-63] and [A-62], the therapy is cancer immunotherapy or autoimmune disease immunotherapy. [A-63a] In a preferred embodiment, the cancer or autoimmune disease of [A-63] is a cancer or autoimmune disease that is sensitive to improvement or prevention by increased or enhanced IL-2R signaling. [A-64] The present invention also relates to the use of the pharmaceutical composition of [A-61] in the manufacture of pharmaceuticals for the treatment of diseases. In preferred embodiments of the use of [A-65] [A-64], the disease is cancer or an autoimmune disease. [A-65a] In a preferred embodiment, the cancer or autoimmune disease of [A-65] is a cancer or autoimmune disease that is sensitive to improvement or prevention by increased or enhanced IL-2R signaling. In a preferred embodiment of either method [A-66], [A-60], or [A-61], a pharmaceutical composition for either use of either [A-62] or [A-63], or any one use of [A-64], [A-65], or [A-65a], the scaffold protein is PD1, the first receptor protein is IL-2Rβ, and the second receptor protein is IL-2Rγ. In a preferred embodiment of either method [A-60] or [A-61], a pharmaceutical composition for either use of either [A-62] or [A-63], or any one use of [A-64], [A-65], or [A-65a], the scaffold protein is IL-2Rα, the first receptor protein is IL-2Rβ, and the second receptor protein is IL-2Rγ. In the methods of [A-67] and [A-61], the pharmaceutical compositions for use of [A-63], or preferred embodiments of the use of [A-65] or [A-65a], cancer or autoimmune disease is characterized by the expression of PD1, IL-2Rα, IL-2Rβ, or IL-2Rγ. [A-68] The present invention also relates to a method for treating a disease in a subject, wherein the subject in need of such treatment A first target-binding molecule comprising a first binding domain capable of binding to a scaffold protein and a second binding domain capable of binding to a first receptor protein, and A second target-binding molecule comprising a third binding domain capable of binding to the scaffold protein and a fourth binding domain capable of binding to a second receptor protein. Includes, The first target-binding molecule and the second target-binding molecule are capable of binding non-competitively to the scaffold protein. The first receptor protein and the second receptor protein are receptor subunits that can associate to form a receptor complex. Therapeutic effective amount of the composition The process includes administering The composition is capable of inducing receptor signaling of the receptor complex. Regarding the said method. [A-69] The present invention also relates to a method for treating a disease in a subject, wherein the subject in need of such treatment A first target-binding molecule comprising a first binding domain capable of binding to a scaffold protein and a second binding domain capable of binding to a first receptor protein, and A second target-binding molecule comprising a third binding domain capable of binding to the scaffold protein and a fourth binding domain capable of binding to a second receptor protein. Includes, The first target-binding molecule and the second target-binding molecule are capable of binding non-competitively to the scaffold protein. The first receptor protein and the second receptor protein are receptor subunits that can associate to form a receptor complex. Therapeutic effective amount of a composition containing a protein complex The process includes administering The protein complex is capable of inducing receptor signaling of the receptor complex. Regarding the said method. [A-70] The present invention also relates to a method for conditionally inducing receptor signaling, (i) a step of identifying a subject that needs to express the scaffold protein, the first receptor protein, and the second receptor protein, and (ii) The step of administering one combination of target-binding molecules from [A-1] to [A-17], or one protein complex from [A-38] to [A-54]. The method, including the method described herein. [A-71] The present invention also relates to a method for screening combinations of target-binding molecules, (i) A step of identifying a scaffold protein, and a first binding domain and a third binding domain that can bind non-competitively to the scaffold protein, (ii) A step of identifying a first receptor protein and a second receptor protein, and a step of identifying a second binding domain that can bind to the first receptor protein and a fourth binding domain that can bind to the second receptor protein, (iii) A step of producing a first target-binding molecule comprising a first binding domain and a second binding domain, and a second target-binding molecule comprising a third binding domain and a fourth binding domain, wherein the first target-binding molecule and the second target-binding molecule are any one of [A-1] to [A-17], (iv) A step of determining the receptor signaling activity of a first receptor protein and a second receptor protein in the presence of a first target-binding molecule, a second target-binding molecule, and the scaffold protein. (v) A step of determining the receptor signaling activity of the first receptor protein and the second receptor protein in the presence of a second target binding molecule and the scaffold protein, and in the absence of the scaffold protein, and (vi)Optionally, if the receptor signaling activity of (iv) is higher than that of (v), the step of selecting a combination of a first target binding molecule and a second target binding molecule if the receptor signaling activity of (iv) is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% higher than that of (v). The method, including the method described herein. [A-72] The present invention also relates to a method for screening protein complexes, (i) A step of identifying a scaffold protein, and a first binding domain and a third binding domain that can bind non-competitively to the scaffold protein, (ii) A step of identifying a first receptor protein and a second receptor protein, and a step of identifying a second binding domain that can bind to the first receptor protein and a fourth binding domain that can bind to the second receptor protein, (iii) A step of producing a protein complex comprising a first binding domain, a second binding domain, a third binding domain, and a fourth binding domain, wherein the protein complex is one of [A-38] to [A-54], (iv) A step of determining the receptor signaling activity of the protein complex in the presence of the scaffold protein, (v) A step of determining the receptor signaling activity of the protein complex in the absence of the scaffold protein, and (vi)Optionally, the step of selecting the protein complex if the receptor signaling activity of (iv) is higher than that of (v), by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% higher than that of (v). The method, including the method described herein. [A-72a] A step to identify a first binding domain and a third binding domain that can each bind non-competitively to the scaffold protein, (a) A step of identifying a neutralizing binding domain that binds to the scaffold protein, (b) A step of identifying a non-neutralizing binding domain that binds to the scaffold protein, (c) A step of selecting a neutralizing binding domain as the first binding domain and a non-neutralizing binding domain as the third binding domain, or vice versa. The method of [A-71] or [A-72], including the above. [A-72b] The present invention also relates to a method for screening non-competitive and / or biparatopic protein-binding domains that bind to a scaffold protein, (i) A step of identifying a neutralizing binding domain that binds to the scaffold protein, (ii) A step of identifying a non-neutralizing binding domain that binds to the scaffold protein, (iii) Selecting neutralizing and non-neutralizing domains as non-competitive and / or biparatopic protein-binding domains. The method, including the method described herein. [A-72c] The present invention also provides a method for screening a combination of target-binding molecules, any one of [A-1] to [A-17], which includes a non-competitive and / or biparatopic scaffold protein-binding domain. (i) A step to identify a target binding molecule containing a neutralizing binding domain that binds to a scaffold protein. (ii) A step of identifying a target binding molecule containing a non-neutralizing binding domain that binds to the scaffold protein, and (iii) A step of selecting a target binding molecule containing a neutralizing binding domain that binds to the scaffold protein and a target binding molecule containing a non-neutralizing binding domain that binds to the scaffold protein. The method, including the method described herein. [A-72d] The present invention also relates to a method for screening any one of the protein complexes [A-38] to [A-54] comprising a non-competitive and / or biparatopic scaffold protein-binding domain, (i) A step to identify a protein complex containing a neutralizing binding domain that binds to a scaffold protein, (ii) A step of identifying a protein complex containing a non-neutralizing binding domain that binds to the scaffold protein, and (iii) A step of selecting a protein complex that includes a neutralizing binding domain as the first binding domain and a non-neutralizing binding domain as the third binding domain, or the reverse. The method, including the method described herein. [A-72e] The present invention also relates to a method for producing any one of the combinations of target-binding molecules from [A-1] to [A-17], (a) A step to identify the neutralizing binding domain that binds to the scaffold protein, (b) A step of identifying a non-neutralizing binding domain that binds to the scaffold protein, (c) A step of selecting a neutralizing binding domain as the first binding domain and a non-neutralizing binding domain as the third binding domain, or vice versa. The method, including the method described herein. In aspects of the method described in [A-72e1] and [A-72e], the method is as follows: (d) A step to obtain a polynucleotide encoding a neutralizing binding domain and a polynucleotide encoding a non-neutralizing binding domain, (e) optionally a step of linking a polynucleotide encoding a neutralization binding domain to a polynucleotide encoding a second binding domain via a linker, (f) Optionally, a step of linking a polynucleotide encoding a non-neutralizing binding domain to a polynucleotide encoding a fourth binding domain via a linker. (g) The step of expressing the polynucleotides of (e) and (f). It also includes. In aspects of the method described in [A-72e2] and [A-72e], the method is as follows: (d) Steps for producing a first target-binding molecule comprising a first binding domain and a second binding domain, and a second target-binding molecule comprising a third binding domain and a fourth binding domain. It also includes. [A-72f] The present invention also relates to a method for producing any one of the protein complexes [A-38] to [A-54], (a) A step to identify the neutralizing binding domain that binds to the scaffold protein, (b) A step of identifying a non-neutralizing binding domain that binds to the scaffold protein, (c) A step of selecting a neutralizing binding domain as the first binding domain and a non-neutralizing binding domain as the third binding domain, or vice versa. The method, including the method described herein. In the manner of the method described in [A-72f1] and [A-72f], the method is as follows: (d) A step to obtain a polynucleotide encoding a neutralizing binding domain and a polynucleotide encoding a non-neutralizing binding domain, (e) optionally, a step of linking a polynucleotide encoding a neutralization binding domain to a polynucleotide encoding at least one fragment of a second binding domain via a linker, (f) optionally, a step of linking a polynucleotide encoding a non-neutralizing binding domain to a polynucleotide encoding at least one fragment of a fourth binding domain via a linker, (g) The process of expressing the polynucleotides of (e) and (f) in order to produce the protein complex. It also includes. In the manner of the method in [A-72f2] [A-72f], the method is: (d) A step to produce one of the protein complexes [A-38] to [A-54], which includes a first binding domain, a second binding domain, a third binding domain, and a fourth binding domain. It also includes. In a preferred embodiment of any one of the methods [A-73] [A-68]~[A-72f2], the first binding domain and the third binding domain are capable of biparatopic binding to the scaffold protein. In a preferred embodiment of any one of the methods of [A-74] [A-68]~[A-73], a combination of target-binding molecules or a protein complex induces receptor signaling at a first concentration or amount of the scaffold protein, and does not induce receptor signaling at a second concentration or amount of the scaffold protein, wherein the first concentration or amount of the scaffold protein is compared to the second concentration or amount of the scaffold protein. If receptor signaling is increased by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, or 500%, then receptor signaling is increased by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, or 500%. In a preferred embodiment of any one of the methods of [A-75] [A-68] to [A-74], the first binding domain is optionally connected to the second binding domain via a linker, and / or the third binding domain is optionally connected to the fourth binding domain via a linker. In a preferred embodiment, the linker is 20, 15, 10 amino acids, or less. In a more preferred embodiment, the linker is 10 amino acids or less. In a preferred embodiment of any one of the methods [A-76] [A-68] to [A-75], the first binding domain and the third binding domain are capable of biparatopically binding to the scaffold protein within the domain of the scaffold protein. In a preferred embodiment of any one of the methods [A-76a] [A-68] to [A-75], the first binding domain and the third binding domain are each selected from the group consisting of neutralizing binding domains and non-neutralizing binding domains. Preferably, the first binding domain is a neutralizing binding domain and the third binding domain is a non-neutralizing binding domain, or vice versa. In a preferred embodiment of any one of the methods of [A-77] [A-68] to [A-76a], the scaffold protein comprises two, three, four, or more subunits. In a preferred embodiment of any one of the methods of [A-78] [A-68] to [A-77], the first binding domain and the third binding domain can each bind to a subunit of a scaffold protein that can associate to form a scaffold protein. In a preferred embodiment of any one of the methods [A-79] [A-68] to [A-78], the scaffold protein is a membrane-bound protein, a soluble protein, or an insoluble protein deposit. In a preferred embodiment of any one of the methods [A-80] [A-68] to [A-79], the scaffold protein is a cell surface marker, an immune cell surface marker, a T cell exhaustion marker, a tumor-specific marker, a tumor-associated marker, or a protein whose expression is characteristic of a disease or disorder. In a preferred embodiment of any one of the methods [A-81] [A-68]~[A-80], the scaffold protein is PD1, PDL1, CSF2RB, CD4, CD5, CD6, CD7, CD8, CD9, CCR4, CD8, CD25, CD27, CD38, CD39, CD45, CD62L, CCR6, CD69, CD103, CD73, CD127, GITR, LRRC32, ICOS, TIGIT, RBD, MUC1, CEA, CTLA4, IL2Rα, CXCR5, neuropilin-1, TIM3, LAG3, The group is selected from TNFα, CD19, CD20, CD22, CD30, CD33, glycoprotein NMB, CD56, CD70, CD79A, CD79B, CD138, PSCA, PSMA, BCMA, FcRH5, GPRC5D, FAP, LRRC15, E-selectin, EphB2, melanotransferrin, HER2, TROP2, nectin 4, EGFRvIII, IL13RA2, TMEFF2, Muc16, EpCAM, FcRH2, amyloid β, AFP, PSA, MBP, and ASGPR. In a more preferred embodiment, the scaffold protein is selected from the group consisting of PD1, PDL1, TNFα, CD25, MUC1, CEA, and CD8. In a preferred embodiment of any one of the methods [A-82] [A-68]~[A-81], the first receptor protein and the second receptor protein are independently IL2Rβ, IL2Rγ, IL4R, IL13RA1, IL7R, IL9R, IL21R, TSLPR, IL3RA, CSF2RB, IL5RA, CSF2RA, IL6R, gp130, IL11RA, IL12RB1, IL12RB2, IL27RA, IL31RA, OSMR, CNTFR, LIFR, IL10RA, IL10RB, IL20RA, IL20RB, IL22RA1, IL28RA, IFNAR1, IFNAR2, IFNGR1, IFNGR2, IL1R1, IL1RAP, IL18R1, IL18RAP, ST2, IL17RA, IL17RC, T LR3, TLR4, TLR7, TLR9, CSF1R, TNFR1, TNFR2, LTBR, ​​HVEM, FAS, CD28, cMET, DR3, DR4, DR5, NGFR, RANK, FN14, CD40, 4-1BB, OX40, GITR, TGFBR1, TGFBR2, ACVRL1, ACVR2A, BMPR2, ACVR2B, ACVR1B, ACVR1 In a more preferred embodiment, the first receptor protein and the second receptor protein are each independently selected from the group consisting of IL2Rβ, IL2Rγ, Fzd1, Fzd2, Fzd3, Fzd4, Fzd5, Fzd6, Fzd7, Fzd8, Fzd9, Fzd10, LRP5, LRP6, and LGR5. In a preferred embodiment of any one of the methods [A-82a] [A-68]~[A-81], the scaffold protein is PD1, the first receptor protein is IL2Rβ, and the second receptor protein is IL2Rγ. In a preferred embodiment of any one of the methods [A-82b] [A-68]~[A-81], the scaffold protein is IL2Rα, the first receptor protein is IL2Rβ, and the second receptor protein is IL2Rγ. In a preferred embodiment of any one of the methods [A-82c] [A-68]~[A-81], the combination of target-binding molecules or protein complex competes for binding with any one of the target-binding molecules or protein complexes from Tables 1 to 17, or binds to the same epitope. In a preferred embodiment of any one of the methods [A-82d] [A-68]~[A-81], the scaffold protein, the first receptor protein, and the second receptor protein are combinations selected from any of (i)~(vi) below: (i) The scaffold protein is PDL1, the first receptor protein is IL2Rβ, and the second receptor protein is IL2Rγ; (ii) The scaffold protein is TNFa, the first receptor protein is IL2Rβ, and the second receptor protein is IL2Rγ; (iii) The scaffold protein is CD25, the first receptor protein is IL2Rβ, and the second receptor protein is IL2Rγ; (iv) The scaffold protein is PDL1, the first receptor protein is CSF2RA, and the second receptor protein is CSF2RB; (v) The scaffold protein is MUC1 or CEA, the first receptor protein is the Fzd receptor, and the second receptor protein is Lrp; or (vi) The scaffold protein is CD8, the first receptor protein is IL2Rβ, and the second receptor protein is IL2Rγ. In a preferred embodiment of any one of the methods [A-82e] [A-68]~[A-81], the receptor complex is IL-2R. In a more preferred embodiment, inducing IL-2 receptor complex signaling involves evaluating the activation of the IL-2 receptor complex using a colorimetric enzyme assay that determines alkaline phosphatase activity. In an even more preferred embodiment, alkaline phosphatase activity is determined by measuring optical density at 620 nm, optionally. In the most preferred embodiment, receptor signaling of the receptor complex is at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180% in the presence of the target binding molecule combination or protein complex compared to the absence of the target binding molecule combination or protein complex. It is induced when there is an increase in optical density of 190%, 200%, 210%, 220%, 230%, 240%, 250%, 260%, 270%, 280%, 290%, 300%, 310%, 320%, 330%, 340%, 350%, 360%, 370%, 380%, 390%, 400%, 410%, 420%, 430%, 440%, 450%, 460%, 470%, 480%, 490%, or 500%. In a preferred embodiment of any one of the methods [A-83] [A-68]~[A-82e], each binding domain is an antigen-binding domain comprising VH and VL, sdAb, VHH, scFv, Fab, scFab, Fab', Fab'-SH, F(ab')2, diabody, triabody, Fv, aptamer, aphibody, cytokine, ligand, or split cytokine. In a preferred embodiment of any one of the methods of [A-84] [A-68] to [A-83], the combination of target-binding molecules or protein complex further comprises a half-life extension domain. In a preferred embodiment of any one of the methods [A-85], [A-68], [A-70], or [A-72] to [A-84], the first target-binding molecule is connected to a half-life extension domain in a first binding domain, and the second target-binding molecule is connected to a half-life extension domain in a third binding domain. In preferred embodiments of the methods of [A-86], [A-84], or [A-85], the half-life extension domain comprises an Fc domain, an FcRn-binding domain, an albumin-binding domain, albumin or a variant thereof, or polyethylene glycol. In a preferred embodiment of any one of the methods of [A-87] [A-84] to [A-86], the half-life extension domain comprises an Fc domain, which optionally includes modifications that reduce antibody-dependent cell-mediated cytotoxicity (ADCC), reduce complement-dependent cytotoxicity (CDC), increase FcRn binding, increase pI, or promote heterodimerization. [A-88] The present invention also relates to a combination of target-binding molecules or protein complexes, comprising any one of the target-binding molecules or protein complexes in Tables 1 to 17. As shown in this application, any one of the target-binding molecules or protein complexes in Tables 1 to 17 is capable of inducing receptor signaling of the respective receptor complex. [B-1] An antigen-binding molecule containing one of the following (a1) to (a4): (a1) Complementary determination region (CDR) 1 having the amino acid sequence SYTMG, CDR2 having the amino acid sequence AIRWSGSITYYADSVKG, and CDR3 having the amino acid sequence SPVAGWGTSPAWYDY; (a2) CDR1 having the amino acid sequence SYGMG, CDR2 having the amino acid sequence TISWNSGSIYYTDSVKG, and CDR3 having the amino acid sequence GPRDWGNMRKFEEYEY; (a3) CDR1 having the amino acid sequence EYGMG, CDR2 having the amino acid sequence TISWDSDSIYYTDSVKG, and CDR3 having the amino acid sequence RPRDWGNMRRFEAYEY; or (a4) CDR1 having the amino acid sequence DYAGS, CDR2 having the amino acid sequence SINWRGDTTYYADSVKG, and CDR3 having the amino acid sequence KATDWSSTLYEYDY. [B-2] An antigen-binding molecule of [B-1] in which at least one of the amino acid sequences of CDR1, CDR2, or CDR3 further comprises one or more conserved amino acid substitutions. [B-3] One antigen-binding molecule, either [B-1] or [B-2], which is either a VHH or a single-domain antibody. [B-4] One antigen-binding molecule from [B-1] to [B-3] containing one of the following (a1) to (a4): (a1) Amino acid sequence Amino acid sequences having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with TIFF2026510624000002.tif18146, (a2) Amino acid sequence Amino acid sequences having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with TIFF2026510624000003.tif18149, (a3) Amino acid sequence An amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with TIFF2026510624000004.tif18149, or (a4) Amino acid sequence An amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with TIFF2026510624000005.tif18149. [B-5] An antigen-binding molecule containing one of the following (a1) to (a4): (a1) Amino acid sequence Amino acid sequences having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with TIFF2026510624000006.tif17147, (a2) Amino acid sequence Amino acid sequences having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with TIFF2026510624000007.tif18150, (a3) Amino acid sequence An amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with TIFF2026510624000008.tif17149, or (a4) Amino acid sequence An amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with TIFF2026510624000009.tif17149. [B-6] One of the antigen-binding molecules [B-1] to [B-5] that binds to CD8. An antigen-binding molecule that binds to the same epitope in CD8 as one of the antigen-binding molecules [B-7] [B-1]~[B-5]. A multispecific antigen-binding molecule containing one of the antigen-binding molecules [B-8], [B-1], to [B-7]. [B-9] A combination of target-binding molecules from any of [A-1] to [A-17] or a protein complex from any of [A-38] to [A-54], wherein the first binding domain and / or the third binding domain is one of (a1) to (a4) as defined in any of [B-1] to [B-7]. A pharmaceutical composition containing one antigen-binding molecule from [B-10] [B-1] to [B-9]. [B-11] A pharmaceutical composition of [B-10] for use in therapy. [B-12] A pharmaceutical composition of [B-10] for use in the therapy of medical conditions characterized by CD8 expression or increased CD8 expression. [B-13] A method for treating an individual having a medical condition, comprising the step of administering to the individual an effective amount of the pharmaceutical composition of [B-10], wherein the medical condition is characterized by CD8 expression or increased CD8 expression. [Brief explanation of the drawing]

[0009] [Figure 1A] Figure 1A shows a schematic diagram of the protein complex in this disclosure. Figure 1A shows two different VHH-VHH fusions, each having a scaffold protein-binding domain and a receptor protein-binding domain. Receptor activation occurs only in the presence of the scaffold protein. [Figure 1B] Figure 1B shows a schematic diagram of the protein complex in this disclosure. Figure 1B shows a promising single-molecule form that can induce receptor activation in the presence of the scaffold protein. [Figure 2A] Figure 2A shows conditional IL-2R activation by expressing a mixture of VHH-VHH fusions that bind to the SARS-CoV2 receptor-binding domain (RBD) and the IL-2 receptor in a biparatopic manner on either RBD-expressing HEK-Blue IL-2 cells or PDL1-expressing HEK-Blue IL-2 cells. [Figure 2B] Figure 2B shows a schematic diagram of the experimental setup in this embodiment. [Figure 3] Figures 3A and 3B show a comparison of the effects of treating RBD-expressing HEK-Blue IL-2 cells (Figure 3A) or PDL1-expressing HEK-Blue IL-2 cells (Figure 3B) with a mixture of recombinant VHH-VHH fusions that bind to the IL-2 receptor and RBD in a biparatopic manner, and a mixture of recombinant VHH-VHH fusions that bind to the IL-2 receptor and RBD in a monoparatopic manner. [Figure 4]Figure 4 shows conditional IL-2R activation by expressing a mixture of VHH-VHH fusions that bind to PDL1 and IL-2 receptors on either PDL1-expressing HEK-Blue IL-2 cells or RBD-expressing HEK-Blue IL-2 cells. [Figure 5] Figures 5A and 5B show a comparison of transfecting HEK-Blue IL-2 cells with RBD-IL2RA or PDL1 with expression plasmids between a fusion protein of Split Neo-2 / 15 and VHH that binds to RBD (Figure 5A) and a mixture of Vhh2b-Nb36 and Nb21-Vhh2g that binds to RBD and the IL-2 receptor (Figure 5B). [Figure 6A] Figure 6A shows conditional IL-2R activation by expressing a mixture of VHH-VHH fusions that bind to TNFα and IL-2 receptors on either TNFα-expressing HEK-Blue IL-2 cells or PDL1-expressing HEK-Blue IL-2 cells. [Figure 6B] Figure 6B shows a schematic diagram of TNFα-dependent IL-2R activation by a mixture of VHH-VHH fusions. [Figure 7] Figures 7A and 7B show conditional Wnt receptor activation on either RBD-expressing HEK293 STF cells (Figure 7A) or PDL1-expressing HEK293 STF cells (Figure 7B) by a mixture of recombinant VHH-VHH and VHH-scFv fusions that can bind to RBD, as well as FZD and LRP, which are Wnt receptors. [Figure 8] Figure 8A shows conditional IL-2R signaling induction by the Vhh2b-Nb36 / Nb21-Vhh2g Fc fusion protein on either RBD-expressing HEK-Blue IL-2 cells or PDL1-expressing HEK-Blue IL-2 cells. Figure 8B shows IL-2R activation by recombinant IL-2 on either RBD-expressing HEK-Blue IL-2 cells or PDL1-expressing HEK-Blue IL-2 cells. [Figure 9A]Figure 9A shows a schematic diagram of IL-2Rα-dependent IL-2R activation by a mixture of IL2_RETR and Vhh2g-BT942 scFv. [Figure 9B] Figure 9B shows the induction of IL-2R signaling on HEK-Blue IL-2 cells with or without daclizumab (anti-IL-2Rα neutralizing agent) treatment, using a mixture of IL2_RETR and Vhh2g-BT942 scFv, which can bind to IL-2Rα as a scaffold protein and to IL-2Rβ or IL-2Rγ as target receptor proteins. [Figure 9C] Figure 9C shows recombinant IL-2-mediated IL-2R activation in HEK-Blue IL-2 cells with and without daclizumab treatment. [Figure 10] Figure 10 shows a comparison between biparatopic and monoparatopic binding to IL-2Rα in IL-2R activation by a mixture of IL-2 variants and / or VHH-scFV that can bind to IL-2Rα as a scaffold protein and to IL-2Rβ or IL-2Rγ as target receptor proteins. [Figure 11] Figures 11A and 11B show conditional induction of IL-2R signaling on either PD1-expressing HEK-Blue IL-2 cells (Figure 11A) or PDL1-expressing HEK-Blue IL-2 cells (Figure 11B) by a mixture of VHH-scFv fusions that bind to PD1 and IL-2 receptors. [Figure 12] Figure 12 shows a comparison between biparatopic and monoparatopic binding to PD1 in IL-2R activation by a mixture of VHH-scFv, which binds to PD1 as a scaffold protein and to the IL-2 receptor as a target receptor protein. [Figure 13] Figure 13 shows the binding activity of anti-PD1 Ab to PD1-expressing cell lines, as evaluated by flow cytometry. [Figure 14] Figure 14 shows the neutralizing activity of anti-PD1 Ab, as evaluated using a PD1+ Jurkat reporter cell line. [Figure 15] Figure 15 shows conditional IL-2 receptor activation on either PD1-expressing HEK-Blue IL-2 cells (Figure 15A) or PDL1-expressing HEK-Blue IL-2 cells (Figure 15B) using a mixture of VHH-IgG fusion proteins that bind to IL-2Rβ or IL-2Rγ as the target receptor protein and PD1 as the target scaffold protein. All mixtures are combinations of neutralizing anti-PD1 Ab and non-neutralizing anti-PD1 Ab. [Figure 16] Figure 16 shows STAT5 activation on either activated PD1-expressing CD4+ T cells (Figure 16A) or PD1-negative NK92 cells (Figure 16B) by a mixture of VHH-IgG or VHH-scFv fusion proteins that bind to IL-2Rβ or IL-2Rγ as the target receptor protein and PD1 as the target scaffold protein. [Figure 17] Figure 17 shows conditional IL-2 receptor activation on either PD1-expressing HEK-Blue IL-2 cells (Figure 17A) or PDL1-expressing HEK-Blue IL-2 cells (Figure 17B) using a mixture of VHH-IgG fusion proteins that bind to IL-2Rβ or IL-2Rγ as the target receptor protein and PD1 as the target scaffold protein. [Figure 18] Figure 18 shows conditional IL-2 receptor activation on either PD1-expressing HEK-Blue IL-2 cells (Figure 18A) or PDL1-expressing HEK-Blue IL-2 cells (Figure 18B) using a mixture of VHH-IgG fusion proteins or scFv-IgG fusion proteins that bind to IL-2Rβ or IL-2Rγ as the target receptor protein and PD1 as the target scaffold protein. [Figure 19] Figure 19 shows the activation of the IL-2 receptor on CD25-expressing CD4+ T cells (Figure 19) by a mixture of VHH-scFv fusion proteins that bind to IL-2Rβ or IL-2Rγ as the target receptor protein and CD25 as the target scaffold protein. [Figure 20] Figure 20 shows the activation of the GM-CSF receptor on either PDL1-expressing HEK-Blue GM-CSF cells (Figure 20A) or RBD-expressing HEK-Blue GM-CSF cells (Figure 20B) by a mixture of VHH-scFv fusion proteins that bind to CSF2RA or CSF2RB as the target receptor protein and PDL1 as the target scaffold protein. [Figure 21] Figure 21 shows the activation of the cMET receptor in the presence or absence of recombinant RBD protein by a VHH-Fab fusion protein that binds to cMET as the target receptor protein and RBD as the target scaffold protein. Figure 21A shows the activation of the cMET receptor by a mixture of anti-RBD biparatopic antibodies fused to the cMET binding domain. Figures 21B and 21C show the activation of the cMET receptor by anti-RBD monoparatopic antibodies fused to the cMET binding domain. [Figure 22] Figure 22 shows conditional Wnt receptor activation on either MUC1-expressing HEK293 STF cells or RBD-expressing HEK293 STF cells by a mixture of bispecific Abs that bind to the Wnt receptor as the target receptor protein and to MUC1 as the target scaffold protein. [Figure 23] Figure 23 shows conditional Wnt receptor activation on either CEA-expressing HEK293 STF cells or RBD-expressing HEK293 STF cells by a mixture of bispecificity abs that bind to the Wnt receptor as the target receptor protein and to CEA as the target scaffold protein. Figure 23A shows Wnt receptor activation by a mixture of anti-CEA biparatopic abs, and Figure 23B shows Wnt receptor activation by a mixture of anti-CEA monoparatopic abs. [Figure 24] Figure 24 shows STAT5 activation in CD8+ T cells or CD4+ T cells by a mixture of VHH-VHH fusion proteins that bind to CD8 as the target scaffold protein and to IL-2Rβ or IL-2Rγ as the target receptor protein. [Figure 25] Figure 25 shows STAT5 activation in CD8+ T cells by a mixture of VHH-VHH fusion proteins that bind to CD8 as the target scaffold protein and to IL-2Rβ or IL-2Rγ as the target receptor protein. [Figure 26] Figure 26 shows the proliferation of CD8+ T cells or CD4+ T cells using a mixture of VHH-VHH fusion proteins that bind to CD8 as the target scaffold protein and to IL-2Rβ or IL-2Rγ as the target receptor protein. [Modes for carrying out the invention]

[0010] Description of the manner General technology Unless otherwise indicated, the implementation of the present invention will utilize the prior art of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology, which are within the scope of the art. Such techniques are well described in literature such as Molecular Cloning: A Laboratory Manual, second edition (Sambrook et al., 1989); Oligonucleotide Synthesis (MJ Gait, ed., 1984); Animal Cell Culture (RI Freshney, ed., 1987); Methods in Enzymology (Academic Press, Inc.); Current Protocols in Molecular Biology (FM Ausubel et al., eds 1987, and regularly updated); PCR: The Polymerase Chain Reaction (Mullis et al., ed., 1994); A Practical Guide to Molecular Cloning (Perbal Bernard V., 1988); and Phase Display: A Laboratory Manual (Barbas et al., 2001).

