Conditionally activated receptor signaling by use of scaffold proteins

Abstract

The present invention relates to one or more binding molecules capable of inducing receptor signaling of a receptor complex under conditions of non-competitive and / or two-site binding to a scaffold protein. The invention also relates to the therapeutic use of said one or more binding molecules in cancer and autoimmune diseases.

Description

TECHNICAL FIELD

[0001] The present invention relates to one or more target binding molecules capable of inducing receptor signaling of a receptor complex as a condition of binding to a scaffold protein. BACKGROUND

[0002] Due to toxicity risks, most receptor ligands, including but not limited to agonistic ligands such as cytokines, have limitations for therapy. Targeting the agonistic activity of ligands is a promising approach to improve the efficacy and safety of therapeutic molecules. Currently, several approaches are being developed, such as protease-activated ligands (Nature Communications 2021; 12: 2768), antibody-ligand conjugation (Nature 2022; 610: 161-172), and split cytokines (WO2020 / 106708 A1). However, the selectivity of these approaches is still limited. In addition, many receptors, including but not limited to cytokine receptors, induce signaling by forming homodimers, heterodimers, or oligomerization on the cell surface for effective signaling. The present disclosure relates to target binding molecules that rely on binding to a scaffold protein to induce effective signaling of a receptor complex, addressing these needs.

[0003] [LIST OF CITATIONS]

[0004] [PATENT LITERATURE]

[0005] [PTL 1] WO 2020 / 106708 A1

[0006] [NON-PATENT LITERATURE]

[0007] [NPL 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)

[0008] [NPL 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)

[0009] [NPL 3] Wang, X., Rickert, M., Garcia, K. C. Structure of the quaternary complex of interleukin-2 with its alpha, beta, and gamma c receptors. Science 310, 1159-1163 (2005)

[0010] [NPL 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)

[0011] [NPL 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).

[0012] [NPL 6] Yen, M., Ren, J., Liu, Q. et al. Facile discovery of surrogate cytokine agonists. Cell 185, 1414-1430.e19 (2022)

[0013] [NPL 7] Sun, D., Sang, Z., Kim, Y. J. et al. Potent neutralizing nanobodies resist convergent circulating variants of SARS-CoV-2 by targeting diverse and conserved epitopes. Nature Communications 12, 4676 (2021)

[0014] [NPL 8] Fenwick, C., Loredo-Varela, J-L., Joo, V. et al. Tumor suppression of novel anti-PD1 antibodies mediated through CD28 costimulatory pathway. Journal of Experimental Medicine 216, 1525-1541 (2019)

[0015] [NPL9] Tan, S., Zhang, H., Chai, Y. et al. An unexpected N-terminal loop in PD-1 dominates binding by nivolumab. Nature Communications 8, 14369 (2017)

[0016] [NPL10] Wang, L., Bi, X., Zhu, Y. et al. IL-2R alpha up-regulation is mediated by latent membrane protein 1 and promotes lymphomagenesis and chemotherapy resistance in natural killer / T-cell lymphoma. Cancer Communications 38, 62 (2018) SUMMARY

[0017] [TECHNICAL PROBLEM]

[0018] Efficient receptor signaling is a key requirement for the effectiveness of agonistic ligands. A well-known example is cytokines, which are key immune mediators present in many pathological sites, whose action when exploited can significantly improve the immune response. This also applies to interleukin-2 (IL-2). Although many therapies exploiting agonistic ligands have been developed, the problems of high toxicity and low efficacy remain a cause for concern.

[0019] [SOLUTION TO THE PROBLEM]

[0020] The inventors have considered that the ability to deliver agonists capable of activating receptor signaling site-specifically would overcome the problems of systemic toxicity and low efficacy. To achieve such site-specific activation of receptor signaling, the inventors have inter alia developed a combination or protein complex of target binding molecules capable of inducing receptor signaling of a receptor complex in the presence of a scaffold protein. The combination or protein complex of target binding molecules of the present invention comprises a first binding domain and a third binding domain, each of which is capable of non-competitive binding to a scaffold protein. The combination or protein complex of target binding molecules 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 a receptor subunit capable of associating to form a receptor complex. Upon binding of all four binding domains, the receptor complex is activated and can induce signaling. By making the presence of the scaffold protein a condition for activation of the receptor complex and specific binding of the present invention, receptor signaling can be induced conditionally, thereby significantly increasing specificity and reducing toxicity.

[0021] [EXEMPLARY EMBODIMENTS]

[0022] The present invention is based on such findings and specifically comprises the exemplary aspects and embodiments described below.

[0023] [A-1] The present invention specifically relates to a combination of target binding molecules, the combination comprising:

[0024] 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

[0025] 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,

[0026] wherein the first target binding molecule and the second target binding molecule are capable of non-competitive binding to the scaffold protein, and

[0027] wherein the first receptor protein and the second receptor protein are each a receptor subunit capable of associating to form a receptor complex, wherein the combination of target binding molecules is capable of inducing receptor signaling of the receptor complex.

[0028] [A-1a] The present invention relates to a combination of target binding molecules, the combination comprising:

[0029] 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

[0030] 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,

[0031] wherein the first target binding molecule and the second target binding molecule are capable of non-competitive binding to the scaffold protein, and

[0032] wherein the first receptor protein and the second receptor protein are each receptor subunits capable of associating to form a receptor complex. Upon binding of the four binding domains as defined above, the receptor complex induces signaling.

[0033] [A-2] In a preferred embodiment of the target binding molecule combination according to [A-1] or [A-1a], the first binding domain and the third binding domain are capable of bi-site binding to the scaffold protein.

[0034] [A-3] In a preferred embodiment of the target binding molecule combination according to any one of [A-1] to [A-2], the target binding molecule combination 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 receptor signaling is increased at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, or 500% when the first concentration or amount of the scaffold protein is increased at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, or 500% compared to the second concentration or amount of the scaffold protein.

[0035] [A-4] In a preferred embodiment of the target binding molecule combination according to any one of [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 an even more preferred embodiment, the linker is 10 amino acids or less.

[0036] [A-5] In a preferred embodiment of the target binding molecule combination according to any one of [A-1] to [A-4], the first binding domain and the third binding domain are capable of bi-site binding to the scaffold protein within a domain of the scaffold protein.

[0037] [A-5a] In a preferred embodiment of the target binding molecule combination according to any one of [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.

[0038] [A-6] In a preferred embodiment of the target binding molecule combination according to any one of [A-1] to [A-5a], the scaffold protein comprises two, three, four or more subunits.

[0039] [A-7] In a preferred embodiment of the target binding molecule combination according to any one of [A-1] to [A-6], the first binding domain and the third binding domain are each capable of binding to a subunit of the scaffold protein, which subunits are capable of associating to form the scaffold protein.

[0040] [A-8] In a preferred embodiment of the target binding molecule combination according to any one of [A-1] to [A-7], the scaffold protein is a membrane-bound protein, a soluble protein or an insoluble protein deposit.

[0041] [A-9] In a preferred embodiment of the target binding molecule combination according to any one of [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 condition.

[0042] [A-10] In a preferred embodiment of the target binding molecule combination according to any one of [A-1] to [A-9], the scaffold protein is selected from the group consisting of 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, neurotransmitter A, B cell carcinoma, leukemia, pancreas, neuraminidase, leukemia, pancreas, cerebral ischemia, thrombin, necrosis factor alpha, leukemia, sclerosing reaction ... Felt protein -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, binding protein -4, EGFRvIII, IL13RA2, TMEFF2, Muc16, EpCAM, FcRH2, AFP, PSA, beta amyloid protein, MBP and ASGPR. In an even more preferred embodiment, the scaffold protein is selected from the group consisting of PD1, PDL1, TNFα, CD25, MUC1, CEA and CD8.

[0043] [A-11] In preferred embodiments of the target binding molecule combination according to any one of [A-1] to [A-10], the first and second receptor proteins are each independently selected from the group consisting of IL2Rβ, IL2Rγ, IL4R, IL13RA1, IL7R, IL9R, IL21R, TSLPR, IL3RA, CSF2RB, IL5RA, CSF2RA, IL6R, gpl30, 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, 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. In even more preferred embodiments, the first and second receptor proteins are each independently selected from the group consisting of IL2Rβ, IL2Rγ, Fzd1, Fzd2, Fzd3, Fzd4, Fzd5, Fzd6, Fzd7, Fzd8 Fzd9, Fzd10, LRP5, LRP6, CSF2RA, CSF2RB, and cMET.

[0044] [A-11a] In preferred embodiments of the target binding molecule combination according to any one of [A-1] to [A-10], the scaffold protein is PD1, the first receptor protein is IL2Rβ, and the second receptor protein is IL2Rγ.

[0045] [A-11b] In preferred embodiments of the target binding molecule combination according to any one of [A-1] to [A-10], the scaffold protein is IL2Ra, the first receptor protein is IL2Rp, and the second receptor protein is IL2Ry.

[0046] [A-11c] In preferred embodiments of the target binding molecule combination according to any one of [A-1] to [A-10], the target binding molecule competes for binding or binds to the same epitope as any one of the target binding molecules or protein complexes of any one of Tables 1 to 17.

[0047] [A-11d] In preferred embodiments of the target binding molecule combination according to any one of [A-1] to [A-10], the scaffold protein, the first receptor protein, and the second receptor protein are in a combination selected from any one of (i) to (vi):

[0048] (i) the scaffold protein is PDL1, the first receptor protein is IL2Rp, and the second receptor protein is IL2Ry;

[0049] (ii) the scaffold protein is TNFa, the first receptor protein is IL2Rp, and the second receptor protein is IL2Ry;

[0050] (iii) the scaffold protein is CD25, the first receptor protein is IL2Rp, and the second receptor protein is IL2Ry;

[0051] (iv) the scaffold protein is PDL1, the first receptor protein is CSF2RA, and the second receptor protein is CSF2RB;

[0052] (v) the scaffold protein is MUC1 or CEA, the first receptor protein is a Fzd receptor, and the second receptor protein is a Lrp; or

[0053] (vi) the scaffold protein is CD8, the first receptor protein is IL2Rp, and the second receptor protein is IL2Ry.

[0054] [A-11e] In preferred embodiments of the target binding molecule combination of any one of [A-1] to [A-11d], the receptor complex is IL-2R. In more preferred embodiments, inducing IL-2 receptor complex signaling comprises assessing IL-2 receptor complex activation using a colorimetric enzyme assay that determines alkaline phosphatase activity. In even more preferred embodiments, alkaline phosphatase activity is determined by measuring optical density, optionally at 620 nm. In most preferred embodiments, the receptor signaling of the receptor complex is induced when the optical density is increased at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 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 the presence of the target binding molecule combination of [A-1] to [A-11c] as compared to in the absence of the target binding molecule combination of [A-1] to [A-11c].

[0055] [A-12] In preferred embodiments of the target binding molecule combination of any one of [A-1] to [A-11e], each binding domain is an antigen binding domain comprising a VH and a VL, a sdAb, a VHH, a scFv, a Fab, a scFab, a Fab', a Fab'-SH, a F(ab')2, a diabody, a triabody, a Fv, an aptamer, an affimer, a cytokine, a ligand, or a split cytokine.

[0056] [A-13] In preferred embodiments of the target binding molecule combination of any one of [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 protein complex.

[0057] [A-14] In preferred embodiments of the target binding molecule combination of any one of [A-1] to [A-13], the first target binding molecule and the second target binding molecule are each optionally linked to a half-life extension domain via a linker.

[0058] [A-15] In a preferred embodiment of the target binding molecule combination according to [A-14], the first target binding molecule is linked to the half-life extension domain at the first binding domain, and the second target binding molecule is linked to the half-life extension domain at the third binding domain.

[0059] [A-16] In a preferred embodiment of the target binding molecule combination according to [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 a polyethylene glycol.

[0060] [A-17] In a preferred embodiment of the target binding molecule combination according to [A-14], the half-life extension domain comprises an Fc domain, optionally wherein the Fc domain comprises a modification that reduces antibody-dependent cellular cytotoxicity (ADCC), reduces complement-dependent cellular cytotoxicity (CDC), increases FcRn binding, increases pi, or facilitates heterodimerization.

[0061] [A-18] The present application also relates to a nucleic acid molecule encoding the target binding molecule combination according to any one of [A-1] to [A-17].

[0062] [A-19] The present application also relates to a plurality of nucleic acid molecules encoding the target binding molecule combination according to any 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, wherein the first nucleic acid molecule encodes a first target binding molecule as defined in any one of [A-1] to [A-17], and wherein the second nucleic acid molecule encodes a second target binding molecule as defined in any one of [A-1] to [A-17].

[0063] [A-20] The present application also relates to a vector comprising one nucleic acid molecule according to [A-18] or a plurality of nucleic acid molecules according to [A-19].

[0064] [A-20a] The present application also relates to a first vector comprising a first nucleic acid molecule according to [A-19]; and a second vector comprising a second nucleic acid molecule according to [A-19].

[0065] [A-21] The present application also relates to a host cell comprising one nucleic acid molecule according to [A-18] or a plurality of nucleic acid molecules according to [A-19] or a vector according to [A-20].

[0066] [A-21a] The present application also relates to a first host cell comprising the first nucleic acid molecule according to [A-19] or the first vector according to [A-20a]; and a second host cell comprising the second nucleic acid molecule according to [A-19] or the second vector according to [A-20a].

[0067] [A-22] The present application also relates to a method for producing a target binding molecule combination, the method comprising the steps of:

[0068] (i) culturing the host cell according to [A-21] or the host cell according to [A-21a] under conditions suitable for protein expression;

[0069] (ii) optionally lysing the host cell; and

[0070] (iii) isolating the target binding molecule combination.

[0071] [A-23] The present application also relates to a method for producing a target binding molecule combination, the method comprising the steps of:

[0072] (i) culturing the host cell according to [A-21] or the first host cell according to [A-21a] under conditions suitable for protein expression to obtain a first target binding molecule;

[0073] (ii) culturing the host cell according to [A-21] or the second host cell according to [A-21a] under conditions suitable for protein expression to obtain a second target binding molecule;

[0074] (iii) isolating the first target molecule and the second target molecule from the host cell; and

[0075] (iv) combining the first target binding molecule with the second target binding molecule to obtain the target binding molecule combination.

[0076] [A-24] The present application also relates to a pharmaceutical composition comprising the target binding molecule combination according to any one of claims [A-1] to [A-17].

[0077] [A-25] The present application also relates to a method for treating a disease in a subject, the method comprising administering to a subject in need thereof the pharmaceutical composition according to [A-24].

[0078] [A-26] In a preferred embodiment of the method for treating a disease in a subject according to [A-25], the disease is a cancer or an autoimmune disease.

[0079] [A-26a] In preferred embodiments, the cancer or autoimmune disease according to [A-25] is a cancer or autoimmune disease susceptible to improvement or prevention by an increase or enhancement of IL-2R signaling.

[0080] [A-27] The present application also relates to a pharmaceutical composition according to [A-24] for use in therapy.

[0081] [A-28] In preferred embodiments of the pharmaceutical composition for use according to [A-27], the therapy is cancer immunotherapy or autoimmune disease immunotherapy.

[0082] [A-28a] In preferred embodiments, the cancer or autoimmune disease according to [A-28] is a cancer or autoimmune disease susceptible to improvement or prevention by an increase or enhancement of IL-2R signaling.

[0083] [A-29] The present application also relates to the use of a pharmaceutical composition according to [A-24] in the manufacture of a medicament for the treatment of a disease.

[0084] [A-30] In preferred embodiments of the use according to claim [A-29], the disease is a cancer or autoimmune disease.

[0085] [A-30a] In preferred embodiments, the cancer or autoimmune disease according to [A-30] is a cancer or autoimmune disease susceptible to improvement or prevention by an increase or enhancement of IL-2R signaling.

[0086] [A-31] In preferred embodiments of the target binding molecule combination according to any one of [A-1] to [A-17], the method according to any one of [A-25] or [A-26], the pharmaceutical composition for use according to any one of [A-27] or [A-28], or the use according to any one of [A-29], [A-30] or [A-30a], the scaffold protein is PD-1, the first receptor protein is IL-2Rß, and the second receptor protein is IL-2Ry.

[0087] [A-31a] In preferred embodiments of the target binding molecule combination according to any one of [A-1] to [A-17], the method according to any one of [A-25] or [A-26], the pharmaceutical composition for use according to any one of [A-27] or [A-28], or the use according to any one of [A-29], [A-30] or [A-30a], the scaffold protein is IL-2Ra, the first receptor protein is IL-2Rß, and the second receptor protein is IL-2Ry.

[0088] [A-32] In preferred embodiments of the target binding molecule combination according to any one of [A-1] to [A-17], the method according to claim [A-26], the pharmaceutical composition for use according to [A-28] or the use according to [A-30] or [A-30a], the cancer or autoimmune disease is characterized by PD1, IL2Ra, IL-2Rb or IL-2Ry expression.

[0089] [A-33] The present application 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 administration of the first target binding molecule in combination with a second target binding molecule as defined in any one of [A-1] to [A-17], optionally wherein the first target binding molecule and the second target binding molecule are to be administered simultaneously, sequentially or separately.

[0090] [A-34] The present application 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 administration of the second target binding molecule in combination with a first target binding molecule as defined in any one of [A-1] to [A-17], optionally wherein the first target binding molecule and the second target binding molecule are to be administered simultaneously, sequentially or separately.

[0091] [A-35] In preferred embodiments of the first target binding molecule for use according to [A-33] or the second target binding molecule for use according to [A-34], the disease is a cancer or an autoimmune disease.

[0092] [A-35a] In preferred embodiments, the cancer or autoimmune disease according to [A-35] is a cancer or autoimmune disease susceptible to improvement or prevention by an increase or enhancement of IL-2R signaling.

[0093] [A-36] In preferred embodiments of the first target binding molecule or the second target binding molecule for use according to [A-35], the cancer or autoimmune disease is characterized by PD1, IL2Ra, IL-2Rb or IL-2Ry expression.

[0094] [A-37] In preferred embodiments of the first target binding molecule or the second target binding molecule for use according to any one of [A-33] to [A-36], the scaffold protein is PD1, the first receptor protein is IL-2Rb and the second receptor protein is IL-2Ry.

[0095] [A-37a] In a preferred embodiment of the first target binding molecule or the second target binding molecule for use according to any one of [A-33] to [A-36], the scaffold protein is IL2Ra, the first receptor protein is IL-2Rß, and the second receptor protein is IL-2Ry.

[0096] [A-38] The present application also relates to a protein complex 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,

[0097] wherein the first target binding molecule and the second target binding molecule are capable of non-competitive binding to the scaffold protein,

[0098] wherein the first receptor protein and the second receptor protein are each a receptor subunit capable of associating to form a receptor complex,

[0099] wherein the protein complex is capable of inducing receptor signaling of the receptor complex.

[0100] [A-38a] The present application also relates to a protein complex 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,

[0101] wherein the first target binding molecule and the second target binding molecule are capable of non-competitive binding to the scaffold protein,

[0102] wherein the first receptor protein and the second receptor protein are each a receptor subunit capable of associating to form a receptor complex.

[0103] In the binding of the four binding domains as defined above, the receptor complex induces signaling.

[0104] [A-39] The present application also relates to a protein complex 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,

[0105] wherein the first binding domain and the third binding domain are capable of non-competitively binding to the scaffold protein,

[0106] wherein the first receptor protein and the second receptor protein are each a receptor subunit capable of associating to form a receptor complex,

[0107] wherein the protein complex is capable of inducing receptor signaling of the receptor complex.