[0011] The following definitions and detailed explanations are provided to facilitate understanding of the disclosure set forth herein. All references referred to herein are incorporated specifically by reference.

[0012] I. Definition Protein / Polypeptide As used herein, the term "polypeptide" refers to a molecule composed of monomers (amino acids) linearly linked by amide bonds (also known as peptide bonds). The term "polypeptide" typically refers to a peptide having a length of approximately four amino acids or more, and does not refer to a product of a specific length. As used herein, the term also includes fragments of polypeptides. Therefore, the definition of "polypeptide" includes peptides, dipeptides, tripeptides, oligopeptides, "proteins," "amino acid chains," or any other terms used to refer to chains of two or more amino acids, and the term "polypeptide" may be used in place of or interchangeably with any of these terms. The term "polypeptide" is also intended to refer to products of post-expression modifications of polypeptides, including, but not limited to, glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting / blocking groups, proteolytic cleavage, or modification with amino acids not naturally occurring. Polypeptides may originate from natural biological sources or may be produced by recombinant technology, but do not necessarily have to be translated from a specified nucleic acid sequence. Polypeptides may be produced in any manner, including chemical synthesis. Polypeptides as described herein may have sizes of approximately 3 or more, 5 or more, 10 or more, 20 or more, 25 or more, 50 or more, 75 or more, 100 or more, 200 or more, 500 or more, 1,000 or more, or 2,000 or more amino acids. Polypeptides may, but do not necessarily, have a defined three-dimensional structure. Polypeptides having a defined three-dimensional structure are referred to as folded, while polypeptides that do not have a defined three-dimensional structure and can rather take on many different conformations are referred to as unfolded.

[0013] Fusion protein As used herein, the term “fusion protein” is sometimes abbreviated as “fusion,” and refers to a single-chain protein, i.e., a protein formed by a single chain of amino acids, formed by a combination of at least two (poly)peptides. In some embodiments, the combination arises via peptide bonds. In some embodiments, the combination arises via peptide bonds provided by a peptide linker. An example of a fusion protein of the present invention is a first target-binding molecule of [A-1] or [A-4] in which a first binding domain is optionally connected or linked to a second binding domain via a linker. A further example of a fusion protein of the present invention is a second target-binding molecule of [A-1] or [A-4] in which a third binding domain is optionally linked to a fourth binding domain via a linker. Another example is contained in a protein complex of [A-39] or [A-42] in which the first binding domain and the second binding domain are linked, or the third binding domain and the fourth binding domain are linked. In some embodiments, all four binding domains are ligated together and therefore part of a single fusion protein.

[0014] protein complex As used herein, the term "protein complex" refers to a molecular construct formed by the association of at least two proteins or polypeptides. The association of polypeptides in a complex does not occur via peptide bonds, and therefore, the polypeptides can be separated without disrupting the primary structure of the proteins involved. For example, polypeptides in a protein complex can associate via one or more disulfide bonds or non-covalently. An exemplary embodiment of the protein complex of the present invention is the protein complex of [A-38] comprising a first target binding molecule comprising a first binding domain capable of binding to a scaffold protein and a second binding domain capable of binding to a first receptor protein, and a second target binding molecule comprising a third binding domain capable of binding to the scaffold protein and a fourth binding domain capable of binding to a second receptor protein, wherein the first target binding molecule and the second target binding molecule are capable of binding non-competitively to the scaffold protein, the first receptor protein and the second receptor protein are receptor subunits capable of associating to form a receptor complex, and the protein complex is capable of inducing receptor signaling of the receptor complex. In one embodiment of the protein complex of [A-38], the first target binding molecule and the second target binding molecule are linked to each other.

[0015] Another embodiment of the protein complex of the present invention is a protein complex of [A-39] comprising a first binding domain, a second binding domain, a third binding domain, and a fourth binding domain, wherein the first binding domain is capable of binding to a scaffold protein, the second binding domain is capable of binding to a first receptor protein, the third binding domain is capable of binding to the scaffold protein, and the fourth binding domain is capable of binding to a second receptor protein, the first binding domain and the third binding domain are capable of binding non-competitively to the scaffold protein, the first receptor protein and the second receptor protein are receptor subunits that can each associate to form a receptor complex, and the protein complex is capable of inducing receptor signaling of the receptor complex. In one embodiment of the protein complex of [A-39], the first binding domain, the second binding domain, the third binding domain, and the fourth binding domain are linked together to form a single polypeptide chain.

[0016] target binding molecule The term "target-binding molecule" refers to a molecule, such as a protein or polypeptide or fusion protein or protein complex, that is capable of eliciting a measurable and reproducible interaction between itself and its target, such as an antigen, e.g., IL-2R or a scaffold protein, in the presence of a heterogeneous population of molecules, including biological molecules. A target-binding molecule binds to its target with greater affinity, avidity, ease, and / or duration than it binds to other targets. In one embodiment, the degree of binding of a target-binding molecule to an unrelated target is less than about 10% of the binding of the target-binding molecule to the target, as measured, for example, by radioimmunoassay (RIA). In a particular embodiment, a target-binding molecule that specifically binds to a target may have concentrations of ≤1 micromolar (μM), ≤100 nM, ≤10 nM, ≤1 nM, ≤0.1 nM, ≤0.01 nM, or ≤0.001 nM (e.g., 10 -8 M or less, for example, 10 -8 M~10 -13 M, for example, 10 -9 M~10 -13 It has a dissociation constant (Kd) of M). The "target-binding molecule" of the present invention may include an antibody, an antibody-like construct, a VHH, or a protein complex comprising a heavy chain and a light chain associated with each other.

[0017] Joint domain The target-binding molecule of the present invention comprises two binding domains, each having a different target. Therefore, the target-binding molecule of the present invention is capable of simultaneously binding to two targets. As used herein, the term "binding domain" refers to a portion of a protein that exhibits target / antigen-binding activity. The binding domain may be an antigen-binding domain including VH and VL, sdAb, VHH, scFv, Fab, scFab, Fab', Fab'-SH, F(ab')2, diabody, triabody, Fv, aptamer, or aphibody. It may be derived from a cytokine (including cytokine variants) or a native ligand (including variants). It may also be a split cytokine, etc.

[0018] Neutralizing binding domain and non-neutralizing binding domain The term "neutralizing binding domain" refers to a binding domain that binds to its target / antigen, resulting in the prevention, inhibition, reduction, delay, or interference of its activity. This may be because the binding domain binds to an epitope involved in the activity of the target / antigen. In non-limiting examples, a neutralizing binding domain may bind to an epitope near or at the ligand-binding site of the target / antigen, thereby preventing, inhibiting, reducing, delaying, or interfering with the activity of the target / antigen. The term "neutralizing binding domain" may also refer to an antagonist-binding domain, such as in an antagonist antibody. The term "non-neutralizing binding domain" refers to a binding domain that binds to its target / antigen, but does not cause the prevention, inhibition, reduction, delay, or interference of its activity. The term "non-neutralizing binding domain" may also refer to a non-antagonist-binding domain, such as in a non-antagonist antibody.

[0019] Multispecific target binding molecules In the present invention, the target-binding molecules provided herein are multispecific target-binding molecules, for example, bispecific target-binding molecules. A multispecific target-binding molecule is a target-binding molecule having binding specificity to at least two different sites. In the present invention, one of the binding specificities is to a scaffold protein and the other is to a receptor protein. A bispecific target-binding molecule can be prepared as a full-length antibody or may include an antibody fragment.

[0020] Methods for constructing multispecific target-binding molecules are not limited to these, but include, recombinant co-expression of two immunoglobulin heavy-light chain pairs with different specificities (see Milstein and Cuello, Nature 305: 537 (1983), WO93 / 08829, and Traunecker et al., EMBO J. 10: 3655 (1991)), and knob-into-hole techniques (see, for example, U.S. Patent No. 5,731,168). Multispecific target-binding molecules can be created by manipulating electrostatic steering effects to produce Fc heterodimer molecules (WO2009 / 089004A1); crosslinking two or more antibodies or fragments (see U.S. Patent No. 4,676,980 and Brennan et al., Science, 229:81 (1985)); creating bispecific antibodies using a leucine zipper (see Kostelny et al., J. Immunol., 148(5):1547-1553 (1992)); creating bispecific antibody fragments using "diabody" technology (see Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993)); and using single-chain Fv (scFv) dimers (Gruber et al., J. Immunol., 152:5368). (See 1994); and may also be prepared by preparing a trispecific antibody as described, for example, in Tutt et al. J. Immunol. 147: 60 (1991).

[0021] Competitive and non-competitive coupling The first and second target-binding molecules of the present invention, or the first and third binding domains of the protein complex of the present invention, are capable of non-competitive binding to a scaffold protein. As used herein, “competitive binding” refers to a process of two binding domains that results in only one binding domain being capable of binding to the scaffold protein by competing for the same epitope or paratope. The epitopes or paratopes for both binding domains can be the same or overlap. In either case, both binding domains cannot bind to their epitope-paratope simultaneously. As used herein, “non-competitive binding” refers, conversely, to a process of two binding domains that can bind to their epitope or paratope without interfering with the binding mechanism of the other binding domain. In one embodiment, the epitopes or paratopes do not overlap, and there is no steric hindrance preventing simultaneous binding of both binding domains. An example of non-competitive binding is biparatopic binding. In another embodiment, epitopes may be identical or overlapping, but there may be more than one epitope on the target. This can be the case when the target is a multimer. For example, the scaffold protein of the present invention may consist of several subunits, each containing an epitope. In this case, the target-binding molecule binds to two distinct but identical epitopes that have the same sequence but are located on different subunits of the multimer. It is essential that both target-binding molecules can bind simultaneously without interfering with the binding mechanisms of the other target-binding molecule.

[0022] Biparatopic bonding As used herein, "biparatopic binding" refers to a binding mechanism that targets two epitopes on a molecular target. In the present invention, the two binding domains targeting the scaffold protein each target a separate epitope on the protein, thereby enabling simultaneous binding of both binding domains to the scaffold protein.

[0023] In one embodiment, two binding domains targeting a scaffold protein can biparatopically bind to the scaffold protein, where the epitopes to which each binding domain binds are close to each other. In another embodiment, two binding domains targeting a scaffold protein can biparatopically bind to the scaffold protein within the domain of the scaffold protein. The epitopes can be determined from the crystal structure of the complex of the scaffold protein binding domains and the scaffold protein. Specifically, the interatomic distance between the non-hydrogen atoms constituting the side chain or back chain of the amino acids forming the scaffold protein and the non-hydrogen atoms constituting the side chain or back chain of the amino acids forming the scaffold protein is calculated. Amino acid residues containing non-hydrogen atoms with interatomic distances below thresholds set at 3.5 angstroms, 4.0 angstroms, 4.2 angstroms, 4.5 angstroms, or 5.0 angstroms were considered to be residues contained within the epitope. Next, the proximity of the epitopes to which each binding domain in this specification binds, or the distance between epitopes, can be determined. In one embodiment, the interatomic distance between any α-carbon of a first epitope on the scaffold protein and any α-carbon of a second epitope on the scaffold protein is at least 2.0 angstroms, 2.5 angstroms, 3.0 angstroms, 3.5 angstroms, 4.0 angstroms, 4.5 angstroms, 5.0 angstroms, 5.5 angstroms, 6.0 angstroms, 6.5 angstroms, 7.0 angstroms, 7.5 angstroms, 8.0 angstroms, 8.5 angstroms, 9.0 angstroms, 9.5 angstroms, 10.0 angstroms, 10.5 angstroms, 11.0 angstroms, 11.5 angstroms, 12.0 angstroms, These are 12.5 angstroms, 13.0 angstroms, 13.5 angstroms, 14.0 angstroms, 14.5 angstroms, 15.0 angstroms, 15.5 angstroms, 16.0 angstroms, 16.5 angstroms, 17.0 angstroms, 17.5 angstroms, 18.0 angstroms, 18.5 angstroms, 19.0 angstroms, 19.5 angstroms, 20.0 angstroms, 20.5 angstroms, 21.0 angstroms, 21.5 angstroms, 22.0 angstroms, 22.5 angstroms, 23.0 angstroms, 23.5 angstroms, 24.0 angstroms, 24.5 angstroms, or 25.0 angstroms. In one embodiment, the interatomic distance between any α-carbon of a first epitope on the scaffold protein and any α-carbon of a second epitope on the scaffold protein is at most 30.0 angstroms, 30.5 angstroms, 31.0 angstroms, 31.5 angstroms, 32.0 angstroms, 32.5 angstroms, 33.0 angstroms, 33.5 angstroms, 34.0 angstroms, 34.5 angstroms, 35.0 angstroms, 35.5 angstroms, 36.0 angstroms, and 36.5 angstroms, 37.0 angstroms, 37.5 angstroms, 38.0 angstroms, 38.5 angstroms, 39.0 angstroms, 39.5 angstroms, 40.0 angstroms, 40.5 angstroms, 41.0 angstroms, 41.5 angstroms, 42.0 angstroms, 42.5 angstroms, 43.0 angstroms, 43.5 angstroms, 44.0 angstroms, 44.5 angstroms, 45. 0 angstroms, 45.5 angstroms, 46.0 angstroms, 46.5 angstroms, 47.0 angstroms, 47.5 angstroms, 48.0 angstroms, 48.5 angstroms, 49.0 angstroms, 49.5 angstroms, 50.0 angstroms, 50.5 angstroms, 51.0 angstroms, 51.5 angstroms, 52.0 angstroms, 52.5 angstroms, 53.0 angstroms, 53.5 Angstrom, 54.0 Angstrom, 54.5 Angstrom, 55.0 Angstrom, 55.5 Angstrom, 56.0 Angstrom, 56.5 Angstrom, 57.0 Angstrom, 57.5 Angstrom, 58.0 Angstrom, 58.5 Angstrom, 59.0 Angstrom, 59.5 Angstrom, 60.0 Angstrom, 60.5 Angstrom, 61.0 Angstrom, 61.5 Angstrom, 62.0 Angstrom These are 6.5 angstroms, 62.5 angstroms, 63.0 angstroms, 63.5 angstroms, 64.0 angstroms, 64.5 angstroms, 65.0 angstroms, 65.5 angstroms, 66.0 angstroms, 66.5 angstroms, 67.0 angstroms, 67.5 angstroms, 68.0 angstroms, 68.5 angstroms, 69.0 angstroms, 69.5 angstroms, or 70.0 angstroms.

[0024] combination of target-binding molecules As used herein, the term “combination of target-binding molecules” means, in one embodiment, a composition comprising both a first target-binding molecule and a second target-binding molecule. In this embodiment, the first target-binding molecule and the second target-binding molecule may be connected or linked to one another, resulting in a single protein or protein complex combining both the first and second target-binding molecules.

[0025] In a further embodiment, a combination means two compositions, the first composition comprising a first target-binding molecule and the second composition comprising a second target-binding molecule. Since both the first and second target-binding molecules must be present at the biological site of action, both compositions must be part of a combination. However, both compositions do not need to be combined into a single composition. Having separate compositions, the first composition comprising a first target-binding molecule and the second composition comprising a second target-binding molecule, allows for separate administration of the compositions, and subsequently, allows for better fine-tuning of the administration method. Thus, as used herein, the term “combining” encompasses adding both target-binding molecules to a single composition before administration, and administering both target-binding molecules separately, whether simultaneously, in parallel, or sequentially, resulting in combinations of target-binding molecules administered separately at the biological site of action, as defined in [A-33] and / or [A-34].

[0026] Scaffold Protein Each target-binding molecule of the present invention includes a binding domain capable of binding to a scaffold protein. As used herein, the term "scaffold protein" refers, in particular, to a cell surface protein, soluble protein, or insoluble protein expressed in a target cell, target tissue, or target organ. As used herein, a scaffold protein is not limited by structure or function. Rather, the scaffold protein acts as an anchor for the first and second target-binding molecules, or the first and third binding domains of a protein complex, to bind non-competitively and / or biparatopically. Scaffold proteins are cell surface markers, specific cell surface markers, immune cell surface markers, T cell exhaustion markers, tumor-related markers, tumor-related antigens or tumor-specific antigens, tissue-specific markers, disease tissue-specific markers, organ-specific markers, soluble proteins, insoluble protein deposits, such as PD1, PDL1, CSF2RB, CD4, CD5, CD6, CD7, CD8, CD9, CD25, CCR4, CD8, CD27, CD38, CD39, CD45, CD62L, CCR6, CD69, CD103, CD73, CD127, GITR, LRRC32, ICOS, TIGIT, RBD, This could include MUC1, CEA, CTLA4, IL2Rα, CXCR5, neuropilin-1, TIM3, LAG3, TNFα, CD19, CD20, CD22, CD30, CD33, glycoprotein NMB, CD56, CD70, CD79A, CD79B, CD138, PSCA, PSMA, BCMA, FcRH5, GPRC5D, FAP, LRRC15, E-selectin, EphB2, melanotransferrin, HER2, TROP2, nectin 4, EGFRvIII, IL13RA2, TMEFF2, Muc16, EpCAM, FcRH2, AFP, PSA, amyloid-beta, MBP, or ASGPR. In a preferred embodiment, the scaffold protein is PD1.

[0027] The scaffold protein of the present invention can be a membrane-bound protein, a soluble protein, or an insoluble protein deposit. It is preferably membrane-bound, which facilitates localization to the intended site of action. In the case of a soluble scaffold protein, localization to the intended site of action can be ensured by targeting a soluble scaffold protein known to be present at a high concentration at that site of action. For example, if the intended site of action is tumor tissue, it may be useful to target a soluble scaffold protein secreted by the tumor tissue. This would achieve localization and avoid systemic activation of the signaling pathway.

[0028] Tumor-specific markers or tumor-related markers As used herein, “tumor-specific marker” or “tumor-associated marker,” as a non-limiting example of a scaffold protein, means an antigen expressed by cancer cells that makes it possible to distinguish cancer cells from healthy cells. For example, “tumor-specific marker” or “tumor-associated marker” includes antigens expressed in conjunction with the malignant transformation of cells, and abnormal glycans that appear on the cell surface or on protein molecules during the carcinogenesis of cells. Specific examples include ALK, pleiotrophin (PTN), EpCAM, CA125, prostatic acid phosphatase (PAP), prostate-specific antigen (PSA), TYRP1, HMW-MAA, prostate-specific membrane antigen (PSMA), CEA, MUC1, HMFG1, TAG-72, GICA (CA19-9), NY-ESO-1, LEA, CD15, CD17, CD19, CD20, CD22, CD30, CD33, CD38, CD77, CD79b, CD147, CD228, GD2, GD3, GM2, GM3, TSTA, and virus-induced tumor antigens. (e.g., envelope antigens of DNA oncoviruses and RNA oncoviruses), EBV-specific antigens, α-fetoprotein (AFP), 5T4, differentiation antigens (e.g., L6 and L20 antigens), CD165, EGFR, ANKRD17, ErbB2, APO-1, SSEA-1, SCP-1, LeY, oligosaccharide antigens, SSEA-3, SSEA-4, CTAGE1, MART-1, sialyl Tn (STn), NY-CO-45, NY-LU-12, ART1, MA2, NOVA2, TSPAN8, MAGE-C1, MAGE-B1, MAGE-B2, MAGE-4A, Examples include MAGE-X2, YKL-40, EREG, CA15-3, CLEC12A, Nectin 4, Trop2, BCMA, Tissue Factor, FRα (FOLR1), ErbB3, Claudin 18 (Claudin 18.2), B7-H3 (CD276), MET, PSCA, PTK7, MSLN (Mesothelin), CCR4, CDH6, IL13RA2, DLL3, GPC3, Claudin 6, Fibroblast-associated protein (FAP), FLT3, GD2, GD3, and any fragments of these polypeptides, as well as their modified structures. Multimeric scaffold proteins The scaffold protein of the present invention may be composed of several subunits. The subunits may be the same or different. Thus, the scaffold protein may be a homomultimer or a heteromultimer. In some embodiments, if the first binding domain and the third binding domain are directed to the same or overlapping epitopes on the homomultimerized scaffold protein, the homomultimerized scaffold protein contains more than one of the same epitopes, and thus non-competitive binding of the first and third binding domains becomes possible.

[0029] receptor Each target-binding molecule of the present invention includes a binding domain capable of binding to a receptor protein. As used herein, the term “receptor” refers to a protein structure in or on the surface of a cell that receives and transmits signals by binding to and / or multimerizing a particular molecule. Receptors can be cytokine receptors; Wnt receptors; or receptors that induce signals by heterodimerization, homodimerization, or oligomerization, which are activated only after the association of homodimers, homotrimers, homomultimers, heterodimers, heterotrimers, or monomers of a heteromultimer. According to the present invention, a first receptor protein and a second receptor protein are each receptor subunits that can associate to form a receptor complex. The receptor complex is activated upon the association of receptor subunits. The association of receptor subunits occurs upon the binding of the first target-binding molecule to both of its targets, and upon the binding of the second target-binding molecule to both of its targets.Examples of receptor subunits include IL2Rβ, IL2Rγ, IL4R, IL13RA1, IL7R, IL9R, IL21R, TSLPR, IL3RA, CSF2RB, IL5RA, CSF2RA, IL6R, gp130, IL11RA, IL12RB1, IL12RB2, IL27RA, IL31RA, OSMR, CNTFR, LIFR, IL10RA, IL10RB, IL20RA, IL20RB, IL22RA1, IL28RA, IFNAR1, IFNAR2, IFNGR1, IFNGR2, IL1R1, IL1RAP, IL18R1, IL18RAP, ST2, IL17RA, IL17RC, TLR3, TLR4, TLR7, TLR9, and CSF1R. , TNFR1, TNFR2, LTBR, ​​HVEM, FAS, CD25, CD28, DR3, DR4, DR5, NGFR, RANK, FN14, CD40, 4-1BB, OX40, GITR, TGFBR1, TGFBR2, ACVRL1, ACVR2A, BMPR2, ACVR2B, ACVR1B, ACVR1C, ACVR1, AMHR2, BMPR1A, BMPR1B, BMPR2, TRA, TRB, CD3E, CD16, TREM2, FGFR1, FGFR2, FGFR3, FGFR4 , Fzd1, Fzd2, Fzd3, Fzd4, Fzd5, Fzd6, Fzd7, Fzd8, Fzd9, Fzd10, LRP5, LRP6, or LGR5. In a preferred embodiment, the first receptor protein is IL-2Rβ, and the second receptor protein is IL-2Rγ.

[0030] Inducing receptor signaling As used herein, the term "inducing receptor signaling" refers to the biological mechanism that proceeds upon the formation of an activated receptor complex. Activation of the receptor complex requires the association of receptor protein subunits of the receptor complex. The receptor protein includes an extracellular portion, as known in the art. This extracellular portion can interact with the target-binding molecules of the present invention. The receptor protein may also include a cytoplasmic portion that can interact with downstream members of the signaling pathway activated by the receptor complex. Upon association of receptor subunits, they are in close proximity to each other and induce receptor signaling, as well as is well known in the art, either by themselves or by facilitating conformational changes in members of the receptor complex. For example, the cytoplasmic portion of a receptor protein subunit can recruit Janus kinase to initiate an intracellular signaling cascade. Janus kinase phosphorylates the cytoplasmic STAT protein. The phosphorylation state of the STAT protein can be readily monitored in assays established in the art. In a particular embodiment, if the receptor protein is IL-2R, activation of the IL-2 receptor complex can be assessed by measuring the optical density at 620 nm using a Multiskan® plate reader with Quanti-Blue solution (InvivoGen, #rep-qbs), a colorimetric enzyme assay that determines alkaline phosphatase activity.In a particular embodiment, receptor signaling of the receptor complex is at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 1% in the presence of the combination of target-binding molecules or protein complex of the present invention compared to the absence of the combination of target-binding molecules or protein complex of the present invention. This is induced when there is an increase in optical density of 80%, 190%, 200%, 210%, 220%, 230%, 240%, 250%, 260%, 270%, 280%, 290%, 300%, 310%, 320%, 330%, 340%, 350%, 360%, 370%, 380%, 390%, 400%, 410%, 420%, 430%, 440%, 450%, 460%, 470%, 480%, 490%, or 500%.

[0031] Close proximity The first receptor protein and the second receptor protein are positioned in close proximity to each other such that when the first, second, third, and fourth binding domains of the combination of target-binding molecules or protein complex of the present invention bind, the receptor complex containing the first receptor subunit and the second receptor subunit is activated and signal transduction is induced. As used herein, the term "proximity" refers to the closeness of the distance between two individual receptor proteins, for example, between IL-2Rβ and the second receptor protein IL-2Rγ.

[0032] Half-life extension domain In the present invention, the first binding domain and / or the third binding domain may each be affixed to a half-life extension domain. One barrier to the application of protein therapies has been the short half-life in circulation of these small proteins. Since protein therapies generally cannot be administered orally and are typically administered by subcutaneous, intramuscular, or intravenous injection or infusion, persistence in circulation is desirable. Therefore, in a preferred embodiment, the present invention may encompass the use of one or more pharmaceutically acceptable half-life extension domains, such as polyethylene glycol (PEG), immunoglobulin Fc domains or CH2 domains of Fc, albumin (e.g., human serum albumin (HSA) or its variants), albumin-binding proteins, FcRn-binding domains, transthyretin, or thyroxine-binding globulin (TBG).

[0033] Exemplary methods for improving the pharmacokinetics (PK) of polypeptides involve the expression of domains in polypeptide chains that bind to receptors that are recycled to the cell's plasma membrane rather than being degraded in lysosomes, such as FcRn receptors and transferrin receptors on endothelial cells. Three proteins, e.g., human IgG, HSA (or fragments), and transferrin, persist in human serum much longer than their size would predict, a function of their ability to bind to receptors that are recycled rather than degraded in lysosomes. These proteins, or their fragments that retain FcRn binding, are routinely ligated to other polypeptides to extend their serum half-lives. In one embodiment, the half-life extension domain is a human serum albumin (HSA) binding domain. HSA may also be directly bound to the pharmaceutical composition or bound via a short linker. Fragments of HSA may also be used. HSA and its fragments can function as both blocking moieties and half-life extension domains. Human IgG can also perform similar functions.

[0034] The serum half-life extension domain can also be an antigen-binding polypeptide that binds to proteins with long serum half-lives, such as serum albumin and transferrin. Examples of such polypeptides include polyclonal antibodies, recombinant antibodies, human antibodies, humanized antibodies, single-chain variable fragments (scFv), single-domain antibodies, such as antibodies and their fragments containing heavy-chain variable domains (VH), light-chain variable domains (VL), and camel-type nanobody variable domains (VHH), dAb, etc. Other suitable antigen-binding domains include non-immunoglobulin proteins that bind to and / or mimic the structure of antibodies, such as anticalin, affilin, afibody molecules, affimer, affitin, alphabody, avimer, DARPin, fynomer, Kunitz domain peptides, monobodies, as well as binding domains based on other manipulated scaffolds such as SpA, GroEL, fibronectin, lipocalin, and the CTLA4 scaffold. Further examples of antigen-binding polypeptides include ligands for desired receptors, ligand-binding moieties of receptors, lectins, and peptides that bind to or associate with one or more target antigens.

[0035] In other embodiments, the serum half-life extension domain may be a water-soluble polymer or a peptide conjugated to a water-soluble polymer, such as PEG. As used herein, “PEG,” “polyethylene glycol,” and “poly(ethylene glycol)” are interchangeable and encompass any non-peptide water-soluble poly(ethylene oxide). The term “PEG” also means a polymer containing a majority, i.e., more than 50%, of the –OCH2CH2– repeating subunit. With respect to a particular form, PEG can take any number of different molecular weights and structural or geometric shapes such as “branched,” “linear,” “forked,” and “polyfunctional,” as described in more detail below. PEG can be linear (e.g., terminally capped, e.g., alkoxyPEG or bifunctional PEG), branched or polyarmed (e.g., forked PEG or PEG attached to a polyol core), dendritic (or star-shaped) configurations, each with or without one or more degradable links. Furthermore, the internal structure of PEG can be organized into any number of different repeating patterns, and can be selected from the group consisting of homopolymers, alternating copolymers, random copolymers, block copolymers, alternating trippolymers, random trippolymers, and block trippolymers. PEG can be conjugated to polypeptides and peptides through any preferred method. Typically, a reactive PEG derivative such as N-hydroxysuccinamidyl ester PEG is reacted with a peptide or polypeptide containing amino acids having side chains containing amine, sulfhydryl, carboxylic acid, or hydroxyl functional groups, such as cysteine, lysine, asparagine, glutamine, theonine, tyrosine, serine, aspartic acid, and glutamic acid.

[0036] Possible Fc variants The target-binding molecule or protein complex of the present invention may further comprise other possible Fc variants. These variants may be suitable for reducing antibody-dependent cell-mediated cytotoxicity (ADCC) or complement-dependent cell-mediated cytotoxicity (CDC), increasing isoelectric point (pI), or increasing FcRn binding to improve the pharmacokinetic profile, or they may be Fc variants for preparing bispecific molecules.

[0037] In one embodiment, the present invention includes an FcγRIIb-binding polypeptide comprising an Fc variant, and a method using the same. In some embodiments, the Fc variant with enhanced FcγRIIb-binding activity of the present invention comprises at least one amino acid modification in the parent Fc region. In a further embodiment, the ratio of [KD value of the parent Fc region to monkey FcγRIIb] / [KD value of the variant Fc region to monkey FcγRIIb] is 2.0 or greater. In a further embodiment, the ratio of [KD value of the parent Fc region to monkey FcγRIIIa] / [KD value of the Fc variant to monkey FcγRIIIa] is 0.5 or less. In a further embodiment, the ratio of [KD value of the parent Fc region to human FcγRIIb] / [KD value of the variant Fc region to human FcγRIIb] is 2.0 or greater. In a further embodiment, the ratio of [KD value of the parent Fc region for human FcγRIIIa] / [KD value of the Fc variant for human FcγRIIIa] is 0.5 or less. In a further embodiment, the ratio of [KD value of the parent Fc region for human FcγRIIa(H type)] / [KD value of the variant Fc region for human FcγRIIa(H type)] is 5.0 or less. In a further embodiment, the ratio of [KD value of the parent Fc region for human FcγRIIa(R type)] / [KD value of the Fc variant for human FcγRIIa(R type)] is 5.0 or less. In another embodiment, the KD value of the variant Fc region for monkey FcγRIIb is 1.0 × 10⁻⁶. -6 It is less than or equal to M. In another embodiment, the KD value of the Fc variant for monkey FcγRIIIa is 5.0 × 10⁻¹⁰ -7 It is M or greater. In another embodiment, the KD value of the variant Fc region for human FcγRIIb is 2.0 × 10⁻⁶.-6 is less than M. In another embodiment, the KD value of the Fc variant for human FcγRIIIa is 1.0×10 -6 or more. In another embodiment, the KD value of the variant Fc region for human FcγRIIa (H type) is 1.0×10 -7 or more. In another embodiment, the KD value of the Fc variant for human FcγRIIa (R type) is 2.0×10 -7 or more.

[0038] In some embodiments, the Fc variant with enhanced FcγRIIb binding activity of the present invention comprises at least one amino acid modification at at least one position selected from the group consisting of 231, 232, 233, 234, 235, 236, 237, 238, 239, 264, 266, 267, 268, 271, 295, 298, 325, 326, 327, 328, 330, 331, 332, 334, and 396 according to EU numbering.

[0039] In a further embodiment, the Fc variant with enhanced FcγRIIb binding activity comprises at least two amino acid modifications including (a) one amino acid modification at position 236 and (b) at least one amino acid modification at at least one position selected from the group consisting of (i) positions 231, 232, 233, 234, 235, 237, 238, 239, 264, 266, 267, 268, 271, 295, 298, 325, 326, 327, 328, 330, 331, 332, 334, and 396; (ii) positions 231, 232, 235, 239, 268, 295, 298, 326, 330, and 396; or (iii) positions 268, 295, 326, and 330 according to EU numbering.

[0040] In a further embodiment, the Fc variant with enhanced FcγRIIb binding activity comprises at least two amino acid modifications, including (a) one amino acid modification at position 236, and (b) at least one amino acid modification at at least one position selected from the group consisting of 231, 232, 233, 234, 235, 237, 238, 239, 264, 266, 267, 268, 271, 295, 298, 325, 326, 327, 328, 330, 331, 332, 334, and 396, according to EU numbering.

[0041] In a further embodiment, the Fc variant with enhanced FcγRIIb binding activity comprises at least two amino acid modifications, including (a) one amino acid modification at position 236, and (b) at least one amino acid modification at at least one position selected from the group consisting of 231, 232, 235, 239, 268, 295, 298, 326, 330, and 396, according to EU numbering.

[0042] In a further embodiment, the Fc variant with enhanced FcγRIIb binding activity comprises at least two amino acid modifications, including (a) one amino acid modification at position 236, and (b) at least one amino acid modification at at least one position selected from the group consisting of 268, 295, 326, and 330, according to EU numbering.

[0043] In some embodiments, the Fc variants of the present invention with enhanced FcγRIIb binding activity are, according to EU numbering, (a) Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, Tyr at position 231; (b) Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, A sn, Gln, Arg, Ser, Thr, Val, Trp, Tyr; (c) Asp at 233rd place; (d) Trp and Tyr at 234th place; (e) Trp at 235th place; (f) Ala, Asp, Glu, His, Ile, Leu, Met, Asn, Gln, Ser, Thr, Val at 236th place; (g) Asp and Tyr at 237th place; (h) Glu, Ile, Met, Gln, Tyr at 238th place; (i) Ile at 239th place , Leu, Asn, Pro, Val; (j) Ile at 264th; (k) Phe at 266th; (l) Ala, His, Leu at 267th; (m) Asp, Glu at 268th; (n) Asp, Glu, Gly at 271st; (o) Leu at 295th; (p) Leu at 298th; (q) Glu, Phe, Ile, Leu at 325th; (r) Thr at 326th; (s) Ile, Asn at 327th; (t) T at 328th hr; (u) Lys at position 330, Arg; (v) Glu at position 331; (w) Asp at position 332; (x) Asp, Ile, Met, Val, Tyr at position 334; and (y) Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp, Tyr at position 396.

[0044] In a further embodiment, the Fc variant with enhanced FcγRIIb binding activity contains at least one amino acid selected from the group consisting of (a) Gly, Thr at position 231; (b) Asp at position 232; (c) Trp at position 235; (d) Asn, Thr at position 236; (e) Val at position 239; (f) Asp, Glu at position 268; (g) Leu at position 295; (h) Leu at position 298; (i) Thr at position 326; (j) Lys, Arg at position 330; and (k) Lys, Met at position 396, according to EU numbering.

[0045] In another embodiment, the present invention provides a polypeptide comprising an Fc variant with an increased isoelectric point (pI), and a method of using the same. In some embodiments, the polypeptide comprising an Fc variant with an increased pI comprises at least two amino acid modifications in the parent Fc region. In a further embodiment, each of the amino acid modifications increases the isoelectric point (pI) of the Fc variant compared to that of the parent Fc region. In a further embodiment, the amino acids can be exposed on the surface of the modified Fc region. In a further embodiment, the polypeptide comprises an Fc variant and an antigen-binding domain. In a further embodiment, the antigen-binding activity of the antigen-binding domain varies according to ion concentration conditions. In a further embodiment, the pI-enhanced modified Fc region of the present invention comprises at least two amino acid modifications at at least two positions selected from the group consisting of 285, 311, 312, 315, 318, 333, 335, 337, 341, 342, 343, 384, 385, 388, 390, 399, 400, 401, 402, 413, 420, 422, and 431, according to EU numbering. In a further embodiment, the pI-enhanced Fc variant comprises Arg or Lys at each of the selected positions.

[0046] In another embodiment, the present invention provides isolated polypeptides comprising Fc variants with increased isoelectric point (pI). In certain embodiments, the Fc variants described herein comprise at least two amino acid modifications in the parent Fc region. In certain embodiments, each amino acid modification increases the isoelectric point (pI) of the variant Fc region compared to that of the parent Fc region. These are based on the finding that antibodies with increased pI due to modifications of at least two amino acid residues can promote antigen elimination from plasma, for example, when the antibody is administered in vivo.

[0047] In one embodiment, pI may be either a theoretical pI or an experimentally determined pI. The value of pI can be determined, for example, by isoelectric focusing, which is known to those skilled in the art. The theoretical value of pI can be calculated, for example, using gene and amino acid sequence analysis software (such as Genetyx).

[0048] In one embodiment, the pI value can be increased by, for example, at least 0.01, 0.03, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5 or more, at least 0.6, 0.7, 0.8, 0.9 or more, at least 1.0, 1.1, 1.2, 1.3, 1.4, 1.5 or more, or at least 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 3.0 or more compared to before modification.

[0049] In certain embodiments, amino acids for increasing pI can be exposed on the surface of the Fc variant. In this invention, amino acids that can be exposed on the surface generally refer to amino acid residues located on the surface of the polypeptide constituting the Fc variant. Amino acid residues located on the surface of a polypeptide are amino acid residues whose side chains can come into contact with solvent molecules (generally, mostly water molecules). However, the entire side chain does not necessarily need to be in contact with solvent molecules; if even a part of the side chain is in contact with solvent molecules, the amino acid is defined as a "surface-located amino acid residue." Surface-located amino acid residues of a polypeptide also include amino acid residues located near the surface, which may be affected by the charge from other amino acid residues whose side chains are in contact with solvent molecules, even if only partially. Those skilled in the art can prepare homology models of polypeptides using, for example, commercially available software. Alternatively, methods known to those skilled in the art, such as X-ray crystallography, can be used. Amino acid residues that can be exposed on the surface are determined using coordinates from a three-dimensional model, for example, using a computer program such as the Insight II program (Accelrys). The surface-exposureable regions can be determined using algorithms known in the art (e.g., Lee and Richards (J. Mol. Biol. 55:379-400 (1971)); Connolly (J. Appl. Cryst. 16:548-558 (1983))). The surface-exposureable regions can be determined using software suitable for protein modeling and three-dimensional structural information. Software available for such purposes includes, for example, the SYBYL Biopolymer Module software (Tripos Associates). If the algorithm requires a user-input size parameter, the "size" of the probe used in the calculation may be set to a radius of approximately 1.4 angstroms (A) or less.Furthermore, a method for determining the surface-exposureable region using personal computer software has been described by Pacios (Comput. Chem. 18(4):377-386 (1994); J. Mol. Model. 1:46-53 (1995)). Based on the above information, appropriate amino acid residues located on the surface of the polypeptide constituting the variant Fc region can be selected.

[0050] In certain embodiments, the polypeptide comprises both an Fc variant and an antigen-binding domain. In further embodiments, the antigen is a soluble antigen. In one embodiment, the antigen is present in the biological fluids of the subject (e.g., plasma, interstitial fluid, lymph, ascites, and pleural fluid). The antigen may also be a membrane antigen.

[0051] In a further embodiment, the antigen-binding activity of the antigen-binding domain varies according to ion concentration conditions. In one embodiment, ion concentration means, without particular limitation, hydrogen ion concentration (pH) or metal ion concentration. In this specification, metal ions mean ions of Group I elements excluding hydrogen, such as alkali metals and copper group elements; Group II elements, such as alkaline earth metals and zinc group elements; Group III elements excluding boron; Group IV elements excluding carbon and silicon; Group VIII elements, such as iron group elements and platinum group elements; elements belonging to subgroups A of Groups V, VI, and VII; and metallic elements such as antimony, bismuth, and polonium. In the present invention, metal ions include calcium ions, as described, for example, in WO2012 / 073992 and WO2013 / 125667. In one embodiment, “ion concentration conditions” may be conditions that focus on the difference in the biological behavior of the antigen-binding domain at low and high ion concentrations. Furthermore, the statement that "the antigen-binding activity of the antigen-binding domain changes according to ion concentration conditions" means that the antigen-binding activity of the antigen-binding domain changes between low and high ion concentrations (such antigen-binding domains are referred to herein as "ion concentration-dependent antigen-binding domains"). The antigen-binding activity of an antigen-binding domain under high ion concentration conditions may be higher (stronger) or lower (weaker) than that under low ion concentration conditions. In one embodiment, ion concentration-dependent antigen-binding domains (such as pH-dependent antigen-binding domains or calcium ion concentration-dependent antigen-binding domains) can be obtained by known methods, for example, as described in WO2009 / 125825, WO2012 / 073992, and WO2013 / 046722.

[0052] In one embodiment, the antigen-binding activity of the antigen-binding domain under high calcium ion concentration conditions may be higher than under low calcium ion concentration conditions. The high calcium ion concentration is not particularly limited, but may be a selected concentration of 100 μM to 10 mM, 200 μM to 5 mM, 400 μM to 3 mM, 200 μM to 2 mM, 400 μM to 1 mM, or 500 μM to 2.5 mM, and is preferably close to the in vivo plasma (blood) calcium ion concentration. On the other hand, the low calcium ion concentration is not particularly limited, but may be a selected concentration of 0.1 μM to 30 μM, 0.2 μM to 20 μM, 0.5 μM to 10 μM, 1 μM to 5 μM, or 2 μM to 4 μM, and is preferably close to the calcium ion concentration in the initial endosomes in vivo.

[0053] In one embodiment, the ratio of antigen-binding activity under low calcium ion concentration conditions to antigen-binding activity under high calcium ion concentration conditions is not limited, but the ratio of the dissociation constant (KD) under low calcium ion concentration conditions to the KD under high calcium ion concentration conditions, i.e., KD(low calcium ion concentration conditions) / KD(high calcium ion concentration conditions), is 2 or greater, 10 or greater, or 40 or greater. The upper limit of the ratio may be 400, 1000, or 10000, provided that such antigen-binding domains can be manufactured by techniques known to those skilled in the art. Alternatively, for example, the dissociation rate constant (kd) can be used instead of KD. In this case, the ratio of kd under low calcium ion concentration conditions to kd under high calcium ion concentration conditions, i.e., kd(low calcium ion concentration conditions) / kd(high calcium ion concentration conditions), is 2 or greater, 5 or greater, 10 or greater, or 30 or greater. The upper limit of the ratio may be 50, 100, or 200, provided that the antigen-binding domains can be manufactured based on the general technical knowledge of those skilled in the art.

[0054] In one embodiment, the antigen-binding activity of the antigen-binding domain may be higher under low hydrogen ion concentrations (neutral pH) than under high hydrogen ion concentrations (acidic pH). The acidic pH may be, for example, selected from pH 4.0 to pH 6.5, selected from pH 4.5 to pH 6.5, selected from pH 5.0 to pH 6.5, or selected from pH 5.5 to pH 6.5, and is preferably close to the in vivo pH in early endosomes. The acidic pH may also be, for example, pH 5.8 or pH 6.0. In a particular embodiment, the acidic pH is pH 5.8. On the other hand, the neutral pH may be, for example, selected from pH 6.7 to pH 10.0, selected from pH 6.7 to pH 9.5, selected from pH 7.0 to pH 9.0, or selected from pH 7.0 to pH 8.0, and is preferably close to the in vivo pH in plasma (blood). The neutral pH may also be, for example, pH 7.4 or pH 7.0. In a particular embodiment, the neutral pH is pH 7.4.

[0055] In one embodiment, the ratio of antigen-binding activity under acidic pH conditions to antigen-binding activity under neutral pH conditions is not limited, but the ratio of dissociation constant (KD) under acidic pH conditions to KD under neutral pH conditions, i.e., KD(acidic pH conditions) / KD(neutral pH conditions), is 2 or greater, 10 or greater, or 40 or greater. The upper limit of the ratio may be 400, 1000, or 10000, provided that such antigen-binding domains can be manufactured by techniques known to those skilled in the art. Alternatively, for example, the dissociation rate constant (kd) can be used instead of KD. In this case, the ratio of kd under acidic pH conditions to kd under neutral pH conditions, i.e., kd(acidic pH conditions) / kd(neutral pH conditions), is 2 or greater, 5 or greater, 10 or greater, or 30 or greater. The upper limit of the ratio may be 50, 100, or 200, provided that the antigen-binding domains can be manufactured based on the general technical knowledge of those skilled in the art.

[0056] In one embodiment, for example, as described in WO2009 / 125825, at least one amino acid residue is substituted with an amino acid residue having a side chain pKa of 4.0–8.0, and / or at least one amino acid having a side chain pKa of 4.0–8.0 is inserted into the antigen-binding domain. The amino acids may be substituted and / or inserted at any site, as long as the antigen-binding activity of the antigen-binding domain is weaker under acidic pH conditions than under neutral pH conditions compared to before substitution or insertion. If the antigen-binding domain has a variable region or CDR, the site may be within the variable region or CDR. The number of amino acids to be substituted or inserted can be appropriately determined by those skilled in the art, and may be one or more. Amino acids having a side chain pKa of 4.0–8.0 can be used to change the antigen-binding activity of the antigen-binding domain according to hydrogen ion concentration conditions. Such amino acids include, for example, natural amino acids such as His(H) and Glu(E), as well as non-natural amino acids such as histidine analogs (US2009 / 0035836), m-NO2-Tyr (pKa 7.45), 3,5-Br2-Tyr (pKa 7.21), and 3,5-I2-Tyr (pKa 7.38) (Heyl et al., Bioorg. Med. Chem. 11(17):3761-3768 (2003)). Amino acids with side-chain pKas of 6.0-7.0 can also be used, including, for example, His(H).

[0057] In another embodiment, preferred antigen-binding domains for Fc variants with increased pI are described and can be obtained by the methods described in Japanese Patent Applications JP2015-021371 and JP2015-185254.

[0058] In a particular embodiment, the Fc variant with increased pI includes at least two amino acid modifications at at least two positions selected from the group consisting of 285, 311, 312, 315, 318, 333, 335, 337, 341, 342, 343, 384, 385, 388, 390, 399, 400, 401, 402, 413, 420, 422, and 431, according to EU numbering.

[0059] In a further embodiment, the Fc variant with increased pI comprises at least two amino acid modifications at at least two positions selected from the group consisting of 311, 341, 343, 384, 399, 400, 401, 402, and 413, according to EU numbering.

[0060] In another aspect, the present invention provides polypeptides comprising Fc variants with increased pI, comprising any one of the following amino acid modifications: (1) at positions 311 and 341; (2) at positions 311 and 343; (3) at positions 311, 343, and 413; (4) at positions 311, 384, and 413; (5) at positions 311 and 399; (6) at positions 311 and 401; (7) at positions 311 and 413; (8) at positions 400 and 413; (9) at positions 401 and 413; and (10) at positions 402 and 413.

[0061] Methods for increasing the pI of a protein include, for example, reducing the number of amino acids with negatively charged side chains (e.g., aspartic acid and glutamic acid) and / or increasing the number of amino acids with positively charged side chains (e.g., arginine, lysine, and histidine) under neutral pH conditions. Amino acids with negatively charged side chains have a negative charge expressed as -1 under pH conditions well above their side chain pKa, a theory well known to those skilled in the art. For example, the theoretical pKa of the side chain of aspartic acid is 3.9, and the side chain has a negative charge expressed as -1 under neutral pH conditions (e.g., in a solution at pH 7.0). Conversely, amino acids with positively charged side chains have a positive charge expressed as +1 under pH conditions well below their side chain pKa. For example, the theoretical pKa of the side chain of arginine is 12.5, and the side chain has a positive charge represented as +1 under neutral pH conditions (e.g., in a solution at pH 7.0). On the other hand, it is known that 15 natural amino acids, namely alanine, cysteine, phenylalanine, glycine, isoleucine, leucine, methionine, asparagine, proline, glutamine, serine, threonine, valine, tryptophan, and tyrosine, have side chains that have no charge under neutral pH conditions (e.g., in a solution at pH 7.0). Naturally, it is understood that amino acids used to increase pI may also be non-natural amino acids.

[0062] From the above, a method for increasing the pI of a protein under neutral pH conditions (for example, in a pH 7.0 solution) is to impart a +1 charge modification to the target protein by substituting aspartic acid or glutamic acid (whose side chains have a negative charge of -1) with an amino acid having an uncharged side chain in the amino acid sequence of the protein. Furthermore, a +1 charge modification can be imparted to the protein by substituting an amino acid whose side chain has no charge with arginine or lysine (whose side chains have a positive charge of +1). Furthermore, a +2 charge modification can be imparted to the protein at once by substituting aspartic acid or glutamic acid (whose side chains have a negative charge of -1) with arginine or lysine (whose side chains have a positive charge of +1). Alternatively, in order to increase the pI of a protein, amino acids having side chains with no charge and / or preferably positively charged side chains can be added or inserted into the amino acid sequence of the protein, or amino acids having side chains with no charge and / or preferably negatively charged side chains that are present in the amino acid sequence of the protein can be deleted. For example, the N-terminal and C-terminal amino acid residues of a protein have a charge derived from the main chain in addition to the charge derived from their side chains (NH3 of the N-terminal amino group). + and the COO of the carbonyl group at the C terminus - It is understood that the protein has the following properties: Therefore, the pI of a protein can also be increased by adding, deleting, substituting, or inserting several functional groups derived from the main chain.

[0063] Amino acid substitutions to increase pI include, for example, substitutions in the amino acid sequence of the parent Fc region of an amino acid whose side chain has no charge with an amino acid that has a negatively charged side chain, substitutions of an amino acid with a positively charged side chain with an amino acid whose side chain has no charge, and substitutions of an amino acid with a positively charged side chain with an amino acid that has a negatively charged side chain, which may be performed individually or in appropriate combinations.

[0064] Amino acid insertions or additions to increase pI include, for example, the insertion or addition of amino acids whose side chains have no charge, and / or the insertion or addition of amino acids with positively charged side chains, which can be done alone or in appropriate combinations.

[0065] Amino acid deletions to increase pI include, for example, deletions of amino acids in the amino acid sequence of the parent Fc region whose side chains have no charge, and / or deletions of amino acids with negatively charged side chains, which are performed alone or in appropriate combinations.

[0066] In one embodiment, the natural amino acids used to increase pI can be classified as follows: (a) amino acids having a negatively charged side chain may be Glu(E) or Asp(D); (b) amino acids whose side chain has no charge may be Ala(A), Asn(N), Cys(C), Gln(Q), Gly(G), His(H), Ile(I), Leu(L), Met(M), Phe(F), Pro(P), Ser(S), Thr(T), Trp(W), Tyr(Y), or Val(V); and (c) amino acids having a positively charged side chain may be His(H), Lys(K), or Arg(R). In one embodiment, the modified amino acid insertion or substitution is Lys(K) or Arg(R).

[0067] Affinity The term "affinity" refers to the strength of the combined non-covalent interactions between one binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise specified, "binding affinity" as used herein refers to the intrinsic binding affinity that reflects the 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of molecule X to its partner Y can generally be expressed by a dissociation constant (Kd). Affinity can be measured by conventional methods known in the art, including those described herein. Specific and exemplary embodiments for measuring binding affinity are described below.

[0068] In certain embodiments, the target-binding molecules provided herein have a minimum size of ≤1 μM, ≤100 nM, ≤10 nM, ≤1 nM, ≤0.1 nM, ≤0.01 nM, or ≤0.001 nM (for example, 10 -8 M or less, for example, 10 -8 M~10 -13 M, for example 10 -9 M~10 -13 It has a dissociation constant (Kd) of M.

[0069] In one embodiment, Kd is measured by a radiolabeled antigen binding assay (RIA). In one embodiment, the RIA is performed using a Fab version of the target-binding molecule of interest and its target. For example, the solution-bound affinity of Fab to an antigen is measured in the presence of a gradual increase in the concentration of the unlabeled antigen. 125I) Fab is equilibrated with a labeled antigen, and then the bound antigen is captured by a plate coated with anti-Fab antibody. (See, for example, Chen et al., J. Mol. Biol. 293:865-881 (1999)). To construct the assay conditions, a MICROTITER® multiwell plate (Thermo Scientific) is coated overnight with 5 μg / ml of capture anti-Fab antibody (Cappel Labs) in 50 mM sodium carbonate (pH 9.6), and then blocked with 2% (w / v) bovine serum albumin in PBS for 2-5 hours at room temperature (approximately 23°C). In a non-adsorbent plate (Nunc #269620), 100 pM or 26 pM [ 125 Mix the [I]-antigen with serial dilutions of the Fab of interest (e.g., as in the evaluation of anti-VEGF antibody, Fab-12, in Presta et al., Cancer Res. 57:4593-4599 (1997)). Then incubate the Fab of interest overnight, although this incubation may be extended for a longer period (e.g., about 65 hours) to ensure equilibrium is achieved. Subsequently, transfer the mixture to a capture plate for incubation at room temperature (e.g., 1 hour). Then remove the solution and wash the plate eight times with 0.1% polysorbate 20 (TWEEN-20®) in PBS. Once the plate is dry, add 150 μl / well of scintillant (MICROSCINT-20®, Packard) and count the plate for 10 minutes on a TOPCOUNT® gamma counter (Packard). Select concentrations of each Fab that give less than 20% of maximum binding for use in competitive binding assays.

[0070] In another embodiment, Kd is measured using a BIACORE® surface plasmon resonance assay. For example, an assay using BIACORE®-2000 or BIACORE®-3000 (BIAcore, Inc., Piscataway, NJ) is performed at 25°C using a CM5 chip immobilized with approximately 10 response units (RUs) of antigen. In one embodiment, a carboxymethylated dextran biosensor chip (CM5, BIACORE, Inc.) is activated with N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to the supplier's instructions. The antigen is diluted to 5 μg / ml (approximately 0.2 μM) with 10 mM sodium acetate, pH 4.8 before being injected at a flow rate of 5 μl / min to achieve binding of approximately 10 response units (RUs) of protein. After antigen injection, 1M ethanolamine is injected to block unreacted groups. For kinetics measurement, two-fold serial dilutions (0.78nM to 500nM) of Fab in PBS (PBST) containing 0.05% polysorbate 20 (TWEEN-20™) surfactant are injected at 25°C and a flow rate of approximately 25 μl / min. Binding rate (k on ) and dissociation rate (k off ) is calculated by simultaneously fitting the coupling and dissociation sensorgrams using a simple one-to-one Langmuir coupling model (BIACORE® evaluation software version 3.2). The equilibrium dissociation constant (Kd) is given by k off / k on It is calculated as a ratio. See, for example, Chen et al., J. Mol. Biol. 293:865-881 (1999). The on velocity was 10 by the surface plasmon resonance assay described above. 6 M -1 s -1If it exceeds this, the ON rate can be determined by measuring the increase or decrease in fluorescence emission intensity (excitation = 295 nm; emission = 340 nm, bandpass 16 nm) at 25°C in the presence of gradually increasing concentrations of antigen using a spectrometer (e.g., a stop-flow spectrophotometer (Aviv Instruments) or an 8000 series SLM-AMINCO® spectrophotometer (ThermoSpectronic) using a stirred cuvette). and / or In this specification, the term "and / or" is used to indicate each subject or any combination thereof that is listed before or after "and / or". For example, "A, B and / or C" includes not only the subjects "A", "B", and "C", but also the combinations "A and B", "A and C", "B and C", and "A and B and C".

[0071] antibody In this specification, the term “antibody” is used in its broadest sense and encompasses a variety of antibody structures, including monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, as long as they exhibit the desired antigen-binding activity.

[0072] antibody fragment An "antibody fragment" refers to a molecule other than the complete antibody that binds to the antigen to which the complete antibody binds, including a portion of that complete antibody. Examples of antibody fragments, but not limited to these, include Fv, Fab, Fab', Fab'-SH, F(ab')2; diabodies; linear antibodies; single-chain antibody molecules (e.g., scFv); single-domain antibodies or VHH; and multispecific antibodies formed from antibody fragments.

[0073] In certain embodiments, the antibodies provided herein are antibody fragments. Antibody fragments include, but are not limited to, Fab, Fab', Fab'-SH, F(ab')2, Fv, and scFv fragments, as well as other fragments described below. For a review of specific antibody fragments, see Hudson et al. Nat. Med. 9:129-134 (2003). For a review of scFv fragments, see, for example, Pluckthun, in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., (Springer-Verlag, New York), pp.269-315 (1994); in addition, see WO93 / 16185; and U.S. Patents 5,571,894 and 5,587,458. For a discussion on Fab and F(ab')2 fragments containing salvage receptor-binding epitope residues and exhibiting extended in vivo half-lives, see U.S. Patent No. 5,869,046.

[0074] A diabody is an antibody fragment containing two antigen-binding sites, which may be bivalent or bispecific. See, for example, EP404,097; WO1993 / 01161; Hudson et al., Nat. Med. 9:129-134 (2003); Hollinger et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993). Triabodies and tetrabodies are also described in Hudson et al., Nat. Med. 9:129-134 (2003).

[0075] A single-domain antibody is an antibody fragment containing all or part of the heavy chain variable domain or all or part of the light chain variable domain of an antibody. In certain embodiments, a single-domain antibody is a human single-domain antibody (see, for example, Domantis, Inc., Waltham, MA; U.S. Patent No. 6,248,516B1).

[0076] Antibody fragments can be produced by various methods, including, but are not limited to, the proteolytic digestion of complete antibodies and production by recombinant host cells (e.g., Escherichia coli or phages) as described herein.

[0077] The term "variable fragment (Fv)," as used in this application, may refer to the smallest unit of antibody-derived portion that binds to an antigen, consisting of a pair of antibody light chain variable region (VL) and antibody heavy chain variable region (VH). In 1988, Skerra and Pluckthun found that homogeneous and active antibodies could be prepared from the E. coli periplasmic fraction by inserting the antibody gene downstream of a bacterial signal sequence and inducing gene expression in E. coli (Science (1988) 240(4855), 1038-1041). In Fv prepared from the periplasmic fraction, VH associates with VL in a manner that binds to the antigen.

[0078] The terms “scFv,” “monoclonal antibody,” and “sc(Fv)2,” as used in this application, refer to a single polypeptide chain antibody fragment that contains variable regions derived from heavy and light chains but does not contain a constant region. Generally, monoclonal antibodies also contain a polypeptide linker between the VH and VL domains, which enables the formation of a desired structure that is thought to allow antigen binding. Monoclonal antibodies are discussed in detail by Pluckthun in “The Pharmacology of Monoclonal Antibodies, Vol. 113, Rosenburg and Moore, eds., Springer-Verlag, New York, 269-315 (1994).” See also International Patent Publication WO1988 / 001649; U.S. Patents 4,946,778 and 5,260,203. In certain embodiments, monoclonal antibodies may be bispecific and / or humanized.

[0079] When used in this application, the term "scFv" may refer to a single-chain polypeptide in which the VH and VL forming Fv are linked to each other by a peptide linker (Proc. Natl. Acad. Sci. USA (1988) 85(16), 5879-5883). The VH and VL can be held in close proximity by the peptide linker.