[0108] [A-39a] The present application also relates to a protein complex 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,

[0109] wherein the first binding domain and the third binding domain are capable of non-competitively binding to the scaffold protein,

[0110] wherein the first receptor protein and the second receptor protein are each a receptor subunit capable of associating to form a receptor complex,

[0111] In the binding of the four binding domains as defined above, the receptor complex induces signaling.

[0112] [A-40] In preferred embodiments of the protein complex according to any one of [A-38] to [A-39a], the first binding domain and the third binding domain are capable of bi-site binding to the scaffold protein.

[0113] [A-41] In preferred embodiments of the protein complex according to any one of [A-38] to [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 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% when the first concentration or amount of the scaffold protein is increased by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, or 500% compared to the second concentration or amount of the scaffold protein.

[0114] [A-42] In preferred embodiments of the protein complex according to any one of [A-38] to [A-41], 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 even more preferred embodiments, the linker is 10 amino acids or less.

[0115] [A-43] In preferred embodiments of the protein complex according to any one of [A-38] to [A-42], the first binding domain and the third binding domain are capable of bi-site binding to the scaffold protein within a domain of the scaffold protein.

[0116] [A-43a] In preferred embodiments of the protein complex according to any one of [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.

[0117] [A-44] In preferred embodiments of the protein complex according to any one of [A-38] to [A-43a], the scaffold protein comprises two, three, four or more subunits.

[0118] [A-45] In preferred embodiments of the protein complex according to any one of [A-38] to [A-44], the first binding domain and the third binding domain are each capable of binding to a subunit of the scaffold protein, which subunits are capable of associating to form the scaffold protein.

[0119] [A-46] In preferred embodiments of the protein complex according to any one of [A-38] to [A-45], the scaffold protein is a membrane-bound protein, a soluble protein or an insoluble protein deposit.

[0120] [A-47] In preferred embodiments of the protein complex according to any one of [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 a condition.

[0121] [A-48] In preferred embodiments of the protein complex according to any one of [A-38] to [A-47], the scaffold protein is selected from the group consisting of 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, IL2R alpha, CXCR5, neuropilin-1, TIM3, LAG3, TNF alpha, 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, beta amyloid, MBP, and ASGPR. In even more preferred embodiments, the scaffold protein is selected from the group consisting of PD1, PDL1, TNF alpha, CD25, MUC1, CEA, and CD8.

[0122] [A-49] In preferred embodiments of the protein complex according to any one of [A-38] to [A-48], wherein the first and second receptor proteins are each independently selected from the group consisting of IL2Rβ, IL2Rγ, IL4R, IL13RA1, IL7R, IL9R, IL21R, TSLPR, IL3RA, CSF2RB, IL5RA, CSF2RA, IL6R, gpl30, 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, 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. In even more preferred embodiments, the first and second receptor proteins are each independently selected from the group consisting of IL2Rβ, IL2Rγ, Fzd1, Fzd2, Fzd3, Fzd4, Fzd5, Fzd6, Fzd7, Fzd8 Fzd9, Fzd10, LRP5, LRP6, CSF2RA, CSF2RB, and cMET.

[0123] [A-49a] In preferred embodiments of the protein complex according to any one of [A-38] to [A-49], the scaffold protein is PD1, the first receptor protein is IL2Rβ, and the second receptor protein is IL2Rγ.

[0124] [A-49b] In preferred embodiments of the protein complex according to any one of [A-38] to [A-49], the scaffold protein is IL2Ra, the first receptor protein is IL2Rp, and the second receptor protein is IL2Ry.

[0125] [A-49c] In preferred embodiments of the protein complex according to any one of [A-38] to [A-49], the protein complex competes for binding or binds to the same epitope as any one of the target binding molecules or protein complexes of any one of Tables 1 to 17.

[0126] [A-49d] In preferred embodiments of the protein complex according to any one of [A-38] to [A-49], the scaffold protein, the first receptor protein, and the second receptor protein are in a combination selected from any one of (i) to (vi):

[0127] (i) the scaffold protein is PDL1, the first receptor protein is IL2Rp, and the second receptor protein is IL2Ry;

[0128] (ii) the scaffold protein is TNFa, the first receptor protein is IL2Rp, and the second receptor protein is IL2Ry;

[0129] (iii) the scaffold protein is CD25, the first receptor protein is IL2Rp, and the second receptor protein is IL2Ry;

[0130] (iv) the scaffold protein is PDL1, the first receptor protein is CSF2RA, and the second receptor protein is CSF2RB;

[0131] (v) the scaffold protein is MUC1 or CEA, the first receptor protein is a Fzd receptor, and the second receptor protein is a Lrp; or

[0132] (vi) the scaffold protein is CD8, the first receptor protein is IL2Rp, and the second receptor protein is IL2Ry.

[0133] [A-49e] In preferred embodiments of the protein complex of [A-38] to [A-49d], the receptor complex is an IL-2R. In more preferred embodiments, inducing IL-2 receptor complex signaling comprises assessing IL-2 receptor complex activation using a colorimetric enzyme assay that determines alkaline phosphatase activity. In even more preferred embodiments, alkaline phosphatase activity is determined by measuring optical density, optionally at 620 nm. In most preferred embodiments, the receptor signaling of the receptor complex is induced when the optical density is increased at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 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 the presence of the protein complex of [A-38] to [A-49c] as compared to in the absence of the protein complex of [A-38] to [A-49c].

[0134] [A-50] In preferred embodiments of the protein complex of any one of [A-38] to [A-49e], each binding domain is an antigen binding domain comprising a VH and a VL, a sdAb, a VHH, a scFv, a Fab, a scFab, a Fab', a Fab'-SH, a F(ab')2, a diabody, a triabody, a Fv, an aptamer, an affimer, a cytokine, a ligand, or a split cytokine.

[0135] [A-51] In preferred embodiments of the protein complex of any one of [A-38] to [A-50], the protein complex further comprises a half-life extension domain.

[0136] [A-52] In preferred embodiments of the protein complex of any one of [A-38] and [A-40] to [A-51], the first target binding molecule is linked to the half-life extension domain at the first binding domain, and the second target binding molecule is linked to the half-life extension domain at the third binding domain.

[0137] [A-53] In preferred embodiments of the protein complex according to any one of [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 a polyethylene glycol.

[0138] [A-54] In preferred embodiments of the protein complex according to [A-53], the half-life extension domain comprises an Fc domain, optionally wherein the Fc domain comprises a modification that reduces antibody-dependent cellular cytotoxicity (ADCC), reduces complement-dependent cellular cytotoxicity (CDC), increases FcRn binding, increases pi, or facilitates heterodimerization.

[0139] [A-55] The present application also relates to a nucleic acid molecule or a plurality of nucleic acid molecules encoding the protein complex according to any one of [A-38] to [A-54].

[0140] [A-56] The present application also relates to a vector comprising the nucleic acid molecule or the plurality of nucleic acid molecules according to [A-55].

[0141] [A-57] The present application also relates to a host cell comprising the nucleic acid molecule or the plurality of nucleic acid molecules according to [A-55] or the vector according to [A-56].

[0142] [A-58] The present application also relates to a method of producing a protein complex, the method comprising the steps of:

[0143] (i) culturing the host cell according to [A-57] under conditions suitable for protein expression

[0144] (ii) optionally lysing the host cell; and

[0145] (iii) isolating the combination of target binding molecules.

[0146] [A-59] The present application also relates to a pharmaceutical composition comprising the protein complex according to any one of [A-38] to [A-54].

[0147] [A-60] The present application also relates to a method for treating a disease in a subject, the method comprising administering to a subject in need thereof the pharmaceutical composition according to [A-59].

[0148] [A-61] In preferred embodiments of the method for treating a disease in a subject according to [A-60], the disease is a cancer or an autoimmune disease.

[0149] [A-61a] In preferred embodiments, the cancer or autoimmune disease according to [A-61] is a cancer or autoimmune disease susceptible to improvement or prevention by an increase or enhancement of IL-2R signaling.

[0150] [A-62] The present application also relates to a pharmaceutical composition according to [A-59] for use in therapy.

[0151] [A-63] In preferred embodiments of the pharmaceutical composition for use according to [A-62], the therapy is cancer immunotherapy or autoimmune disease immunotherapy.

[0152] [A-63a] In preferred embodiments, the cancer or autoimmune disease according to [A-63] is a cancer or autoimmune disease susceptible to improvement or prevention by an increase or enhancement of IL-2R signaling.

[0153] [A-64] The present application also relates to the use of a pharmaceutical composition according to [A-61] in the manufacture of a medicament for the treatment of a disease.

[0154] [A-65] In preferred embodiments of the use according to [A-64], the disease is a cancer or autoimmune disease.

[0155] [A-65a] In preferred embodiments, the cancer or autoimmune disease according to [A-65] is a cancer or autoimmune disease susceptible to improvement or prevention by an increase or enhancement of IL-2R signaling.

[0156] [A-66] In preferred embodiments of the method according to any one of [A-60] or [A-61], the pharmaceutical composition for use according to any one of [A-62] or [A-63], or the use according to any one of [A-64], [A-65] or [A-65a], the scaffold protein is PD1, the first receptor protein is IL-2R beta, and the second receptor protein is IL-2R gamma.

[0157] [A-66a] In preferred embodiments of the method according to any one of [A-60] or [A-61], the pharmaceutical composition for use according to any one of [A-62] or [A-63], or the use according to any one of [A-64], [A-65] or [A-65a], the scaffold protein is IL2R alpha, the first receptor protein is IL-2R beta, and the second receptor protein is IL-2R gamma.

[0158] [A-67] In a preferred embodiment of the method according to [A-61], the pharmaceutical composition for use according to [A-63] or the use according to [A-65] or [A65a], the cancer or autoimmune disease is characterized by PD1, IL2Rα, IL-2Rβ or IL-2Rγ expression.

[0159] [A-68] The present application also relates to a method for treating a disease in a subject, the method comprising administering to a subject in need thereof a therapeutically effective amount of a composition comprising:

[0160] 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

[0161] 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 non-competitive binding to the scaffold protein,

[0162] wherein the first receptor protein and the second receptor protein are each a receptor subunit capable of associating to form a receptor complex,

[0163] wherein the composition is capable of inducing receptor signaling of the receptor complex.

[0164] [A-69] The present application also relates to a method for treating a disease in a subject, the method comprising administering to a subject in need thereof a therapeutically effective amount of a composition comprising:

[0165] a protein complex 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 non-competitive binding to the scaffold protein,

[0166] wherein the first receptor protein and the second receptor protein are each a receptor subunit capable of associating to form a receptor complex,

[0167] wherein the protein complex is capable of inducing receptor signaling of the receptor complex.

[0168] [A-70] The present application also relates to a method of conditionally inducing receptor signaling, the method comprising:

[0169] (i) identifying a subject in need thereof expressing a scaffold protein, a first receptor protein and a second receptor protein, and

[0170] (ii) administering a target binding molecule combination according to any one of [A-1] to [A-17] or a protein complex according to any one of [A-38] to [A-54].

[0171] [A-71] The present application also relates to a method of screening a target binding molecule combination, the method comprising the steps of:

[0172] (i) identifying a scaffold protein and a first binding domain and a third binding domain that can each non-competitively bind to the scaffold protein,

[0173] (ii) identifying a first receptor protein and a second receptor protein and 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,

[0174] (iii) generating a first target binding molecule comprising the first binding domain and the second binding domain and a second target binding molecule comprising the third binding domain and the fourth binding domain, optionally wherein the first target binding molecule and the second target binding molecule are a first target binding molecule and a second target binding molecule according to any one of [A-1] to [A-17],

[0175] (iv) determining a receptor signaling activity of the first receptor protein and the second receptor protein in the presence of the first target binding molecule, the second target binding molecule and the scaffold protein,

[0176] (v) determining a receptor signaling activity of the first receptor protein and the second receptor protein in the absence of the scaffold protein in the presence of the second target binding molecule and the scaffold protein, and

[0177] (vi) selecting the combination of the first target binding molecule and the second target binding molecule if the receptor signaling activity of (iv) is higher than (v), optionally wherein the receptor signaling activity of (iv) is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% higher than (v).

[0178] [A-72] The present application also relates to a method of screening a protein complex, the method comprising the steps of:

[0179] (i) identifying a scaffold protein and a first binding domain and a third binding domain that can each non-competitively bind to the scaffold protein,

[0180] (ii) identifying a first receptor protein and a second receptor protein, and 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,

[0181] (iii) producing a protein complex comprising the first binding domain, the second binding domain, the third binding domain and the fourth binding domain, optionally wherein the protein complex is a protein complex according to any one of [A-38] to [A-54],

[0182] (iv) determining the receptor signaling activity of the protein complex in the presence of the scaffold protein,

[0183] (v) determining the receptor signaling activity of the protein complex in the absence of the scaffold protein, and

[0184] (vi) selecting the protein complex if the receptor signaling activity of (iv) is higher than (v), optionally wherein the receptor signaling activity of (iv) is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% higher than (v).

[0185] [A-72a] The method according to [A-71] or [A-72], wherein the identifying a first binding domain and a third binding domain that can each bind non-competitively to the scaffold protein comprises the steps of:

[0186] (a) identifying a neutralizing binding domain that binds to the scaffold protein,

[0187] (b) identifying a non-neutralizing binding domain that binds to the scaffold protein, and

[0188] (c) selecting the neutralizing binding domain as the first binding domain and the non-neutralizing binding domain as the third binding domain, or vice versa.

[0189] [A-72b] The present application also relates to a method for screening non-competitive and / or biparatopic protein binding domains that bind to a scaffold protein, the method comprising the steps of:

[0190] (i) identifying a neutralizing binding domain that binds to the scaffold protein,

[0191] (ii) identifying a non-neutralizing binding domain that binds to the scaffold protein, and

[0192] (iii) selecting a neutralizing binding domain and a non-neutralizing binding domain as the non-competitive and / or dual-site protein binding domain.

[0193] [A-72c] The present application also relates to a method of screening a combination of target binding molecules according to any one of [A-1] to [A-17] comprising a non-competitive and / or dual-site scaffold protein binding domain, the method comprising the steps of:

[0194] (i) identifying a target binding molecule comprising a neutralizing binding domain that binds to the scaffold protein,

[0195] (ii) identifying a target binding molecule comprising a non-neutralizing binding domain that binds to the scaffold protein, and

[0196] (iii) selecting a target binding molecule comprising a neutralizing binding domain that binds to the scaffold protein and a target binding molecule comprising a non-neutralizing binding domain that binds to the scaffold protein.

[0197] [A-72d] The present application also relates to a method of screening a protein complex according to any one of [A-38] to [A-54] comprising a non-competitive and / or dual-site scaffold protein binding domain, the method comprising the steps of:

[0198] (i) identifying a protein complex comprising a neutralizing binding domain that binds to the scaffold protein,

[0199] (ii) identifying a protein complex comprising a non-neutralizing binding domain that binds to the scaffold protein, and

[0200] (iii) selecting a protein complex comprising a neutralizing binding domain as the first binding domain and a non-neutralizing binding domain as the third binding domain, or vice versa.

[0201] [A-72e] The present application also relates to a method for producing a combination of target binding molecules according to any one of [A-1] to [A-17], the method comprising the steps of:

[0202] (a) identifying a neutralizing binding domain that binds to the scaffold protein,

[0203] (b) identifying a non-neutralizing binding domain that binds to the scaffold protein, and

[0204] (c) selecting a neutralizing binding domain as the first binding domain and a non-neutralizing binding domain as the third binding domain, or vice versa.

[0205] [A-72el] In one embodiment of the method according to [A-72e], the method further comprises the steps of:

[0206] (d) obtaining a polynucleotide encoding the neutralizing binding domain and a polynucleotide encoding the non-neutralizing binding domain,

[0207] (e) optionally linking the polynucleotide encoding the neutralizing binding domain to the polynucleotide encoding the second binding domain via a linker,

[0208] (f) optionally linking the polynucleotide encoding the non-neutralizing binding domain to a polynucleotide encoding the fourth binding domain via a linker,

[0209] (g) expressing the polynucleotides of (e) and (f).

[0210] [A-72e2] In one embodiment of the method according to [A-72e], the method further comprises the steps of:

[0211] (d) producing a first target binding molecule comprising the first binding domain and the second binding domain and a second target binding molecule comprising the third binding domain and the fourth binding domain.

[0212] [A-72f] The present application also relates to a method for producing a protein complex according to any one of [A-38] to [A-54], the method comprising the steps of:

[0213] (a) identifying a neutralizing binding domain that binds to the scaffold protein,

[0214] (b) identifying a non-neutralizing binding domain that binds to the scaffold protein, and

[0215] (c) selecting the neutralizing binding domain as the first binding domain and the non-neutralizing binding domain as the third binding domain, or vice versa.

[0216] [A-72fl] In one embodiment of the method according to [A-72f], the method further comprises the steps of:

[0217] (d) obtaining a polynucleotide encoding the neutralizing binding domain and a polynucleotide encoding the non-neutralizing binding domain,

[0218] (e) optionally linking the polynucleotide encoding the neutralizing binding domain to the polynucleotide encoding at least one fragment of the second binding domain via a linker,

[0219] (f) linking the polynucleotide encoding the non-neutralizing binding domain to a polynucleotide encoding at least one fragment of the fourth binding domain, optionally via a linker,

[0220] (g) expressing the polynucleotides of (e) and (f) to produce the protein complex.

[0221] [A-72f2] In one embodiment of the method according to [A-72f], the method further comprises the step of:

[0222] (d) producing the protein complex according to any one of [A-38] to [A-54] comprising the first binding domain, the second binding domain, the third binding domain and the fourth binding domain.

[0223] [A-73] In a preferred embodiment of the method according to any one of [A-68] to [A-72f2], the first binding domain and the third binding domain are capable of dual-site binding to the scaffold protein.

[0224] [A-74] In a preferred embodiment of the method according to any one of [A-68] to [A-73], the target binding molecule combination or 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 receptor signaling is increased at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, or 500% when the first concentration or amount of the scaffold protein is increased at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, or 500% compared to the second concentration or amount of the scaffold protein.

[0225] [A-75] In a preferred embodiment of the method according to any one of [A-68] to [A-74], the first binding domain is linked to the second binding domain, and / or the third binding domain is linked to the fourth binding domain, optionally via a linker. In a preferred embodiment, the linker is 20, 15, 10 amino acids or less. In an even more preferred embodiment, the linker is 10 amino acids or less.

[0226] [A-76] In preferred embodiments of the method according to any one of [A-68] to [A-75], the first binding domain and the third binding domain are capable of dual-site binding to the scaffold protein within a domain of the scaffold protein.

[0227] [A-76a] In preferred embodiments of the method according to any one of [A-68] to [A-75], 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.

[0228] [A-77] In preferred embodiments of the method according to any one of [A-68] to [A-76a], the scaffold protein comprises two, three, four or more subunits.

[0229] [A-78] In preferred embodiments of the method according to any one of [A-68] to [A-77], the first binding domain and the third binding domain are each capable of binding to a subunit of the scaffold protein, which subunits are capable of associating to form the scaffold protein.

[0230] [A-79] In preferred embodiments of the method according to any one of [A-68] to [A-78], the scaffold protein is a membrane-bound protein, a soluble protein or an insoluble protein deposit.

[0231] [A-80] In preferred embodiments of the method according to any one of [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 condition.

[0232] [A-81] In preferred embodiments of the method according to any one of [A-68] to [A-80], the scaffold protein is selected from the group consisting of 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, IL2Ra, CXCR5, neuropilin-1, TIM3, LAG3, TNFa, 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, beta amyloid, AFP, PSA, MBP, and ASGPR. In even more preferred embodiments, the scaffold protein is selected from the group consisting of PD1, PDL1, TNFa, CD25, MUC1, CEA, and CD8.

[0233] [A-82] In preferred embodiments of the method of any one of [A-68] to [A-81], wherein the first and second receptor proteins are each independently selected from the group consisting of: IL2Rβ, IL2Rγ, IL4R, IL13RA1, IL7R, IL9R, IL21R, TSLPR, IL3RA, CSF2RB, IL5RA, CSF2RA, IL6R, gpl30, 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, 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. In even more preferred embodiments, the first and second receptor proteins are each independently selected from the group consisting of: IL2Rβ, IL2Rγ, Fzd1, Fzd2, Fzd3, Fzd4, Fzd5, Fzd6, Fzd7, Fzd8 Fzd9, Fzd10, LRP5, LRP6, CSF2RA, CSF2RB, and cMET.

[0234] [A-82a] In preferred embodiments of the method of any one of [A-68] to [A-81], the scaffold protein is PD1, the first receptor protein is IL2Rβ, and the second receptor protein is IL2Rγ.

[0235] [A-82b] In preferred embodiments of the method of any one of [A-68] to [A-81], the scaffold protein is IL2Rα, the first receptor protein is IL2Rβ, and the second receptor protein is IL2Rγ.

[0236] [A-82c] In preferred embodiments of the method according to any one of [A-68] to [A-81], the target binding molecule combination or the protein complex competes for binding to or binds to the same epitope as any one of the target binding molecules or protein complexes of any one of Tables 1 to 17.

[0237] [A-82d] In preferred embodiments of the method according to any one of [A-68] to [A-81], the scaffold protein, the first receptor protein and the second receptor protein are in a combination selected from any one of (i) to (vi):

[0238] (i) the scaffold protein is PDL1, the first receptor protein is IL2Rβ, and the second receptor protein is IL2Rγ;

[0239] (ii) the scaffold protein is TNFa, the first receptor protein is IL2Rβ, and the second receptor protein is IL2Rγ;

[0240] (iii) the scaffold protein is CD25, the first receptor protein is IL2Rβ, and the second receptor protein is IL2Rγ;

[0241] (iv) the scaffold protein is PDL1, the first receptor protein is CSF2RA, and the second receptor protein is CSF2RB;

[0242] (v) the scaffold protein is MUC1 or CEA, the first receptor protein is a Fzd receptor, and the second receptor protein is a Lrp; or

[0243] (vi) the scaffold protein is CD8, the first receptor protein is IL2Rβ, and the second receptor protein is IL2Rγ.

[0244] [A-82e] In preferred embodiments of the method of any one of [A-68] to [A-81], the receptor complex is IL-2R. In more preferred embodiments, inducing IL-2 receptor complex signaling comprises assessing IL-2 receptor complex activation using a colorimetric enzyme assay that determines alkaline phosphatase activity. In even more preferred embodiments, alkaline phosphatase activity is determined by measuring optical density, optionally at 620 nm. In most preferred embodiments, the receptor signaling of the receptor complex is induced when the optical density is increased at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 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 the presence of the target binding molecule combination or the protein complex as compared to in the absence of the target binding molecule combination or the protein complex.

[0245] [A-83] In preferred embodiments of the method of any one of [A-68] to [A-82e], each binding domain is an antigen binding domain comprising a VH and a VL, a sdAb, a VHH, a scFv, a Fab, a scFab, a Fab', a Fab'-SH, a F(ab')2, a diabody, a triabody, a Fv, an aptamer, an affimer, a cytokine, a ligand, or a split cytokine.

[0246] [A-84] In preferred embodiments of the method of any one of [A-68] to [A-83], the target binding molecule combination or the protein complex further comprises a half-life extension domain.

[0247] [A-85] In preferred embodiments of the method of any one of [A-68], [A-70], or [A-72] to [A-84], the first target binding molecule is linked to the half-life extension domain at the first binding domain, and the second target binding molecule is linked to the half-life extension domain at the third binding domain.

[0248] [A-86] In preferred embodiments of the method according to [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 a polyethylene glycol.

[0249] [A-87] In preferred embodiments of the method according to any one of [A-84] to [A-86], the half-life extension domain comprises an Fc domain, optionally wherein the Fc domain comprises a modification that reduces antibody-dependent cellular cytotoxicity (ADCC), reduces complement-dependent cytotoxicity (CDC), increases FcRn binding, increases pi, or facilitates heterodimerization.

[0250] [A-88] The present application also relates to a target binding molecule combination or a protein complex comprising a target binding molecule or protein complex of any one of Tables 1 to 17. As shown herein, a target binding molecule or protein complex of any one of Tables 1 to 17 is capable of inducing receptor signaling of the respective receptor complex.

[0251] [B-1] An antigen binding molecule comprising any one of the following (a1) to (a4):

[0252] (a1) a complementarity determining region (CDR) 1 having the amino acid sequence SYTMG, a CDR 2 having the amino acid sequence AIRWSGSITYYADSVKG, and a CDR 3 having the amino acid sequence SPVAGWGTSPAWYDY;

[0253] (a2) a CDR 1 having the amino acid sequence SYGMG, a CDR 2 having the amino acid sequence TISWNSGSIYYTDSVKG, and a CDR 3 having the amino acid sequence GPRDWGNMRKFEEYEY;

[0254] (a3) a CDR 1 having the amino acid sequence EYGMG, a CDR 2 having the amino acid sequence TISWDSDSIYYTDSVKG, and a CDR 3 having the amino acid sequence RPRDWGNMRRFEAYEY; or

[0255] (a4) a CDR 1 having the amino acid sequence DYAGS, a CDR 2 having the amino acid sequence SINWRGDTTYYADSVKG, and a CDR 3 having the amino acid sequence KATDWSSTLYEYDY.

[0256] [B-2] The antigen binding molecule according to [B-1], wherein at least one of the CDR 1, CDR 2, or CDR 3 amino acid sequences further comprises one or more conservative amino acid substitutions.

[0257] [B-3] The antigen-binding molecule according to any one of [B-1] or [B-2], wherein the antigen-binding molecule is a VHH or a single domain antibody.

[0258] [B-4] The antigen-binding molecule according to any one of [B-1] to [B-3], wherein the antigen-binding molecule comprises any one of the following (a1) to (a4):

[0259] (a1) an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence QLQLVESGGGLVQAGGSLRLSCAASGRGFSSYTMGWFRQAPGKEREFVS AIRWSGSITYYADSVKGRFTISRDNAKNTMYLQMNSLKPEDTAVYYCAASPVAGWGTSPAWYDYWGQGTQVTVSS,

[0260] (a2) an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence QLQLVESGGGLVQPGGSLTLSCAASGRSFSSYGMGWFRQAPGKEREFVA TISWNSGSIYYTDSVKGRFTISRDDAKNTMSLQMNSLRPEDTAVYSCAAG PRDWGNMRKFEEYEYRGQGTQVTVSS,

[0261] (a3) an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence QVQLVESGGGLVQPGGSLTLSCAASGRSFSEYGMGWFRQAPGKEREFVA TISWDSIYYTDSVKGRFTISRDNAKNTMSLQMNSLKPEDTAVYSCAARPR DWGNMRRFEAYYYRGQGTQVTVSS, or

[0262] (a4) an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence QVQLVESGGGLVQTGGSLRLSCAASEPTFSDYAGSWFRQAPGKEREFLASINWRGDTTTYYADSVKGRFTISRDNAKNMYLEMNNLEPEDTAVYRCA AKATDWSSTLYEYDYWGQGTQVTVSS.

[0263] [B-5] An antigen-binding molecule comprising any one of the following (a1) to (a4):

[0264] (a1) an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence QLQLVESGGGLVQAGGSLRLSCAASGRGFSSYTMGWFRQAPGKEREFVS AIRWSGSITYYADSVKGRFTISRDNAKNTMYLQMNSLKPEDTAVYYCAA SPVAGWGTSPAWYDYWGQGTQVTVSS.

[0265] (a2) an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence QLQLVESGGGLVQPGGSLTLSCAASGRSFSSYGMGWFRQAPGKEREFVA TISWNSGSIYYTDSVKGRFTISRDDAKNTMSLQMNSLRPEDTAVYSCAAG PRDWGNMRKFEEYEYRGQGTQVTVSS.

[0266] (a3) an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence QVQLVESGGGLVQPGGSLTLSCAASGRSFSEYGMGWFRQAPGKEREFVA TISWDSIYYTDSVKGRFTISRDNAKNTMSLQMNSLKPEDTAVYSCAARPR DWGNMRRFEAYYYRGQGTQVTVSS, or

[0267] (a4) an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence QVQLVESGGGLVQTGGSLRLSCAASEPTFSDYAGSWFRQAPGKEREFLASINWRGDTTTYYADSVKGRFTISRDNAKNMYLEMNNLEPEDTAVYRCA AKATDWSSTLYEYDYWGQGTQVTVSS.

[0268] [B-6] The antigen binding molecule of any one of [B-1] to [B-5], wherein the antigen binding molecule binds CD8.

[0269] [B-7] An antigen binding molecule that binds to the same epitope in CD8 as the antigen binding molecule of any one of [B-1] to [B-5]

[0270] [B-8] A multispecific antigen binding molecule comprising the antigen binding molecule of any one of [B-1] to [B-7].

[0271] [B-9] The target binding molecule combination of any one of [A-1] to [A-17] or the protein complex of any one of [A-38] to [A-54], wherein the first binding domain and / or the third binding domain is any one of (a1) to (a4) as defined in any one of [B-1] to [B-7]

[0272] [B-10] A pharmaceutical composition comprising the antigen binding molecule of any one of [B-1] to [B-9].

[0273] [B-11] The pharmaceutical composition of [B-10] for use in therapy.

[0274] [B-12] The pharmaceutical composition of [B-10] for use in the therapy of a medical condition, wherein the medical condition is characterized by CD8 expression or an increase in CD8 expression.

[0275] [B-13] A method of treating an individual having a medical condition, the method comprising administering to the individual an effective amount of the pharmaceutical composition of [B-10], wherein the medical condition is characterized by CD8 expression or an increase in CD8 expression. BRIEF DESCRIPTION OF DRAWINGS

[0276] [ Figure 1 A] Figure 1 A shows a schematic representation of a protein complex in the present disclosure. Figure 1A shows two different VHH-VHH fusions, each with a scaffold protein binding domain and a receptor protein binding domain. Receptor activation occurs only in the presence of the scaffold protein.

[0277] [ Figure 1 B] Figure 1 B shows a schematic diagram of the protein complex in the present disclosure. Figure 1 B shows a possible single-molecule format that can induce receptor activation in the presence of a scaffold protein.

[0278] [ Figure 2 A] Figure 2 A shows conditional IL-2R activation by expressing a mixture of VHH-VHH fusions that bind to the SARS-CoV2 receptor binding domain (RBD) in a two-site manner and to the IL-2 receptor on HEK-Blue IL-2 cells expressing RBD or PDL1.

[0279] [ Figure 2 B] Figure 2 B shows a schematic diagram of the experimental system of this example.

[0280] [ Figure 3 ] Figure 3 A and 3B show the comparison between a mixture of recombinant VHH-VHH fusions that bind to the IL-2 receptor and bind to the RBD in a two-site manner and a mixture of recombinant VHH-VHH fusions that bind to the IL-2 receptor and bind to the RBD in a single-site manner by treating these recombinant VHH-VHH fusions to HEK-Blue IL-2 cells expressing RBD ( Figure 3 A) or HEK-Blue IL-2 cells expressing PDL1 ( Figure 3 B) to make the comparison.

[0281] [ Figure 4 ] Figure 4 Shown is conditional IL-2R activation by expressing a mixture of VHH-VHH fusions binding to PDL1 and the IL-2 receptor on HEK-Blue IL-2 cells expressing PDL1 or RBD.

[0282] [ Figure 5 ] Figure 5 A and 5B show the fusion protein of Split Neo-2 / 15 and VHH binding to RBD by transfecting the expression plasmid together with RBD-IL2RA or PDL1 into HEK-BlueIL-2 cells ( Figure 5 A) A mixture of Vhh2b-Nb36 and Nb21-Vhh2g that binds to RBD and IL-2 receptor ( Figure 5 Comparison between A) and B).

[0283] [ Figure 6 A] Figure 6 A shows conditional IL-2R activation by a mixture of VHH-VHH fusions that bind to TNFa and IL-2 receptor expressed on HEK-Blue IL-2 cells expressing TNFa or PDL1.

[0284] [ Figure 6 B] Figure 6 B shows a schematic of the activation of TNFa-dependent IL-2R by a mixture of VHH-VHH fusions.

[0285] [ Figure 7 ] Figure 7 A and 7B show conditional Wnt receptor activation by a mixture of recombinant VHH-VHH and VHH-scFv fusions that can bind to RBD and Wnt receptors, FZD and LRP, on HEK293 STF cells expressing RBD (A) or HEK293 STF cells expressing PDL1 (B). Figure 7 A) or HEK293 STF cells expressing PDL1 (B). Figure 7

[0286] [ Figure 8 ] Figure 8 A shows conditional IL-2R signal induction by Vhh2b-Nb36 / Nb21-Vhh2g Fc fusion proteins on HEK-Blue IL-2 cells expressing RBD or PDL1. Figure 8 B shows IL-2R activation by recombinant IL-2 on HEK-Blue IL-2 cells expressing RBD or PDL1.

[0287] [ Figure 9 A] Figure 9 A shows a schematic of the activation of IL-2Ra-dependent IL-2R by a mixture of IL2_RETR and Vhh2g-BT942 scFv.

[0288] [ Figure 9 B] Figure 9 B shows IL-2R signal induction by a mixture of IL2_RETR and Vhh2g-BT942 scFv that can bind to IL-2Ra as a scaffold protein and IL-2Rb or IL-2Ry as target receptor proteins on HEK-Blue IL-2 cells with or without treatment of daclizumab (anti-IL-2Ra neutralizing Ab).

[0289] [ Figure 9 C] Figure 9 ​C shows IL-2R activation by recombinant IL-2 on HEK-Blue IL-2 cells with or without treatment of daclizumab.

[0290] [Fig. 10] Figure 10 shows a comparison between two-site binding to IL-2Ra and unit point binding in activation of IL-2R by mixtures of IL-2 mutants and / or VHH-scFV that can bind to IL-2Ra as a scaffold protein and IL-2Rb or IL-2Ry as a target receptor protein.

[0291] [Fig. 11] Figures 11A and 11B show conditional IL-2R signal induction by mixtures of VHH-scFv fusions that bind to PD1 and IL-2 receptor on HEK-Blue IL-2 cells expressing PD1 (Fig. 11A) or HEK-Blue IL-2 cells expressing PDL1 (Fig. 11B).

[0292] [Fig. 12] Figure 12 shows a comparison between two-site binding to PD1 and unit point binding in activation of IL-2R by mixtures of VHH-scFv that bind to PD1 as a scaffold protein and IL-2 receptor as a target receptor protein.

[0293] [Fig. 13] Figure 13 shows binding activity of anti-PD1 antibodies on cell lines expressing PD1 as assessed by flow cytometry.

[0294] [Fig. 14] Figure 14 shows neutralization activity of anti-PD1 Abs as assessed by using PD1+Jurkat reporter cell lines.

[0295] [Fig. 15] Figure 15 shows conditional IL-2R receptor activation by mixtures of VHH-IgG fusion proteins that bind to IL-2Rb or IL-2Ry as a target receptor protein and PD1 as a target scaffold protein on HEK-Blue IL-2 cells expressing PD1 (Fig. 15A) or HEK-Blue IL-2 cells expressing PDL1 (Fig. 15B). All mixtures are combinations of neutralizing and non-neutralizing anti-PD1 Abs.

[0296] [Fig. 16] Figure 16 shows STAT5 activation by mixtures of VHH-IgG or VHH-scFv fusion proteins that bind to IL-2Rb or IL-2Ry as a target receptor protein and PD1 as a target scaffold protein on CD4+T cells expressing PD1 (Fig. 16A) or NK92 cells that are negative for PD1 (Fig. 16B).

[0297] [Fig. 17] Figure 17 shows conditional IL-2R receptor activation by a mixture of VHH-IgG fusion proteins binding to IL-2R beta or IL-2R gamma as target receptor protein and PD1 as target scaffold protein on HEK-Blue IL-2 cells expressing PD1 (Fig. 17A) or HEK-Blue IL-2 cells expressing PDL1 (Fig. 17B).

[0298] [Fig. 18] Figure 18 shows conditional IL-2R receptor activation by a mixture of VHH-IgG or scFv-IgG fusion proteins binding to IL-2R beta or IL-2R gamma as target receptor protein and PD1 as target scaffold protein on HEK-Blue IL-2 cells expressing PD1 (Fig. 18A) or HEK-Blue IL-2 cells expressing PDL1 (Fig. 18B).

[0299] [Fig. 19] Figure 19 shows activation of the IL-2 receptor by a mixture of VHH-scFv fusion proteins binding to IL-2R beta or IL-2R gamma as target receptor protein and CD25 as target scaffold protein on activated CD4+ T cells expressing CD25 (Fig. 19).

[0300] [Fig. 20] Figure 20 shows activation of the GM-CSF receptor by a mixture of VHH-scFv fusion proteins binding to CSF2RA or CSF2RB as target receptor protein and PDL1 as target scaffold protein on HEK-Blue GM-CSF cells expressing PDL1 (Fig. 20A) or HEK-Blue GM-CSF cells expressing RBD (Fig. 20B).

[0301] [Fig. 21] Figure 21 shows activation of the cMET receptor by VHH-Fab fusion proteins binding to cMET as target receptor protein and RBD as target scaffold protein in the presence or absence of recombinant RBD protein. Fig. 21A shows activation of the cMET receptor by a mixture of anti-RBD bispecific Abs fused to a cMET binding domain. Fig. 21B and 21C show activation of the cMET receptor by anti-RBD monospecific antibodies fused to a cMET binding domain.

[0302] [Fig. 22] Figure 22 shows conditional Wnt receptor activation by a mixture of bispecific Abs binding to Wnt receptor as target receptor protein and MUC1 as target scaffold protein on HEK293 STF cells expressing MUC1 or RBD.

[0303] [Figure 23] Figure 23 shows conditional Wnt receptor activation on HEK293 STF cells expressing CEA or RBD by a mixture of bispecific Abs that bind to Wnt receptors as target receptor proteins and CEA as target scaffold proteins. Figure 23A shows Wnt receptor activation by a mixture of anti-CEA dual-site Abs, and Figure 23B shows Wnt receptor activation by a mixture of anti-CEA single-site Abs.

[0304] [ Fig. 24] Fig. 24 shows STAT5 activation in CD8+ or CD4+ T cells by a mixture of VHH-VHH fusion proteins that bind to CD8 as a target scaffold protein and IL-2Rβ or IL-2Rγ as a target receptor protein.

[0305] [ Fig. 25] Fig. 25 shows STAT5 activation in CD8+ T cells by a mixture of VHH-VHH fusion proteins that bind to CD8 as a target scaffold protein and IL-2Rβ or IL-2Rγ as a target receptor protein.