[0080] When used in this application, the term "sc(Fv)2" may refer to a single-chain antibody in which four variable regions, two VLs and two VHs, are linked by a linker such as a peptide linker to form a single chain (J Immunol. Methods (1999) 231(1-2), 177-189). The two VHs and two VLs may be derived from different monoclonal antibodies. Such sc(Fv)2 preferably includes a bispecific sc(Fv)2 that recognizes two epitopes present in a single antigen, for example, as disclosed in Journal of Immunology (1994) 152(11), 5368-5374. sc(Fv)2 can be manufactured by methods known to those skilled in the art. For example, sc(Fv)2 can be manufactured by linking scFv with a linker such as a peptide linker.

[0081] In this specification, sc(Fv)2 takes the form in which two VH units and two VL units of the antibody are arranged in the order VH, VL, VH, and VL ([VH]-linker-[VL]-linker-[VH]-linker-[VL]) starting from the N-terminus of the single-chain polypeptide. The order of the two VH units and two VL units is not limited to the above form and can be arranged in any order. Exemplary configurations are listed below. [VL]-Linker-[VH]-Linker-[VH]-Linker-[VL] [VH]-Linker-[VL]-Linker-[VL]-Linker-[VH] [VH]-Linker-[VH]-Linker-[VL]-Linker-[VL] [VL]-Linker-[VL]-Linker-[VH]-Linker-[VH] [VL]-Linker-[VH]-Linker-[VL]-Linker-[VH]

[0082] The terms "Fab," "F(ab')2," and "Fab'" may have the following meanings when used in this application.

[0083] "Fab" consists of a single light chain, as well as a CH1 region and a variable region derived from a single heavy chain. The heavy chain of the wild-type Fab molecule cannot form disulfide bonds with other heavy-chain molecules. Fab variants are also included, in which amino acid residues in the wild-type Fab molecule may be modified by substitution, addition, or deletion, depending on the purpose. In specific embodiments, the mutated amino acid residues in the Fab variant (e.g., cysteine ​​or lysine residues after substitution, addition, or insertion) can form disulfide bonds with other heavy-chain molecules or parts thereof (e.g., the Fab molecule).

[0084] scFab is an antigen-binding domain in which a single light chain forming the Fab, as well as CH1 regions and variable regions derived from a single heavy chain, are linked to each other by a peptide linker. The light chain, as well as the CH1 regions and variable regions derived from the heavy chain, can be kept in close proximity by the peptide linker.

[0085] "F(ab')2" or "Fab" refers to an antibody fragment produced by treating immunoglobulin (monoclonal antibody) with proteases such as pepsin and papain, by digesting the immunoglobulin (monoclonal antibody) near the disulfide bond located between the hinge regions in each of the two heavy chains. For example, papain cleaves IgG upstream of the disulfide bond located between the hinge regions in each of the two heavy chains, producing two homologous antibody fragments in which the light chain, containing VL (variable light chain region) and CL (constant light chain region), is linked via a disulfide bond at its C-terminus to a heavy chain fragment containing VH (variable heavy chain region) and CHγ1 (γ1 region in the constant heavy chain region). Each of these two homologous antibody fragments is called Fab'.

[0086] "F(ab')2" consists of two light chains and two heavy chains containing a constant region comprising a CH1 domain and a portion of the CH2 domain, thereby forming a disulfide bond between the two heavy chains. For example, the F(ab')2 disclosed herein can be prepared as follows: A whole monoclonal antibody or such containing the desired antigen-binding domain is partially digested with a protease such as pepsin; and the Fc fragment is removed by adsorption onto a protein A column. The protease is not particularly limited as long as it can selectively cleave the whole antibody to produce F(ab')2 under appropriate setting enzymatic reaction conditions such as pH. Such proteases include, for example, pepsin and ficin.

[0087] When used in this application, the term "single-domain antibody" (sdAb) does not particularly limit the structure of the domain, as long as the domain itself can exert antigen-binding activity. Conventional antibodies, exemplified by IgG antibodies, exert antigen-binding activity when a variable region is formed by the pairing of VH and VL domains. In contrast, single-domain antibodies are known to exert antigen-binding activity through their own domain structure alone, without pairing with another domain. Single-domain antibodies typically have a relatively low molecular weight and exist in monomeric form.

[0088] Examples of single-domain antibodies include, but are not limited to, naturally occurring light-chain-less antigen-binding molecules such as camelid VHH and shark VNAR, as well as antibody fragments containing all or part of an antibody VH domain or all or part of an antibody VL domain. Examples of single-domain antibodies that are antibody fragments containing all or part of an antibody VH / VL domain include, but are not limited to, artificially prepared single-domain antibodies derived from human antibody VH or human antibody VL, such as those described in U.S. Patent No. 6,248,516B1. In some aspects of the present invention, a single-domain antibody has three CDRs (CDR1, CDR2, and CDR3).

[0089] Single-domain antibodies can be obtained from animals capable of producing single-domain antibodies, or by immunizing animals capable of producing single-domain antibodies. Examples of animals capable of producing single-domain antibodies include, but are not limited to, camelids and transgenic animals into which a gene capable of producing single-domain antibodies has been introduced. Camelids include camels, llamas, alpacas, dromedaries, and guanacos. Examples of transgenic animals into which a gene capable of producing single-domain antibodies has been introduced include, but are not limited to, the transgenic animals described in International Publication No. WO2015 / 143414 or U.S. Patent Application Publication No. 2011 / 0123527A1. Humanized single-chain antibodies can also be obtained by replacing the framework sequence of a single-domain antibody obtained from an animal with a human germline sequence or a similar sequence. A humanized single-domain antibody (e.g., humanized VHH) is one embodiment of the single-domain antibody of the present invention.

[0090] Alternatively, single-domain antibodies can be obtained from polypeptide libraries containing single-domain antibodies by methods such as ELISA or panning. Examples of polypeptide libraries containing single-domain antibodies include naive antibody libraries obtained from various animals or humans (e.g., Methods in Molecular Biology 2012 911 (65-78) and Biochimica et Biophysica Acta - Proteins and Proteomics 2006 1764:8 (1307-1319)), antibody libraries obtained by immunizing various animals (e.g., Journal of Applied Microbiology 2014 117:2 (528-536)), and synthetic antibody libraries prepared from antibody genes of various animals or humans (e.g., Journal of Biomolecular Screening 2016 21:1 (35-43), Journal of Biological Chemistry 2016 291:24 (12641-12657), and AIDS 2016 30:11) This includes, but is not limited to, the period (1691-1701).

[0091] The same epitope-binding antibody An antibody that "binds to the same epitope as the reference antibody" is defined as an antibody that blocks the binding of the reference antibody to its antigen by 50% or more in a competitive assay; conversely, the reference antibody blocks the binding of other antibodies to its antigen by 50% or more in a competitive assay. An exemplary competitive assay is provided herein.

[0092] Chimera The term "chimeric" refers to an antibody in which a portion of the heavy chain and / or light chain originates from a specific source or species, while the remaining heavy chain and / or light chain originates from a different source or species.

[0093] class The "class" of an antibody refers to the type of constant domain or constant region present in the antibody's heavy chain. There are five main classes of antibodies: IgA, IgD, IgE, IgG, and IgM. Some of these may be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains corresponding to different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively.

[0094] Combination with existing technologies The combination of target-binding molecules or protein complexes of the present invention can be combined with various existing technologies. A non-limiting aspect of such combinations is illustrated by the generation of cells expressing chimeric antigen receptors (CARs) using the combination of target-binding molecules or protein complexes of the present invention. Cells as used herein include, for example, T cells, γδT cells, NK cells, NKT cells, cytokine-induced killer (CIK) cells, and macrophages (Int J Mol Sci. (2019) 20(11), 2839, Nat Rev Drug Discov. (2020) 19(5), 308). One non-limiting method for generating CAR-expressing T cells (CAR-T) includes, for example, introducing a CAR containing the combination of target-binding molecules or protein complexes of the present invention, which specifically binds to the intracellular signaling domain of a costimulatory molecule such as a scaffold protein, tumor-associated antigen, the transmembrane domain of the TCR, and CD28, into effector cells such as T cells by genetic modification technology to enhance T cell activation. Another non-limiting method for combining with CAR-T cell technology includes, for example, expressing scaffold proteins on CAR-T cells and using the combination of target-binding molecules or protein complexes of the present invention to deliver cytokines or costimulatory signals to CAR-T cells in a specific manner.

[0095] Effector function "Effector function" refers to the biological activity that varies depending on the antibody isotype, stemming from the Fc region of the antibody. Examples of antibody effector functions include: C1q binding and complement-dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g., B cell receptors); and B cell activation.

[0096] Antibody-dependent cell-mediated cytotoxicity Antibody-dependent cell-mediated cytotoxicity (ADCC) is a form of cytotoxicity in which secreted IgG binds to Fc receptors (FcRs) present on specific cytotoxic cells (e.g., NK cells, neutrophils, and macrophages), thereby enabling these cytotoxic effector cells to specifically bind to target cells containing antigens and subsequently kill those target cells cytotoxicly. NK cells, the primary cells mediating ADCC, express only FcγRIII, while monocytes express FcγRI, FcγRII, and FcγRIII. FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991). To evaluate the ADCC activity of the molecule of interest, in vitro ADCC assays, such as those described in U.S. Patent No. 5,500,362, No. 5,821,337, or U.S. Patent No. 6,737,056 (Presta), may be performed. Effector cells useful for such assays include PBMCs and NK cells. Alternatively, the ADCC activity of the molecule of interest may be evaluated in vivo in animal models, such as the animal models disclosed in Clynes et al. PNAS (USA) 95:652-656 (1998).

[0097] Complement-dependent cell injury Complement-dependent cell injury, or CDC, refers to the lysis of target cells in the presence of complement. Activation of the classical complement pathway is initiated by the binding of the first element of the complement system (C1q) to an antibody (bound to the corresponding antigen) (of the appropriate subclass). To assess complement activation, a CDC assay can be performed, for example, as described in Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996). Polypeptide variants with modified Fc region amino acid sequences (polypeptides with Fc variants) and increased or decreased C1q binding ability are described, for example, in U.S. Patent No. 6,194,551B1 and WO1999 / 51642. See also, for example, Idusogie et al. J. Immunol. 164: 4178-4184 (2000).

[0098] Effective amount The “effective dose” of a drug (for example, a pharmaceutical formulation) refers to the amount in the required dosage and over the required period of time that is effective in achieving the desired therapeutic or prophylactic outcome.

[0099] Fc receptor An "Fc receptor" or "FcR" refers to a receptor that binds to the Fc region of an antibody. In some embodiments, the FcR is a native human FcR. In some embodiments, the FcR is one that binds to an IgG antibody (gamma receptor) and includes the FcγRI, FcγRII, and FcγRIII subclass receptors, including allelic variants and alternative splicing forms of these receptors. The FcγRII receptor includes FcγRIIA ("activating receptor") and FcγRIIB ("inhibiting receptor"), which have similar amino acid sequences that differ primarily in their cytoplasmic domains. The activating receptor FcγRIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain. The inhibitory receptor FcγRIIB contains an immunoreceptor tyrosine-based inhibition motif (ITIM) in its cytoplasmic domain. (See, for example, Daeron, Annu. Rev. Immunol. 15:203-234 (1997).) FcRs have been reviewed, for example, in Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991); Capel et al., Immunomethods 4:25-34 (1994); and de Haas et al., J. Lab. Clin. Med 126:330-41 (1995). Other FcRs, including those to be identified in the future, are also included in the term “FcR” as used herein.

[0100] The term “Fc receptor” or “FcR” also includes the neonatal receptor FcRn, which is responsible for the transfer of maternal IgG to the fetus (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)) and the regulation of immunoglobulin homeostasis. Methods for measuring binding to FcRn are publicly known (see, e.g., Ghetie and Ward., Immunol. Today 18(12):592-598 (1997); Ghetie et al., Nature Biotechnology, 15(7):637-640 (1997); Hinton et al., J. Biol. Chem. 279(8):6213-6216 (2004); WO2004 / 92219 (Hinton et al.)).

[0101] In vivo binding to human FcRn and the plasma half-life of human FcRn high-affinity binding polypeptides can be assayed, for example, in transgenic mice expressing human FcRn or transfected human cell lines, or in primates administered with polypeptides containing Fc variants. WO2000 / 42072 (Presta) describes antibody variants with increased or decreased binding to FcR. See also, for example, Shields et al. J. Biol. Chem. 9(2):6591-6604 (2001).

[0102] Fc area In this specification, the term “Fc region” is used to define the C-terminal region of an immunoglobulin heavy chain, including at least a portion of the constant region. This term includes the Fc region of the native sequence and variant Fc regions. In one embodiment, the human IgG heavy chain Fc region extends from Cys226 or Pro230 to the carboxyl terminus of the heavy chain, provided that the lysine (Lys447) or glycine-lysine (Gly446-Lys447) at the C-terminus of the Fc region is present or absent. Unless otherwise specified herein, the numbering of amino acid residues in the Fc region or constant region follows the EU numbering system (also known as the EU index) described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD 1991.

[0103] Variant Fc region A "variant Fc region" includes an amino acid sequence that differs from that of the native sequence Fc region by at least one amino acid modification, preferably one or more amino acid substitutions. Preferably, the variant Fc region has at least one amino acid substitution, e.g., about 1 to about 10 amino acid substitutions, preferably about 1 to about 5 amino acid substitutions, compared to the native sequence Fc region or the parent polypeptide Fc region. The variant Fc regions described herein preferably have at least about 80% homology with the native sequence Fc region and / or the parent polypeptide Fc region, most preferably at least about 90% homology, and more preferably at least about 95% homology.

[0104] Fc region containing antibody The term "Fc region-containing antibody" refers to an antibody that contains an Fc region. The C-terminal lysine of the Fc region (residue 447 according to the EU numbering system) or the C-terminal glycine-lysine of the Fc region (residues 446-447) can be removed, for example, during antibody purification or by recombination of the nucleic acid encoding the antibody. Therefore, a composition containing an antibody having an Fc region according to the present invention may include an antibody with G446-K447, an antibody with G446 but without K447, an antibody from which G446-K447 has been completely removed, or a mixture of the above three types of antibodies.

[0105] Functional Fc region A "functional Fc region" possesses "effector functions" of a native sequence Fc region. Exemplary "effector functions" include C1q binding; CDC; Fc receptor binding; ADCC; phagocytosis; and downregulation of cell surface receptors (e.g., B cell receptors: BCRs). Such effector functions generally require the Fc region to be combined with a binding domain (e.g., an antibody-variable domain) and can be evaluated using various assays disclosed, for example, in the definitions herein.

[0106] Natural-type sequence Fc region The "natural-type sequence Fc region" contains amino acid sequences identical to those of Fc regions found in nature. The natural-type sequence human Fc region includes the natural-type sequence human IgG1 Fc region (non-A and A allotypes); the natural-type sequence human IgG2 Fc region; the natural-type sequence human IgG3 Fc region; and the natural-type sequence human IgG4 Fc region, as well as naturally occurring variants thereof.

[0107] Full-length antibodies, complete antibodies, and whole antibodies The terms "full-length antibody," "complete antibody," and "whole antibody" are used interchangeably herein and refer to antibodies having a structure substantially similar to that of a native antibody, or having a heavy chain containing an Fc region as defined herein.

[0108] Host cells, host cell lines, and host cell cultures The terms “host cell,” “host cell line,” and “host cell culture” refer to cells (including their offspring) that are interchangeably used and into which foreign nucleic acids have been introduced. Host cells include “transformed organisms” and “transformed cells,” which include primary transformed cells and their offspring, regardless of passage number. Offspring do not have to be completely identical to the parent cells in terms of nucleic acid content and may contain mutations. Mutant offspring that have the same function or biological activity as those used when the original transformed cells were screened or selected are also included herein.

[0109] Human antibodies A "human antibody" is an antibody that possesses an amino acid sequence corresponding to the amino acid sequence of an antibody produced by a human or human cell, or an antibody derived from a non-human source that uses the human antibody repertoire or other human antibody coding sequences. This definition of a human antibody explicitly excludes humanized antibodies that contain non-human antigen-binding residues.

[0110] Humanized antibodies A “humanized” antibody is a chimeric antibody that contains amino acid residues from a non-human HVR and amino acid residues from a human FR. In some embodiments, a humanized antibody contains substantially all of at least one, typically two, variable domains, in which all or substantially all HVRs (e.g., CDRs) correspond to those of a non-human antibody, and all or substantially all FRs correspond to those of a human antibody. A humanized antibody may optionally contain at least a portion of the antibody constant region derived from a human antibody. The “humanized form” of an antibody (e.g., a non-human antibody) refers to an antibody that has undergone humanization.

[0111] Individual or subject The “individual” or “subject” is a mammal. Mammals include, but are not limited to, domesticated animals (e.g., cattle, sheep, cats, dogs, horses), primates (e.g., humans, and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In certain embodiments, the individual or subject is a human.

[0112] Isolated nucleic acids "Isolated" nucleic acids are nucleic acid molecules that have been separated from the components of their original environment. Isolated nucleic acids include nucleic acid molecules that would normally be found in the cell containing them, but these nucleic acid molecules are located outside the chromosome or in a chromosomal location different from their original chromosomal location.

[0113] Isolated nucleic acids encoding target-binding molecules or protein complexes or combinations. "Isolated nucleic acids encoding a target-binding molecule or protein complex or combination" means one or more nucleic acid molecules encoding a binding domain, such as the heavy and light chains (or fragments thereof) of an antibody, and includes nucleic acid molecules mounted on one or more vectors, and nucleic acid molecules present at one or more locations in a host cell.

[0114] Percentage (%) Amino acid sequence identity "Percent (%) amino acid sequence identity" with respect to a reference polypeptide sequence is defined as the percentage ratio of amino acid residues in a candidate sequence that are identical to amino acid residues in the reference polypeptide sequence, after the sequences have been aligned to obtain the greatest possible percentage sequence identity and gaps have been introduced where necessary, and no conservative substitutions are considered part of the sequence identity. Alignment for the purpose of determining percentage amino acid sequence identity can be achieved by using various methods within the scope of the art, such as publicly available computer software, including BLAST, BLAST-2, ALIGN, Megalign (DNASTAR) software, or GENETYX® (Genetics Co., Ltd.). A person skilled in the art can determine appropriate parameters for sequence alignment, including any algorithm necessary to achieve the greatest possible alignment over the entire length of the sequences being compared.

[0115] The ALIGN-2 sequence comparison computer program is copyrighted by Genentech, Inc., and its source code, along with user documentation, is filed with the U.S. Copyright Office (Washington DC, 20559) and registered under U.S. Copyright Registration Number TXU510087. The ALIGN-2 program is publicly available from Genentech, Inc., South San Francisco, California, and may also be compiled from the source code. The ALIGN-2 program is compiled for use on UNIX operating systems, including Digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not change. In situations where ALIGN-2 is used for amino acid sequence comparison, the % amino acid sequence identity of a given amino acid sequence A to, or with, or relative to, a given amino acid sequence B (or, a given amino acid sequence A having or containing a certain % amino acid sequence identity to, or with, or relative to, a given amino acid sequence B) is calculated as follows: 100 times the fraction X / Y, where X is the number of amino acid residues scored as identical in the alignment of A and B by the sequence alignment program ALIGN-2, and Y is the total number of amino acid residues in B. It will be understood that if the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % amino acid sequence identity of A to B is not equal to the % amino acid sequence identity of B to A. Unless otherwise specified, all % amino acid sequence identity values ​​used herein are obtained using the ALIGN-2 computer program as described in the preceding paragraph.

[0116] Pharmaceutical preparations / pharmaceutical compositions The term "pharmaceutical preparation" or "pharmaceutical composition" refers to a preparation in which the biological activity of the active ingredient contained herein can exert its effect, and which does not contain additional elements that are toxic to an extent unacceptable to the subject to whom the preparation is administered.

[0117] Pharmacologically acceptable carriers "Pharmaceutically acceptable carrier" refers to components other than the active ingredient in a pharmaceutical preparation that are non-toxic to the subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

[0118] treatment As used herein, "treatment" (and its grammatical derivatives, such as "treat," "treating," etc.) means a clinical intervention intended to modify the natural course of the individual being treated and can be implemented for prevention or during the course of a clinical condition. Desirable effects of treatment include, but are not limited to, preventing the onset or recurrence of a disease, alleviating symptoms, attenuating any direct or indirect pathological effects of the disease, preventing metastasis, reducing the rate of disease progression, restoring or alleviating the disease state, and achieving remission or improved prognosis. In some embodiments, the antibodies of the invention are used to delay the onset of a disease or slow its progression.

[0119] vector As used herein, the term "vector" refers to a nucleic acid molecule that can amplify another nucleic acid to which it is ligated. This term includes vectors as self-replicating nucleic acid structures and vectors that are incorporated into the genome of the host cell into which they are introduced. A vector can effect the expression of a nucleic acid operably linked thereto. Such a vector is also referred to herein as an "expression vector." Vectors can be introduced into host cells by methods such as using viruses or electroporation, but the introduction of vectors is not limited to in vitro introduction; it is also possible to directly introduce vectors into a living body.

[0120] cancer The combination of target-binding molecules or protein complexes of the present invention can be used for treating or preventing cancer. As used herein, the terms "cancer" and "cancerous" refer to or describe a physiological state in a mammal typically characterized by unregulated cell growth / proliferation. Examples of cancer include, but are not limited to, carcinomas, lymphomas (e.g., Hodgkin and non-Hodgkin lymphomas), blastomas, sarcomas, and leukemias. More detailed examples of such cancers include squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, squamous carcinoma of the lung, peritoneal cancer, hepatocellular carcinoma, gastrointestinal cancer, pancreatic cancer, glioma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, colorectal cancer, endometrial or uterine cancer, salivary gland cancer, kidney cancer, liver cancer, prostate cancer, vulvar cancer, thyroid cancer, hepatocellular carcinoma, leukemia and other lymphoproliferative disorders, and various types of head and neck cancer.

[0121] Cell proliferation disorder As used herein, the terms "cell proliferative disorder" and "proliferative disorder" refer to a disorder associated with a degree of abnormal cell proliferation. In one aspect, the cell proliferative disorder is cancer.

[0122] In vitro and in vivo assays for inhibition of cell proliferation and tumor growth In certain embodiments, a combination of the target-binding molecules of the present invention or a protein complex of the present invention, or a target-binding molecule of the present invention as defined in [A-1], combined with other target-binding molecules as defined in [A-1] of the present invention, is tested for its ability to inhibit cell growth or proliferation in vitro. Assays for inhibiting cell growth or proliferation are well known in the art. Certain cell proliferation assays, exemplified by the “cell-killing” assays described herein, measure cell viability. One such assay is the CellTiter-Glo® Luminescent Cell Viability Assay, commercially available from Promega (Madison, WI). This assay determines the number of viable cells in a culture based on the abundance of ATP, an indicator of metabolically active cells. See Crouch et al (1993) J. Immunol. Meth. 160:81-88, U.S. Patent No. 6,602,677. The assay may be performed in a 96 or 384-well format to accommodate automated high-throughput screening (HTS). See Cree et al (1995) AntiCancer Drugs 6:398-404. The assay procedure involves directly adding a single reagent (CellTiter-Glo® reagent) to cultured cells. This lyses the cells, and a luciferase reaction generates a luminescence signal. The luminescence signal is proportional to the amount of ATP present, which is directly proportional to the number of viable cells in the culture. Data can be recorded using a luminometer or CCD camera imaging device. The luminescence value is expressed in relative light units (RLU).

[0123] Another cell proliferation assay is the "MTT" assay, a colorimetric assay that measures the oxidation of 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide to formazan by mitochondrial reductase. Similar to the CellTiter-Glo® assay, this assay indicates the number of metabolically active cells present in the cell culture. See, for example, Mosmann (1983) J. Immunol. Meth. 65:55-63 and Zhang et al. (2005) Cancer Res. 65:3877-3882.

[0124] In one aspect, a combination of the target-binding molecules of the present invention or the protein complex of the present invention, or the target-binding molecules of the present invention as defined in [A-1], combined with other target-binding molecules as defined in [A-1] of the present invention, is tested for its ability to induce cell death in vitro. Assays for the induction of cell death are well known in the art. In some embodiments, such assays measure loss of membrane integrity, indicated by, for example, the uptake of propidium iodide (PI), trypan blue (see Moore et al. (1995) Cytotechnology, 17:1-11), or 7AAD. In an exemplary PI uptake assay, cells are cultured in Dulbecco's modified Eagle medium (D-MEM): Ham's F-12 (50:50) supplemented with 10% thermally inactivated FBS (Hyclone) and 2 mM L-glutamine. Thus, the assay is performed in the absence of complement and immunoeffector cells. The cells are placed in a 100x20mm dish, with 3x10 cells per dish. 6 Seeds are seeded at a density of 1,000 cells and allowed to adhere overnight. The medium is removed and replaced with fresh medium alone or with medium containing varying concentrations of antibodies or immunoconjugates. Cells are incubated for 3 days. After treatment, the monolayer is washed with PBS and detached by trypsin treatment. Then, to remove cell aggregates, the cells are centrifuged at 4°C and 1200 rpm for 5 minutes, and the pellet is treated with 3 ml of cold Ca25. 2+The molecules are resuspended in binding buffer (10 mM Hepes, pH 7.4, 140 mM NaCl, 2.5 mM CaCl2) and divided into 12x75 mm test tubes with 35 mm strainer caps (1 ml per test tube, 3 test tubes per treatment group). PI (10 μg / ml) is then added to the test tubes. The samples are analyzed using a FACSCAN® flow cytometer and FACSCONVERT® CellQuest software (Becton Dickinson). In this manner, a combination of the target binding molecules of the present invention or the protein complex of the present invention, or the target binding molecules of the present invention as defined in [A-1], in combination with other target binding molecules as defined in [A-1] of the present invention, is identified that induce a statistically significant level of cell death determined by PI uptake.

[0125] In one aspect, a combination of the target-binding molecules of the present invention or a protein complex of the present invention, or a target-binding molecule of the present invention as defined in [A-1] combined with other target-binding molecules as defined in [A-1] of the present invention, is tested for its ability to induce apoptosis (programmed cell death) in vitro. An exemplary assay for a combination of the target-binding molecules of the present invention or a protein complex of the present invention, or a target-binding molecule of the present invention as defined in [A-1] combined with other target-binding molecules as defined in [A-1] of the present invention, or an immunoconjugate that induces apoptosis, is the annexin binding assay. In the exemplary annexin binding assay, cells are cultured and seeded in a dish as discussed in the previous paragraph. The medium is removed and replaced with fresh medium alone or medium containing 0.001–10 μg / ml of antibody or immunoconjugate. After a 3-day incubation period, the monolayer is washed with PBS and detached by trypsin treatment. The cells are then centrifuged as discussed in the previous paragraph, resuspended in Ca2+ binding buffer, and divided into test tubes. Labeled annexin (e.g., annexin V-FITC) (1 μg / ml) is then added to the test tubes. The samples are analyzed using a FACSCAN® flow cytometer and FACSCONVERT® CellQuest software (Becton Dickinson). In this way, antibodies that induce statistically significant levels of annexin binding compared to the control are identified. Another exemplary assay for an antibody or immunoconjugate that induces apoptosis is a histone DNA ELISA colorimetric assay that detects intranucleosomal degradation of genomic DNA. Such assays can be performed, for example, using the Cell Death Detection ELISA Kit (Roche, Palo Alto, CA).

[0126] Cells for use in any of the in vitro assays described above include cells or cell lines that naturally express the scaffold protein or receptor protein as defined in [A-1], or cells that have been engineered to express the scaffold protein or to contain all components of the receptor complex, including the receptor protein, as defined in [A-1]. Such cells include tumor cells that overexpress the scaffold protein compared to normal cells of the same tissue origin. Such cells also include cell lines that express the scaffold protein (including tumor cell lines), and cell lines that do not normally express the scaffold protein but have been transfected with nucleic acids that encode the scaffold protein.

[0127] In one aspect, the ability to inhibit cell growth or proliferation in vivo is tested for a combination of the target-binding molecules of the present invention, or a protein complex of the present invention, or a target-binding molecule of the present invention as defined in [A-1], in combination with other target-binding molecules as defined in [A-1] of the present invention. In a particular embodiment, the ability to inhibit tumor growth in vivo is tested for a combination of the target-binding molecules of the present invention, or a protein complex of the present invention, or a target-binding molecule of the present invention as defined in [A-1], in combination with other target-binding molecules as defined in [A-1] of the present invention. In vivo model systems such as syngeneic tumor cell transplantation models can be used for such tests. In an exemplary syngeneic tumor cell transplantation model, mouse tumor cells are introduced into a suitable strain of mouse, e.g., C57BL / 6 mice. Additionally, if the target-binding molecules do not exhibit any cross-reactivity with the animals or cell lines used, it is a possible option to introduce human gene expression by genetic engineering of scaffold proteins or receptor proteins. A combination of the target-binding molecules of the present invention or a protein complex of the present invention, or a target-binding molecule of the present invention as defined in [A-1], combined with other target-binding molecules as defined in [A-1] of the present invention, is administered to an animal. The ability of the combination of the target-binding molecules of the present invention or a protein complex of the present invention, or a target-binding molecule of the present invention as defined in [A-1], combined with other target-binding molecules as defined in [A-1] of the present invention, to inhibit or reduce tumor growth is measured. In certain embodiments, mouse tumor cells are mouse tumor-derived cells such as MC38, CT26, A20, B16-F10, or Pan02. In certain embodiments, mouse tumor cells are introduced into a preferred strain of mouse by subcutaneous injection or by transplantation into a preferred site such as a mammary gland fat pad.

[0128] B-cell neoplasm / Hodgkin's disease "B-cell neoplasms" include Hodgkin's disease, including lymphocyte-dominant Hodgkin's disease (LPHD); non-Hodgkin's lymphoma (NHL); follicular center cell (FCC) lymphoma; acute lymphocytic leukemia (ALL); chronic lymphocytic leukemia (CLL); and hairy cell leukemia. Non-Hodgkin lymphomas include low-grade / follicular non-Hodgkin lymphoma (NHL), small lymphocytic (SL) NHL, intermediate-grade / follicular NHL, intermediate-grade diffuse NHL, high-grade immunoblastic NHL, high-grade lymphoblastic NHL, high-grade small non-cleavage cell NHL, giant lesion NHL, plasmacytoid lymphocytic lymphoma, mantle cell lymphoma, AIDS-associated lymphoma, and Valdenström macroglobulinemia. Treatment of recurrence of these cancers is also considered. LPHD is a type of Hodgkin's disease that tends to recur frequently even with radiation or chemotherapy. CLL is one of the four major types of leukemia. CLL, a cancer of mature B cells called lymphocytes, develops from the progressive accumulation of cells in the blood, bone marrow, and lymphoid tissues. Slow-growing, incurable lymphoma is a disease in which the average patient survives for 6 to 10 years after periods of multiple remissions and relapses.

[0129] Breast tumor The term “breast tumor” or “breast cancer” means any tumor or cancer of the breast, including, for example, adenocarcinomas such as invasive or non-invasive ductal carcinoma, invasive or non-invasive lobular carcinoma, medullary carcinoma, colloidal carcinoma, and papillary carcinoma; as well as less common forms such as phyllodes cyssarcoma, sarcoma, squamous cell carcinoma, and carcinosarcoma.

[0130] Colon tumor The term "colon tumor" or "colon cancer" refers to any tumor or cancer of the colon (the large intestine from the cecum to the rectum).

[0131] Colorectal tumors The term “colorectal tumor” or “colorectal cancer” refers to any tumor or cancer of the large intestine, including the colon (from the cecum to the rectum) and the rectum, including, for example, adenocarcinoma, and less common forms such as lymphoma and squamous cell carcinoma.

[0132] Non-Hodgkin lymphoma As used herein, the term “non-Hodgkin lymphoma” or “NHL” (non-Hodgkin’s lymphoma) refers to lymphatic cancers other than Hodgkin lymphoma. Hodgkin lymphoma can generally be distinguished from non-Hodgkin lymphoma by the presence of Reed-Sternberg cells in Hodgkin lymphoma and the absence of these cells in non-Hodgkin lymphoma. Examples of non-Hodgkin lymphomas as used herein include any that would be identified as such by a person skilled in the art (e.g., an oncologist or pathologist) according to classification schemes known in the art, such as the Revised European-American Lymphoma (REAL) scheme described in Colour Atlas of Clinical Haematology, Third Edition; A. Victor Hoffbrand and John E. Pettit (eds.) (Harcourt Publishers Limited 2000) (see especially Fig. 11.57, 11.58 and / or 11.59).More specific examples, though not limited to these, include: relapsed or refractory NHL, newly diagnosed low-grade NHL, stage III / IV NHL, chemotherapy-resistant NHL, progenitor B-lymphoblastic leukemia and / or lymphoma, small lymphocytic lymphoma, B-cell chronic lymphocytic leukemia and / or pre-lymphoblastic leukemia and / or small lymphocytic lymphoma, B-cell pre-lymphoblastic lymphoma, immunocytoma and / or lymphoplasmacytic lymphoma, marginal zone B-cell lymphoma, splenic marginal zone lymphoma, extranodal marginal zone / MALT lymphoma, nodal marginal zone Marginal zone lymphoma, hairy cell leukemia, plasmacytoma and / or plasmacytic myeloma, low-grade / follicular lymphoma, intermediate-grade / follicular NHL, mantle cell lymphoma, follicular central lymphoma (follicular), intermediate-grade diffuse NHL, diffuse large B-cell lymphoma, aggressive NHL (including aggressive primary NHL and aggressive relapsed NHL), relapsed or autologous stem cell transplantation NHL for which autologous cell transplantation is ineffective, primary mediastinal large B-cell lymphoma, primary exudative lymphoma, high-grade immunoblastic NHL, high-grade lymphoblastic NHL, high-grade small uncleaved cell NHL, giant lesion NHL, Burkitt lymphoma, progenitor (peripheral) T-cell lymphoblastic leukemia and / or lymphoma, adult T-cell lymphoma and / or leukemia, T-cell chronic lymphocytic leukemia and / or pre- Lymphocytic leukemia, large granular lymphocytic leukemia, mycosis fungoides and / or Sézary syndrome, extranodal natural killer / T-cell (nasal type) lymphoma, enteropathy-type T-cell lymphoma, hepatosplenic T-cell lymphoma, subcutaneous panniculitis-like T-cell lymphoma, cutaneous lymphoma, anaplastic large cell lymphoma, vascular central lymphoma, enteric T-cell lymphoma, peripheral T-cell (unspecified type) lymphoma, and angioimmunoblastic T-cell lymphoma.

[0133] Ovarian cancer "Ovarian cancer" refers to a heterogeneous group of malignant tumors derived from the ovary. Approximately 90% of malignant ovarian tumors are of epithelial origin; the remainder are germ cell and stromal tumors. Epithelial ovarian tumors are classified into the following histological subtypes: serous adenocarcinoma (accounting for approximately 50% of epithelial ovarian tumors); endometrioid adenocarcinoma (approximately 20%); mucinous adenocarcinoma (approximately 10%); clear cell carcinoma (approximately 5-10%); Brenner (transitional cell) tumors (less common). The prognosis of ovarian cancer, the sixth most common cancer in women, is generally poor, with a 5-year survival rate ranging from 5 to 30%. For a review of ovarian cancer, see Fox et al. (2002) "Pathology of epithelial ovarian cancer," in Ovarian Cancer ch. 9 (Jacobs et al., eds., Oxford University Press, New York); Morin et al. (2001) "Ovarian Cancer," in Encyclopaedic Reference of Cancer, pp.654-656 (Schwab, ed., Springer-Verlag, New York). Methods for diagnosing or treating any of the above epithelial ovarian tumor subtypes, particularly the serous adenocarcinoma subtype, are within the contemplation of the present invention.

[0134] recurrence "Recurrence" refers to the return of a patient's disease to its previous disease state, particularly the recurrence of symptoms after apparent recovery or partial recovery. Unless otherwise indicated, the recurrence state refers to the process or return to the disease prior to any previous treatment (including, but not limited to, chemotherapy and stem cell transplantation therapy).

[0135] refractory "Refractory" refers to the resistance or non-response of a disease or condition to treatment (e.g., the number of neoplastic plasma cells increases even when treatment is administered). Unless otherwise indicated, the term "refractory" refers to resistance or non-response to any previous treatment (including, but not limited to, chemotherapy and stem cell transplantation therapy).

[0136] Gastric tumor As used herein, the terms “gastric tumor” or “gastric cancer” refer to any tumor or cancer of the stomach, including, for example, adenocarcinoma (e.g., diffuse and intestinal types) as well as less common forms such as lymphoma, leiomyosarcoma, and squamous cell carcinoma.

[0137] tumor As used herein, the term “tumor” (or “tumor”) refers to all neoplastic cell growth and proliferation, whether malignant or benign, as well as all precancerous and cancerous cells and tissues. The terms “cancer,” “cancerous,” “proliferative disorder,” “proliferative disorder,” and “tumor” are not mutually exclusive as used herein.

[0138] Inhibition of cell growth or proliferation / Suppression of cell growth "Inhibition of cell growth or proliferation" or "suppression of cell growth" means reducing cell growth or proliferation by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%, and includes inducing cell death.

[0139] oligonucleotides As used herein, “oligonucleotide” refers to a synthetic polynucleotide, usually single-stranded, typically less than approximately 200 nucleotides in length, but not necessarily so. The terms “oligonucleotide” and “polynucleotide” are not mutually exclusive. The above description of polynucleotides is equally and fully applicable to oligonucleotides.

[0140] In effect, it decreased As used herein, the expressions “substantially reduced” or “substantially different” mean that the difference between two numerical values ​​(usually between one relating to a molecule and one relating to a reference / comparative molecule) is sufficiently large that a person skilled in the art would consider the difference between the two numerical values ​​to be statistically significant in terms of the biological characteristic measured by the numerical value (e.g., the Kd value).

[0141] Substantially similar As used herein, the terms “substantially similar” or “substantially identical” mean that the similarity between two numerical values ​​(e.g., between the antibody of the present invention and the reference / comparative antibody) is sufficiently high that a person skilled in the art would consider the difference between the two numerical values ​​to be little or no biological and / or statistically significant in terms of the biological characteristics measured by the numerical value (e.g., the Kd value).

[0142] autoimmune disease The combination of target-binding molecules or protein complexes of the present invention can be used to treat or prevent autoimmune diseases. “Autoimmune disease” means a non-malignant disease or disorder that originates from and is directed toward the tissues of an individual. In this specification, autoimmune disease explicitly excludes malignant or cancerous diseases or conditions, particularly B-cell lymphoma, acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), hairy cell leukemia, and chronic myeloblastic leukemia.Examples of autoimmune diseases or disorders include, but are not limited to, the following: inflammatory reactions such as inflammatory skin diseases including psoriasis and dermatitis (e.g., atopic dermatitis); systemic scleroderma and sclerosis; reactions associated with inflammatory bowel disease (e.g., Crohn's disease and ulcerative colitis); respiratory distress syndromes (including adult respiratory distress syndrome: ARDS); dermatitis; meningitis; encephalitis; uveitis; colitis; glomerulonephritis; allergic conditions such as eczema and asthma and other conditions with T-cell infiltration and chronic inflammatory reactions; atherosclerosis; leukocyte adhesion deficiency; rheumatoid arthritis; and systemic lupus erythematosus (SLE). (Including but not limited to lupus nephritis and cutaneous lupus); diabetes mellitus (e.g., type 1 diabetes or insulin-dependent diabetes mellitus); multiple sclerosis; Raynaud's syndrome; autoimmune thyroiditis; Hashimoto's thyroiditis; allergic encephalomyelitis; Sjögren's syndrome; juvenile-onset diabetes mellitus; and immune responses associated with acute and delayed hypersensitivity mediated by cytokines and T lymphocytes, typically seen in tuberculosis, sarcoidosis, polymyositis, granulomatosis, and vasculitis; pernicious anemia (Addison's disease); diseases involving leukocyte leakage; central nervous system (CNS) Inflammatory disorders; multiple organ injury syndromes; hemolytic anemia (including but not limited to cryoglobulinemia or Coombs-positive anemia); myasthenia gravis; antigen-antibody complex-mediated disorders; anti-glomerular basement membrane disorders; antiphospholipid syndromes; allergic neuritis; Graves' disease; Lambert-Eaton myasthenic syndrome; bullous pemphigoid; pemphigus; autoimmune polyglandular endocrine disorders; Reiter's disease; Stiffman syndrome; Behçet's disease; giant cell arteritis; immune complex nephritis; IgA nephropathy; IgM polyneuropathy; immune thrombocytopenic purpura (ITP) or autoimmune thrombocytopenia.

[0143] Immunosuppressants / Anti-inflammatory drugs As used herein in relation to adjunctive therapy, the term “immunosuppressant” refers to a substance that suppresses or shields the immune system of the mammal being treated. This includes substances that suppress cytokine production, downregulate or suppress the expression of autoantigens, or shield MHC antigens. Examples of such agents include: 2-amino-6-aryl-5-substituted pyrimidines (see U.S. Patent No. 4,665,077); non-steroidal anti-inflammatory drugs: NSAIDs); ganciclovir, tacrolimus, glucocorticoids (e.g., cortisol or aldosterone), anti-inflammatory agents (e.g., cyclooxygenase inhibitors, 5-lipoxygenase inhibitors, or leukotriene receptor antagonists); purine antagonists such as azathioprine or mycophenolate mofetil (MMF); alkylating agents such as cyclophosphamide; bromocriptine; danazol; dapsone; glutaraldehyde (which shields MHC antigens as described in U.S. Patent No. 4,120,649); anti-idiologics against MHC antigens and MHC fragments Type antibodies; cyclosporine A; steroids such as corticosteroids or glucocorticosteroids or glucocorticoid analogs (e.g., prednisone, methylprednisolone including SOLU-MEDROL® methylprednisolone sodium succinate, and dexamethasone); dihydrofolate reductase inhibitors such as methotrexate (oral or subcutaneous); antimalarial agents such as chloroquine and hydroxychloroquine; sulfasalazine; leflunomide; anti-interferon-alpha, -beta, or -gamma antibodies; antitumor necrosis factor (tumor Cytokines or cytokine receptor antibodies, including necrosis factor: TNF-alpha antibody (infliximab (REMICADE®) or adalimumab), anti-TNF-alpha noadhesin (etanercept), anti-TNF-beta antibody, anti-interleukin-2 (IL-2) antibody and anti-IL-2 receptor antibody, and anti-interleukin-6 (IL-6) receptor antibody and antagonist (e.g., ACTEMA® (tocilizumab));Anti-LFA-1 antibodies including anti-CD11a and anti-CD18 antibodies; anti-L3T4 antibodies; heterologous anti-lymphocyte globulins; pan-T antibodies, preferably anti-CD3 or anti-CD4 / CD4a antibodies; soluble peptides containing an LFA-3 binding domain (WO90 / 08187, published July 26, 1990); streptokinase; transforming growth factor-beta (TGF-beta); streptodolase; host-derived RNA or DNA; FK506; RS-61443; chlorambucil; deoxysperguarin; rapamycin; T cell receptor (Cohen et al., U.S. Patent No. 5,114,721); T cell receptor fragments (Offner et al., Science, 251: 430-432 (1991); WO90 / 11294; Ianeway, Nature, 341: 482) (1989); and WO91 / 01133); BAFF antagonists such as BAFF antibodies, BR3 antibodies, and zTNF4 antagonists (see Mackay and Mackay, Trends Immunol., 23:113-5 (2002) for a review, and also see the definition below); inhibitory antibodies against CD40-CD40 ligands (e.g., Durie et al., Science, 261: 1328-30 (1993); Mohan et al., J. Immunol., 154: 1470-80 (1995)) and CTLA4-Ig (Finck et al., Science, 265: 1225-7 Bioagents that interfere with T cell helper signaling, such as anti-CD40 receptors or anti-CD40 ligands (CD154), including (1994); as well as T cell receptor antibodies (EP340, 109), such as T10B9. Some preferred immunosuppressants herein include cyclophosphamide, chlorambucil, azathioprine, leflunomide, MMF, or methotrexate.

[0144] II. Compositions and Methods In one aspect, the present invention is A first target-binding molecule comprising a first binding domain capable of binding to a scaffold protein and a second binding domain capable of binding to a first receptor protein, and A second target-binding molecule comprising a third binding domain capable of binding to the scaffold protein and a fourth binding domain capable of binding to a second receptor protein. A combination of target-binding molecules, including The first target-binding molecule and the second target-binding molecule are capable of binding non-competitively to the scaffold protein, and The first receptor protein and the second receptor protein are receptor subunits that can associate to form a receptor complex. This relates to a combination of target-binding molecules. This combination of target-binding molecules can induce receptor signaling in receptor complexes.

[0145] In another aspect, the present invention relates to a protein complex comprising a first target-binding molecule having a first binding domain capable of binding to a scaffold protein and a second binding domain capable of binding to a first receptor protein, and a second target-binding molecule having a third binding domain capable of binding to the scaffold protein and a fourth binding domain capable of binding to a second receptor protein, The first target-binding molecule and the second target-binding molecule are capable of binding non-competitively to the scaffold protein. The first receptor protein and the second receptor protein are receptor subunits that can associate to form a receptor complex. This relates to the protein complex. Protein complexes can induce receptor signaling in receptor complexes.

[0146] The present invention also relates to a protein complex comprising a first binding domain, a second binding domain, a third binding domain, and a fourth binding domain, The first binding domain is capable of binding to the scaffold protein, the second binding domain is capable of binding to the first receptor protein, the third binding domain is capable of binding to the scaffold protein, and the fourth binding domain is capable of binding to the second receptor protein. The first binding domain and the third binding domain are capable of binding non-competitively to the scaffold protein. The first receptor protein and the second receptor protein are receptor subunits that can associate to form a receptor complex. This relates to the protein complex. Protein complexes can induce receptor signaling in receptor complexes.

[0147] The present invention also relates to nucleic acids that encode a combination of target-binding molecules or a protein complex of the present invention.

[0148] The present invention also relates to a method for producing a combination of target-binding molecules or a protein complex of the present invention.

[0149] The present invention also relates to the medical use of a combination of target-binding molecules or protein complexes of the present invention, or a pharmaceutical composition comprising a combination of target-binding molecules or protein complexes of the present invention.

[0150] The present invention also relates to a combination of target-binding molecules or protein complexes, comprising any one of the target-binding molecules or protein complexes in Tables 1 to 17. As shown in this application, any one of the target-binding molecules or protein complexes in Tables 1 to 17 is capable of inducing receptor signaling in their respective receptor complexes.

[0151] Recombination method and configuration For example, as described in U.S. Patent No. 4,816,567, the combination of target-binding molecules or the protein complex of the present invention can be produced using recombinant methods or configurations. In one embodiment, an isolated nucleic acid encoding the combination of target-binding molecules or the protein complex of the present invention as described herein is provided. Such nucleic acid may encode an amino acid sequence containing VL and / or VH of the target-binding molecule (e.g., the light chain and / or heavy chain of the antibody). In a further embodiment, one or more vectors (e.g., expression vectors) containing such nucleic acid are provided. In a further embodiment, a host cell containing such nucleic acid is provided. In one such embodiment, the host cell comprises (1) a vector containing nucleic acid encoding an amino acid sequence containing VL of the antibody and an amino acid sequence containing VH of the antibody, or (2) a first vector containing nucleic acid encoding an amino acid sequence containing VL of the antibody and a second vector containing nucleic acid encoding an amino acid sequence containing VH of the antibody (e.g., transformed). In one embodiment, the host cells are eukaryotic (e.g., Chinese hamster ovary (CHO) cells) or lymphoid cells (e.g., Y0, NS0, Sp2 / 0 cells). In one embodiment, a method is provided for producing a combination of target-binding molecules or a protein complex of the present invention, comprising the steps of: culturing host cells containing nucleic acids encoding the combination of target-binding molecules or the protein complex of the present invention, as provided above, under conditions suitable for the expression of the combination of target-binding molecules or the protein complex of the present invention; and optionally, recovering the combination of target-binding molecules or the protein complex of the present invention from the host cells (or host cell culture medium).

[0152] For the recombinant production of the target-binding molecule combinations or protein complexes of the present invention as described herein, for example, nucleic acids encoding the target-binding molecules, such as those described above, are isolated and inserted into one or more vectors for further cloning and / or expression in host cells. Such nucleic acids would be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that can specifically bind to genes encoding the heavy and light chains of antibodies).

[0153] Suitable host cells for cloning or expressing antibody-encoding vectors include prokaryotic or eukaryotic cells as described herein. For example, target-binding molecules may be produced in bacteria, particularly when glycosylation and Fc effector function are not required. For bacterial expression of antibody fragments and polypeptides, see, for example, U.S. Patents 5,648,237, 5,789,199, and 5,840,523. (See also Charlton, Methods in Molecular Biology, Vol. 248 (BKC Lo, ed., Humana Press, Totowa, NJ, 2003), pp. 245-254, for a description of antibody fragment expression in Escherichia coli.) After expression, target-binding molecules may be isolated from bacterial cell paste into a soluble fraction and further purified.

[0154] In addition to prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeasts, including strains of fungi and yeasts whose glycosylation pathways have been "humanized" to produce antibodies with partial or complete human glycosylation patterns, are suitable cloning or expression hosts for antibody-coding vectors. See Gerngross, Nat. Biotech. 22:1409-1414 (2004) and Li et al., Nat. Biotech. 24:210-215 (2006).

[0155] Cells derived from multicellular organisms (invertebrates and vertebrates) are also suitable host cells for the expression of glycosylated antibodies. Examples of invertebrate cells include plant and insect cells. Numerous baculovirus strains have been identified for use in conjugation with insect cells, particularly for the transformation of Spodoptera frugiperda cells.

[0156] Plant cell cultures can also be used as hosts. See, for example, U.S. Patents 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (which describe PLANTIBODIES® technology for antibody production in transgenic plants).

[0157] Vertebrate cells can also be used as hosts. For example, mammalian cell lines adapted to grow in a suspension state would be useful. Other examples of useful mammalian host cell lines include SV40-transformed monkey kidney CV1 cell line (COS-7); human embryonic kidney cell line (293 or 293 cells as described in Graham et al., J. Gen Virol. 36:59 (1977), etc.); baby hamster kidney cells (BHK); mouse Sertoli cells (TM4 cells as described in Mather, Biol. Reprod. 23:243-251 (1980), etc.); monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical cancer cells (HELA); canine kidney cells (MDCK); Buffalo rat hepatocytes (BRL 3A); human lung cells (W138); human hepatocytes (Hep G2); mouse mammary cancer cells (MMT 060562); and TRI cells (e.g., Mather et al., Annals NY Acad. Sci. 383:44-68 (1982)). These include MRC5 cells and FS4 cells, as described in [reference]. Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR-CHO cells (Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)), and myeloma cell lines such as Y0, NS0, and Sp2 / 0. For a review of specific mammalian host cell lines suitable for antibody production, see, for example, Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (BKC Lo, ed., Humana Press, Totowa, NJ), pp. 255-268 (2003).

[0158] In a particular embodiment, the present invention provides a combination of target-binding molecules or a protein complex of the present invention that can be obtained by the method described above.

[0159] Assay The combinations of target-binding molecules or protein complexes of the present invention provided herein can be identified, screened, or characterized for their physical / chemical properties and / or biological activity by various assays known in the art.

[0160] Binding assays and other assays In one aspect, the target-binding molecule of the present invention is tested for its target-binding activity by known methods such as ELISA and Western blotting.

[0161] In another context, a competition assay may be used to identify target-binding molecules that bind to the scaffold protein but do not compete with further target-binding molecules of the present invention that bind to the scaffold protein. In certain embodiments, such non-competitive first target-binding molecules do not bind to the same epitopes (e.g., linear or structural epitopes) to which the second target-binding molecule binds. Detailed exemplary methods for mapping the epitopes to which antibodies bind are provided in Morris (1996) "Epitope Mapping Protocols," in Methods in Molecular Biology vol. 66 (Humana Press, Totowa, NJ).

[0162] In an exemplary competitive assay, an immobilized scaffold protein is incubated in a solution containing a first labeled target-binding molecule that binds to the scaffold protein, and a second unlabeled target-binding molecule being tested for its ability to compete with the first target-binding molecule for binding to the scaffold protein. The second target-binding molecule may be present in the hybridoma supernatant. As a control, the immobilized target-binding molecule is incubated in a solution containing the first labeled target-binding molecule but not the second unlabeled target-binding molecule. After incubation under conditions that allow the first target-binding molecule to bind to the scaffold protein, any excess unbound target-binding molecules are removed, and the amount of label associated with the immobilized scaffold protein is measured. If the amount of label associated with the immobilized scaffold protein is substantially reduced in the test sample compared to the control sample, it indicates that the second target-binding molecule is competing with the first target-binding molecule for binding to the scaffold protein. If the amount of label associated with the immobilized scaffold protein is not substantially reduced in the test sample compared to the control sample, it indicates that the second target-binding molecule is not competing with the first target-binding molecule for binding to the scaffold protein. (See Harlow and Lane (1988) Antibodies: A Laboratory Manual ch.14 (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY).)

[0163] Pharmaceutical preparations / pharmaceutical compositions Pharmaceutical formulations of the target-binding molecule combination or protein complex of the present invention as described herein are prepared in the form of lyophilized formulations or aqueous solutions by mixing the target-binding molecule combination or protein complex of the present invention having the desired purity with one or more pharmaceutically acceptable carriers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)). Pharmaceutically acceptable carriers are generally non-toxic to the recipient at the doses and concentrations used, and include, but are not limited to, the following: buffers such as phosphates, citrates, and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives (octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl, or benzyl alcohol; alkylparabens such as methyl or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and Examples of pharmaceutically acceptable carriers herein include: m-cresol, etc.; low molecular weight (less than approximately 10 residues) polypeptides; proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrin; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, and sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or nonionic surfactants such as polyethylene glycol (PEG). Exemplary pharmaceutically acceptable carriers herein further include interstitial drug dispersants such as soluble neutral active hyaluronidase glycoproteins (sHASEGP) (e.g., human soluble PH-20 hyaluronidase glycoprotein such as rHuPH20 (HYLENEX®, Baxter International, Inc.)).Specific exemplary sHASEGP and its uses (including rHuPH20) are described in U.S. Patent Application Publications 2005 / 0260186 and 2006 / 0104968. In one aspect, sHASEGP is combined with one or more additional glycosaminoglycans, such as chondroitinase.

[0164] An exemplary lyophilized formulation is described in U.S. Patent No. 6,267,958. Aqueous aqueous formulations include those described in U.S. Patent No. 6,171,586 and WO2006 / 044908, the latter of which contains a histidine-acetate buffer.

[0165] The formulations described herein may contain one or more active ingredients if necessary for the specific indication being treated. Preferably, these active ingredients have complementary activities that do not adversely affect each other. Such active ingredients are preferably present in combination in amounts effective for the intended purpose.

[0166] The active ingredient may be incorporated into microcapsules (e.g., hydroxymethylcellulose or gelatin microcapsules and poly(methyl methacrylate) microcapsules, respectively) prepared by, for example, a droplet formation (coacervation) technique or interfacial polymerization, or into a colloidal drug delivery system (e.g., liposomes, albumin spheres, microemulsions, nanoparticles, and nanocapsules), or into a macroemulsion. Such techniques are disclosed in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

[0167] A sustained-release formulation may be prepared. A preferred example of a sustained-release formulation is one comprising a semipermeable matrix of a solid hydrophobic polymer containing an antibody, the matrix being in the form of a fabricated product such as a film or microcapsule.

[0168] Preparations used for in vivo administration are typically sterile. Sterility can be easily achieved, for example, by filtering through a sterile filtration membrane.

[0169] Therapeutic methods and compositions The combination of target-binding molecules or protein complexes of the present invention provided herein may be used in therapeutic methods.

[0170] In one aspect, a combination of target-binding molecules or a protein complex of the present invention is provided for use as a pharmaceutical. In a further aspect, a combination of target-binding molecules or a protein complex of the present invention is provided for use in the treatment of cancer or autoimmune diseases. In a particular embodiment, a target-binding molecule as defined in [A-1] is provided for medical use in combination with other target-binding molecules as defined in [A-1]. In a particular embodiment, a combination of target-binding molecules or a protein complex of the present invention, or a target-binding molecule as defined in [A-1] combined with other target-binding molecules as defined in [A-1], is provided for use in methods of treatment. In a particular embodiment, the present invention provides for use in a method for treating an individual having cancer or an autoimmune disease, comprising the step of administering to the individual an effective amount of a combination of the target-binding molecules of the present invention or a protein complex of the present invention, or a target-binding molecule of the present invention as defined in [A-1] combined with other target-binding molecules as defined in [A-1].

[0171] The therapeutic mechanism of the target-binding molecules of the present invention, the protein complex of the present invention, or the target-binding molecules of the present invention as defined in [A-1], when combined with other target-binding molecules as defined in [A-1], is the conditional activation of the receptor complex upon binding of the four binding domains to their respective targets. The signal induced by the activated receptor complex results in a therapeutic effect.

[0172] In a further aspect, the present invention provides the use of a combination of the target-binding molecules of the present invention or a protein complex of the present invention, or the target-binding molecules of the present invention as defined in [A-1], in combination with other target-binding molecules as defined in [A-1], in the manufacture or preparation of a pharmaceutical product. In one embodiment, the pharmaceutical product is for the treatment of cancer or an autoimmune disease. In a further embodiment, the pharmaceutical product is for use in a method of treating cancer or an autoimmune disease, the treatment comprising the step of administering an effective amount of the pharmaceutical product to an individual having cancer or an autoimmune disease.

[0173] In one embodiment, the method further includes the step of administering to an individual an effective amount of at least one additional therapeutic agent, such as the following:

[0174] In a further aspect, the present invention provides pharmaceutical formulations comprising, for example, a combination of the target-binding molecules of the present invention or a protein complex of the present invention, or a target-binding molecule of the present invention as defined in [A-1] combined with other target-binding molecules as defined in [A-1] provided herein, for use in any of the therapeutic methods described above. In one embodiment, the pharmaceutical formulation comprises any combination of the target-binding molecules of the present invention or a protein complex of the present invention, or a target-binding molecule of the present invention as defined in [A-1] combined with other target-binding molecules as defined in [A-1] provided herein, and a pharmaceutically acceptable carrier. In another embodiment, the pharmaceutical formulation comprises any combination of the target-binding molecules of the present invention or a protein complex of the present invention, or a target-binding molecule of the present invention as defined in [A-1] combined with other target-binding molecules as defined in [A-1] provided herein, and at least one additional therapeutic agent, for example, as described below.

[0175] A combination of the target-binding molecules of the present invention or a protein complex of the present invention, or a target-binding molecule of the present invention as defined in [A-1] combined with other target-binding molecules as defined in [A-1], can be used in therapy either alone or in combination with other agents. For example, a combination of the target-binding molecules of the present invention or a protein complex of the present invention, or a target-binding molecule of the present invention as defined in [A-1] combined with other target-binding molecules as defined in [A-1], can be administered concurrently with at least one additional therapeutic agent.

[0176] The combination therapies described above include combination administration (two or more therapeutic agents contained in the same or separate formulations) and individual administration, in which case, administration of the combination of target-binding molecules of the present invention or the protein complex of the present invention, or the target-binding molecules of the present invention as defined in [A-1] in combination with other target-binding molecules as defined in [A-1] of the present invention, may be performed prior to, simultaneously with, and / or subsequently to the administration of one or more additional therapeutic agents. In one embodiment, the administration of the combination of target-binding molecules of the present invention or the protein complex of the present invention, or the target-binding molecules of the present invention as defined in [A-1] in combination with other target-binding molecules as defined in [A-1] of the present invention, and the administration of additional therapeutic agents may be performed within about one month, or within about one, two, or three weeks, or within about one, two, three, four, five, or six days. The combination of target-binding molecules of the present invention or the protein complex of the present invention, or the target-binding molecules of the present invention as defined in [A-1] in combination with other target-binding molecules as defined in [A-1] of the present invention, may also be used in combination with radiotherapy.