[0306] [ Fig. 26] Fig. 26 shows the proliferation of CD8+ or CD4+ T cells by a mixture of VHH-VHH fusion proteins that bind to CD8 as a target scaffold protein and IL-2Rβ or IL-2Rγ as a target receptor protein. DETAILED DESCRIPTION

[0307] General techniques

[0308] The practice of the present application will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are within the skill of the art. Such techniques are explained fully in the literature, such as, Molecular Cloning: A Laboratory Manual, second edition (Sambrook et al., 1989); Oligonucleotide Synthesis (M. J. Gait, ed., 1984); Animal Cell Culture (R. I. Freshney, ed., 1987); Methods in Enzymology (Academic Press, Inc.); Current Protocols in Molecular Biology (F. M. Ausubel et al., eds 1987, and periodic updates); PCR: The Polymerase Chain Reaction (Mullis et al., ed., 1994); A Practical Guide to Molecular Cloning (Perbal Bernard V., 1988); Phage Display: A Laboratory Manual (Barbas et al., 2001).

[0309] The following definitions and detailed description are provided to assist in understanding the present disclosure shown herein. All references mentioned herein are specifically incorporated by reference.

[0310] I. Definitions

[0311] Protein / Peptide

[0312] 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" generally refers to a peptide of about 4 amino acids in length or more, rather than to a specific length of the product. As used herein, the term also includes fragments of polypeptides. Thus, a peptide, dipeptide, tripeptide, oligopeptide, "protein," "amino acid chain," or any other term used to refer to a chain having two or more amino acids is included within the definition of "polypeptide," and the term "polypeptide" can be used in place of, or interchangeably with, any of these terms. The term "polypeptide" is also intended to refer to post-expression modifications of the polypeptide, including but not limited to, glycosylation, acetylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage, or modification with non-naturally occurring amino acids. A polypeptide can be derived from a natural biological source or produced by recombinant techniques, without necessarily being translated from a specified nucleic acid sequence. It can be generated in any manner, including by chemical synthesis. A polypeptide as described herein can be about 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 in size. Polypeptides can have a defined three-dimensional structure, but they do not necessarily have such a structure. Polypeptides that have a defined three-dimensional structure are referred to as folded; and polypeptides that do not have a defined three-dimensional structure, but can adopt a large number of different conformations, are referred to as unfolded.

[0313] Fusion protein

[0314] As used herein, the term "fusion protein", sometimes abbreviated as "(fusion(s))", refers to a single chain protein, i.e. a protein formed from a combination of at least two (poly)peptides, formed from single chain amino acids. In embodiments, the combination occurs via a peptide bond. In embodiments, the combination occurs via a peptide bond provided by a peptide linker. An example of a fusion protein of the application is the first target binding molecule of [A-1] or [A-4], wherein the first binding domain is optionally linked or joined to the second binding domain via a linker. Another example of a fusion protein of the application is the second target binding molecule of [A-1] or [A-4], wherein the third binding domain is optionally joined to the fourth binding domain by a linker. Another example includes the protein complex of [A-39] or [A-42], wherein the first binding domain and the second binding domain are joined, or wherein the third binding domain and the fourth binding domain are joined. In some embodiments, all four binding domains are joined, and thus part of one single fusion protein.

[0315] protein complexes

[0316] 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 the polypeptides in the complex does not occur via a peptide bond and thus allows the polypeptides to be separated without destroying the primary structure of the proteins involved. For example, the polypeptides in the protein complex can be associated via one or more disulfide bonds or non-covalently. An exemplary embodiment of a protein complex of the present invention includes 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 non-competitive binding to the scaffold protein, wherein the first receptor protein and the second receptor protein are each a receptor subunit capable of associating to form a receptor complex, wherein 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.

[0317] Another embodiment of a protein complex of the present invention includes the 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, wherein the first binding domain and the third binding domain are capable of non-competitive binding to the scaffold protein, wherein the first receptor protein and the second receptor protein are each a receptor subunit capable of associating to form a receptor complex, wherein 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 to each other to form one single polypeptide chain.

[0318] target binding molecules

[0319] 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 the protein or polypeptide or fusion protein or protein complex and its target, the target binding molecule determining the presence of 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. The target binding molecule binds its target with higher affinity, avidity, more readily and / or for a longer duration than it binds another target. In one embodiment, the extent of binding of a target binding molecule to an unrelated target is less than about 10% of the extent of binding of the target binding molecule to the target, e.g., as measured by a radioimmunoassay (RIA). In certain embodiments, a target binding molecule that specifically binds to a target has a dissociation constant (Kd) of 1 micromolar (pM) or less, 100 nM or less, 10 nM or less, 1 nM or less, 0.1 nM or less, 0.01 nM or less, or 0.001 nM or less (e.g., 10"8M or less, e.g., 10"8M to 10"13M, e.g., 10"9M to 10"13M). A "target binding molecule" of the application can comprise an antibody or antibody-like construct, a VHH, or a protein complex comprising a heavy chain and a light chain associated with each other.

[0320] Binding domain

[0321] A target binding molecule of the application comprises two binding domains, wherein each binding domain has a different target. Thus, a target binding molecule of the application is capable of binding two targets simultaneously. The term "binding domain" as used herein refers to a portion of a protein that exhibits target / antigen binding activity. The binding domain can be an antigen binding domain comprising a VH and a VL, a sdAb, a VHH, a scFv, a Fab, a scFab, a Fab', a Fab'-SH, a F(ab')2, a diabody, a triabody, a Fv, an aptamer, or an affimer. It can be derived from a cytokine (including a cytokine mutant), or from a natural ligand (including a mutant). It can also be a split cytokine, etc.

[0322] Neutralizing and non-neutralizing binding domains

[0323] The term "neutralizing binding domain" refers to a binding domain as described above that binds to its target / antigen and causes prevention, inhibition, reduction, delay or interference with an activity of the target / antigen. This can be because the binding domain binds to an epitope that is involved in the activity of the target / antigen. In non-limiting examples, a neutralizing binding domain can bind to an epitope near or at a ligand binding site of the target / antigen, thereby preventing, inhibiting, reducing, delaying or interfering with an activity of the target / antigen. The term "neutralizing binding domain" can also refer to an antagonistic binding domain, such as an antagonistic antibody. The term "non-neutralizing binding domain" refers to a binding domain as described above that binds to its target / antigen and does not cause prevention, inhibition, reduction, delay or interference with an activity of the target / antigen. The term "non-neutralizing binding domain" can also refer to a non-antagonistic binding domain, such as a non-antagonistic antibody.

[0324] Multispecific target binding molecules

[0325] In the present application, the target binding molecules provided herein are multispecific target binding molecules, e.g., bispecific target binding molecules. A multispecific target binding molecule is a target binding molecule that has binding specificity for at least two different sites. In the present application, one of the binding specificities is for a scaffold protein and the other is for a receptor protein. Bispecific target binding molecules can be prepared as full-length antibodies or as comprising antibody fragments.

[0326] Techniques for making multispecific target binding molecules include, but are not limited to, recombinant co- expression of two immunoglobulin heavy chain-light chain pairs having different specificities (see, Milstein and Cuello, Nature 305:537 (1983), WO 93 / 08829, and Traunecker et al., EMBO J. 10:3655 (1991)), and "knobs-in-holes" engineering (see, e.g., U.S. Patent No. 5,731,168). Multispecific target binding molecules can also be made by engineering electrostatic steering effects for the production of antibody Fc-heterodimeric molecules (WO 2009 / 089004 Al); cross-linking two or more antibodies or fragments (see, e.g., U.S. Patent No. 4,676,980, and Brennan et al., Science, 229:81 (1985)); using leucine zippers to produce bispecific antibodies (see, e.g., Kostelny et al., J. Immunol., 148(5): 1547-1553 (1992)); using "diabody" technology (see, e.g., Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993)); and using single-chain Fv (scFv) dimers (see, e.g., Gruber et al., J. Immunol., 152:5368 (1994)); and according to, e.g., Tutt et al. J. Immunol. 147:60 (1991).

[0327] Competitive binding and noncompetitive binding

[0328] The first target binding molecule and the second target binding molecule of the present application, or the first binding domain and the third binding domain of the protein complex of the present application, are capable of non-competitive binding to the scaffold protein. As used herein, "competitive binding" refers to a process in which two binding domains compete for the same epitope or paratope, resulting in only one binding domain capable of binding to the scaffold protein. The epitopes or paratopes of the two binding domains can be identical or can be overlapping. In any case, the two binding domains cannot bind to their epitope paratope simultaneously. As used herein, "non-competitive binding" refers, in turn, to a process in which two binding domains can bind to their epitope or paratope without interfering with the binding mechanism of the corresponding other binding domain. In one embodiment, the epitopes or paratopes are not overlapping and there is no steric hindrance that simultaneously prevents the simultaneous binding of both binding domains. One example of non-competitive binding is bivalent binding. In another embodiment, the epitopes can be identical or overlapping, but there are more than one epitope on the target. This can be the case if the target is a multimer. For example, the scaffold protein of the present application can be composed of several subunits, each of which contains an epitope. In this case, the target binding molecules bind to two distinct but identical epitopes, which have the same sequence but are on different subunits of the multimer. It is essential that the two target binding molecules can bind simultaneously without interfering with the binding mechanism of the corresponding other target binding molecule.

[0329] Two-site binding

[0330] As used herein, "bivalent binding" refers to a binding mechanism in which two epitopes on the target molecule are targeted. In the present application, the two binding domains targeting the scaffold protein each target a different epitope on the protein, allowing the two binding domains to bind to the scaffold protein simultaneously.

[0331] In one embodiment, the two binding domains targeting the scaffold protein are capable of bivalent binding to the scaffold protein, wherein the epitopes bound by each binding domain are in proximity to each other. In one embodiment, the two binding domains targeting the scaffold protein are capable of bivalent binding to the scaffold protein within a domain of the scaffold protein. The epitopes can be determined from the crystal structure of the complex of the scaffold protein binding domain and the scaffold protein. Specifically, the interatomic distances between the non-hydrogen atoms constituting the side chains or main chains of the amino acids forming the scaffold protein binding domain and the non-hydrogen atoms constituting the side chains or main chains of the amino acids forming the scaffold protein are calculated. The amino acid residues containing non-hydrogen atoms with interatomic distances below a threshold set at 3.5 angstroms, 4.0 angstroms, 4.2 angstroms, 4.5 angstroms, or 5.0 angstroms are considered to be residues included within the epitopes.

[0332] The proximity of epitopes, or the distance between epitopes bound by each binding domain, in the present specification can then be determined. In one embodiment, the interatomic distance between any alpha-carbon of a first epitope on the scaffold protein and any alpha-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, 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 alpha-carbon of a first epitope on the scaffold protein and any alpha-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, 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 Angstroms, 54.0 Angstroms, 54.5 Angstroms, 55.0 Angstroms, 55.5 Angstroms, 56.0 Angstroms, 56.5 Angstroms, 57.0 Angstroms, 57.5 Angstroms, 58.0 Angstroms, 58.5 Angstroms, 59.0 Angstroms, 59.5 Angstroms, 60.0 Angstroms, 60.5 Angstroms, 61.0 Angstroms, 61.5 Angstroms, 62.0 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.

[0333] Target binding molecule combination

[0334] As used herein, the term "target binding molecule combination" refers, in one embodiment, to 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 can be linked or joined to each other, thereby resulting in a single protein or protein complex that combines both the first target binding molecule and the second target binding molecule.

[0335] In a further embodiment, the combination refers to two compositions, wherein a first composition comprises a first target binding molecule and a second composition comprises a second target binding molecule. Since both the first target binding molecule and the second target binding molecule need to be present at the site of biological action, both compositions need to be part of the combination. However, it is not necessary to combine the two compositions into one single composition. Separating the compositions, wherein a first composition comprises a first target binding molecule and a second composition comprises a second target binding molecule, allows for separate administration of the compositions, which in turn allows for better fine-tuning of the method of administration. Thus, as used herein, the term "combination" encompasses adding both target binding molecules to a single composition prior to administration, as well as separately (simultaneously, concurrently or consecutively) administering the two target binding molecules, which results in a combination of target binding molecules administered separately at the site of biological action as defined in [A-33] and / or [A-34].

[0336] Scaffold proteins

[0337] Each target binding molecule of the present application comprises a binding domain capable of binding to a scaffold protein. As used herein, the term "scaffold protein" refers, inter alia, to a cell surface protein, a soluble protein or an insoluble protein expressed in a target cell, a target tissue or a target organ. As used herein, a scaffold protein is not limited by structure or function. Rather, a scaffold protein serves as an anchor for the first binding domain and the third binding domain of the first target binding molecule and the second target binding molecule or protein complex for non-competitive and / or two-site binding. The scaffold protein can be a cell surface marker, a specific cell surface marker, an immune cell surface marker, a T cell exhaustion marker, a tumor associated marker, a tumor associated antigen or a tumor specific antigen, a tissue specific marker, a disease tissue specific marker, an organ specific marker, a soluble protein, an insoluble protein deposit, 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, MUC1, CEA, CTLA4, IL2R alpha, CXCR5, neuropilin-1, TIM3, LAG3, TNF alpha, 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, beta amyloid, MBP or ASGPR, etc. In a preferred embodiment, the scaffold protein is PD1.

[0338] The scaffold protein of the present application 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 biological site of action. In case of a soluble scaffold protein, localization to the intended biological site of action can be ensured by targeting a soluble scaffold protein known to be present at high concentrations at the intended biological site of action. For example, when the intended biological site of action is a tumor tissue, it can be useful to target a soluble scaffold protein secreted by the tumor tissue. Thus, localization is achieved and systemic activation of the signaling pathway can be avoided.

[0339] Tumor-specific or tumor-related markers

[0340] As used herein, "tumor-specific marker" or "tumor-associated marker" as a non-limiting example of a scaffold protein means an antigen expressed by a cancer cell that allows the cancer cell to be distinguished from a healthy cell. For example, "tumor-specific marker" or "tumor-associated marker" includes an antigen that expresses as a malignant or abnormal sugar chain present on the cell surface or protein molecule when a cell becomes a cancer cell. Specific examples thereof include ALK, Pleiotrophin (PTN), EpCAM, CA125, Prostate 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, viral-induced tumor antigens (e.g., envelope antigens of DNA and RNA tumor viruses), EBV-specific antigens, alpha-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, MAGE-X2, YKL-40, EREG, CA15-3, CLEC12A, Nectin 4, Trop2, BCMA, Tissue factor, FRa (FOLR1), ErbB3, Claudin18 (Claudin18.2), B7-H3 (CD276), MET, PSCA, PTK7, MSLN (Mesothelin), CCR4, CDH6, IL13RA2, DLL3, GPC3, Claudin6, Fibroblast associated protein (FAP), FLT3, GD2, GD3, and any fragments of these polypeptides, and modified structures thereof.

[0341] Multimeric scaffold proteins

[0342] The scaffold protein of the application can be composed of several subunits. The subunits can be identical or different. Thus, the scaffold protein can be a homo- or hetero-multimer. In some embodiments, wherein the first and third binding domains are directed to the same or overlapping epitopes on a homo-multimeric scaffold protein, the homo-multimeric scaffold protein comprises more than one of the same epitope, thereby allowing non-competitive binding of the first and third binding domains.

[0343] receptors

[0344] Each target binding molecule of the present application comprises a binding domain capable of binding a receptor protein. As used herein, the term“receptor” refers to a protein structure in or on a cell that receives and transduces signals by binding to a particular molecule and / or multimerization. The receptor can be a cytokine receptor, a Wnt receptor, a receptor that induces a signal through heterodimerization, homodimerization, or oligomerization (activation only upon association of monomers of a homodimer, homotrimer, homomultimer, heterodimer, heterotrimer, or heteromultimer). According to the present application, the first receptor protein and the second receptor protein are each a receptor subunit capable of associating to form a receptor complex. The receptor complex is activated upon association of the receptor subunits. The association of the receptor subunits occurs when the first target binding molecule binds to its two targets, and when the second target binding molecule binds to its two targets. Examples of receptor subunits are IL2R beta, IL2R gamma, 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, 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 preferred embodiments, the first receptor protein is IL-2R beta and the second receptor protein is IL-2R gamma.

[0345] Induced receptor signaling

[0346] As used herein, the term "inducing receptor signaling" refers to the biological mechanism that occurs upon formation of an activated receptor complex. Activation of the receptor complex requires association of receptor protein subunits of the receptor complex. The receptor protein comprises an extracellular portion as known in the art. This extracellular portion can interact with the target binding molecule of the present application. The receptor protein can further comprise a cytoplasmic portion that can interact with downstream members of the signaling pathway activated by the receptor complex. As is well known in the art, when the receptor subunits associate, the receptor subunits are in close proximity to each other, or induce receptor signaling by themselves or by facilitating a conformational change of the receptor complex members. For example, the cytoplasmic portion of the receptor protein subunit can recruit Janus kinases to initiate an intracellular signaling cascade. The Janus kinases phosphorylate cytoplasmic STAT proteins. The phosphorylation status of the STAT proteins can be readily monitored in assays established in the art. In certain embodiments, when the receptor protein is IL-2R, IL-2 receptor complex activation can be assessed using Quanti-Blue solution (InvivoGen, #rep-qbs), which is a colorimetric enzyme assay that determines alkaline phosphatase activity by measuring the optical density at 620 nm using a Multiskan™ plate reader. In certain embodiments, receptor signaling of the receptor complex is induced when the optical density is increased at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 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 the presence of the target binding molecule combination or protein complex of the present application as compared to in the absence of the target binding molecule combination or protein complex of the present application.

[0347] Proximity

[0348] When the first binding domain, the second binding domain, the third binding domain, and the fourth binding domain of the target binding molecule combination or protein complex of the present application bind, the first receptor protein and the second receptor protein are brought into proximity with each other, such that a receptor complex comprising the first receptor subunit and the second receptor subunit is activated and induces signaling. As used herein, the term "proximity" refers to the proximity of the distance between two individual receptor proteins (e.g., between IL-2Rβ), and the second receptor protein is IL-2Rγ.

[0349] Half-life extension domain

[0350] In the present application, the first binding domain and / or the third binding domain can each be linked to a half-life extension domain. One obstacle to the use of protein therapeutics is the short half-life of these small proteins in circulation. Persistence in circulation is desirable because protein therapeutics are typically not administered orally, and are typically administered by subcutaneous, intramuscular, or intravenous injection or infusion. Thus, in preferred embodiments, the present application can encompass the use of one or more pharmaceutical half-life extension domains, such as polyethylene glycol (PEG), an immunoglobulin Fc domain or CH2 domain of Fc, albumin (e.g., human serum albumin (HSA) or variants thereof), an albumin binding protein, an FcRn binding domain, transthyretin, or a thyroid binding globulin (TBG).

[0351] An exemplary method of improving the pharmacokinetics (PK) of a polypeptide is by expressing in the polypeptide chain a domain that binds to a receptor that is recycled to the plasma membrane of a cell rather than being degraded in a lysosome, such as the FcRn receptor and the transferrin receptor on endothelial cells. Three types of proteins, e.g., human IgG, HSA (or fragments), and transferrin, persist in human serum much longer than would be predicted based on their size alone, as a function of their ability to bind to receptors that are recycled rather than degraded in lysosomes. These proteins or fragments of proteins that retain FcRn binding are often linked to other polypeptides to extend their serum half-life. In one embodiment, the half-life extension domain is a human serum albumin (HSA) binding domain. HSA can also be bound directly to the pharmaceutical composition or via a short linker. HSA fragments can also be used. HSA and fragments thereof can be used both as blocking moieties and as half-life extension domains. Human IgG can also perform similar functions.

[0352] The serum half-life prolonging domain can also be an antigen binding polypeptide that binds to a protein with long serum half-life such as serum albumin, transferrin, and the like. Examples of such polypeptides include antibodies and fragments thereof, including polyclonal antibodies, recombinant antibodies, human antibodies, humanized antibodies, single chain variable fragments (scFv), single domain antibodies such as heavy chain variable domains (VH), light chain variable domains (VL), and variable domains of camelid-type nanobodies (VHH), dAbs, and the like. Other suitable antigen binding domains include non-immunoglobulin proteins that mimic antibody binding and / or structure, such as anticalins, affilins, affibody molecules, affimers, affitins, alphabodies, avimers, DARPins, fynomers, kunitz domain peptides, monobodies, and binding domains based on other engineered scaffolds such as SpA, GroEL, fibronectin, lipocallin, and CTLA4 scaffolds. Further examples of antigen binding polypeptides include ligands for a desired receptor, ligand binding portions of receptors, lectins, and peptides that bind or associate with one or more target antigens.

[0353] In other embodiments, the serum half-life prolonging domain can 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-peptidic water-soluble poly(ethylene oxide). The term “PEG” also means a polymer containing a majority (i.e., more than 50%) of -OCH2CH2- repeating units. With regard to particular forms, PEGs can have any number of various molecular weights, as well as structures or geometries, such as “branched,” “linear,” “forked,” “multi-functional,” and the like, which will be described in more detail below. PEGs are not limited to a particular structure, and can be linear (e.g., end-capped, e.g., alkoxy PEG or bifunctional PEG), branched or multi-armed (e.g., forked PEG or PEG attached to a polyol core), dendritic (or star-shaped) structures, each with or without one or more degradable linkages. Furthermore, the internal structure of the PEG can be organized in any number of different repeating patterns, and can be selected from the group consisting of homopolymers, alternating copolymers, random copolymers, block copolymers, alternating terpolymers, random terpolymers, and block terpolymers. PEGs can be conjugated to polypeptides and peptides by any suitable method. Typically, a reactive PEG derivative, such as a N-hydroxysuccinmide ester PEG, is reacted with a peptide or polypeptide comprising an amino acid with a side chain containing an amine, thiol, carboxylic acid, or hydroxyl functional group, such as cysteine, lysine, asparagine, glutamine, threonine, tyrosine, serine, aspartic acid, and glutamic acid.

[0354] Possible Fc modifications

[0355] The target binding molecules of the invention or the protein complexes of the invention can further comprise other possible Fc modifications. These modifications can be suitable to reduce antibody-dependent cellular cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC), increase the isoelectric point (pi), or increase Fc Rn binding to improve pharmacokinetic characteristics, or they can be Fc mutants for the preparation of bispecific molecules.

[0356] In one embodiment, the invention comprises FcyRIIB binding polypeptides comprising Fc modifications and methods of use thereof. In some embodiments, the Fc modifications of the invention having enhanced FcyRIIb binding activity comprise at least one amino acid alteration in a parent Fc region. In further embodiments, the ratio of [KD value of parent Fc region of monkey FcyRIIb] / [KD value of variant Fc region of monkey FcyRIIb] is 2.0 or greater. In further embodiments, the ratio of [KD value of parent Fc region of monkey FcyRIIIa] / [KD value of Fc modification of monkey FcyRIIIa] is 0.5 or less. In further embodiments, the ratio of [KD value of parent Fc region of human FcyRIIb] / [KD value of variant Fc region of human FcyRIIb] is 2.0 or greater. In further embodiments, the ratio of [KD value of parent Fc region of human FcyRIIIa] / [KD value of Fc modification of human FcyRIIIa] is 0.5 or less. In further embodiments, the ratio of [KD value of parent Fc region of human FcyRIIa (H type)] / [KD value of variant Fc region of human FcyRIIa (H type)] is 5.0 or less. In further embodiments, the ratio of [KD value of parent Fc region of human FcyRIIa (R type)] / [KD value of Fc modification of human FcyRIIa (R type)] is 5.0 or less. In another embodiment, the KD value of the variant Fc region of monkey FcyRIIb is 1.0 x 10 -6 M or less. In another embodiment, the KD value of the Fc modification of monkey FcyRIIIa is 5.0 x 10 -7 M or greater. In another embodiment, the KD value of the variant Fc region of human FcyRIIb is 2.0 x 10 -6 M or less. In another embodiment, the KD value of the Fc modification of human FcyRIIIa is 1.0 x 10 -6 M or greater. In another embodiment, the KD value of the variant Fc region of human FcyRIIa (H type) is 1.0 x 10 -7 M or greater. In another embodiment, the KD value of the Fc modification of human FcyRIIa (R type) is 2.0 x 10 -7 M or greater.

[0357] In some embodiments, the Fc modification of the application having enhanced FcyRIIb binding activity comprises at least one amino acid alteration of 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.

[0358] In further embodiments, the Fc modification having enhanced FcyRIIb binding activity comprises at least two amino acid alterations comprising: (a) one amino acid alteration at position 236, and (b) at least one amino acid alteration of 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.

[0359] In further embodiments, the Fc modification having enhanced FcyRIIb binding activity comprises at least two amino acid alterations comprising: (a) one amino acid alteration at position 236, and (b) at least one amino acid alteration of 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.

[0360] In further embodiments, the Fc modification having enhanced FcyRIIb binding activity comprises at least two amino acid alterations comprising: (a) one amino acid alteration at position 236, and (b) at least one amino acid alteration of 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.

[0361] In further embodiments, the Fc modification having enhanced FcyRIIb binding activity comprises at least two amino acid alterations comprising: (a) one amino acid alteration at position 236, and (b) at least one amino acid alteration of at least one position selected from the group consisting of: 268, 295, 326, and 330, according to EU numbering.

[0362] In some embodiments, the Fc modification having enhanced FcyRIIb binding activity of the present application comprises at least one amino acid selected from the group consisting of: (a) Asp, Glu, Phe, Gly, His, lie, Lys, Leu, Met, Asn, Pro, Gin, Arg, Ser, Thr, Val, Trp, Tyr at position 231 ; (b) Ala, Asp, Glu, Phe, Gly, His, lie, Lys, Leu, Met, Asn, Gin, Arg, Ser, Thr, Val, Trp, Tyr at position 232; (c) Asp at position 233; (d) Trp, Tyr at position 234; (e) Trp at position 235; (f) Ala, Asp, Glu, His, lie, Leu, Met, Asn, Gin, Ser, Thr, Val at position 236; (g) Asp, Tyr at position 237; (h) Glu, lie, Met, Gin, Tyr at position 238; (i) lie, Leu, Asn, Pro, Val at position 239; (j) lie at position 264; (k) Phe at position 266; (1) Ala, His, Leu at position 267; (m) Asp, Glu at position 268; (n) Asp, Glu, Gly at position 271 ; (o) Leu at position 295; (p) Leu at position 298; (q) Gin, Phe, lie, Leu at position 325; (r) Thr at position 326; (s) lie, Asn at position 327; (t) Thr at position 328; (u) Lys, Arg at position 330; (v) Gin at position 331 ; (w) Asp at position 332; (x) Asp, lie, Met, Val, Tyr at position 334; and (y) Ala, Asp, Glu, Phe, Gly, His, lie, Lys, Leu, Met, Asn, Gin, Arg, Ser, Thr, Val, Trp, Tyr at position 396; according to EU numbering.

[0363] In further embodiments, the Fc modification having enhanced FcyRIIb binding activity comprises 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.

[0364] In another embodiment, the present application provides polypeptides comprising Fc modifications having increased isoelectric point (pi) and methods of use thereof. In some embodiments, the polypeptides comprising Fc modifications having increased pi comprise at least two amino acid changes in a parent Fc region. In further embodiments, each of the amino acid changes increases the isoelectric point (pi) of the Fc modification as compared to the isoelectric point (pi) of the parent Fc region. In further embodiments, the amino acids can be exposed on the surface of the modified Fc region. In further embodiments, the polypeptides comprise the Fc modification and an antigen binding domain. In further embodiments, the antigen binding activity of the antigen binding domain varies as a function of ionic concentration conditions. In further embodiments, the modified Fc region having increased pi of the present application comprises at least two amino acid changes 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 further embodiments, the Fc modification having increased pi comprises an Arg or Lys at each of the selected positions.

[0365] In another embodiment, the present application provides isolated polypeptides comprising Fc modifications having increased isoelectric point (pi). In certain embodiments, the Fc modifications described herein comprise at least two amino acid changes in a parent Fc region. In certain embodiments, each of the amino acid changes increases the isoelectric point (pi) of the variant Fc region as compared to the isoelectric point (pi) of the parent Fc region. They are based on the finding that antigen elimination from plasma can be facilitated with antibodies whose pi has been increased by modifying at least two amino acid residues, for example when the antibodies are administered in vivo.

[0366] In one embodiment, the pi can be a theoretically determined or experimentally determined pi. The value of the pi can be determined, for example, by isoelectric focusing, as known to one of skill in the art. For example, the value of the theoretical pi can be calculated using genetic and amino acid sequence analysis software (Genetyx, etc.).

[0367] In one embodiment, the pi value can be increased, compared to before the modification, by, for example, at least 0.01, 0.03, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, or more, by at least 0.6, 0.7, 0.8, 0.9, or more, by at least 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, or more, or by 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.

[0368] In certain embodiments, the amino acid used to increase pi can be exposed on the surface of the Fc modification. In the present invention, the amino acid exposed on the surface generally refers to an amino acid residue located on the surface of the polypeptide constituting the Fc modification. The amino acid residue located on the surface of the polypeptide refers to an amino acid residue whose side chain can be in contact with solvent molecules (usually, primarily water molecules). However, the side chain does not necessarily have to be completely in contact with the solvent molecules, and even when a part of the side chain is in contact with the solvent molecules, the amino acid is defined as "an amino acid residue located on the surface." The amino acid residue located on the surface of the polypeptide also includes an amino acid residue located near the surface, and thus can have a charge influence from another amino acid residue whose side chain (even partially) is in contact with the solvent molecules. One of ordinary skill in the art can prepare a homology model of the polypeptide, for example, using a commercially available software. Alternatively, methods known to one of ordinary skill in the art, such as X-ray crystallography, can be used. For example, using a computer program such as the Insight II program (Accelrys), the coordinates from the three-dimensional model are used to determine the amino acid residues that can be exposed on the surface. The surface-exposed sites 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-exposed sites can be determined using software suitable for protein modeling and three-dimensional structural information. Software that can be used for such purposes include, for example, the SYBYL Biopolymer Module software (Tripos Associates). When the algorithm requires user input of a size parameter, the "size" of the probe used in the calculation can be set to a radius of about 1.4 angstroms (A) or less. Furthermore, a method for determining surface-exposed sites 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 such information as described above, a suitable amino acid residue located on the surface of the polypeptide constituting the variant Fc region can be selected.

[0369] In certain embodiments, the polypeptide comprises both the Fc modification and an antigen binding domain. In further embodiments, the antigen is a soluble antigen. In one embodiment, the antigen is present in a biological fluid (e.g., plasma, interstitial fluid, lymph fluid, ascites fluid, and pleural effusion) of a subject. The antigen can also be a membrane antigen.

[0370] In further embodiments, the antigen binding activity of the antigen binding domain varies depending on the ion concentration condition. In one embodiment, the ion concentration is not particularly limited, and refers to the concentration of hydrogen ions (pH) or the concentration of metal ions. In the present context, metal ions refer to: ions of Group I elements other than 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 and platinum group elements; elements belonging to Group V, VI, and VII A subgroups; and metal elements, such as antimony, bismuth, and plutonium. In the present application, metal ions include, for example, calcium ions, as described in WO 2012 / 073992 and WO 2013 / 125667. In one embodiment, the "ion concentration condition" can be a condition in which the difference in the biological behavior of the antigen binding domain between low ion concentration and high ion concentration is of interest. Furthermore, "the antigen binding activity of the antigen binding domain varies depending on the ion concentration condition" means that the antigen binding activity of the antigen binding domain varies between low ion concentration and high ion concentration (such antigen binding domains are referred to herein as "ion concentration-dependent antigen binding domains"). The antigen binding activity of the antigen binding domain under high ion concentration conditions can be higher (stronger) or lower (weaker) than the antigen binding activity 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, the methods described in WO 2009 / 125825, WO 2012 / 073992, and WO 2013 / 046722.

[0371] In one embodiment, the antigen binding activity of the antigen binding domain under high calcium ion concentration conditions can be higher than the antigen binding activity under low calcium ion concentration conditions. The high calcium ion concentration is not particularly limited, but can be a selected concentration of between 100 μΜ and 10 mM, between 200 μΜ and 5 mM, between 400 μΜ and 3 mM, between 200 μΜ and 2 mM, between 400 μΜ and 1 mM, or between 500 μΜ and 2.5 mM, which is preferably close to the plasma (blood) concentration of calcium ions in vivo. Meanwhile, the low calcium ion concentration is not particularly limited, but can be a selected concentration of between 0.1 μΜ and 30 μΜ, between 0.2 μΜ and 20 μΜ, between 0.5 μΜ and 10 μΜ, between 1 μΜ and 5 μΜ, or between 2 μΜ and 4 μΜ, which is preferably close to the concentration of calcium ions in early endosomes in vivo.

[0372] In one embodiment, the ratio between the antigen-binding activity under low calcium ion concentration conditions and the antigen-binding activity under high calcium ion concentration conditions is not limited, but the ratio of dissociation constants (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 more, 10 or more, or 40 or more. The upper limit of this ratio can be 400, 1000, or 10000, as long as such an antigen-binding domain can be produced by techniques known to one of skill in the art. Alternatively, for example, the off-rate constant (kd) can be used instead of KD. In this case, the ratio between the kd under low calcium ion concentration conditions and 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 more, 5 or more, 10 or more, or 30 or more. The upper limit of this ratio can be 50, 100, or 200, as long as the antigen-binding domain can be produced based on the common technical knowledge of one of skill in the art.

[0373] In one embodiment, the antigen-binding activity of the antigen-binding domain under low hydrogen ion concentration (neutral pH) can be higher than the antigen-binding activity under high hydrogen ion concentration (acidic pH). The acidic pH can be, for example, a pH selected from the group consisting of pH 4.0 to pH 6.5, selected from the group consisting of pH 4.5 to pH 6.5, selected from the group consisting of pH 5.0 to pH 6.5, or selected from the group consisting of pH 5.5 to pH 6.5, which is preferably close to the in vivo pH in early endosomes. The acidic pH can also be, for example, pH 5.8 or pH 6.0. In a particular embodiment, the acidic pH is pH 5.8. Meanwhile, the neutral pH can be, for example, a pH selected from the group consisting of pH 6.7 to pH 10.0, selected from the group consisting of pH 6.7 to pH 9.5, selected from the group consisting of pH 7.0 to pH 9.0, or selected from the group consisting of pH 7.0 to pH 8.0, which is preferably close to the in vivo pH in blood plasma (blood). The neutral pH can also be, for example, pH 7.4 or pH 7.0. In a particular embodiment, the neutral pH is pH 7.4.

[0374] In one embodiment, the ratio between antigen binding activity under acidic pH conditions and antigen binding activity under neutral pH conditions is not limited, but the ratio between dissociation constant (KD) under acidic pH conditions and KD under neutral pH conditions (i.e., KD(acidic pH conditions) / KD(neutral pH conditions)) is 2 or more, 10 or more, or 40 or more. The upper limit of the ratio can be 400, 1000, or 10000, as long as such an antigen binding domain can be produced by techniques known to those skilled in the art. Alternatively, for example, the off rate constant (kd) can be used instead of KD. In this case, the ratio of kd under acidic pH conditions and kd under neutral pH conditions (i.e., kd(acidic pH conditions) / kd(neutral pH conditions)) is 2 or more, 5 or more, 10 or more, or 30 or more. The upper limit of the ratio can be 50, 100, or 200, as long as the antigen binding domain can be produced based on the common technical knowledge of those skilled in the art.

[0375] For example, in one embodiment, 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, as described in WO 2009 / 125825. The amino acid can be substituted and / or inserted at any site, as long as the antigen binding activity of the antigen binding domain under acidic pH conditions becomes weaker than the antigen binding activity under neutral pH conditions compared to before the substitution or insertion. When the antigen binding domain has a variable region or a CDR, the site can be within the variable region or the CDR. The number of substituted or inserted amino acids can be appropriately determined by those skilled in the art; and the number can 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 depending on the hydrogen ion concentration conditions. Such amino acids include, for example, natural amino acids such as His (H) and Glu (E), and unnatural amino acids such as histidine analogs (US 2009 / 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 having a side chain pKa of 6.0-7.0, which include, for example, His (H), can also be used.

[0376] In another embodiment, preferred antigen binding domains for Fc modifications having increased pi are described, and can be obtained by the methods described in Japanese Patent Applications JP 2015-021371 and JP 2015-185254.

[0377] In certain embodiments, the Fc modification having an increased pi comprises at least two amino acid changes 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.

[0378] In further embodiments, the Fc modification having an increased pi comprises at least two amino acid changes 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.

[0379] In another aspect, the present application provides a polypeptide comprising an Fc modification having an increased pi, the modification comprising amino acid changes of any one of (1)-(10): (1) positions 311 and 341; (2) positions 311 and 343; (3) positions 311, 343, and 413; (4) positions 311, 384, and 413; (5) positions 311 and 399; (6) positions 311 and 401; (7) positions 311 and 413; (8) positions 400 and 413; (9) positions 401 and 413; and (10) positions 402 and 413; according to EU numbering.

[0380] Methods for increasing the pi of a protein, for example, are 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 of -1 under pH conditions sufficiently above their side chain pKa, which is a well-known theory to those skilled in the art. For example, the theoretical pKa of an aspartic acid side chain is 3.9, and the side chain has a negative charge of -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 of +1 under pH conditions sufficiently below their side chain pKa. For example, the theoretical pKa of an arginine side chain is 12.5, and the side chain has a positive charge of +1 under neutral pH conditions (e.g., in a solution at pH 7.0). Meanwhile, amino acids known to be uncharged under neutral pH conditions (e.g., in a solution at pH 7.0) include 15 types of natural amino acids, namely alanine, cysteine, phenylalanine, glycine, isoleucine, leucine, methionine, asparagine, proline, glutamine, serine, threonine, valine, tryptophan, and tyrosine. Of course, it will be appreciated that amino acids for increasing pi can be non-natural amino acids.

[0381] As described above, the method for increasing the pi of a protein under neutral pH conditions (e.g., in a solution at pH 7.0) can, for example, impart a +1 charge change to the target protein by replacing an aspartic acid or a glutamic acid (which has a side chain with a negative charge of -1) with an amino acid having a side chain that is uncharged in the amino acid sequence of the protein. In addition, a protein can be imparted a +1 charge change, for example, by replacing an amino acid whose side chain is uncharged with an arginine or a lysine (which has a side chain with a positive charge of +1). In addition, a protein can be imparted a +2 charge change at one time by replacing an aspartic acid or a glutamic acid (which has a side chain with a negative charge of -1) with an arginine or a lysine (which has a side chain with a positive charge of +1). Alternatively, in order to increase the pi of a protein, an amino acid having a side chain that is uncharged and / or preferably an amino acid having a side chain that is positively charged can be added or inserted into the amino acid sequence of the protein, or an amino acid having a side chain that is uncharged and / or preferably an amino acid having a side chain that is negatively charged present in the amino acid sequence of the protein can be deleted. It should be understood that, 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 the side chain (NH3 + and COO - at the amino group at the N-terminal and the carbonyl group at the C-terminal, respectively). Thus, the pi of a protein can also be increased by some addition, deletion, substitution, or insertion to the functional group derived from the main chain.