[0177] A combination of the target-binding molecules of the present invention or the protein complex of the present invention, or a combination of the target-binding molecules of the present invention as defined in [A-1], or with other target-binding molecules as defined in [A-1] of the present invention (and any additional therapeutic agents), may be administered by any preferred means, including parenteral administration, intrapulmonary administration, and nasal administration, and, if desired for topical treatment, intralesional administration. Parenteral administration includes intramuscular, intravenous, intra-arterial, intraperitoneal, or subcutaneous administration. Dosage may be made by any preferred route, including injection, such as intravenous or subcutaneous injection, depending in part whether the administration is short-term or long-term. Various dosing schedules, including single doses or repeated doses over various time points, bolus administration, and pulse infusion, are within consideration herein, but are not limited to these.

[0178] The combination of target-binding molecules of the present invention or the protein complex of the present invention, or the target-binding molecules of the present invention as defined in [A-1], combined with other target-binding molecules as defined in [A-1] of the present invention, are formulated, administered, and given in a manner consistent with good medical practice. Factors to be considered from this perspective include the specific disorder being treated, the specific mammal being treated, the clinical symptoms of the individual patient, the cause of the disorder, the site to which the agent is delivered, the method of administration, the schedule of administration, and other factors known to healthcare professionals. The combination of target-binding molecules of the present invention or the protein complex of the present invention, or the target-binding molecules of the present invention as defined in [A-1], combined with other target-binding molecules as defined in [A-1] of the present invention, are formulated, but may not be, together with one or more agents currently used to prevent or treat the disorder in question. The effective amount of such other agents depends on the combination of target-binding molecules of the present invention or protein complexes of the present invention present in the formulation, or the amount of target-binding molecules of the present invention as defined in [A-1] in combination with other target-binding molecules as defined in [A-1] of the present invention, the type of disorder or treatment, and other factors discussed above. These are typically used in the same doses and routes of administration as described herein, or in about 1 to 99% of the doses described herein, or in any dose and route as deemed empirically / clinically appropriate.

[0179] For the prevention or treatment of a disease, the appropriate dose of the combination of target-binding molecules of the present invention or the protein complex of the present invention, or the target-binding molecules of the present invention as defined in [A-1], in combination with other target-binding molecules as defined in [A-1] of the present invention (when used alone or with one or more additional therapeutic agents) will depend on the type of disease being treated, the type of target-binding molecule, the severity and course of the disease, whether the combination of target-binding molecules of the present invention or the protein complex of the present invention, or the target-binding molecules of the present invention as defined in [A-1], in combination with other target-binding molecules as defined in [A-1] of the present invention, is administered for prophylactic or therapeutic purposes, the patient's medical history, clinical history and response to the target-binding molecules, and the discretion of the attending physician. The combination of target-binding molecules of the present invention or the protein complex of the present invention, or the target-binding molecules of the present invention as defined in [A-1], in combination with other target-binding molecules as defined in [A-1] of the present invention, is preferably administered to the patient in a single dose or over a series of treatments. Depending on the type and severity of the disease, for example, a combination of the target-binding molecules of the present invention or the protein complex of the present invention, or the target-binding molecules of the present invention as defined in [A-1] combined with other target-binding molecules as defined in [A-1] of the present invention, in doses ranging from approximately 1 μg / kg to 15 mg / kg (e.g., 0.1 mg / kg to 10 mg / kg), whether administered as a single or multiple separate doses or as a continuous infusion, may be the initial candidate dose for administration to a patient. A typical daily dose may range from approximately 1 μg / kg to 100 mg / kg or more, depending on the factors described above. In the case of repeated administrations over several days or longer, treatment is usually maintained, depending on the situation, until the desired suppression of disease symptoms occurs. One exemplary dose of the combination of the target-binding molecules of the present invention or the protein complex of the present invention, or the target-binding molecules of the present invention as defined in [A-1] combined with other target-binding molecules as defined in [A-1] of the present invention, is in the range of approximately 0.05 mg / kg to approximately 10 mg / kg.Therefore, one or more doses (or any combination thereof) of approximately 0.5 mg / kg, 2.0 mg / kg, 4.0 mg / kg, or 10 mg / kg may be administered to the patient. Such doses may be administered intermittently, for example, every week or every three weeks (for example, so that the patient receives approximately 2 to approximately 20, or for example, approximately 6 doses of the combination of target-binding molecules of the present invention or the protein complex of the present invention, or the target-binding molecules of the present invention as defined in [A-1] in combination with other target-binding molecules as defined in [A-1] of the present invention). The course of this therapy can be easily monitored by conventional methods and assays.

[0180] product In another aspect of the present invention, a product is provided comprising equipment useful for the treatment, prevention, and / or diagnosis of the above-mentioned disorders. The product comprises a container and a label on the container or accompanying documentation attached to the container. Preferred containers include, for example, bottles, vials, syringes, and IV solution bags. Containers may be formed from a variety of materials, such as glass or plastic. Containers may hold a composition alone or in combination with another composition effective for the treatment, prevention, and / or diagnosis of a symptom, and may have a sterile access port (for example, the container may be an intravenous solution bag or vial with a stopper that can be punctured by a subcutaneous injection needle). At least one active ingredient in the composition is a combination of target-binding molecules of the present invention or a protein complex of the present invention, or a target-binding molecule of the present invention as defined in [A-1] in combination with other target-binding molecules as defined in [A-1] of the present invention. The label or accompanying documentation indicates that the composition is used to treat a selected symptom. The product may further include (a) a first container containing a composition comprising a combination of the target-binding molecules of the present invention or a protein complex of the present invention, or a target-binding molecule of the present invention as defined in [A-1], in combination with other target-binding molecules as defined in [A-1] of the present invention; and (b) a second container containing a composition comprising a further cytotoxic agent or other therapeutic agent. The product in this embodiment of the present invention may further include a package insert indicating that the composition may be used to treat a particular condition. Alternatively, the product may further include a second (or third) container containing a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution, and dextrose solution. It may further include other equipment desirable from a commercial or user perspective, such as other buffers, diluents, filters, needles, and syringes. [Examples]

[0181] III. Examples The following are examples of the methods and compositions of the present invention. It will be understood that various other embodiments may be carried out in light of the general description provided above.

[0182] Example 1: Concept of Disclosure

[0183] A schematic diagram of the protein complex in this disclosure is shown in Figure 1A. Conditional receptor signaling is achieved using a first target-binding molecule comprising a first binding domain that binds to a scaffold protein and a second binding domain that binds to a first receptor protein, and a second target-binding molecule comprising a third binding domain that binds to the scaffold protein and a fourth binding domain that binds to a second receptor protein. The first and third binding domains (S1 and S2) of the target-binding molecule can simultaneously, non-competitively, and biparatopically bind to the scaffold protein. The second binding domain (R1) binds to the first receptor protein, and the fourth binding domain (R2) binds to the second receptor protein (R1 binds to receptor A, and R2 binds to receptor B), where receptors A and B can associate to form a receptor complex when they are in close proximity to each other or when they associate with each other, thereby inducing receptor activation and / or signaling activity. Adding linker sequences between each binding domain is an option for optimizing agonist activity.

[0184] In the presence of receptor proteins and in the absence of scaffold proteins, the two target-binding molecules simply bind to their respective receptor proteins (receptor A and receptor B) through their first and third binding domains (R1 and R2), but no signaling activity is induced because the receptor proteins (receptor A and receptor B) are neither close to nor associated with each other. In the presence of both receptor proteins and scaffold proteins, the first and third binding domains (S1 and S2) of the target-binding molecules bind to the scaffold protein non-competitively and / or biparatopically, while the second binding domain binds to the first receptor protein and the fourth binding domain binds to the second receptor protein. As a result, the receptor proteins come into close proximity to each other or associate with each other, thereby inducing receptor activation and / or signaling activity. Adding linker sequences between each binding domain and half-life extension domains such as Fc domains is an option to improve functionality.

[0185] As a non-limiting example, this concept of conditional activation is illustrated using two VHH-VHH fusions. Each VHH-VHH fusion contains a scaffold protein-binding domain and a receptor protein-binding domain. As a non-limiting example, the SARS-CoV-2 receptor-binding protein (RBD) or human PDL1 may be the scaffold protein, and IL-2Rβ, IL-2Rγ, or Wnt receptor may be the receptor protein.

[0186] We identified VHHs that can simultaneously bind to RBD in a biparatopic manner, or to human PDL1 in a biparatopic manner, and VHHs that can bind to IL-2Rβ or IL-2Rγ. Nb21 and Nb36 are VHHs that recognize different epitopes of RBD, respectively, and can therefore simultaneously bind to RBD in a biparatopic manner (Nature Communications 2021;12:4676).

[0187] Figure 1B shows a promising single-molecule form that can induce conditional receptor activation by controlling receptor distance through binding to scaffold proteins. In the presence of receptor proteins and in the absence of scaffold proteins, the target-binding molecule simply binds to its respective receptor proteins (receptor A and receptor B) through its first and third binding domains (R1 and R2), but because the receptor proteins (receptor A and receptor B) are neither close to nor associated with each other, only weak signaling activity is induced, or no signaling activity is induced at all. In the presence of both receptor proteins and scaffold proteins, the first and third binding domains (S1 and S2) of the target-binding molecule biparatopically bind to the scaffold proteins, while the second binding domain binds to the first receptor protein, and the fourth binding domain binds to the second receptor protein. As a result, the receptor proteins come close to or associate with each other, forming a receptor complex, thereby inducing receptor activation and / or signaling activity. Adding linker sequences between each binding domain and half-life extension domains such as Fc domains is an option for improving functionality.

[0188] Example 2: Plasmid preparation for VHH-VHH fusions that bind to RBD or PDL1 as a scaffold protein and to IL-2Rβ or IL-2Rγ as a receptor protein.

[0189] VHH-VHH fusions (SEQ ID NOs: 1-10) were constructed by ligating each of the following proteins, along with a signal sequence: a VHH capable of binding to human IL-2Rβ or human IL-2Rγ, along with a scaffold-binding protein (RBD or VHH capable of binding to human PDL1). To be used as a scaffold protein, RBD was fused along with a signal sequence to the transmembrane and intracellular domains of human IL-2Rα (RBD-IL2RA) (SEQ ID NO: 11). DNA encoding the VHH-VHH fusion, RBD-IL2RA, or human PDL1 (SEQ ID NO: 12) was cloned into mammalian expression vectors.

[0190] While the native SARS-CoV-2 spike protein, including RBD, is a homotrimeric protein, RBD-IL2RA lacks the domain for trimer formation and can be expressed as a monomer. Additionally, IL2RA (IL-2Rα) is part of the high-affinity IL-2 receptor complex but is not involved in IL-2R signaling (Cancer Communications 2018; 38: 62).

[0191] [Table 1] TIFF2026510624000011.tif208170TIFF2026510624000012.tif87170

[0192] Sequence IDs 1 and 2 represent fusion proteins of the RBD-binding domain (Nb21) and the IL-2Rγ-binding domain (Vhh2g). The binding of Nb21 to RBD does not compete with Nb36. In Table 1, the sequence of Vhh2g is underlined and the sequence of Nb21 is double-underlined.

[0193] Sequence IDs 3 and 4 represent fusion proteins of the RBD-binding domain (Nb36) and the IL-2Rβ-binding domain (Vhh2b). The binding of Nb36 to RBD does not compete with that of Nb21. In Table 1, the sequence of Vhh2b is underlined and the sequence of Nb36 is double-underlined.

[0194] Sequence ID: 5 represents a fusion protein of the RBD-binding domain (Nb21) and the IL-2Rβ-binding domain (Vhh2b). In Table 1, the sequence of Vhh2b is underlined and the sequence of Nb21 is double-underlined.

[0195] Sequence ID: 7 represents a fusion protein of the IL-2Rγ binding domain (Vhh2g) and the PDL1 binding domain (VhhPL1). In Table 1, the sequence of Vhh2g is underlined and the sequence of VhhPL1 is double-underlined.

[0196] Sequence ID: 8 represents a fusion protein of the IL-2Rβ binding domain (Vhh2b) and the PDL1 binding domain (VhhPL1-12). In Table 1, the sequence of Vhh2b is underlined and the sequence of VhhPL1-12 is double-underlined.

[0197] Sequence ID: 9 represents a fusion protein of the IL-2Rβ binding domain (Vhh2b) and the PDL1 binding domain (VhhPL1-50). In Table 1, the sequence of Vhh2b is underlined and the sequence of VhhPL1-50 is double-underlined.

[0198] Sequence ID: 10 represents a fusion protein of the IL-2Rβ binding domain (Vhh2b) and the PDL1 binding domain (VhhPL1). In Table 1, the sequence of Vhh2b is underlined and the sequence of VhhPL1 is double-underlined.

[0199] Sequence ID: 11 represents a fusion protein of the SARS-CoV-2 receptor-binding domain (RBD) and the transmembrane and intracellular domains of IL-2Rα (IL-2RA).

[0200] Sequence ID: 12 represents human PDL1.

[0201] Example 3: Induction of IL-2 receptor signaling by a mixture of VHH-VHH fusions that bind to RBD in a biparatopic manner and also bind to the IL-2 receptor protein is dependent on RBD expression.

[0202] Conditional receptor activation in the presence of a scaffold protein was demonstrated in this example using RBD as the scaffold protein and IL-2Rβ and IL-2Rγ as the conditionally activated receptor proteins.

[0203] As described in Example 2, the VHH-VHH fusion was constructed by linking a VHH (either Nb21 or Nb36) that binds to RBD to either a VHH (Vhh2b) that binds to human IL-2Rβ or a VHH (Vhh2g) that binds to human IL-2Rγ. Nb21 and Nb36 are biparatopic antibodies that recognize different epitopes of RBD and can bind to RBD simultaneously (Nature Communications 2021;12: 4676). Vhh2b (PDB:7S2S) and Vhh2g (PDB:7S2R) have been described (Cell 2022; 185: 1414-1430.e19), and their structures and binding epitopes are shown in the RCSB Protein Data Bank (7S2S and 7S2R).

[0204] Plasmids for VHH-VHH fusions (SEQ ID NOs: 1-4) were expressed in HEK-Blue IL-2 cells (InvivoGen, #hkb-il2) using the SF Cell Line 4D-Nucleofector X Kit L (Lonza, #V4XC-2024) along with the RBD-IL2RA (SEQ ID NO: 11) or PDL1 (SEQ ID NO: 12) plasmid. The pmaxGFP® vector was used as a negative control. Cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin. These transfectants were seeded in 96-well plates at 37°C for 3 days in the presence of 5% CO2. After 3 days of culture, the activation of the IL-2 receptor complex was evaluated by measuring the optical density at 620 nm using a Multiskan® plate reader with Quanti-Blue solution (InvivoGen, #rep-qbs) (Figure 2A).

[0205] Figure 2A shows the results of IL-2R signal induction. The VHH-VHH fusion was able to activate the IL-2 receptor complex in HEK-Blue IL-2 cells in the presence of RBD-IL2RA, but not in the presence of PDL1. This indicates that IL-2R signal induction by the VHH-VHH fusion mixture depends on the expression of the scaffold protein RBD. Figure 2B shows a schematic diagram of the experimental setup in this example.

[0206] Example 4: Expression and purification of VHH-VHH fusions that bind to RBD and IL-2 receptor proteins.

[0207] VHH-VHH fusions (SEQ ID NOs: 1, 4, 5, and 6) were transiently expressed in the Expi293 cell line (Thermo Fisher, Carlsbad, CA, USA). Antibody purification was performed using protein A affinity chromatography. The absorbance of the purified antibody at 280 nm was measured using a spectrophotometer. From the obtained values, the antibody concentration was calculated using the extinction coefficient calculated by methods such as PACE (Protein Science 1995; 4: 2411-2423).

[0208] Example 5: Activation of the IL-2 receptor complex by a mixture of VHH-VHH fusions targeting RBD as a scaffold protein depends on biparatopic binding to RBD.

[0209] To demonstrate that biparatopic binding to scaffold proteins is a condition for IL-2R activation, the effects of VHH-VHH fusion combinations that can biparatopically bind to scaffold proteins were compared to VHH-VHH fusion combinations that cannot biparatopically bind to scaffold proteins.

[0210] RBD-IL2RA (SEQ ID NO: 11) or PDL1 (SEQ ID NO: 12) was first expressed in HEK-Blue IL-2 cells (InvivoGen, #hkb-il2) by electroporation using SF Cell Line 4D-Nucleofector X Kit L (Lonza, #V4XC-2024). After electroporation, these transfectants were seeded in 96-well plates and incubated overnight at 37°C in the presence of 5% CO2. These cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin. On day 1 after electroporation, cells were treated with various mixtures of recombinant VHH-VHH fusions (SEQ ID NOs: 1, 4, 5, and 6) or recombinant human IL-2 (Peprotech, #200-02), and incubated for another day at 37°C in the presence of 5% CO2. On day 2 after electroporation, activation of the IL-2 receptor complex was evaluated by measuring the optical density at 620 nm using a Multiskan® plate reader with Quanti-Blue solution (InvivoGen, #rep-qbs) (Figures 3A and 3B).

[0211] As shown in Figures 3A and 3B, combinations of VHH-VHH fusions containing biparatopic RBD antibodies, Vhh2b-Nb36 and Nb21-Vhh2g, were able to induce IL-2 receptor signaling in the presence of RBD expression. However, combinations of monoparatopic RBD antibodies, Vhh2b-Nb36 and Nb36-Vhh2g or Vhh2b-Nb21 and Nb21-Vhh2g, were unable to induce IL-2 receptor signaling even in the presence of RBD. This result confirms that activation of the IL-2 receptor complex by VHH-VHH fusion mixtures is dependent on biparatopic binding to the scaffold protein, which is RBD.

[0212] Example 6: Induction of IL-2 receptor complex signaling by a mixture of VHH-VHH fusions that bind to PDL1 and IL-2 receptor proteins is dependent on PDL1 expression.

[0213] Conditional activation of the IL-2 receptor complex by a mixture of VHH-VHH fusions that bind to PDL1 as the target scaffold protein and to the IL-2 receptor protein as the target receptor protein was demonstrated in HEK-Blue IL-2 cells. A VHH that biparatopically binds to PDL1 was identified. VhhPL1 is a VHH that can achieve biparatopic binding by simultaneously binding to PDL1 with either VhhPL1-12 or VHHPL1-50.

[0214] Plasmids for VHH-VHH fusions (SEQ ID NOs: 7-10) that bind to PDL1 and human IL-2Rβ or human IL-2Rγ were expressed in HEK-Blue IL-2 cells (InvivoGen, #hkb-il2) using the SF Cell Line 4D-Nucleofector X Kit L (Lonza, #V4XC-2024) along with the PDL1 (SEQ ID NO: 12) or RBD-IL2RA (SEQ ID NO: 11) plasmid. The VHH-VHH fusion combinations tested were Vhh2g-VhhPL1, which is a combination of Vhh2b-VhhPL1-12, Vhh2b-VhhPL1-50, or Vhh2b-VhhPL1. The pmaxGFP® vector was used as a negative control. Cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin. These transfectants were seeded in 96-well plates at 37°C for 2 days in the presence of 5% CO2. After 2 days of culture, IL-2 receptor complex activation was evaluated by measuring the optical density at 620 nm using a Multiskan® plate reader with Quanti-Blue solution (InvivoGen, #rep-qbs) (Figure 4).

[0215] As shown in Figure 4, a mixture of VHH-VHH fusions that bind to PDL1 in a biparatopic manner and also bind to the IL-2 receptor protein can induce IL-2R signaling under conditions of PDL1 expression. On the other hand, monoparatopic VHH-VHH fusion mixtures, Vhh2b-VhhPL1 and Vhh2g-VhhPL1, could not induce clear IL-2R signaling even in the presence of PDL1. This indicates that IL-2R signaling by a mixture of VHH-VHH fusions targeting PDL1 and the IL-2 receptor protein depends on the expression of the PDL1 scaffold protein and its biparatopic binding.

[0216] Example 7: Plasmid preparation for Split Neo-2 / 15 fused with VHH that binds to RBD

[0217] The present invention's concept of conditional receptor signaling is compared to Split Neo-2 / 15 (WO2020 / 106708A1), a split version of a known IL-2 / IL-15 mimetic, which is active only when two separated fragments co-localize at a site on the target cell.

[0218] VHH, which can bind to RBD, was fused with either Neo2A or Neo2B, specifically Split Neo-2 / 15 (WO2020 / 106708A1), along with its signal sequence and GS linker (SEQ ID NOs: 13-15). The DNA encoding these fusion proteins was cloned into mammalian expression vectors.

[0219] [Table 2]

[0220] Sequence ID: 13 represents a fusion protein of the RBD-binding domain (Nb21) and Neo2A from Split Noe-2 / 15. In Table 2, the sequence of Nb21 is underlined and the sequence of Neo2A is double-underlined.

[0221] Sequence ID: 14 ​​represents a fusion protein of the RBD-binding domain (Nb21) and Neo2B from Split Neo-2 / 15. In Table 2, the sequence of Nb21 is underlined and the sequence of Neo2B is double-underlined.

[0222] Sequence ID: 15 represents a fusion protein of the RBD-binding domain (Nb36) and Neo2A from Split Neo-2 / 15. In Table 2, the sequence of Nb36 is underlined and the sequence of Neo2A is double-underlined.

[0223] Example 8: The combination of Vhh2b-Nb36 and Nb21-Vhh2g demonstrated superior selectivity compared to Split Neo-2 / 15.

[0224] Activation of the IL-2 receptor complex with a mixture of Vhh2b-Nb36 and Nb21-Vhh2g showed superior selectivity for RBD expression compared to the fusion protein of Split Neo-2 / 15 and the RBD-binding domain.

[0225] To prevent unintended heterodimerization of Split Neo-2 / 15 within the endosomes of expressing cells, plasmids for Split Neo-2 / 15, which are conjugations (SEQ ID NOs: 13-15) of VHH that can bind to RBD and Neo2A or Neo2B, were individually expressed in HEK-Blue IL-2 cells (InvivoGen, #hkb-il2) using SF Cell Line 4D-Nucleofector X Kit L (Lonza, #V4XC-2024) along with either the RBD-IL2RA (SEQ ID NO: 11) or PDL1 (SEQ ID NO: 12) plasmid. To match these conditions, plasmids Vhh2b-Nb36 or Nb21-Vhh2g (SEQ ID NOs: 1 and 4) were individually expressed in HEK-Blue IL-2 cells along with either the RBD-IL2RA or PDL1 plasmid. The pmaxGFP® vector was used as a negative control. After electroporation, these transfectants were mixed in equivolume in various combinations and seeded in 96-well plates at 37°C for 2 days in the presence of 5% CO2. These transfectant mixtures were cultured in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin. After 2 days of culture, IL-2 receptor complex activation was evaluated by measuring the optical density at 620 nm using a Multiskan® plate reader with Quanti-Blue solution (InvivoGen, #rep-qbs) (Figures 5A and 5B).

[0226] As shown in Figure 5A, the conjugation of Split-Neo2 / 15 with the RBD-binding domain showed limited dependence on RBD expression. On the other hand, a mixture of Vhh2b-Nb36 and Nb21-Vhh2g, which bind to RBD in a biparatopic manner, demonstrated complete dependence on RBD expression (Figure 5B). Therefore, the present invention's concept of conditional receptor signaling has superior selectivity compared to the Split Neo-2 / 15 method known in the art.

[0227] Example 9: Plasmid preparation for a VHH-VHH fusion that binds to TNFα as a target scaffold protein and to IL-2Rβ or IL-2Rγ as a target receptor protein.

[0228] The present invention's concept of conditional receptor signaling is further illustrated using TNFα as the scaffold protein and IL-2Rβ or IL-2Rγ as the target receptor protein.

[0229] VHH-VHH fusions (SEQ ID NOs: 16-19) were constructed by ligating a scaffold-binding protein, Vhhtnfa3, along with its signal sequence, to another VHH capable of binding to human IL-2Rβ or human IL-2Rγ. These VHH-VHH fusions or DNA encoding human TNFα (SEQ ID NO: 20) were cloned into mammalian expression vectors.

[0230] [Table 3]

[0231] Sequence IDs 16 and 17 represent fusion proteins of a TNFα-binding domain (Vhhtnfa3) and an IL-2Rβ-binding domain (Vhh2b). In Table 3, the sequence of Vhh2b is underlined and the sequence of Vhhtnfa3 is double-underlined.

[0232] Sequence IDs 18 and 19 represent fusion proteins of a TNFα-binding domain (Vhhtnfa3) and an IL-2Rγ-binding domain (Vhh2g). In Table 3, the sequence of Vhh2g is underlined and the sequence of Vhhtnfa3 is double-underlined.

[0233] Sequence ID: 20 represents human TNFα.

[0234] Example 10: IL-2R signaling induction by a mixture of VHH-VHH fusions capable of binding to the TNFα and IL-2 receptor complex could be induced by monoparatopic binding to the trimer antigen TNFα.

[0235] Since TNFα is a homotrimeric antigen, each VHH-VHH fusion can bind to the same epitope on each TNFα monomer (i.e., monoparatopic binding). When TNFα monomers form a trimer, each scaffold protein-binding domain binds to a TNFα monomer and is located very close to one another. Therefore, the receptor protein-binding domains connected to each scaffold protein-binding domain can also bring receptor proteins into close proximity, thereby enabling receptor activation and / or induction of signaling activity. Exemplary VHH-VHH fusions were constructed: Vhh2b connected to Vhhtnfa3 and Vhh2g connected to Vhhtnfa3.

[0236] Plasmids of VHH-VHH fusions (SEQ ID NOs: 16-19) that bind to TNFα as the target scaffold protein and to human IL-2Rβ or human IL-2Rγ as the target receptor protein were expressed in HEK-Blue IL-2 cells (InvivoGen, #hkb-il2) using SF Cell Line 4D-Nucleofector X Kit L (Lonza, #V4XC-2024) along with TNFa (SEQ ID NO: 20) or PDL1 (SEQ ID NO: 12). The pmaxGFP® vector was used as a negative control. Cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin. These transfectants were seeded in 96-well plates at 37°C for 2 days in the presence of 5% CO2. After 2 days of culture, the activation of the IL-2 receptor complex was evaluated by measuring the optical density at 620 nm using a Multiskan® plate reader with Quanti-Blue solution (InvivoGen, #rep-qbs) (Figure 6A).

[0237] As shown in Figure 6A, a monoparatopic TNFα-binding domain targeting the same epitope on the monomer of the TNFα trimer was able to induce IL-2 receptor signaling in the presence of TNFα. A schematic diagram of the mechanism of conditional IL-2R signaling induction by a mixture VHH-VHH fusion that can bind to IL-2Rβ or IL-2Rγ and TNFα in a monoparatopic manner is shown in Figure 6B.

[0238] Example 11: Expression and purification of VHH-VHH fusions or VHH-scFv fusions that bind to RBD and Wnt receptors, which are LRP or FZD.

[0239] VHH (Nb21 or Nb36) capable of binding to RBD was fused with a signal sequence to one or more VHH or scFv-containing receptor protein-binding domains (Vhhlrp36 and R2M3, respectively) that can bind to human lipoprotein receptor-associated protein (LRP) or human Frizzled receptor (FZD) (SEQ ID NOs: 21-23). ​​The DNA encoding these fusion proteins was cloned into mammalian expression vectors. These plasmids were transiently expressed using the Expi293 cell line (Thermo Fisher, Carlsbad, CA, USA). Antibody purification was performed using protein A affinity chromatography. The absorbance of the purified antibody at 280 nm was measured using a spectrophotometer. From the obtained values, the antibody concentration was calculated using the extinction coefficient calculated by methods such as PACE (Protein Science 1995; 4: 2411-2423).

[0240] [Table 4]

[0241] Sequence ID: 21 represents a fusion protein of two LRP-binding domains (Vhhlrp36) and an RBD-binding domain (Nb21). In Table 4, the sequence of Vhhlrp36 is underlined and the sequence of Nb21 is double-underlined.

[0242] Sequence ID: 22 represents a fusion protein of two LRP-binding domains (Vhhlrp36) and an RBD-binding domain (Nb36). In Table 4, the sequence of Vhhlrp36 is underlined and the sequence of Nb36 is double-underlined.

[0243] Sequence ID: 23 represents a fusion protein of the FZD-binding domain (R2M3) and the RBD-binding domain (Nb21). In Table 4, the sequence of R2M3 is underlined and the sequence of Nb21 is double-underlined.

[0244] Example 12: Activation of the Wnt receptor complex by a mixture of VHH-VHH and VHH-scFv fusions depends on biparatopic binding to RBD.

[0245] The present invention's concept of conditional receptor signaling is further illustrated using RBD as the scaffold protein and the Wnt receptor, which includes LRP and FZD, as the conditionally activated receptor proteins. This example also demonstrates that other binding proteins, such as scFv, can also be used as scaffold protein-binding domains or receptor protein-binding domains.

[0246] RBD-IL2RA (SEQ ID NO: 11) or PDL1 (SEQ ID NO: 12) was first expressed in HEK293 STF cells (ATCC, CRL-3249) by electroporation using SF Cell Line 4D-Nucleofector X Kit L (Lonza, #V4XC-2024). These transfectants were seeded in white 96-well plates and incubated overnight at 37°C / 5% CO2 after adding 200 nM RSPO1 protein (in-house, #PPU5200) and 50 nM LGK974 (Cayman Chemical, #14072). These cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) F-12, HEPES, supplemented with 20% fetal bovine serum and 200 μg / mL Geneticin. On day 1 after electroporation, these cells were treated with various mixtures of VHH-VHH fusions or VHH-scFv fusions (SEQ ID NOs: 21-23) that bind to RBD and Wnt receptor proteins (FZD and LRP), and then incubated for another day at 37°C / 5% CO2. On day 2 after electroporation, Wnt signaling activation was evaluated by measuring luminescence using a Glomax Explorer with the ONE-Glo luciferase assay system (Promega, #E6120) (Figures 7A and 7B).

[0247] As shown in Figures 7A and 7B, only the combinations of R2M3-Nb21 and Vhhlrp36-Vhhlrp36-Nb36 were able to induce Wnt signaling in the presence of RBD expression. Consistent with the present invention's concept of conditional receptor signaling, only when both Nb21 and Nb36 bind to RBD in a biparatopic manner were the LRP-binding domain (Vhhlrp36) and FZD-binding domain (R2M3) able to bind to their respective antigens, bringing them into close proximity and inducing Wnt receptor signaling. In contrast, for monoparatopic VHH-VHH combinations in which both VHH-VHH fusions contain Nb21 (Figure 7A), and in the absence of the scaffold protein RBD (Figure 7B), no Wnt receptor signaling was observed. Therefore, activation of Wnt receptor signaling by a mixture of VHH-VHH and VHH-scFv fusions that bind to RBD and Wnt receptor proteins depends on RBD expression and biparatopic binding to RBD.

[0248] Example 13: Expression and purification of Vhh2b-Nb36 / Nb21-Vhh2g Fc fusion protein

[0249] The present invention's concept of conditional receptor activation can also be achieved in a single-molecule form, as shown in Figure 1B. The two target-binding molecules may be ligated to form a single molecule or associate with each other as a protein complex, with one or more domains extending the molecular half-life. In one example, the VHH-VHH fusion can be ligated to an Fc domain via a knob-into-hole technique (Protein Engineering 1996; 9: 617).

[0250] VHH-VHH fusions, Vhh2b-Nb36 (SEQ ID NO: 4) or Nb21-Vhh2g (SEQ ID NO: 1), were fused with Fc molecules containing mutations to facilitate heterodimerization of Vhh2b-Nb36 and Nb21-Vhh2g. The DNA encoding these Fc fusion proteins was cloned into mammalian expression vectors (SEQ ID NOs: 24 and 25). These plasmids were transiently expressed using the Expi293 cell line (Thermo Fisher, Carlsbad, CA, USA). Antibody purification was performed using protein A affinity chromatography. The absorbance of the purified antibody at 280 nm was measured using a spectrophotometer. The antibody concentration was calculated from the obtained values ​​using extinction coefficients calculated by methods such as PACE (Protein Science 1995; 4: 2411-2423).

[0251] [Table 5]

[0252] Sequence ID: 24 represents the Vhh2b-Nb36 Fc fusion for preparing the Vhh2b-Nb36 / Nb21-Vhh2g Fc heterodimer. In Table 5, the Vhh2b-Nb36 sequence is underlined and the Fc sequence is double-underlined.

[0253] Sequence ID: 25 represents the Nb21-Vhh2g Fc fusion for preparing the Vhh2b-Nb36 / Nb21-Vhh2g Fc heterodimer. In Table 5, the Nb21-Vhh2g sequence is underlined and the Fc sequence is double-underlined.

[0254] Example 14: The Vhh2b-Nb36 / Nb21-Vhh2g Fc fusion protein can induce stronger IL-2 receptor signaling in the presence of RBD compared to the absence of RBD.

[0255] Activation of the IL-2 receptor complex by the heterodimer Fc fusion of Vhh2b-Nb36 and Nb21-Vhh2g was more pronounced in HEK-Blue IL-2 cells in the presence of RBD compared to PDL1 expression.

[0256] RBD-IL2RA (SEQ ID NO: 11) or PDL1 (SEQ ID NO: 12) was first expressed in HEK-Blue IL-2 cells (InvivoGen, #hkb-il2) by electroporation using SF Cell Line 4D-Nucleofector X Kit L (Lonza, #V4XC-2024). After electroporation, these transfectants were seeded in 96-well plates and incubated overnight at 37°C in the presence of 5% CO2. These cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin. On day 1 after electroporation, cells were treated with Vhh2b-Nb36 / Nb21-Vhh2g Fc fusion protein (heterodimer of SEQ ID NOs: 24 and 25) or recombinant human IL-2 (Peprotech, #200-02), and incubated for another day at 37°C in the presence of 5% CO2. On day 2 after electroporation, activation of the IL-2 receptor complex was evaluated by measuring the optical density at 620 nm using a Multiskan® plate reader with Quanti-Blue solution (InvivoGen, #rep-qbs) (Figures 8A and 8B).

[0257] The Vhh2b-Nb36 / Nb21-Vhh2g Fc fusion protein induced stronger IL-2 receptor complex signaling in the presence of RBD compared to its absence (Figure 8A). Unlike conventional conjugations of recombinant IL-2 with targeting antibodies (Nature 2022; 610: 161-172), the Vhh2b-Nb36 / Nb21-Vhh2g Fc fusion not only showed stronger IL-2R signaling from lower concentrations compared to the absence of RBD, but also induced higher maximal STAT5 activation. This indicates that expression of the scaffold protein RBD is a condition for inducing strong IL-2 receptor complex signaling using the Vhh2b-Nb36 / Nb21-Vhh2g Fc fusion protein. On the other hand, direct administration of recombinant human IL-2, an IL-2R ligand, showed no selectivity between RBD-expressing HEK-Blue IL-2 cells and PDL1-expressing HEK-Blue IL-2 cells (Figure 8B).

[0258] Example 15: Expression and purification of VHH-scFv fusions or IL-2 mutants that bind to IL2Rα as a scaffold protein, and to IL-2Rβ and IL-2Rγ as receptor proteins.

[0259] The IL-2 mutant IL2_RETR was fused with a His tag sequence (SEQ ID NO: 26). VHH (Vhh2g), which can bind to human IL-2Rγ, was fused with the IL-2Rα binding domain BT942 scFv (SEQ ID NO: 27) or with Dac scFv (SEQ ID NO: 28). VHH (Vhh2b), which can bind to human IL-2Rβ, was ligated to BT942 scFv (SEQ ID NO: 29). The DNA encoding these proteins was cloned into mammalian expression vectors. These plasmids were transiently expressed using the Expi293 cell line (Thermo Fisher, Carlsbad, CA, USA). Protein purification was performed using protein A or immobilized metal ion affinity chromatography. The concentration of the purified protein was measured by its absorbance at 280 nm using a spectrophotometer. The antibody concentration was calculated using the extinction coefficient, which was determined by methods such as PACE, based on the obtained values ​​(Protein Science 1995; 4: 2411-2423).

[0260] [Table 6]

[0261] Sequence ID: 26 represents an IL-2 mutant that lacks binding to IL-2Rγ but retains binding to IL-2Rα and IL-2Rβ.

[0262] Sequence ID: 27 represents a fusion protein of the IL-2Rγ binding domain (Vhh2g) and the IL-2Rα binding domain (BT942 scFv). The binding of BT942 scFv to IL-2Rα does not compete with the binding of IL2_RETR to IL-2Rα. In Table 6, the sequence of BT942 scFv is underlined, and the sequence of Vhh2g is double-underlined.

[0263] Sequence ID: 28 represents a fusion protein of the IL-2Rγ binding domain (Vhh2g) and the IL-2Rα binding domain (Dac scFv). The binding of Dac scFv to IL-2Rα competes with the binding of IL2_RETR to IL-2Rα. In Table 6, the sequence of Dac scFv is underlined and the sequence of Vhh2g is double-underlined.

[0264] Sequence ID: 29 represents a fusion protein of the IL-2Rβ binding domain (Vhh2b) and the IL-2Rα binding domain (BT942 scFv). The binding of BT942 scFv to IL-2Rα does not compete with the binding of IL2_RETR to IL-2Rα. In Table 6, the sequence of BT942 scFv is underlined, and the sequence of Vhh2b is double-underlined.

[0265] Example 16: Activation of IL-2Rβ and IL-2Rγ by a mixture of IL-2 mutants that bind to IL-2Rα in a biparatopic manner as a target scaffold protein and to IL-2Rβ / γ as target receptor proteins, and a VHH-scFv fusion, is dependent on IL-2Rα binding.

[0266] The present invention's concept of conditional receptor signaling is further illustrated using IL-2Rα as a scaffold protein that brings IL-2Rβ and IL-2Rγ into proximity for conditional receptor signaling. This example also demonstrates that other non-antibody-binding proteins can also be used as scaffold protein-binding domains or receptor protein-binding domains. The structures of the quaternary complexes of IL-2 and IL-2Rα, IL-2Rβ, and IL-2Rγ have been described (Science 2005; 310: 1159-1163), and their structures and binding epitopes are shown in the RCSB Protein Data Bank (2B5I).

[0267] For example, IL2_RETR is a non-agonist IL-2 mutant that binds to IL-2Rα as a scaffold protein and to IL-2Rβ as a target receptor, but lacks binding activity to IL-2Rγ (Immunity 2015; 42: 826-838). Vhh2g-BT942 scFv is a fusion protein of VHH, which binds to IL-2Rγ, and BT942 scFv, which binds to IL-2Rα as a scaffold protein. The binding epitope of BT942 on IL2-Rα has been described (Scientific Reports 2021; 11: 22966), and its structure and binding epitope are shown in the RCSB Protein Data Bank (7F9W). Since BT942 binding does not affect the binding of IL-2 and IL-2Rα, IL2_RETR and BT942 scFv do not compete with each other for binding to IL-2Rα. Together, the mixture of IL2_RETR and Vhh2g-BT942 scFv can enable colocalization of IL-2Rα, IL-2Rβ, and IL-2Rγ, and induce IL-2R signaling in an IL-2Rα binding-dependent manner. A schematic diagram of the mechanism of IL-2Rα-dependent IL-2R activation by the mixture of IL2_RETR and Vhh2g-BT942 scFv is shown in Figure 9A.

[0268] HEK-Blue IL-2 cells (InvivoGen, #hkb-il2) were seeded in 96-well plates overnight at 37°C in the presence of 5% CO2. The cells were cultured in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin. On day 1 post-seeding, the cells were treated with or without 5 μg / mL daclizumab (RnD Systems, #MAB9927) at 37°C for 30 minutes in the presence of 5% CO2. IL-2Rα binding-dependent IL-2R activation was evaluated using daclizumab, an anti-IL-2Rα antibody capable of inhibiting the binding of IL2_RETR to IL-2Rα. A mixture of IL2_RETR (SEQ ID NO: 26) and Vhh2g-BT942 scFv (SEQ ID NO: 27) or recombinant human IL-2 (Peprotech, #200-02) was added, and the mixture was incubated for another day at 37°C / 5% CO2. On day 2 after seeding, the activation of the IL-2 receptor complex was evaluated by measuring the optical density at 620 nm using a Multiskan® plate reader with Quanti-Blue solution (InvivoGen, #rep-qbs) (Figures 9B and 9C).

[0269] As shown in Figures 9B and 9C, the combination of IL2_RETR and Vhh2g-BT942 scFv, which targets IL2Rα as a scaffold protein and IL-2Rβ and IL-2Rγ as receptor proteins, induced IL-2R signaling in HEK-Blue IL-2 cells constitutively expressing IL2Rα. Additionally, this IL-2R signaling induction was suppressed by treatment with daclizumab (5 μg / mL), an anti-IL-2Rα neutralizing antibody (Figure 9B). On the other hand, daclizumab treatment did not have a significant effect on recombinant IL-2-induced IL-2R signaling (Figure 9C). The significant inhibitory effect of daclizumab on IL-2R signaling suggests that IL-2R activation by IL2_RETR and Vhh2g-BT942 scFv is dependent on IL-2Rα binding.

[0270] Example 17: Activation of the IL-2 receptor complex by IL-2 variants or a mixture of VHH-VHH fusions targeting IL-2Rα as a scaffold protein is dependent on biparatopic binding to IL-2Rα.

[0271] In this case, to demonstrate that biparatopic binding to the scaffold protein IL-2Rα is a condition for IL-2Rβ / γ activation, the effect of combinations of proteins that can biparatopically bind to IL-2Rα was compared to combinations that cannot biparatopically bind to IL-2Rα.

[0272] HEK-Blue IL-2 cells (InvivoGen, #hkb-il2) were seeded in 96-well plates overnight at 37°C in the presence of 5% CO2. The cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin. After seeding, these cells were treated with various mixtures of recombinant IL2_RETR and / or VHH-scFv fusions (SEQ ID NOs: 26-29) and incubated at 37°C / 5% CO2 for 3 days. Activation of the IL-2 receptor complex was evaluated by measuring the optical density at 620 nm using a Multiskan® plate reader with Quanti-Blue solution (InvivoGen, #rep-qbs) (Figure 10).

[0273] Figure 10 shows that induction of IL-2R signaling by IL-2 mutants or VHH-ScFv mixtures targeting IL-2Rα as the scaffold protein and IL-2Rβ / γ as the receptor proteins was dependent on biparatopic binding to IL-2Rα. In HEK-Blue IL-2 cells constitutively expressing IL-2Rα, biparatopic binding combinations of IL2_RETR and Vhh2g-BT942 scFv, or Vhh2b-BT942 scFv and Vhh2g-Dac scFv, induced IL-2R signaling, but monoparatopic binding combinations of IL2_RETR and Vhh2g-Dac scFv, or Vhh2b-BT942 scFv and Vhh2g-BT942 scFv, did not induce IL-2R signaling. Dac scFv is a single-chain variable fragment version of daclizumab that binds competitively to IL-2Rα with IL2_RETR, but not to BT942 scFv.

[0274] Example 18: Expression and purification of a VHH-scFv fusion that binds to PD1 as the target scaffold protein and to the IL-2 receptor as the target receptor protein.

[0275] VHH proteins capable of binding to human IL-2Rβ (Vhh2b or Vhh2b3) or human IL-2Rγ (Vhh2g or Vhh2g6) were ligated to human PD1-binding domains, namely Nivo scFvLH, Nivo scFvHL, NB01a scFvLH, or NB01a scFvHL (SEQ ID NOs: 30-36). The DNA encoding these proteins was cloned into mammalian expression vectors. These plasmids were transiently expressed using the Expi293 cell line (Thermo Fisher, Carlsbad, CA, USA). Antibody purification was performed using Protein A. The absorbance of the purified protein at 280 nm was measured using a spectrophotometer. From the obtained values, the antibody concentration was calculated using the extinction coefficient calculated by methods such as PACE (Protein Science 1995; 4: 2411-2423).

[0276] [Table 7] TIFF2026510624000019.tif181170TIFF2026510624000020.tif105170

[0277] Sequence ID: 30 represents a fusion protein of the IL-2Rγ binding domain (Vhh2g) and the PD1 binding domain (Nivo scFvLH). In Table 7, the sequence of Nivo scFvLH is underlined, and the sequence of Vhh2g is double-underlined.

[0278] Sequence ID: 31 represents a fusion protein of the IL-2Rγ binding domain (Vhh2g) and the PD1 binding domain (Nivo scFvHL). In Table 7, the sequence of Nivo scFvHL is underlined and the sequence of Vhh2g is double-underlined.

[0279] Sequence ID: 32 represents a fusion protein of the IL-2Rβ binding domain (Vhh2b) and the PD1 binding domain (NB01a scFvLH). In Table 7, the sequence of NB01a scFvLH is underlined, and the sequence of Vhh2b is double-underlined.

[0280] Sequence ID: 33 represents the IL-2Rβ binding domain (Vhh2b3) and the PD1 binding domain (NB01a scFvLH). In Table 7, the sequence of NB01a scFvLH is underlined, and the sequence of Vhh2b3 is double-underlined.

[0281] Sequence ID: 34 represents a fusion protein of the IL-2Rγ binding domain (Vhh2g6) and the PD1 binding domain (Nivo scFvHL). In Table 7, the sequence of Nivo scFvHL is underlined and the sequence of Vhh2g6 is double-underlined.

[0282] Sequence ID: 35 represents a fusion protein of the IL-2Rγ binding domain (Vhh2g) and the PD1 binding domain (NB01a scFvLH). In Table 7, the sequence of NB01a scFvLH is underlined, and the sequence of Vhh2g is double-underlined.

[0283] Sequence ID: 36 represents a fusion protein of the IL-2Rγ binding domain (Vhh2g) and the PD1 binding domain (NB01a scFvHL). In Table 7, the sequence of NB01a scFvHL is underlined, and the sequence of Vhh2g is double-underlined.

[0284] Sequence ID: 37 represents human PD1.

[0285] Example 19: Induction of IL-2R signaling by a mixture of recombinant VHH-scFv fusions that bind to PD1 in a biparatopic manner and also bind to the IL-2 receptor protein is dependent on PD1 expression.

[0286] The present invention's concept of conditional receptor signaling is further illustrated using PD1 as the scaffold protein and IL-2Rβ and IL-2Rγ as the receptor proteins for conditional receptor signaling.

[0287] For example, Nivo scFvLH or Nivo scFvHL and NB01a scFvLH are biparatopic antibodies that recognize different epitopes of PD1 and can bind to PD1 simultaneously. The epitope on PD1 to which NB01a binds has also been described (J. Exp. Med 2019; 216: 1525-1541), and its structure and binding epitope are shown in the RCSB Protein Data Bank (6HIG). Since the binding of Nivo scFvLH or Nivo scFvHL does not compete with the binding of NB01a scFvLH to PD1, a mixture of these proteins may enable co-localization of PD1, IL-2Rβ, and IL-2Rγ, thereby inducing PD1-binding-dependent IL-2R signaling. The epitope on PD1 to which Nivo binds has been described (Nature Communications 2017; 08: 14369), and its structure and binding epitope are shown in the RCSB Protein Data Bank (5WT9).

[0288] Human PD1 (SEQ ID NO: 37) or PDL1 (SEQ ID NO: 12) was first expressed in HEK-Blue IL-2 cells (InvivoGen, #hkb-il2) by electroporation using SF Cell Line 4D-Nucleofector X Kit L (Lonza, #V4XC-2024). After electroporation, these transfectants were seeded in 96-well plates and incubated overnight at 37°C / 5% CO2. These cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin. On day 1 after electroporation, these cells were treated with various mixtures of recombinant VHH-scFv fusions (SEQ ID NOs: 30-33) or recombinant human IL-2 (Peprotech, #200-02), and further incubated for another day at 37°C / 5% CO2. Two days after electroporation, the activation of the IL-2 receptor complex was evaluated by measuring the optical density at 620 nm using a Multiskan® plate reader with Quanti-Blue solution (InvivoGen, #rep-qbs) (Figures 11A and 11B).

[0289] As shown in Figures 11A and 11B, a mixture of VHH-scFv fusions that bind to PD1 as a scaffold protein in a biparatopic manner and to IL-2Rβ or IL-2Rγ as a target receptor protein can induce IL-2R signaling under conditions of PD1 scaffold protein expression. In the absence of the PD1 scaffold protein, IL-2R signaling was not induced.

[0290] Example 20: Activation of the IL-2 receptor complex by a mixture of VHH-VHH fusions targeting PD1 as a scaffold protein depends on biparatopic binding to PD1.

[0291] In this case, to demonstrate that biparatopic binding to the scaffold protein PD1 is a condition for IL-2Rβ / γ activation, the effect of combinations of VHH-VHH fusions that can biparatopically bind to PD1 was compared to combinations of VHH-VHH fusions that cannot biparatopically bind to PD1.

[0292] Human PD1 (SEQ ID NO: 37) was first expressed in HEK-Blue IL-2 cells (InvivoGen, #hkb-il2) by electroporation using SF Cell Line 4D-Nucleofector X Kit L (Lonza, #V4XC-2024). After electroporation, these transfectants were seeded in 96-well plates and incubated overnight at 37°C / 5% CO2. These cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin. On day 1 after electroporation, these cells were treated with various mixtures of recombinant VHH-scFv fusions (SEQ ID NOs: 30, 32-36) and incubated for another day at 37°C / 5% CO2. Two days after electroporation, the activation of the IL-2 receptor complex was evaluated by measuring the optical density at 620 nm using a Multiskan® plate reader with Quanti-Blue solution (InvivoGen, #rep-qbs) (Figure 12).

[0293] As shown in Figure 12, IL-2R signaling induction by a mixture of VHH-scFv fusions that bind to PD1 as a scaffold protein and to IL-2Rβ or IL-2Rγ as target receptor proteins depends on biparatopic binding to PD1. In PD1-expressing HEK-Blue IL-2 cells, biparatopic PD1 binding combinations using Nivo scFvHL or Nivo scFvLH and NB01a scFvHL and NB01a scFvLH as PD1-binding domains induced IL-2R signaling, while monoparatopic binding combinations using only NB01a scFvHL or NB01a scFvLH as the PD1-binding domain did not induce IL-2R signaling.

[0294] Example 21: Expression and purification of anti-PD1 antibody (Ab)

[0295] DNA encoding the heavy chain (Hch) and light chain (Lch) (SEQ ID NOs: 38-59) of anti-PD1 antibodies was cloned into mammalian expression vectors. To prepare purified anti-PD1 antibodies, plasmids expressing the corresponding Hch and Lch were transiently expressed using the Expi293 cell line (Thermo Fisher, Carlsbad, CA, USA). The Hch and Lch pairs for each antibody are listed in Table 8. Protein purification was performed using Protein A. The absorbance of the purified protein at 280 nm was measured using a spectrophotometer. From the obtained values, the antibody concentration was calculated using the extinction coefficient calculated by methods such as PACE (Protein Science 1995; 4: 2411-2423). [Table 8] TIFF2026510624000022.tif231170TIFF2026510624000023.tif231170

[0296] Example 22: Binding activity of anti-PD1 Ab

[0297] The binding activity of anti-PD1 Ab was evaluated by flow cytometry. Anti-PD1 Ab (10 μg / mL) and human PD1-expressing cell lines (NFAT-luc2 / PD1 Jurkat cell line, Promega, #CS187102) were incubated in staining buffer (Biolegend, #420201) for 20 minutes. Unbound Ab was removed by washing twice with staining buffer. Human IgG Ab bound to PD1 was detected using Goat F(ab')2 anti-Human IgG, Mouse ads-PE (Southern Biotech, #2043-09). Data acquisition was performed using LSRFortessa X-20 (Becton Dickinson) (Figure 13).

[0298] As shown in Figure 13, all anti-PD1 Ab strains showed clear binding to human PD1-expressing cell lines.

[0299] Example 23: Neutralizing activity of anti-PD1 Ab

[0300] The neutralizing activity of anti-PD1 Ab was evaluated using a PD1 / PD-L1 inhibition assay system (Promega, #CS187109). PD-L1+ CHOK1 cells (Promega, #CS187108) were seeded in a 384-well white plate overnight at 37°C in the presence of 5% CO2. The cells were cultured in RPMI1640 containing 5% fetal bovine serum and 1% penicillin-streptomycin. On day 1 post-seeding, anti-PD1 Ab (final concentration 5 μg / mL) and PD1+ Jurkat cells (NFAT-luc2 / PD1 Jurkat cell line, Promega, #CS187102) were added to the PD-L1+ CHOK1 seeded wells and incubated for a further 6 hours. Nivolumab (Selleck, #A2002, final concentration 50 μg / mL) was used as a positive control. Six hours later, TCR activation was detected using the Bio-Glo luciferase assay system (Promega, #G7940) and the GloMax multiplate reader (Promega, #GM3500) (Figure 14).

[0301] As shown in Figure 14, PDA0041, PDA0070, PDA0107, PDA0138, GY5, and GY14 demonstrated PD1 inhibitory activity, while PDA0044, PDA0129, PDC0037, PDC0053, and PDE0171 did not.

[0302] Example 24: Expression and purification of a VHH-IgG fusion protein that binds to PD1 as the target scaffold protein and to IL-2Rβ and IL-2Rγ as target receptor proteins. Anti-IL-2RγVHH or anti-IL-2RβVHH were fused to the Lch (SEQ ID NOs: 60-65, 71-76) or Hch (SEQ ID NOs: 66-70, 77-81) of neutralizing anti-PD1 IgG Ab via a linker. Anti-IL-2RβVHH or IL-2RγVHH were also fused to anti-PD1 scFv via a linker (SEQ ID NOs: 82, 83). The DNA encoding these proteins was cloned into mammalian expression vectors. To prepare purified Ab, plasmids expressing the corresponding Hch and Lch were transiently expressed using the Expi293 cell line (Thermo Fisher, Carlsbad, CA, USA). The Hch and Lch pairs for each antibody are listed in Table 9. Protein purification was performed using Protein A. The absorbance of the purified protein at 280 nm was measured using a spectrophotometer. The antibody concentration was calculated using the extinction coefficient, which was determined by methods such as PACE, based on the obtained values ​​(Protein Science 1995; 4: 2411-2423). [Table 9] TIFF2026510624000025.tif233170TIFF2026510624000026.tif233170TIFF202 6510624000027.tif233170TIFF2026510624000028.tif233170TIFF20265106240 00029.tif233170TIFF2026510624000030.tif233170TIFF2026510624000031.t if233170TIFF2026510624000032.tif233170TIFF2026510624000033.tif233170

[0303] Example 25: Combinations of neutralizing anti-PD1 Ab and non-neutralizing anti-PD1 Ab fused with anti-IL-2RβVHH or anti-IL-2RγVHH efficiently induce IL-2 receptor activation in PD1+ HEK-Blue IL-2 cells.

[0304] Human PD1 (SEQ ID NO: 37) or PDL1 (SEQ ID NO: 12) was first expressed in HEK-Blue IL-2 cells (InvivoGen, #hkb-il2) by electroporation using SF Cell Line 4D-Nucleofector X Kit L (Lonza, #V4XC-2024). After electroporation, these transfectants were seeded in 384-well plates and incubated overnight at 37°C / 5% CO2. These cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin. On day 1 after electroporation, these cells were further treated for 1 day at 37°C / 5% CO2 with various mixtures of neutralizing anti-PD1 Ab fused with anti-IL-2RγVHH (final concentration 12.5 nM) and non-neutralizing anti-PD1 Ab fused with anti-IL-2RβVHH (final concentration 12.5 nM). Recombinant human IL-2 (Peprotech, #200-02) was used as a positive control. On day 2 after electroporation, activation of the IL-2 receptor complex was evaluated by measuring the optical density at 620 nm using Quanti-Blue solution (InvivoGen, #rep-qbs) (Figures 15A and B).

[0305] As shown in Figures 15A and B, mixtures of neutralizing anti-PD1 Ab and non-neutralizing anti-PD1 Ab containing an IL2Rβ-binding domain or an IL2Rγ-binding domain (Vhh2b or Vhh2g) efficiently induced IL-2 receptor activation in PD1+ HEK-Blue IL-2 cells, but not in the absence of PD1 expression. These data suggest that it is possible to identify combinations of neutralizing anti-PD1 Ab and non-neutralizing anti-PD1 Ab that can demonstrate PD1-dependent IL-2R agonist activity. This is an effective method for screening non-competitive or biparatopic conjugates to construct combinations of target-binding molecules or protein complexes of this application.

[0306] Example 26: Combinations of neutralizing anti-PD1 Ab and non-neutralizing anti-PD1 Ab fused with anti-IL-2RβVHH or anti-IL-2RγVHH induce IL-2 receptor activation in PD1+ primary T cells, but not in PD1-negative NK92 cells.

[0307] Human CD4+ T cells were isolated from human PBMCs (STEMCELL, #70025.2) using the EasySep® Human CD4+ T Cell Isolation Kit (STEMCELL, #17952). After isolation, the CD4+ T cells were treated with plate-coated anti-CD3 Ab (Biolegend, #317347, 5 μg / mL) and anti-CD28 Ab (Biolegend, #302943, 5 μg / mL) and recombinant human IL-2 (Peprotech, #200-02) at 37°C / 5% CO2 for 3 days. Penicillin / streptomycin (GIBCO, #15140122), GlutaMax (GIBCO, #35050061), and CTS Optimizer T cell expansion SFM (GIBCO, #A1048501) containing 10% fetal bovine serum were used as the culture medium. After 3 days of culture, CD4+ T cells were collected, washed twice with culture medium, and rested overnight in culture medium alone. After resting, T cells were stimulated at 37°C for 30 minutes with a mixture of Vhh2g-PDA0041 and Vhh2b-PDE0171, Vhh2g-GY5 and Vhh2b-PDA0129, or Vhh2b-Nivo scFv and Vhh2g4-NB01a scFv. Recombinant human IL-2 (Peprotech, #200-02) was used as a positive control, and NK92 cells (ATCC, #CRL-2407) were used as PD1-negative IL-2 responsive cells. These cells were immediately fixed with CytoFix (BD Biosciences, #554655) at room temperature for 20 minutes. The cells were washed once with staining buffer (Biolegend, #420201), and then chilled permeabilization buffer (BD Biosciences, #558050) was added to the sample and incubated on ice for 30 minutes. After washing the cells twice with staining buffer, they were stained with Alexa Fluor® 647 mouse anti-Stat5 (pY694) (BD Biosciences, #562076) at room temperature for at least 60 minutes. The cells were washed once with staining buffer before analysis. Data acquisition was performed using an LSRFortessa X-20 (Becton Dickinson) (Figures 16A and B).

[0308] As shown in Figures 16A and B, Vhh2g-PDA0041 and Vhh2b-PDE0171, Vhh2g-GY5 and Vhh2b-PDA0129, or mixtures of Vhh2b-Nivo scFv and Vhh2g4-NB01a scFv induced significant STAT5 activation in activated human CD4+ T cells that highly express PD1, but not in NK92 cells that are negative for PD1 expression. These data suggest that combinations of neutralizing anti-PD1 Ab and non-neutralizing anti-PD1 Ab fused with anti-IL-2Rβ-binding domains or anti-IL-2Rγ-binding domains can induce PD1-dependent IL-2 receptor activation not only in reporter cells but also in primary T cells.

[0309] Example 27: Expression and purification of a VHH-IgG fusion protein that binds to PD1 as a scaffold protein and to IL-2Rβ and IL-2Rγ as receptor proteins.

[0310] Anti-IL-2RβVHH, specifically Vhh2b1, Vhh2b3, or Vhh2b4, was fused to the Hch of PDE0171 using a linker (SEQ ID NOs: 84-86), and anti-IL-2RγVHH, specifically Vhh2g1, Vhh2g2, Vhh2g4, Vhh2g6, Vhh2g8, or Vhh2g10, was fused to the Lch of PDA0041 (SEQ ID NOs: 87-92). These anti-IL-2Rβ / γVHH proteins are described in WO2022 / 032006A2, WO2022 / 031884A2, or Cell (2022; 185: 1414-1430.e19). The DNA encoding these proteins was cloned into mammalian expression vectors. To prepare purified antibodies, plasmids expressing the corresponding Hch and Lch were transiently expressed using the Expi293 cell line (Thermo Fisher, Carlsbad, CA, USA). The Hch and Lch pairs for each antibody are listed in Table 10. Protein purification was performed using Protein A. The absorbance of the purified protein at 280 nm was measured using a spectrophotometer. From the obtained values, the antibody concentration was calculated using the extinction coefficient calculated by methods such as PACE (Protein Science 1995; 4: 2411-2423). [Table 10] TIFF2026510624000035.tif231170

[0311] Example 28: A mixture of anti-PD1 Ab (PDA0041) fused with various IL-2Rγ binding domains and anti-PD1 Ab (PDE00171) fused with various IL-2Rβ binding domains induces IL-2 receptor activation in PD1+ HEK-Blue IL-2 cells.