[0382] The substitution of amino acids to increase the pi includes, for example, in the amino acid sequence of the parent Fc region, replacing an amino acid having a side chain with a negative charge with an amino acid whose side chain is uncharged, replacing an amino acid whose side chain is uncharged with an amino acid having a side chain with a positive charge, and replacing an amino acid having a side chain with a negative charge with an amino acid having a side chain with a positive charge, alone or in appropriate combinations.

[0383] The insertion or addition of amino acids to increase the pi includes, for example, in the amino acid sequence of the parent Fc region, inserting or adding an amino acid whose side chain is uncharged and / or inserting or adding an amino acid having a side chain with a positive charge, alone or in appropriate combinations.

[0384] The deletion of amino acids to increase the pi includes, for example, in the amino acid sequence of the parent Fc region, deleting an amino acid whose side chain is uncharged and / or deleting an amino acid having a side chain with a negative charge, alone or in appropriate combinations.

[0385] In one embodiment, the natural amino acids used to increase pi can be classified as follows: (a) amino acids with a side chain that carries a negative charge can be Glu (E) or Asp (D); (b) amino acids whose side chain does not carry a charge can be Ala (A), Asn (N), Cys (C), Gin (Q), Gly (G), His (H), He (I), Leu (L), Met (M), Phe (F), Pro (P), Ser (S), Thr (T), Trp (W), Tyr (Y), or Val (V); and (c) amino acids with a side chain that carries a positive charge can be His (H), Lys (K), or Arg (R). In one embodiment, the modified amino acid insertion or substitution is Lys (K) or Arg (R).

[0386] Affinity

[0387] The term "affinity" refers to the strength of the sum total of noncovalent interactions between individual binding sites of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless indicated otherwise, "binding affinity," as used herein, refers to intrinsic binding affinity, which reflects a 1 : 1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of a molecule X for its partner Y can be generally represented by the dissociation constant (Kd). Affinity can be measured by routine methods known in the art, including those described herein. Specific illustrative and exemplary embodiments for measuring binding affinity are described below.

[0388] In certain embodiments, the target binding molecules provided herein have a dissociation constant (Kd) of 1 mM or less, 100 nM or less, 10 nM or less, 1 nM or less, 0.1 nM or less, 0.01 nM or less, or 0.001 nM or less (e.g., 10 -8 M or less, e.g., 10 -8 M to 10 -13 M, e.g., 10 -9 M to 10 -13 M) for its target.

[0389] In one embodiment, Kd is measured by a radiolabeled antigen binding assay (RIA). In one embodiment, the RIA is performed with the Fab form of the target binding molecule of interest and its target. For example, the Kd is determined by incubating a minimum concentration of the Fab with a constant concentration of the target in a series of unlabelled antigen titrations. The concentration of Fab is chosen to be saturating at the lowest antigen concentration. The amount of Fab bound to the target is determined by the amount of radioactivity in the complex. The Kd is calculated from the dissociation of the complex at various antigen concentrations. 125The solution binding affinity of Fab for antigen was measured by equilibrating Fab with 1) labeled antigen and then capturing the bound antigen with an anti-Fab antibody coated plate (see, for example, Chen et al., J. Mol. Biol. 293:865-881 (1999)). To establish the assay conditions, MICROTITER (registered trademark) multiwell plates (Thermo Scientific) were coated with 5 μg / ml capture anti-Fab antibody (Cappel Labs) in 50 mM sodium carbonate (pH 9.6) overnight and then blocked with 2% (w / v) bovine serum albumin in PBS at room temperature (about 23°C) for two to five hours. In non-adsorbent plates (Nunc #269620), 100 pM or 26 pM [ 125 I] -antigen is mixed with a serial dilution of the Fab of interest (e.g., consistent with the assessment of the anti-VEGF antibody, Fab-12 in Presta et al., Cancer Res. 57:4593-4599 (1997)). The Fab of interest is then incubated overnight; however, the incubation can be continued for a longer period (e.g., about 65 hours) to ensure that equilibrium is reached. Thereafter, the mixture is transferred to a capture plate for incubation at room temperature (e.g., one hour). The solution is then removed and the plate is washed eight times with 0.1% polysorbate 20 (TWEEN-20 (registered trademark)) in PBS. When the plate has dried, 150 μl / well of scintillant (MICROSCINT-20 TM ; Packard), and in TOPCOUNT TM The plates were counted for tens of minutes on a gamma counter (Packard).The concentration of each Fab that gave less than or equal to 20% of maximal binding was chosen for use in the competitive binding assay.

[0390] According to another embodiment, Kd is measured using a BIACORE (registered trademark) surface plasmon resonance assay. For example, assays using a BIACORE (registered trademark)-2000 or BIACORE (registered trademark)-3000 (BIAcore, Inc., Piscataway, NJ) are performed at 25°C with immobilized antigen CM5 chips at approximately 10 response units (RU). In one embodiment, carboxymethylated dextran biosensor chips (CM5, BIACORE, Inc.) are activated with N-ethyl-N'-(3-dimethylaminopropyl)- carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to the supplier's instructions. Antigen is diluted with 10 mM sodium acetate, pH 4.8, to 5 μg / ml (~0.2 μM) before injection at a flow rate of 5 μl / minute to achieve approximately 10 response units (RU) of coupled protein. Following the injection of antigen, 1 M ethanolamine is injected to block unreacted groups. For kinetics measurements, two-fold serial dilutions of Fab (0.78 nM to 500 nM) are injected in PBS with 0.05% polysorbate 20 (TWEEN-20 TM surface active agent (PBST) at 25 μl / min. Association rates (k on ) and dissociation rates (k off ) are calculated using a simple one-to-one Langmuir binding model (BIACORE (registered trademark) Evaluation Software version 3.2) by simultaneously fitting the association and dissociation sensorgrams. The equilibrium dissociation constant (Kd) is calculated as the ratio k off / k on . See, e.g., Chen et al., J. Mol. Biol. 293:865-881 (1999). If the association rate exceeds 10 6 M -1 s -1 -1, then the association rate can be determined by using the fluorescence quenching technique, i.e., measuring the increase or decrease in fluorescence emission intensity (excitation = 295 nm; emission = 340 nm, 16 nm band-pass) of a 20 nM anti-antigen antibody (in the Fab form) in PBS pH 7.2 at 25°C in the presence of increasing concentrations of antigen, as measured in a spectrometer, such as a stop-flow equipped spectrophometer (Aviv Instruments) or a 8000-series SLM-AMINCO TM spectrophotometer (ThermoSpectronic) with stirred cuvettes.

[0391] and / or

[0392] The term "and / or," as used herein, is used to indicate any of the subject matter following the term in the list of elements, or any combination thereof. For example, "A, B, and / or C" includes single subject matter A, B, and C, as well as combinations of A and B, A and C, B and C, and A and B and C.

[0393] Antibody

[0394] The term "antibody" herein is used in the broadest sense and includes various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments so long as they exhibit the desired antigen-binding activity.

[0395] Antibody fragments

[0396] "Antibody fragment" refers to a molecule other than an intact antibody that comprises a portion of an intact antibody that binds the antigen to which the intact antibody binds. Examples of antibody fragments include but are not limited to Fv, Fab, Fab', Fab'-SH, F(ab')2; diabodies; linear antibodies; single-chain antibody molecules (e.g., scFv); single-domain antibodies or VHHs; and multispecific antibodies formed from antibody fragments.

[0397] 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, and other fragments as described infra. For a review of certain antibody fragments, see Hudson et al. Nat. Med. 9:129-134 (2003). For a review of scFv fragments, see e.g., Pluckthun, in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., (Springer-Verlag, New York), pp. 269- 315 (1994); see also WO 93 / 16185; and U.S. Patent Nos. 5,571,894 and 5,587,458. For discussion of Fab and F(ab')2 fragments comprising salvage receptor binding epitope residues and having increased in vivo half-life, see U.S. Patent No. 5,869,046.

[0398] Diabodies are antibody fragments with two antigen-binding sites which can be bivalent or bispecific. See, e.g., EP 404,097; WO 1993 / 01161; Hudson et al., Nat. Med. 9: 129-134 (2003); and Hollinger et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993). See also Hudson et al., Nat. Med. 9: 129-134 (2003) for a description of triabodies and tetrabodies.

[0399] Single-domain antibodies are antibody fragments which comprise all or a portion of the heavy chain variable domain or all or a portion of the light chain variable domain of an antibody. In certain embodiments, a single-domain antibody is a human single-domain antibody (Domantis, Inc., Waltham, MA; see, e.g., U.S. Patent 6,248,516 Bl).

[0400] Antibody fragments can be made by various techniques, including but not limited to proteolytic digestion of whole antibodies, and production by recombinant host cells, e.g., E. coli or phage, as described herein.

[0401] If the term "variable fragment (Fv)" is used in this application, it can mean the smallest unit of an antibody-derived portion that binds to an antigen with an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH) pair. In 1988, Skerra and Pluckthun found that uniform and active antibodies can be prepared from the periplasmic fraction of E. coli by inserting antibody genes downstream of a bacterial signal sequence and inducing expression of the genes in E. coli (Science (1988) 240(4855), 1038-1041). In the Fv prepared from the periplasmic fraction, VH is associated with VL in a manner that binds to an antigen.

[0402] If the terms "scFv", "single chain antibody" and "sc(Fv)2" are used in the present application, they refer to antibody fragments containing a single polypeptide chain derived from the variable regions of the heavy and light chains, but not the constant regions. Generally, single chain antibodies also contain a polypeptide linker between the VH and VL domains, which enables the formation of the desired structure that is believed to allow antigen binding. Single chain 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 WO 1988 / 001649; U.S. Patent Nos. 4,946,778 and 5,260,203. In particular embodiments, the single chain antibodies can be bispecific and / or humanized.

[0403] If the term "scFv" is used in the present application, it can mean a single chain polypeptide in which VH and VL forming Fv are linked together by a peptide linker (Proc. Natl. Acad. Sci. U.S.A. (1988) 85(16), 5879-5883). The VH and VL can be kept in a very close position by a peptide linker.

[0404] If the term "sc(Fv)2" is used in the present application, it can mean a single chain antibody in which four variable regions of two VL and two VH 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 VH and two VL can be derived from different monoclonal antibodies. Such sc(Fv)2 preferably includes, for example, bispecific sc(Fv)2, which recognizes two epitopes present in a single antigen, as disclosed in Journal of Immunology (1994) 152(11), 5368-5374. The sc(Fv)2 can be produced by methods known to those skilled in the art. For example, the sc(Fv)2 can be produced by linking scFv with a linker such as a peptide linker.

[0405] In the present context, sc(Fv)2 takes the form in which two VH units and two VL units of an antibody are arranged in the order of VH, VL, VH and VL ([VH]-linker-[VL]-linker-[VH]-linker-[VL]) starting from the N-terminus of a single chain polypeptide. The order of the two VH units and the two VL units is not limited to the above form, and they can be arranged in any order. Example orders of the form are listed as follows.

[0406] [VL]-linker-[VH]-linker-[VH]-linker-[VL]

[0407] [VH]-linker-[VL]-linker-[VL]-linker-[VH]

[0408] [VH]-linker-[VH]-linker-[VL]-linker-[VL]

[0409] [VL]-linker-[VL]-linker-[VH]-linker-[VH]

[0410] [VL]-linker-[VH]-linker-[VL]-linker-[VH]

[0411] If the terms "Fab", "F(ab')2", and "Fab'" are used in the present application, they can mean the following.

[0412] A "Fab" consists of a single light chain and a CH1 region and a variable region from a single heavy chain. The heavy chain of a wild-type Fab molecule is unable to form a disulfide bond with another heavy chain molecule. Depending on any purpose, Fab variants in which the amino acid residues in the wild-type Fab molecule can be altered by substitution, addition, or deletion are also included. In specific embodiments, the mutated amino acid residues (e.g., cysteine residues or lysine residues after substitution, addition, or insertion) included in the Fab variants can form a disulfide bond with another heavy chain molecule or a portion thereof (e.g., a Fab molecule).

[0413] An scFab is an antigen-binding domain in which a single light chain and a CH1 region and a variable region from a single heavy chain forming a Fab are connected together by a peptide linker. The light chain, the CH1 region from the heavy chain, and the variable region can be kept in close proximity by the peptide linker.

[0414] An "F(ab')2" or "Fab" is produced by treating an immunoglobulin (monoclonal antibody) with a protease such as pepsin and papain, and refers to an antibody fragment generated by digesting an immunoglobulin (monoclonal antibody) near a disulfide bond present between the hinge region of each of the two H chains. For example, papain cleaves IgG upstream of the disulfide bond present between the hinge region of each of the two H chains to generate two homologous antibody fragments, in which a L chain including a VL (L chain variable region) and a CL (L chain constant region) is connected to a H chain fragment including a VH (H chain variable region) and a CHγ1 (γ1 region in the H chain constant region) via a disulfide bond at the C-terminal region thereof. Each of the two homologous antibody fragments is called a Fab'.

[0415] An "F(ab')2" is composed of two light chains and two heavy chains, which heavy chain comprises a constant region of a CH1 domain and a part of a CH2 domain, thereby forming a disulfide bond between the two heavy chains. For example, the F(ab')2 disclosed herein can be produced as follows. A whole monoclonal antibody or a monoclonal antibody comprising a desired antigen binding domain is partially digested with a protease such as pepsin; and Fc fragments are removed by adsorption onto a protein A column. The protease is not particularly limited as long as it can cleave the whole antibody in a selective manner under suitable set enzyme reaction conditions such as pH to produce F(ab')2. Such proteases include, for example, pepsin and ficin.

[0416] If the term "single domain antibody" (sdAb) is used in the present application, the structure thereof is not particularly limited as long as the domain can exert antigen binding activity by itself. A general antibody exemplified by an IgG antibody exerts antigen binding activity in a state of forming a variable region by pairing of VH and VL. In contrast, a single domain antibody is known to be capable of exerting antigen binding activity by itself without pairing with another domain. A single domain antibody generally has a relatively low molecular weight and exists in a monomeric form.

[0417] Examples of the single domain antibody include, but are not limited to, antigen binding molecules naturally lacking light chains such as VHH of Camelidae and VNAR of sharks, and antibody fragments comprising all or a part of an antibody VH domain or all or a part of an antibody VL domain. Examples of the single domain antibody as the antibody fragment comprising all or a 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, as described in, for example, U.S. Patent No. 6,248,516B1. In some embodiments of the present application, one single domain antibody has three CDRs (CDR1, CDR2, and CDR3).

[0418] The single domain antibody can be obtained from an animal capable of producing a single domain antibody, or by immunizing an animal capable of producing a single domain antibody. Examples of the animal capable of producing a single domain antibody include, but are not limited to, Camelidae and transgenic animals into which a gene capable of producing a single domain antibody has been introduced. Camelidae includes camels, llamas, alpacas, dromedaries, guanacos, and the like. Examples of the transgenic animal into which a gene capable of producing a single domain antibody has been introduced include, but are not limited to, the transgenic animals described in International Publication No. WO2015 / 143414 or U.S. Patent Publication No. US2011 / 0123527A1. A humanized single chain antibody 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 sequence similar thereto. The humanized single domain antibody (e.g., humanized VHH) is one embodiment of the single domain antibody of the present application.

[0419] Alternatively, a single domain antibody can be obtained from a polypeptide library containing a single domain antibody by ELISA, panning, etc. Examples of polypeptide libraries containing a single domain antibody include, but are not limited to, an initial antibody library 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)), an antibody library obtained by immunizing various animals (e.g., Journal of Applied Microbiology 2014 117:2 (528-536)), and a synthetic antibody library 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 (1691-1701))

[0420] Antibodies that bind to the same epitope

[0421] An "antibody that binds to the same epitope" as a reference antibody refers to an antibody that blocks binding of the reference antibody to its antigen in a competition assay by 50% or more, and conversely, the reference antibody blocks binding of this antibody to its antigen in a competition assay by 50% or more. An exemplary competition assay is provided herein.

[0422] Chimerism

[0423] The term "chimeric" antibody refers to an antibody in which a portion of the heavy and / or light chain is of one origin or species, while the remainder of the heavy and / or light chain is of different origin or species.

[0424] category

[0425] The "class" of an antibody refers to the type of constant domain or constant region possessed by its heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and several of these can be further divided into subclasses (isotypes) e.g., IgGl, IgG2, IgG3, IgG4, IgAl and IgA2. The heavy chain constant domains that correspond to the different classes of immunoglobulins are called a, d, e, g, and m, respectively.

[0426] Combination with existing technologies

[0427] The target binding molecule combination of the application or the protein complex of the application can be combined with various prior art technologies. As non-limiting examples of such technology combinations, the generation of cells expressing chimeric antigen receptors (CARs) utilizing the target binding molecule combination of the application or the protein complex of the application is exemplified. Cells herein include, for example, T cells, gd T 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 of the non-limiting methods for generating T cells expressing CARs (CAR-T) involves, for example, a method wherein a CAR comprising the target binding molecule combination of the application or the protein complex of the application specifically bound to intracellular signaling domains of a scaffold protein, a tumor-associated antigen, a transmembrane domain of a TCR, and a costimulatory molecule such as CD28 is introduced into an effector cell such as a T cell by genetic modification techniques to enhance T cell activation. Another non-limiting method in combination with CAR-T cell technology involves, for example, a method wherein a scaffold protein is expressed on a CAR-T cell, and the target binding molecule combination of the application or the protein complex of the application is used to deliver a cytokine or a costimulatory signal to the CAR-T cell in a specific manner.

[0428] Effector function

[0429] “Effector function” refers to those biological activities attributable to the Fc region of an antibody, which vary with the antibody isotype. Examples of antibody effector functions include: Clq binding and complement dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; down regulation of cell surface receptors (e.g., B cell receptor); and B cell activation.

[0430] Antibody-dependent cell-mediated toxicity

[0431] "Antibody-dependent cell-mediated cytotoxicity" or "ADCC" refers to a form of cytotoxicity in which secreted Ig binds to Fc receptors (FcRs) on certain cytotoxic cells (e.g., NK cells, neutrophils, and macrophages) that leads to the elimination of the target. The primary cells for mediating ADCC are NK cells, which express FcγRIII. Monocytes and macrophages express FcγRI, FcγRII and FcγRIII. FcRs are heterodimers composed of an extracellular Ig binding domain and a cytoplasmic tail. There are several FcRs expressed by leukocytes, including FcγRI (a high affinity receptor for IgG), FcγRII (an intermediate affinity receptor for IgG), and FcγRIII (a low affinity receptor for IgG). See, e.g., Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991). To assess ADCC activity of a molecule of interest, an in vitro ADCC assay, such as that described in U.S. Patent No. 5,500,362 or 5,821,337 or U.S. Patent No. 6,737,056 (Presta), can be performed. Useful effector cells for such assays include PBMC and NK cells. Alternatively, or additionally, ADCC activity of the molecule of interest can be assessed in vivo, e.g., in a animal model such as that disclosed in Clynes et al. PNAS (USA) 95:652-656 (1998).

[0432] Complement-dependent cytotoxicity

[0433] "Complement dependent cytotoxicity" or "CDC" refers to the lysis of a target cell in the presence of complement. Activation of the classical complement pathway begins by the binding of the first component of the complement system (Clq) to antibody (of the appropriate subclass) which is bound to its specific antigen. To assess complement activation, a CDC assay, e.g., as described in Gazzano-Santoro et al. J. Immunol. Methods 202:163 (1996), can be performed. Polypeptide variants with altered Fc region amino acid sequences (polypeptides with Fc modifications) and increased or decreased Clq binding capacity are described, e.g., in U.S. Patent No. 6,194,551 Bl and WO 1999 / 51642. See also, e.g., Idusogie et al. J. Immunol. 164:4178-4184 (2000).

[0434] effective dose

[0435] An "effective amount" of an agent (e.g., a pharmaceutical formulation) refers to an amount effective at necessary dosages and for a period of time necessary to achieve the desired therapeutic or prophylactic result.

[0436] Fc receptors

[0437] The term "Fc receptor" or "FcR" herein refers to a receptor for the Fc region of an antibody. In some embodiments, the FcR is a native human FcR. In some embodiments, the FcR is one which binds an IgG antibody (a gamma receptor) and includes receptors of the FcyRI, FcyRII, and FcyRIII subclasses, including allelic variants and alternatively spliced forms of these receptors. FcyRII receptors include FcyRIIA (an "activating receptor") and FcyRIIB (an "inhibiting receptor"), which have similar amino acid sequences except in their cytoplasmic domains. The activating receptor FcyRIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain. The inhibiting receptor FcyRIIB contains an immunoreceptor tyrosine-based inhibition motif (ITIM) in its cytoplasmic domain. (See, e.g., Daeron, Annu. Rev. Immunol. 15:203-234 (1997)). FcRs are reviewed, e.g., 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). The term "FcR" herein also encompasses other FcRs including those to be identified in the future.

[0438] The term "Fc receptor" or "FcR" also includes the neonatal receptor FcRn, which is responsible for the transfer of maternal IgGs to the fetus (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)) and the homeostatic regulation of immunoglobulin levels. Methods of measuring binding to FcRn are 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); WO 2004 / 92219 (Hinton et al.).

[0439] In vivo binding to human FcRn and plasma half-life of human FcRn high affinity binding polypeptides can be determined, for example, in transgenic mice expressing human FcRn or in transfected human cell lines or in primates administered with polypeptides having Fc modifications. WO 2000 / 42072 (Presta) describes antibody variants with increased or decreased binding to FcRs. See, also, e.g., Shields et al. J. Biol. Chem. 9(2):6591-6604 (2001).

[0440] Fc region

[0441] The term "Fc region" herein is used to define a C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region. The term includes native sequence Fc regions and variant Fc regions. In one embodiment, a human IgG heavy chain Fc region extends from Cys226, or from Pro230, to the carboxy-terminus of the heavy chain. However, the C-terminal lysine (Lys447) or glycine-lysine (residues 446-447) of the Fc region can or can not be present. Unless otherwise indicated, the numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, also called the EU index, as in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991.

[0442] Variant Fc region

[0443] A "variant Fc region" comprises an amino acid sequence that differs from that of a native sequence Fc region by virtue of at least one amino acid modification, preferably one or more amino acid substitutions. Preferably, a variant Fc region has at least one amino acid substitution, e.g., from about one to about ten amino acid substitutions, and preferably from about one to about five amino acid substitutions, in the native sequence Fc region or the Fc region of a parent polypeptide. A variant Fc region described herein preferably has at least about 80% homology with the native sequence Fc region and / or with the Fc region of a parent polypeptide, and most preferably at least about 90% homology therewith, and more preferably at least about 95% homology therewith.

[0444] Antibodies containing Fc regions

[0445] The term "Fc region-containing antibody" refers to an antibody comprising an Fc region. The C-terminal lysine (residue 447 according to EU numbering system) or C-terminal glycine lysine (residues 446-447) of an Fc region can be removed, for example, during purification of the antibody or by design of the nucleic acid encoding the antibody by recombination. Thus, the composition comprising an antibody having an Fc region according to the present application can comprise an antibody having G446 to K447, an antibody having G446 but not K447, an antibody having all of G446 to K447 removed, or a mixture of all three types of antibodies described above.

[0446] Functional Fc region

[0447] A "functional Fc region" has an "effector function" of a native sequence Fc region. Exemplary "effector functions" include Clq binding; CDC; Fc receptor binding; ADCC; phagocytosis; down regulation of cell surface receptors (e.g., B cell receptor; BCR), etc. Such effector functions generally require the Fc region to be combined with a binding domain (e.g., an antibody variable domain), and can be assessed using, for example, the various assays disclosed herein in the Definitions section.

[0448] native sequence Fc region

[0449] A "native sequence Fc region" comprises an amino acid sequence that is identical to the amino acid sequence of a Fc region found in nature. Native sequence human Fc regions include a native sequence human IgGl Fc region (non-A and A allotypes); native sequence human IgG2 Fc region; native sequence human IgG3 Fc region; and native sequence human IgG4 Fc region, as well as naturally occurring variants thereof.

[0450] Full-length antibodies, intact antibodies, and whole antibodies

[0451] The terms "full-length antibody," "intact antibody," and "whole antibody" are used interchangeably herein to refer to an antibody with a structure substantially similar to a native antibody structure or with heavy chains that have an Fc region as defined herein.

[0452] Host cells, host cell lines and host cell cultures

[0453] The terms "host cell," "host cell line," and "host cell culture" are used interchangeably and refer to cells into which an exogenous nucleic acid has been introduced, including the progeny of such a cell. Host cells include "transformants" and "transformed cells," which include the primary transformed cell and progeny derived therefrom, regardless of the number of passages. Progeny can not be completely identical to the parent cell in nucleic acid content, for example, mutations can occur during replication. Mutant progeny that have the same function or biological activity as screened or selected for in the original transformed cell are included herein.

[0454] Human antibodies

[0455] A "human antibody" is one which possesses an amino acid sequence that corresponds to that of an antibody produced by a human or a human cell, or derived from a non-human source that utilizes human antibody repertoires or other human antibody-encoding sequences. This definition of a human antibody specifically excludes a humanized antibody comprising non-human antigen-binding residues.

[0456] Humanized antibodies

[0457] A "humanized" antibody refers to a chimeric antibody comprising amino acid residues from non-human HVRs and amino acid residues from human FRs. In certain embodiments, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the HVRs (e.g., CDRs) correspond to those of a non-human antibody, and all or substantially all of the FRs correspond to those of a human antibody. A humanized antibody optionally can comprise at least a portion of an antibody constant region derived from a human antibody. A "humanized form" of an antibody, e.g., a non-human antibody, refers to an antibody that has undergone humanization.

[0458] Individual or subject

[0459] An "individual" or "subject" is a mammal. Mammals include, but are not limited to, domesticated animals (e.g., cows, sheep, cats, dogs, and 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.

[0460] Isolated nucleic acid

[0461] An "isolated" nucleic acid refers to a nucleic acid molecule that has been separated from a component of its natural environment. An isolated nucleic acid includes a nucleic acid molecule contained in cells that typically comprise the nucleic acid molecule, but the nucleic acid molecule is present extrachromosomally or at a chromosomal location that is different from its natural chromosomal location.

[0462] Isolated nucleic acid encoding a target binding molecule or protein complex or combination

[0463] An "isolated nucleic acid encoding a target binding molecule or protein complex or combination" refers to one or more nucleic acid molecules that encode binding domains, e.g., antibody heavy and light chains (or fragments thereof), including such nucleic acid molecules in a single vector or separate vectors, and such nucleic acid molecules present at one or more locations in a host cell.

[0464] Amino acid sequence identity percentage (%)

[0465] "Percentage (%) amino acid sequence identity" with respect to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the reference polypeptide sequence, after aligning the candidate sequence with the reference polypeptide sequence and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance using publicly available computer software such as BLAST, BLAST-2, ALIGN, Megalign (DNA STAR software) or GENETYX (registered trademark) (Genetyx Co., Ltd.). Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.

[0466] The ALIGN-2 sequence comparison computer program was written by Genentech, Inc., and the source code has been filed with the U.S. Copyright Office, Washington D.C., 20559 where it is registered under U.S. Copyright Registration No. TXU510087. The ALIGN-2 program is publicly available from Genentech, Inc., South San Francisco, California, or can be compiled from the source code. The ALIGN-2 program should be compiled for use on UNIX operating systems, including Digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and are not variable. In the case of comparing amino acid sequences using ALIGN-2, the % amino acid sequence identity of a given amino acid sequence A to a given amino acid sequence B (which can alternatively be expressed as a given amino acid sequence A has, or comprises, a certain % amino acid sequence identity to a given amino acid sequence B) is calculated as follows:

[0467] 100 times the fraction X / Y

[0468] where X is the number of amino acid residues scored as identical matches by the sequence alignment program ALIGN-2 in that program's alignment of A and B, and where Y is the total number of amino acid residues in B. It will be appreciated that where 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 will not equal 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 immediately preceding paragraph.

[0469] Pharmaceutical preparations / pharmaceutical compositions

[0470] The term "pharmaceutical formulation" or "pharmaceutical composition" refers to a preparation which is in a form to be administered to a subject and which is in a form in which it is biologically active and which does not contain additional components which are unacceptably toxic to the subject to which the formulation is to be administered.

[0471] Pharmaceutical carriers

[0472] A "pharmaceutically acceptable carrier" refers to a component of a pharmaceutical formulation other than the active ingredient, which is nontoxic to the subject to which it is administered. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizing agents, or preservatives.

[0473] treat

[0474] As used herein, "treatment" (and grammatical variations thereof such as "treat" or "treating") refers to clinical intervention in an attempt to alter the natural course of the individual being treated, and can be performed either for prophylaxis or during the course of clinical pathology. Desirable effects of treatment include, but are not limited to, preventing or reducing incidence or severity of a disease, alleviating symptoms made directly or indirectly by the disease, preventing recurrence of a disease, decreasing or delaying onset of a disease, decreasing or delaying disease progression, ameliorating or palliating a disease state, and remission or improved prognosis. In some embodiments, the antibodies of the present application are used to delay development of a disease or to slow the progression of a disease.

[0475] carrier

[0476] The term "vector", as used herein, refers to a nucleic acid molecule capable of carrying another nucleic acid with which it is associated. The term includes the vector as a self-replicating nucleic acid structure as well as the vector incorporated into the genome of a host cell into which it has been introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as "expression vectors". The vector can be introduced into a host cell by a variety of methods, including methods using viruses, electroporation methods, and the like, but the introduction of the vector is not limited to in vitro introduction, and the vector can also be introduced directly into the body.

[0477] cancer

[0478] The target binding molecule combinations or protein complexes of the application can be used to treat or prevent cancer. As used herein, the terms "cancer" and "cancerous" refer to or describe the physiological condition in mammals that is typically characterized by uncontrolled growth / proliferation of cells. Examples of cancer include, but are not limited to, carcinoma, lymphoma (e.g., Hodgkin's and non-Hodgkin's lymphoma), blastoma, sarcoma, and leukemia. More particular examples of such cancers include squamous cell cancer, small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, squamous carcinoma of the lung, cancer of the peritoneum, hepatocellular cancer, gastrointestinal cancer, pancreatic cancer, glioma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney cancer, liver cancer, prostate cancer, skin cancer, thyroid cancer, hepatic carcinoma, leukemia and other lymphatic cancers, and various types of head and neck cancers.

[0479] Cell proliferative disorders

[0480] As used herein, the terms "cell proliferative disorder" and "proliferative disorder" refer to a condition associated with some degree of abnormal cell proliferation. In one embodiment, the cell proliferative disorder is cancer.

[0481] In vitro and in vivo assays for inhibition of cell proliferation and tumor growth

[0482] In certain embodiments, the ability of a target binding molecule combination of the application or a protein complex of the application or a target binding molecule of the application as defined in [A-1] in combination with other target binding molecules of the application as defined in [A-1] of the application to inhibit cell growth or proliferation in vitro is tested. Assays for inhibiting cell growth or proliferation are well known in the art. Certain assays of cell proliferation, such as the "cell killing" assays described herein, measure cell viability. One such test is the CellTiter-Glo® Luminescent Cell Viability Assay, which is commercially available from Promega (Madison, WI). The ability of a target binding molecule combination of the application or a protein complex of the application or a target binding molecule of the application as defined in [A-1] in combination with other target binding molecules of the application as defined in [A-1] of the application to inhibit cell growth or proliferation in vivo is tested. Assays for inhibiting cell growth or proliferation in vivo are well known in the art. In certain embodiments, the ability of a target binding molecule combination of the application or a protein complex of the application or a target binding molecule of the application as defined in [A-1] in combination with other target binding molecules of the application as defined in [A-1] of the application to inhibit tumor growth in vivo is tested. Assays for inhibiting tumor growth in vivo are well known in the art. TMLuminescent cell viability assay. This assay determines the number of viable cells in a culture based on the quantitation of ATP present, which indicates metabolically active cells. See Crouch et al. (1993) J. Immunol. Meth. 160:81-88, U.S. Patent No. 6602677. The assay can be performed in 96-well or 384-well format, making it suitable for automated high-throughput screening (HTS). See Cree et al. (1995) Anti Cancer Drugs 6:398-404. The assay procedure involves the direct addition of a single reagent (CellTiter-Glo (registered trademark) reagent) to the cultured cells. This causes cell lysis and generates a luminescent signal produced by a luciferase reaction. The luminescent signal is directly proportional to the amount of ATP present, which in turn is directly proportional to the number of viable cells present in the culture. Data can be recorded by a luminometer or a CCD camera imaging device. Luminescent output is expressed in relative light units (RLU).

[0483] Another assay for cell proliferation is the "MTT" assay, which is a colorimetric assay that measures the oxidation of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide to formazan by mitochondrial reductases. Like the CellTiter-Glo TM assay, this assay indicates the number of metabolically active cells present in a cell culture. See, e.g., Mosmann (1983) J. Immunol. Meth. 65:55-63, and Zhang et al. (2005) Cancer Res. 65:3877-3882.

[0484] In one aspect, a combination of target binding molecules of the application or a protein complex of the application or a target binding molecule of the application as defined in [A-1] in combination with another target binding molecule as defined in [A-1] of the application is tested for its ability to induce cell death in vitro. Assays for inducing cell death are well known in the art. In some embodiments, such assays measure, e.g., loss of membrane integrity, as indicated by 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% heat-inactivated FBS (Hyclone) and 2 mM L-glutamine. Thus, the assay is performed in the absence of complement and immune effector cells. Cells are plated at 3 x 10 6Individuals were plated at a density of 100 x 20 mm dishes and allowed to adhere overnight. The media was removed and replaced with fresh media alone or media containing various concentrations of antibody or immunoconjugate. Cells were incubated for a period of three days. Following treatment, the monolayers were washed with PBS and detached by trypsinization. Cells were then centrifuged at 1200 rpm for 5 minutes at 4°C and the pellet resuspended in 3 ml cold Ca 2+ Binding buffer (10 mM Hepes, pH 7.4, 140 mM NaCl, 2.5 mM CaCl2) and aliquoted into 35 mm filter-screen capped 12 x 75 mm tubes (1 ml per tube, 3 tubes per treatment group) to remove cell clumps. Tubes then received PI (10 μg / ml). FACSCAN TM Flow cytometer and FACSCONVERT TM CellQuest software (Becton Dickinson) was used to analyze the samples. Thus, combinations of target binding molecules of the application or protein complexes of the application or combinations of target binding molecules of the application as defined in [A-1] and other target binding molecules as defined in [A-1] of the application that induce statistically significant levels of cell death, as determined by PI uptake, were identified.

[0485] In one aspect, combinations of target binding molecules of the application or protein complexes of the application or combinations of target binding molecules of the application as defined in [A-1] and other target binding molecules as defined in [A-1] of the application are tested for their ability to induce apoptosis (programmed cell death) in vitro. An exemplary assay for a combination of target binding molecules of the application or protein complexes of the application or combinations of target binding molecules of the application as defined in [A-1] and other target binding molecules as defined in [A-1] of the application or immunoconjugates that induce apoptosis is an annexin binding assay. In the exemplary annexin binding assay, cells are cultured and plated in dishes as described in the preceding paragraph. The media is removed and replaced with fresh media alone or media containing 0.001 to 10 μg / ml of antibody or immunoconjugate. After a three day incubation period, the monolayers are washed with PBS and detached by trypsinization. Cells are then centrifuged, resuspended in Ca 2+ Binding buffer and aliquoted into tubes as discussed in the preceding paragraph. Tubes then receive labeled annexin (e.g., annexin V-FITC) (1 μg / ml). FACSCAN TM Flow cytometer and FACSCONVERT TMCellQuest software (BD Biosciences) is used to analyze the samples. Thus, antibodies that induce a statistically significant level of annexin binding relative to controls are identified. Another exemplary assay for antibodies or immunoconjugates that induce apoptosis is a histone DNA ELISA colorimetric assay for detecting internucleosomal degradation of genomic DNA. Such assays can be performed using, for example, the Cell Death Detection ELISA kit (Roche, Palo Alto, CA).

[0486] Cells for use in any of the above in vitro assays include cells or cell lines that naturally express a scaffold protein or a receptor protein as defined in [A-1], or that have been engineered to express a scaffold protein, or that comprise all components of a receptor complex containing a receptor protein as defined in [A-1]. Such cells include tumor cells that overexpress a scaffold protein relative to normal cells of the same tissue origin. Such cells also include cell lines (including tumor cell lines) that express a scaffold protein and cell lines that do not normally express a scaffold protein but have been transfected with a nucleic acid encoding a scaffold protein.

[0487] In one aspect, a target binding molecule combination of the application or a protein complex of the application or a target binding molecule of the application as defined in [A-1] in combination with other target binding molecules as defined in [A-1] is tested for its ability to inhibit cell growth or proliferation in vivo. In certain embodiments, a target binding molecule combination of the application or a protein complex of the application or a target binding molecule of the application as defined in [A-1] in combination with other target binding molecules as defined in [A-1] is tested for its ability to inhibit tumor growth in vivo. In vivo model systems, such as syngeneic tumor cell transplantation models, can be used for such testing. In an exemplary syngeneic tumor cell transplantation model, mouse tumor cells are introduced into a suitable mouse strain, e.g., C57BL / 6 mice. Furthermore, if the target binding molecules are not cross-reactive to the animal or cell line used, it is a possible option to introduce human gene expression against the scaffold protein or the receptor protein by genetic engineering. The target binding molecule combination of the application or the protein complex of the application or the target binding molecule of the application as defined in [A-1] in combination with other target binding molecules as defined in [A-1] is administered to the animal. The target binding molecule combination of the application or the protein complex of the application or the target binding molecule of the application as defined in [A-1] in combination with other target binding molecules as defined in [A-1] is tested for its ability to inhibit or reduce tumor growth. In certain embodiments, the mouse tumor cells are cells from a mouse tumor, such as MC38, CT26, A20, B16-F10, or Pan02. In certain embodiments, the mouse tumor cells are introduced into a suitable mouse strain by subcutaneous injection or transplantation into a suitable site, such as the mammary fat pad.