[0312] Human PD1 (SEQ ID NO: 37) or PDL1 (SEQ ID NO: 12) was first expressed in HEK-Blue IL-2 cells (InvivoGen, #hkb-il2) by electroporation using SF Cell Line 4D-Nucleofector X Kit L (Lonza, #V4XC-2024). After electroporation, these transfectants were seeded in 384-well plates and incubated overnight at 37°C / 5% CO2. These cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin. On day 1 after electroporation, these cells were treated with various mixtures of anti-PD1 Ab (PDA0041) fused with various IL-2Rγ-binding domains (Vhh2g, Vhh2g1, Vhh2g2, Vhh2g4, Vhh2g6, Vhh2g8, or Vhh2g10) and anti-PD1 Ab (PDE0171) fused with various IL-2Rβ-binding domains (Vhh2b, Vhh2b1, Vhh2b3, or Vhh2b4), and further incubated at 37°C / 5% CO2 for another day. The concentration of each Ab was 12.5 nM. Recombinant human IL-2 (Peprotech, #200-02) was used as a positive control. Three days after electroporation, the activation of the IL-2 receptor complex was evaluated by measuring the optical density at 620 nm using Quanti-Blue solution (InvivoGen, #rep-qbs) (Figures 17A and B).

[0313] As shown in Figures 17A and B, mixtures of anti-PD1 Ab(PDA0041) fusion proteins with various IL-2Rγ binding domains and anti-PD1 Ab(PDEA00171) fusion proteins with various IL-2Rβ binding domains induced IL-2 receptor activation in PD1+ HEK-Blue IL-2 cells, but not in the absence of PD1 expression. These data suggest that various IL-2Rβ / γ binding domains can be used to induce PD1-dependent IL-2 receptor activation.

[0314] Example 29: Expression and purification of scFv-IgG or VHH-IgG fusion proteins that bind to PD1 as a scaffold protein and to IL-2Rβ and IL-2Rγ as receptor proteins.

[0315] The anti-PD1 VHH (Vhhpc13) was fused to the Hch of anti-IL2Rγ (AM3) using a linker (SEQ ID NO: 93), and the anti-PD1 scFv (PDA0129 scFv) was fused to the Hch of anti-IL2Rβ (AL1, AL2, AL3, AL4, or AL5) (SEQ ID NO: 94). These Hch fusion proteins and their corresponding Lch (SEQ ID NOs: 95-100) were cloned into mammalian expression vectors. These anti-IL-2Rβ or anti-IL-2Rγ antibodies were reported in WO2023 / 139293A1. To prepare purified antibodies, plasmids expressing the corresponding Hch and Lch were transiently expressed using the Expi293 cell line (Thermo Fisher, Carlsbad, CA, USA). The Hch and Lch pairs for each antibody are listed in Table 11. Protein purification was performed using Protein A. The absorbance of purified protein at 280 nm was measured using a spectrophotometer. The antibody concentration was calculated from the obtained values ​​using the extinction coefficient, which was determined by methods such as PACE (Protein Science 1995; 4: 2411-2423). [Table 11]

[0316] Example 30: A mixture of Vhhpc13-AM3 and PDA0129 scFv-AL1, 2, 3, 4, or 5, which binds to PD1 as the target scaffold protein and to IL-2Rβ or IL-2Rγ as the target receptor protein, induces IL-2R activation in HEK-Blue IL-2 cells.

[0317] Human PD1 (SEQ ID NO: 37) or PDL1 (SEQ ID NO: 12) was first expressed in HEK-Blue IL-2 cells (InvivoGen, #hkb-il2) by electroporation using SF Cell Line 4D-Nucleofector X Kit L (Lonza, #V4XC-2024). After electroporation, these transfectants were seeded in 384-well plates and incubated overnight at 37°C / 5% CO2. These cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin. On day 1 after electroporation, these cells were treated with various mixtures of fusion proteins (final concentration 25 nM) that bind to PD1 as a scaffold protein and IL-2Rβ or IL-2Rγ as a receptor protein, and incubated for another day at 37°C / 5% CO2. Recombinant human IL-2 (Peprotech, #200-02) was used as a positive control. Two days after electroporation, the activation of the IL-2 receptor complex was evaluated by measuring the optical density at 620 nm using Quanti-Blue solution (InvivoGen, #rep-qbs) (Figures 18A and B).

[0318] As shown in Figures 18A and B, mixtures of Vhhpc13-AM3 with PDA0129 scFv-AL1, PDA0129 scFv-AL2, PDA0129 scFv-AL3, PDA0129 scFv-AL4, or PDA0129 scFv-AL5 induced IL-2 receptor activation in PD1+ HEK-Blue IL-2 cells, but not in the absence of PD1 expression. These data suggest that other binding domains, such as Fab, as well as anti-IL-2Rβ / γVHH, can also be used to induce PD1-dependent IL-2 receptor activation.

[0319] Example 31: Expression and purification of scFv-VHH fusion proteins that bind to CD25 as the target scaffold protein and to IL-2Rβ and IL-2Rγ as target receptor proteins.

[0320] Anti-CD25 scFv was fused with VHH (vhh2g) capable of binding to human IL-2Rγ or VHH (Vhh2b) capable of binding to human IL-2Rβ (SEQ ID NOs: 101-109). RG6292 scFv (Front. Oncol. 2023; 13: 1150149), BT942 scFv (Scientific Reports 2021; 11: 22966), or 7G7 / B6 scFv (Cancer Research 2000; 60: 6977-6984) were used as non-neutralizing anti-CD25 antibodies, while Gen scFv (US8,961,968 B2) or Dac scFv (Biomedicines 2019; 7: 18) were used as neutralizing anti-CD25 antibodies. The DNA encoding these proteins was cloned into mammalian expression vectors. These plasmids were transiently expressed using the Expi293 cell line (Thermo Fisher, Carlsbad, CA, USA). Protein purification was performed using protein A or immobilized metal ion affinity chromatography. The absorbance of the purified protein at 280 nm was measured using a spectrophotometer. From the obtained values, the antibody concentration was calculated using the extinction coefficient calculated by methods such as PACE (Protein Science 1995; 4: 2411-2423). [Table 12] TIFF2026510624000038.tif232170

[0321] Example 32: A mixture of neutralizing anti-CD25 scFv containing an IL-2Rβ / γ binding domain and non-neutralizing anti-CD25 scFv containing an IL-2Rβ / γ binding domain induced IL-2 receptor activation in activated primary CD4+ T cells.

[0322] Human CD4+ T cells were isolated from human PBMCs (STEMCELL, #70025.2) using the EasySep® Human CD4+ T Cell Isolation Kit (STEMCELL, #17952). After isolation, the CD4+ T cells were treated with plate-coated anti-CD3 Ab (Biolegend, #317347, 5 μg / mL) and anti-CD28 Ab (Biolegend, #302943, 5 μg / mL) and recombinant human IL-2 (Peprotech, #200-02) at 37°C / 5% CO2 for 3 days. Penicillin / streptomycin (GIBCO, #15140122), GlutaMax (GIBCO, #35050061), and CTS Optimizer T cell expansion SFM (GIBCO, #A1048501) containing 10% fetal bovine serum were used as culture media. After 3 days of culture, CD4+ T cells were collected, washed twice with culture medium, and rested overnight under culture medium conditions. After resting, T cells were stimulated at 37°C for 25 minutes with a mixture of VHH-scFv fusion protein (final concentration 25 nM) that binds to CD25 as the target scaffold protein and IL-2Rβ or IL-2Rγ as the target receptor protein. Immediately after stimulation, these cells were fixed at room temperature for 20 minutes with CytoFix (BD Biosciences, #554655). Cells were washed once with staining buffer (Biolegend, #420201), then chilled permeabilization buffer (BD Biosciences, #558050) was added to the cells and incubated on ice for 30 minutes. Cells were washed twice with staining buffer, and Alexa Fluor® 647 mouse anti-Stat5 (pY694) (BD Biosciences, #562076) was added and incubated at room temperature for at least 60 minutes. Cells were washed once with staining buffer before analysis. Data acquisition was performed using an LSRFortessa X-20 (Becton Dickinson) (Figure 19).

[0323] As shown in Figure 19, a combination of neutralizing anti-CD25 scFv and non-neutralizing anti-CD25 scFv, namely Vhh2g-Gen scFvHL / LH, and a mixture of Vhh2b-RG6292 scFv, Vhh2b-BT942 scFv, or Vhh2b-7G7 / B6 scFv, induced significant STAT5 activation in activated CD4+ T cells. Similarly, a combination of neutralizing anti-CD25 scFv and non-neutralizing anti-CD25 scFv, namely Vhh2g-BT942 scFvHL / LH, and a mixture of Vhh2b-Gen scFv or Vhh2b-Dac scFv, also induced significant STAT5 activation in activated CD4+ T cells. Again, this demonstrates that identifying combinations of neutralizing and non-neutralizing agents for scaffold proteins is an effective method for screening non-competitive or biparatopic conjugates to construct combinations of target-binding molecules or protein complexes of this application.

[0324] Example 33: Expression and purification of a VHH-scFv fusion protein that binds to PDL1 as the target scaffold protein and to CSF2RA or CSF2RB as the target receptor protein.

[0325] Linkers were used to fuse the anti-PDL1 VHH, VhhPL1, with anti-CSF2RA scFv (116.08 scFv, 116.18 scFv) (SEQ ID NOs: 110-113), and another anti-PDL1 VHH, VhhPL1-1, with anti-CSF2RB scFv (131.16 scFv, 131.B2 scFv) (SEQ ID NOs: 114-117). These anti-CSF2RA Abs or anti-CSF2RB Abs were reported in WO2023 / 027177A1. The DNA encoding these proteins was cloned into mammalian expression vectors. These plasmids were transiently expressed using the Expi293 cell line (Thermo Fisher, Carlsbad, CA, USA). Protein purification was performed using protein A or immobilized metal ion affinity chromatography. The absorbance of purified protein at 280 nm was measured using a spectrophotometer. The antibody concentration was calculated from the obtained values ​​using the extinction coefficient, which was determined by methods such as PACE (Protein Science 1995; 4: 2411-2423). [Table 13] TIFF2026510624000040.tif232170

[0326] Example 34: A mixture of VHH-scFv fusion proteins that bind to PDL1 as a scaffold protein and to CSF2RA or CSF2RB as a target receptor protein induces CSF2RA / B activation in HEK-Blue GM-CSF cells.

[0327] Human PDL1 (SEQ ID NO: 12) or RBD-IL2RA (SEQ ID NO: 11) was first expressed in HEK-Blue GM-CSF cells (InvivoGen, #hkb-gmcsfr) by electroporation using SF Cell Line 4D-Nucleofector X Kit L (Lonza, #V4XC-2024). After electroporation, these transfectants were seeded in 96-well plates and incubated overnight at 37°C / 5% CO2. These cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin. On day 1 after electroporation, these cells were treated for an additional day at 37°C / 5% CO2 with various mixtures of VHH-scFv fusion proteins (SEQ ID NOs: 110-113) capable of binding to PDL1(VHH) and CSF2RA(scFv), and VHH-scFv fusion proteins (SEQ ID NOs: 114-117) capable of binding to PDL1(VHH) and CSF2RB(scFv). The final concentration of each Ab was 10 nM. On day 2 after electroporation, the activation of GM-CSF receptors, CSF2RA and CSF2RB, was evaluated by measuring the optical density at 620 nm using Quanti-Blue solution (InvivoGen, #rep-qbs) (Figures 20A and B).

[0328] As shown in Figures 20A and B, a mixture of VHH-scFv fusion proteins binding to PDL1 as the target scaffold protein and to CSF2RA or CSF2RB as the target receptor protein induced significant GM-CSF receptor activation in the presence of PDL1 expression, but not in the absence of PDL1. These data suggest that not only the IL-2 receptor, but also the GM-CSF receptor or other cytokine receptors can be activated by scaffold-dependent mechanisms.

[0329] Example 35: Expression and purification of a VHH-Fab fusion protein that binds to RBD as the target scaffold protein and to cMET as the target receptor protein.

[0330] VHH (Nb21 or Nb36) capable of binding to SARS-CoV-2 RBD was fused to the Lch of anti-human cMET Fab (SEQ ID NO: 119, 120). The DNA encoding these VHH-Lch fusion proteins and the Hch of anti-human cMET Fab (SEQ ID NO: 118) was cloned into mammalian expression vectors. Anti-cMET Fab was reported in the Protein Data Bank (PDB: 6I04). To prepare purified antibodies, plasmids expressing the corresponding Hch and Lch were transiently expressed using the Expi293 cell line (Thermo Fisher, Carlsbad, CA, USA). The Hch and Lch pairs for each antibody are listed in Table 14. Antibody purification was performed using protein A or immobilized metal ion affinity chromatography. The absorbance of the purified antibodies at 280 nm was measured using a spectrophotometer. The antibody concentration was calculated using the extinction coefficient, which was determined by methods such as PACE, based on the obtained values ​​(Protein Science 1995; 4: 2411-2423). [Table 14]

[0331] Example 36: Activation of the cMET receptor by a mixture of VHH-Fab fusions that bind to RBD and cMET depends on biparatopic binding to RBD.

[0332] Saos-2 cells (ATCC, #HTB-85) were used and maintained in RPMI1640 supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin. Saos-2 cells were seeded in 96-well plates and stimulated for 2 days with VHH-Fab fusion proteins (Nb21-Met6I04 Fab, Nb36-Met6I04 Fab) that bind to RBD as the target scaffold protein and to cMET as the target receptor protein, in the presence of recombinant SARS-CoV-2 spike RBD protein (R&D systems, #10523-CV-100, final concentration 640 pM). Recombinant human HGF protein (R&D systems, #294-HGN / CF) was used as a positive control. Two days after stimulation, the culture supernatant was collected, and IL-11 induced by cMET activation was detected using the human IL-11 DuoSet ELISA kit (R&D systems, #DY218) (Figures 21A, 21B, and 21C).

[0333] As shown in Figures 21A, 21B, and 21C, a mixture of VHH-Fab fusion proteins of anti-RBD and anti-cMET Fab, which bind to RBD in a biparatopic manner, induced significant cMET activation in the presence of recombinant SARS-CoV-2 spike RBD protein (Figure 21A), but not monoparatopic Ab (Figures 21B and 21C). These data suggest that not only the IL-2 receptor, but also other cytokine receptors can be activated by scaffold-dependent mechanisms.

[0334] Example 37: Expression and purification of bispecific antibodies that bind to MUC1 or CEA as the target scaffold protein and to FZD or LRP as the target receptor protein.

[0335] DNA encoding Hch and Lch of R2M3-R2M3 (anti-FZD Ab, SEQ ID NO: 121, 123), Hch and Lch of AR20.5 (anti-MUC1 Ab, SEQ ID NO: 124, 127), and Hch and Lch of CEA.Mab3 (anti-CEA Ab, SEQ ID NO: 126, 128) was cloned into mammalian expression vectors. DNA encoding Vhhlrp36-Vhhlrp36 (anti-LRP Ab, SEQ ID NO: 122) and CEA.VHH (anti-CEA Ab, SEQ ID NO: 125) was also cloned into mammalian expression vectors. To prepare purified Abs, plasmids expressing the corresponding Hch and Lch were transiently expressed using the Expi293 cell line (Thermo Fisher, Carlsbad, CA, USA). The Hch and Lch pairs for each antibody are listed in Table 15. From these purified antibodies, bispecific antibodies against FZD and MUC1 (R2M3-R2M3 / / AR20.5), or bispecific antibodies against FZD and CEA (R2M3-R2M3 / / CEA.VHH, R2M3-R2M3 / / CEA.MAb3) were prepared using the Fab arm exchange technique (as described in WO2015 / 046467). Similarly, bispecific antibodies against LRP and MUC1 (Vhhlrp36-Vhhlrp36 / / AR20.5), or bispecific antibodies against LRP and CEA (Vhhlrp36-Vhhlrp36 / / CEA.VHH, Vhhlrp36-Vhhlrp36 / / CEA.MAb3) were also prepared using the Fab arm exchange technique. The absorbance of the purified antibodies at 280 nm was measured using a spectrophotometer. The antibody concentration was calculated using the extinction coefficient, which was determined by methods such as PACE, based on the obtained values ​​(Protein Science 1995; 4: 2411-2423). [Table 15] TIFF2026510624000043.tif232170

[0336] Example 38: A mixture of bispecific Abs that bind to MUC1 and Wnt receptors (FZD or LRP) induced Wnt receptor activation in the presence of MUC1 expression.

[0337] MUC1 (SEQ ID NO: 129) or RBD-IL2RA (SEQ ID NO: 11) was first expressed in HEK293 STF cells (ATCC, #CRL-3249) by electroporation using SF Cell Line 4D-Nucleofector X Kit L (Lonza, #V4XC-2024). These transfectants were seeded in white 384-well plates and incubated overnight at 37°C / 5% CO2 after adding 200 nM RSPO1 protein (in-house, #PPU5200) and 50 nM LGK974 (Cayman Chemical, #14072). These cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) F-12, HEPES, supplemented with 20% fetal bovine serum and 200 μg / mL Geneticin. On day 1 after electroporation, these cells were treated with a mixture of bispecific antibodies (R2M3-R2M3 / / AR20.5 and Vhhlrp36-Vhhlrp36 / / AR20.5, each at a final concentration of 25 nM) that bind to MUC1 as the target scaffold protein and to the Wnt receptor protein (FZD or LRP) as the target receptor protein, and were further incubated at 37°C / 5% CO2 for another day. On day 2 after electroporation, Wnt signaling activation was evaluated using the ONE-Glo luciferase assay system (Promega, #E6120) (Figure 22). AR20.5 recognizes the repeat sequence of the MUC1 protein (Hybrid Hybridomics 2001; 20: 313-24), and therefore this antibody can bind to multiple sites on MUC1.

[0338] As shown in Figure 22, a mixture of bispecific Abs that bind to MUC1 and the Wnt receptor can induce Wnt receptor activation in the presence of MUC1.

[0339] Example 39: A bispecific Ab mixture that binds to CEA and Wnt receptors induces Wnt receptor activation in the presence of CEA.

[0340] CEA (SEQ ID NO: 130) or RBD-IL2RA (SEQ ID NO: 11) was first expressed in HEK293 STF cells (ATCC, #CRL-3249) by electroporation using SF Cell Line 4D-Nucleofector X Kit L (Lonza, #V4XC-2024). These transfectants were seeded in white 384-well plates and incubated overnight at 37°C / 5% CO2 after adding 200 nM RSPO1 protein (in-house, #PPU5200) and 50 nM LGK974 (Cayman Chemical, #14072). These cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) F-12, HEPES, supplemented with 20% fetal bovine serum and 200 μg / mL Geneticin. On day 1 after electroporation, these cells were treated with a mixture of bispecific Ab (final concentration 25 nM) that binds to CEA as the target scaffold protein and to the Wnt receptor (FZD or LRP) as the target receptor protein, and then incubated for another day at 37°C / 5% CO2. On day 2 after electroporation, Wnt signaling activation was evaluated using the ONE-Glo luciferase assay system (Promega, #E6120) (Figures 23A and 23B).

[0341] As shown in Figures 23A and 23B, a mixture of biparatopic anti-CEA Ab fused with a Wnt receptor binding domain induced Wnt receptor activation in the presence of CEA. [Table 16]

[0342] Example 40: Expression and purification of a VHH-VHH fusion protein that binds to CD8 as a scaffold protein and to IL-2Rβ and IL-2Rγ as receptor proteins.

[0343] Anti-CD8a VHH or anti-CD8b VHH was fused with another VHH capable of binding to human IL-2Rγ or IL-2Rβ using a linker (SEQ ID NOs: 131-141). The DNA encoding these proteins was cloned into mammalian expression vectors. These plasmids were transiently expressed using the Expi293 cell line (Thermo Fisher, Carlsbad, CA, USA). Protein purification was performed using protein A or immobilized metal ion affinity chromatography. The absorbance of the purified protein at 280 nm was measured using a spectrophotometer. Antibody concentrations were calculated from the obtained values ​​using extinction coefficients calculated by methods such as PACE (Protein Science 1995; 4: 2411-2423). [Table 17] TIFF2026510624000046.tif234170TIFF2026510624000047.tif234170

[0344] Example 41: A VHH-VHH fusion protein combination targeting CD8 as the target scaffold protein and IL-2Rβ / γ as the target receptor protein induces IL-2 receptor activation in CD8+ T cells but not in CD4+ T cells.

[0345] Human CD8+ T cells or human CD4+ T cells were isolated from human PBMCs (STEMCELL, #70025.2) using the EasySep® Human CD8+ T Cell Isolation Kit (STEMCELL, #19053) or the EasySep® Human CD4+ T Cell Isolation Kit (STEMCELL, #17952). After isolation, the CD8+ T cells or CD4+ T cells were treated with plate-coated anti-CD3 / CD28 Ab (Biolegend, #317347, #302943, 5 μg / mL each) and rhIL-2 (Peprotech, #200-02) at 37°C / 5% CO2 for 3 days. Penicillin / streptomycin (GIBCO, #15140122), GlutaMax (GIBCO, #35050061), and CTS Optimizer T cell expansion SFM (GIBCO, #A1048501) containing 10% fetal bovine serum were used as the culture medium. After 3 days of culture, CD8+ T cells or CD4+ T cells were collected, washed twice with culture medium, and rested overnight in culture medium. After resting, T cells were stimulated at 37°C for 30 minutes with a 100 nM concentration of a mixture of Vhh2g-VhhC8.2 (SEQ ID NO: 131) and VhhC8.4-Vhh2b, VhhC8.8-Vhh2b, or VhhC8.9-Vhh2b (SEQ ID NO: 134-136). rhIL-2 (Peprotech, #200-02) was used as a positive control. These cells were immediately fixed with CytoFix (BD Biosciences, #554655) at room temperature for 20 minutes. The cells were washed once with staining buffer (Biolegend, #420201), then chilled permeabilization buffer (BD Biosciences, #558050) was added, and the cells were incubated on ice for 30 minutes. The cells were washed twice with staining buffer, and Alexa Fluor® 647 mouse anti-Stat5 (pY694) (BD Biosciences, #562076) was added, and the cells were incubated at room temperature for at least 60 minutes. The cells were washed once with staining buffer before analysis. Data acquisition was performed using an LSRFortessa X-20 (Becton Dickinson) (Figures 24A and 24B).

[0346] As shown in Figures 24A and B, mixtures of Vhh2g-VhhC8.2 and VhhC8.4-Vhh2b, VhhC8.8-Vhh2b, or VhhC8.9-Vhh2b induced significant STAT5 activation in activated CD8+ T cells, but not in CD4+ T cells. These data demonstrate that various CD8-binding domains can be used for CD8-dependent IL-2 receptor activation.

[0347] Example 42: A VHH-VHH fusion protein combination targeting CD8 as the target scaffold protein and IL-2Rβ / γ as the target receptor induces IL-2 receptor activation in CD8+ T cells.

[0348] Human CD8+ T cells were isolated from human PBMCs (STEMCELL, #70025.2) using the EasySep® Human CD8+ T Cell Isolation Kit (STEMCELL, #19053). After isolation, CD8+ T cells were treated with plate-coated anti-CD3 / CD28 Ab (Biolegend, #317347, #302943, 5 μg / mL each) and rhIL-2 (Peprotech, #200-02) at 37°C / 5% CO2 for 3 days. Penicillin / streptomycin (GIBCO, #15140122), GlutaMax (GIBCO, #35050061), and CTS Optimizer T cell expansion SFM (GIBCO, #A1048501) containing 10% fetal bovine serum were used as the culture medium. After 3 days of culture, CD8+ T cells were collected, washed in culture medium, and continued to be cultured in the presence of rhIL-2. One day prior to the assay, these CD8+ T cells were washed twice with culture medium and rested overnight in culture medium. After resting, the T cells were stimulated at 37°C for 30 minutes with a mixture of VHH-VHH fusions that bind to CD8 and IL-2Rγ (SEQ ID NOs: 131-133) and VHH-VHH fusions that bind to CD8 and IL-2Rβ (SEQ ID NOs: 134, 137-141). rhIL-2 (Peprotech, #200-02) was used as a positive control. These cells were immediately fixed at room temperature for 20 minutes with CytoFix (BD Biosciences, #554655). Cells were washed with staining buffer (Biolegend, #420201), then chilled permeabilization buffer (BD Biosciences, #558050) was added to the cells, and they were incubated on ice for 30 minutes. Cells were washed twice with staining buffer, and Alexa Fluor® 647 mouse anti-Stat5 (pY694) (BD Bioscience...

Claims

1. A first target-binding molecule comprising a first binding domain capable of binding to a scaffold protein and a second binding domain capable of binding to a first receptor protein, and A second target-binding molecule comprising a third binding domain capable of binding to the scaffold protein and a fourth binding domain capable of binding to a second receptor protein. A combination of target-binding molecules, including The first target-binding molecule and the second target-binding molecule are capable of binding non-competitively to the scaffold protein, and The first receptor protein and the second receptor protein are receptor subunits that can associate to form a receptor complex. The combination of the target-binding molecules.

2. A first target-binding molecule comprising a first binding domain capable of binding to a scaffold protein and a second binding domain capable of binding to a first receptor protein, and A second target-binding molecule comprising a third binding domain capable of binding to the scaffold protein and a fourth binding domain capable of binding to a second receptor protein. A protein complex comprising, The first target-binding molecule and the second target-binding molecule are capable of binding non-competitively to the scaffold protein. The first receptor protein and the second receptor protein are receptor subunits that can associate to form a receptor complex. The protein complex.

3. The combination of target-binding molecules or protein complex according to claim 1 or 2, wherein the first binding domain and the third binding domain are capable of biparatopically binding to the scaffold protein.

4. A combination of target-binding molecules or a protein complex according to any one of claims 1 to 3, wherein the first binding domain and the third binding domain can each bind to a subunit of the scaffold protein that can associate to form the scaffold protein.

5. The aforementioned scaffold proteins include PD1, PDL1, CSF2RB, CD4, CD5, CD6, CD7, CD8, CD9, CCR4, CD8, CD25, CD27, CD38, CD39, CD45, CD62L, CCR6, CD69, CD103, CD73, CD127, GITR, LRRC32, ICOS, TIGIT, RBD, MUC1, CEA, CTLA4, IL2Rα, CXCR5, neuropilin-1, TIM3, LAG3, TNFα, CD19, CD20, CD22, CD30, CD33, and glycoprotein NM. A combination of target-binding molecules or protein complexes according to any one of claims 1 to 4, selected from the group consisting of B, CD56, CD70, CD79A, CD79B, CD138, PSCA, PSMA, BCMA, FcRH5, GPRC5D, FAP, LRRC15, E-selectin, EphB2, melanotransferrin, HER2, TROP2, nectin 4, EGFRvIII, IL13RA2, TMEFF2, Muc16, EpCAM, FcRH2, AFP, PSA, amyloid β, MBP, and ASGPR.

6. The first receptor protein and the second receptor protein independently correspond to IL2Rβ, IL2Rγ, IL4R, IL13RA1, IL7R, IL9R, IL21R, TSLPR, IL3RA, CSF2RB, IL5RA, CSF2RA, IL6R, gp130, IL11RA, IL12RB1, IL12RB2, IL27RA, IL31RA, OSMR, CNTFR, LIFR, IL10RA, IL10RB, IL20RA, IL20RB, IL22RA1, IL28RA, IFNAR1, IFNAR2, IFNGR1, IFNGR2, IL1R1, IL1RAP, IL18R1, IL18RAP, ST2, IL17RA, IL17RC, TLR3, TLR4, TLR7, TLR9, CSF1R, TNFR1, TNFR2, A combination of target-binding molecules or protein complexes according to any one of claims 1 to 5, selected from the group consisting of LTBR, ​​HVEM, FAS, CD28, cMET, DR3, DR4, DR5, NGFR, RANK, FN14, CD40, 4-1BB, OX40, GITR, TGFBR1, TGFBR2, ACVRL1, ACVR2A, BMPR2, ACVR2B, ACVR1B, ACVR1C, ACVR1, AMHR2, BMPR1A, BMPR1B, BMPR2, TRA, TRB, CD3E, CD16, TREM2, FGFR1, FGFR2, FGFR3, FGFR4, Fzd1, Fzd2, Fzd3, Fzd4, Fzd5, Fzd6, Fzd7, Fzd8, Fzd9, Fzd10, LRP5, LRP6, and LGR5.

7. A combination of target-binding molecules or a protein complex according to any one of claims 1 to 6, wherein the scaffold protein is PD1, the first receptor protein is IL2Rβ, and the second receptor protein is IL2Rγ.

8. A combination of target-binding molecules or protein complex according to any one of claims 1 to 7, wherein each binding domain is an antigen-binding domain comprising VH and VL, sdAb, VHH, scFv, Fab, scFab, Fab', Fab'-SH, F(ab')2, diabody, triabody, Fv, aptamer, aphibody, cytokine, ligand, or split cytokine.

9. A nucleic acid molecule or a plurality of nucleic acid molecules that encode a combination or protein complex of target-binding molecules according to any one of claims 1 to 8.

10. A vector or a plurality of vectors comprising the nucleic acid molecule or plurality of nucleic acid molecules described in claim 9.

11. A host cell or a plurality of host cells comprising the vector or plurality of vectors according to claim 10.

12. A method for producing a combination of target-binding molecules or a protein complex according to any one of claims 1 to 8, (iii) A step of culturing the host cell or a plurality of host cells according to claim 11 under conditions suitable for protein expression; (iv) optionally a step of lysing the host cells; and (iii) The step of isolating the combination of target-binding molecules or protein complex. The method, including the method.

13. A pharmaceutical composition comprising a combination of target-binding molecules or a protein complex according to any one of claims 1 to 8.

14. A pharmaceutical composition according to claim 13 for use in therapy.

15. The pharmaceutically acceptable composition for use according to claim 14, wherein the therapy is cancer immunotherapy or autoimmune disease immunotherapy.

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