[0488] B-cell neoplasms / Hodgkin's disease

[0489] “B cell neoplasm” includes Hodgkin’s disease, which includes lymphocyte predominant Hodgkin’s disease (LPHD); non-Hodgkin’s lymphoma (NHL); follicular center cell (FCC) lymphoma; acute lymphoblastic leukemia (ALL); chronic lymphocytic leukemia (CLL); and hairy cell leukemia. Non-Hodgkin’s lymphoma includes low grade / follicular non-Hodgkin’s 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-cleaved cell NHL, bulky disease NHL, lymphoma plasmocytal lymphoma, mantle cell lymphoma, AIDS-related lymphoma, and Waldenstrom’s macroglobulinemia. Treatment of relapse of these cancers is also contemplated. LPHD is a form of Hodgkin’s disease that tends to relapse frequently despite treatment with radiation or chemotherapy. CLL is one of the four major types of leukemia. CLL is a cancer of mature B cells called lymphocytes, which manifests as a gradual accumulation of cells in the blood, bone marrow, and lymphoid tissue. Indolent lymphoma is a slow-growing, incurable disease in which patients survive an average of between six and 10 years after multiple remission and relapse periods.

[0490] Breast tumors

[0491] The term “breast tumor” or “breast cancer” refers to any tumor or cancer of the breast, including, for example, adenocarcinomas such as invasive or in situ ductal carcinoma, invasive or in situ lobular carcinoma, medullary carcinoma, colloid carcinoma, and papillary carcinoma; and less common forms such as phyllodes tumors, sarcomas, squamous cell carcinomas, and carcinosarcomas.

[0492] Colon tumors

[0493] The term “colonic tumor” or “colonic cancer” refers to any tumor or cancer of the colon (large intestine from the cecum to the rectum).

[0494] Colorectal cancer

[0495] The term “colorectal tumor” or “colorectal cancer” refers to any tumor or cancer of the large intestine (including the colon (large intestine from the cecum to the rectum) and rectum), including, for example, adenocarcinomas and less common forms such as lymphomas and squamous cell carcinomas.

[0496] Non-Hodgkin lymphoma

[0497] As used herein, the term "non-Hodgkin's lymphoma" or "NHL" refers to a cancer of the lymphatic system other than Hodgkin's lymphoma. Hodgkin's lymphoma can generally be distinguished from non-Hodgkin's lymphoma by the presence of Reed-Sternberg cells in Hodgkin's lymphoma and the absence of said cells in non-Hodgkin's lymphoma. Examples of non-Hodgkin's lymphoma encompassed by the term as used herein include any non-Hodgkin's lymphoma identified by a person skilled in the art (e.g., an oncologist or pathologist) according to a classification scheme 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 in particular Figures 11.57, 11.58 and / or 11.59). More particular examples include, but are not limited to, relapsed or refractory NHL, frontline low grade NHL, stage III / IV NHL, chemotherapy resistant NHL, precursor B-lymphoblastic leukemia and / or lymphoma, small lymphocytic lymphoma, B-cell chronic lymphocytic leukemia and / or prolymphocytic leukemia and / or small lymphocytic lymphoma, B-cell prolymphocytic lymphoma, immunocytoma and / or lymphoplasmacytic lymphoma, marginal zone B-cell lymphoma, splenic marginal zone lymphoma, extranodal marginal zone-MALT lymphoma, nodal marginal zone lymphoma, hairy cell leukemia, plasmacytoma and / or plasma cell myeloma, low grade / follicular lymphoma, intermediate grade / follicular NHL, mantle cell lymphoma, follicle center lymphoma (follicular), intermediate diffuse NHL, diffuse large B-cell lymphoma, aggressive NHL (including aggressive frontline NHL and aggressive relapsed NHL), NHL relapsing or refractory after autologous stem cell transplantation, primary mediastinal large B-cell lymphoma, primary effusion lymphoma, high grade immunoblastic NHL, high grade lymphoblastic NHL, high grade small non-cleaved cell NHL, bulky disease NHL, Burkitt's lymphoma, precursor (peripheral) T-cell lymphoblastic leukemia and / or lymphoma, adult T-cell lymphoma and / or leukemia, T-cell chronic lymphocytic leukemia and / or prolymphocytic leukemia, large granular lymphocytic leukemia, mycosis fungoides and / or Sezary syndrome, extranodal natural killer / T-cell (nasal type) lymphoma, enteropathy-type T-cell lymphoma, hepatosplenic T-cell lymphoma, subcutaneous panniculitis-like T-cell lymphoma, skin (cutaneous) lymphomas, anaplastic large cell lymphoma, angiocentric lymphoma, intestinal T-cell lymphoma, peripheral T-cell (unspecified) lymphoma, and angioimmunoblastic T-cell lymphoma.

[0498] Ovarian cancer

[0499] “Ovarian cancer” refers to a group of heterogeneous malignancies that originate in the ovary. Approximately 90% of malignant ovarian tumors arise from the epithelium; the remainder are germ cell and stromal tumors. Epithelial ovarian tumors are classified into the following histological subtypes: serous adenocarcinoma (approximately 50% of epithelial ovarian tumors); endometrioid adenocarcinoma (approximately 20%); mucinous adenocarcinoma (approximately 10%); clear cell carcinoma (approximately 5-10%); Brenner (transitional cell) tumors (relatively rare). Ovarian cancer is the sixth most common cancer in women, and its prognosis is generally poor, with a five-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). The present application encompasses methods of diagnosing or treating any of the above epithelial ovarian tumor subtypes and in particular the serous adenocarcinoma subtype.

[0500] relapse

[0501] “Relapse” refers to the return of a patient’s disease to a previous baseline state, particularly after a significant or partial recovery. Unless otherwise specified, the relapse state refers to the process of returning to or returning to the disease prior to a previous treatment, including but not limited to chemotherapy and stem cell transplant therapy.

[0502] Refractory

[0503] “Relapse” refers to the return of a patient’s disease to a previous baseline state, particularly after a significant or partial recovery. Unless otherwise specified, the relapse state refers to the process of returning to or returning to the disease prior to a previous treatment, including but not limited to chemotherapy and stem cell transplant therapy.

[0504] Gastric tumors

[0505] As used herein, the term “gastric tumor” or “gastric cancer” refers to any tumor or cancer of the stomach, including, for example, adenocarcinomas (such as diffuse and intestinal types) and less common forms such as lymphoma, leiomyosarcoma, and squamous cell carcinoma.

[0506] tumor

[0507] As used herein, the term "tumor" refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues. The terms "cancer," "cancerous," "cell proliferative disease," "proliferative disease," and "tumor" are not mutually exclusive as referred to herein.

[0508] Inhibit cell growth or proliferation / suppress cell growth

[0509] "Inhibiting cell growth or proliferation" or "suppressing cell growth" means reducing the growth or proliferation of a cell by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%, and includes inducing cell death.

[0510] Oligonucleotides

[0511] As used herein, "oligonucleotide" refers to a generic single-stranded synthetic polynucleotide, usually but not necessarily less than about 200 nucleotides in length. The terms "oligonucleotide" and "polynucleotide" are not mutually exclusive. The above description regarding polynucleotides applies equally and fully to oligonucleotides.

[0512] Basically reduce

[0513] As used herein, the phrase "substantially reduced" or "substantially different" means that there is a sufficiently high difference between two values (typically one value is associated with a molecule and the other value is associated with a reference / comparator molecule) such that one of skill in the art would believe that the difference between the two values is statistically significant in the context of the biological characteristic measured by the values (e.g., Kd values).

[0514] Basically similar

[0515] As used herein, the term "substantially similar" or "substantially the same" means that there is a sufficiently high degree of similarity between two values (e.g., one value is associated with an antibody of the application and the other value is associated with a reference / comparator antibody) such that one of skill in the art would believe that the difference between the two values is of little biological and / or statistical significance in the context of the biological characteristic measured by the values (e.g., Kd values).

[0516] Autoimmune diseases

[0517] The target binding molecule combinations or protein complexes of the present application can be used to treat or prevent autoimmune diseases. An "autoimmune disease" refers to a non-malignant disease or condition that is caused by and directed against the individual's own tissues. Autoimmune diseases herein expressly exclude malign or cancerous diseases or conditions, particularly B-cell lymphoma, acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), hairy cell leukemia, and chronic myelogenous leukemia. Examples of autoimmune diseases or conditions include, but are not limited to, inflammatory responses such as inflammatory skin diseases, including psoriasis and dermatitis (e.g., atopic dermatitis); systemic scleroderma and sclerosis; responses associated with inflammatory bowel disease (such as Crohn's disease and ulcerative colitis); respiratory distress syndrome (including adult respiratory distress syndrome; ARDS); dermatitis; meningitis; encephalitis; uveitis; colitis; glomerulonephritis; allergic conditions such as eczema and asthma and other conditions involving T cell infiltration and chronic inflammatory responses; atherosclerosis; leukocyte adhesion deficiency; rheumatoid arthritis; systemic lupus erythematosus (SLE) (including but not limited to lupus nephritis, cutaneous lupus); diabetes (e.g., Type I diabetes or insulin-dependent diabetes mellitus); multiple sclerosis; Raynaud's syndrome; autoimmune thyroiditis; Hashimoto's thyroiditis; allergic encephalomyelitis; Sjogren's syndrome; juvenile diabetes; and immune responses associated with acute and delayed hypersensitivity reactions mediated by cytokines and T lymphocytes, commonly seen in tuberculosis, sarcoidosis, polymyositis, granulomatosis, and vasculitis; pernicious anemia (Addison's disease); diseases involving leukocyte diapedesis; central nervous system (CNS) inflammatory conditions; multiple organ injury syndrome; hemolytic anemia (including but not limited to cryoglobulinemia or Coombs-positive anemia); myasthenia gravis; antigen-antibody complex-mediated diseases; anti-glomerular basement membrane disease; antiphospholipid syndrome; allergic neuritis; Graves' disease; Lambert-Eaton myasthenic syndrome; bullous pemphigoid; pemphigus; autoimmune polyendocrinopathy; Rett's disease; stiff person syndrome; Behcet's disease; giant cell arteritis; immune complex nephritis; IgA nephropathy; IgM polyneuropathies; immune thrombocytopenic purpura (ITP) or autoimmune thrombocytopenia.

[0518] Immunosuppressants / anti-inflammatory agents

[0519] The term "immunosuppressants" for adjunctive therapy herein refers to agents that act to suppress or mask the immune system of the mammal being treated herein. This would include agents that suppress cytokine production, down-regulate or suppress self-antigen expression or mask 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 such as cortisone or aldosterone; anti-inflammatory agents such as 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 masks MHC antigens as described in U.S. Patent No. 4,120,649); anti-idiotypic antibodies to MHC antigens and MHC fragments; cyclosporin A; steroids such as corticosteroids or glucocorticosteroids or glucocorticoid analogs, e.g., prednisone, methylprednisolone, including SOLU-MEDROL (registered trademark) methylprednisolone sodium succinate and dexamethasone; dihydrofolate reductase inhibitors such as methotrexate (oral or subcutaneous); antimalarials such as chloroquine and hydroxychloroquine; sulfasalazine; leflunomide; cytokine or cytokine receptor antibodies including anti-interferon-α, -β or -γ antibodies, anti-tumor necrosis factor (TNF)-α antibodies (infliximab (REMICADE (registered trademark)) or adalimumab), anti-TNF-α immunoadhesins (etanercept), anti-TNF-β antibodies, anti-interleukin-2 (IL-2) antibodies and anti-IL-2 receptor antibodies, and anti-interleukin-6 (IL-6) receptor antibodies and antagonists (such as ACTEMRA™ (tocilizumab)); anti-LFA-1 antibodies including anti-CDl la and anti-CD18 antibodies; anti-L3T4 antibodies; heterologous anti-lymphocyte globulin; pan T antibodies, preferably anti-CD3 or anti-CD4 / CD4a antibodies; soluble peptides containing LFA-3 binding domains (WO 90 / 08187, published 7 / 26 / 90); streptokinase; transforming growth factor-β (TGF-β); streptodornase; RNA or DNA from the host; FK506; RS-61443; chlorambucil; deoxyspergualin; rapamycin; T cell receptors (Cohen et al., U.S. Patent No. 5,114,721); T cell receptor fragments (Offner et al., Science, 251 :430-432 (1991); WO 90 / 11294; Ianeway, Nature, 341 :482 (1989); and WO 91 / 01133); BAFF antagonists such as BAFF antibodies and BR3 antibodies and zTNF4 antagonists (for a review see Mackay and Mackay, Trends Immunol., 23: 113-5 (2002), see also definition below); biological agents that interfere with T cell costimulatory signals, such as anti-CD40 receptor or anti-CD40 ligand (CD154), including blocking antibodies to CD40-CD40 ligand (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 (1994)); and T cell receptor antibodies (EP 340, 109), such as T10B9. Some preferred immunosuppressive agents herein include cyclophosphamide, chlorambucil, azathioprine, leflunomide, MMF, or methotrexate.

[0520] II. Compositions and Methods

[0521] In one aspect, the present application relates to a target binding molecule combination comprising:

[0522] 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

[0523] 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,

[0524] wherein the first target binding molecule and the second target binding molecule are capable of non-competitively binding to the scaffold protein, and

[0525] wherein the first receptor protein and the second receptor protein are each a receptor subunit capable of associating to form a receptor complex. The target binding molecule combination is capable of inducing receptor signaling of the receptor complex.

[0526] In another aspect, the present application relates to a protein complex 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,

[0527] wherein the first target binding molecule and the second target binding molecule are capable of non-competitively binding to the scaffold protein,

[0528] wherein the first receptor protein and the second receptor protein are each a receptor subunit capable of associating to form a receptor complex.

[0529] The protein complex is capable of inducing receptor signaling of a receptor complex.

[0530] The present application also relates to a protein complex 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,

[0531] wherein the first binding domain and the third binding domain are capable of non-competitive binding to the scaffold protein,

[0532] wherein the first receptor protein and the second receptor protein are each a receptor subunit capable of associating to form a receptor complex.

[0533] The protein complex is capable of inducing receptor signaling of a receptor complex.

[0534] The present application also relates to a nucleic acid encoding the target binding molecule combination of the present application or the protein complex of the present application.

[0535] The present application also relates to a method of producing the target binding molecule combination of the present application or the protein complex of the present application.

[0536] The present application also relates to a medical use of the target binding molecule combination of the present application or the protein complex of the present application or a pharmaceutical composition comprising the target binding molecule combination of the present application or the protein complex of the present application.

[0537] The present application also relates to a target binding molecule combination or a protein complex comprising the target binding molecule or the protein complex of any one of Tables 1 to 17. As shown in the present application, the target binding molecule or the protein complex of any one of Tables 1 to 17 is capable of inducing receptor signaling of a corresponding receptor complex.

[0538] Recombinant methods and compositions

[0539] The target binding molecule combinations of the application or the protein complexes of the application can be produced using recombinant methods and compositions, e.g., as described in U.S. Patent No. 4,816,567. In one embodiment, an isolated nucleic acid encoding a target binding molecule combination of the application or a protein complex of the application described herein is provided. Such a nucleic acid can encode an amino acid sequence comprising a VL of a target binding molecule (e.g., a light chain and / or a heavy chain of an antibody) and / or an amino acid sequence comprising a VH of a target binding molecule (e.g., a light chain and / or a heavy chain of an antibody). In a further embodiment, one or more vectors (e.g., expression vectors) comprising such a nucleic acid are provided. In a further embodiment, a host cell comprising such a nucleic acid is provided. In one such embodiment, a host cell comprises (e.g., has been transformed with): (1) a vector comprising a nucleic acid encoding an amino acid sequence comprising a VL of an antibody and an amino acid sequence comprising a VH of an antibody; or (2) a first vector comprising a nucleic acid encoding an amino acid sequence comprising a VL of an antibody and a second vector comprising a nucleic acid encoding an amino acid sequence comprising a VH of an antibody. In one embodiment, the host cell is a eukaryotic cell, e.g., a Chinese Hamster Ovary (CHO) cell or a lymphoid cell (e.g., a Y0, NS0, Sp2 / 0 cell). In one embodiment, a method of making a target binding molecule combination of the application or a protein complex of the application is provided, wherein the method comprises culturing a host cell comprising a nucleic acid encoding a target binding molecule combination of the application or a protein complex of the application under conditions suitable for the expression of the target binding molecule combination of the application or the protein complex of the application, and optionally recovering the target binding molecule combination of the application or the protein complex of the application from the host cell (or host cell culture medium).

[0540] To recombinantly produce a target binding molecule combination of the application or a protein complex of the application described herein, a nucleic acid encoding a target binding molecule, e.g., as described above, is isolated and inserted into one or more vectors for further cloning and / or expression in a host cell. Such nucleic acids can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are specific to genes encoding the heavy and light chains of an antibody).

[0541] Suitable host cells for cloning or expression of antibody-encoding vectors include prokaryotic or eukaryotic cells described herein. For example, target binding molecules can be produced in bacteria, particularly when large quantities are desired for use as therapeutics or reagents. For expression of antibody fragments and polypeptides in bacteria, see, e.g., U.S. Patent Nos. 5,648,237, 5,789,199, and 5,840,523. (See also Charlton, Methods in Molecular Biology, Vol. 248 (B.K.C. Lo, ed. Humana Press, Totowa, NJ, 2003), pp. 245-254 describing expression of antibody fragments in E. coli.) After expression, the target binding molecules can be isolated from the bacterial cell paste in a soluble fraction and can be further purified.

[0542] In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeast are also suitable hosts for cloning or expression of antibody-encoding vectors. Among fungi, for example, Aspergillus species such as Aspergillus niger, Aspergillus awamori, Aspergillus foetidus, Aspergillus fumigatus and Aspergillus terreus are suitable. See, e.g., Powell, et al., Bio / Technology 10: 629-633 (1992), and Campbell, et al., Bio / Technology 7: 213-217 (1989) (describing Aspergillus

[0543] Suitable host cells for the expression of glycosylated antibody are also derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant cells and insect cells. Numerous baculoviral strains have been identified which can be used in conjunction with insect cells, particularly for transfection of Spodoptera frugiperda cells.

[0544] Plant cell cultures can also be utilized as hosts. See, e.g., U.S. Patent Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (describing PLANTIBODIES TM technology) for production of antibodies in transgenic plants.

[0545] Vertebrate cells can also be used as hosts. For example, mammalian cell lines that are adapted to grow in suspension can be useful. Other examples of useful mammalian host cell lines are: monkey kidney CV1 line transformed by SV40 (COS-7); human embryonic kidney line (293 or 293 cells as described, e.g., in Graham et al., J. Gen Virol. 36:59 (1977)); baby hamster kidney cells (BHK); mouse Sertoli cells (TM4 cells as described, e.g., in Mather, Biol. Reprod. 23:243-251 (1980)); monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical carcinoma cells (HELA); canine kidney cells (MDCK); buffalo rat liver cells (BRL 3A); human lung cells (W138); human liver cells (Hep G2); mouse mammary tumor (MMT 060562); TRI cells, as described, e.g., in Mather et al., Annals N.Y. Acad. Sci. 383:44-68 (1982); MRC 5 cells; and FS4 cells. 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 certain mammalian host cell lines suitable for antibody production, see, e.g., Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (B.K.C. Lo, ed., Humana Press, Totowa, NJ), pp. 255-268 (2003).

[0546] In certain embodiments, the present application provides a target binding molecule combination of the present application or a protein complex of the present application obtainable by the above-mentioned methods.

[0547] Determination

[0548] The target binding molecule combination of the present application or the protein complex of the present application provided herein can be identified, screened or characterized for its physical / chemical properties and / or biological activities by various assays known in the art.

[0549] Binding assays and other assays

[0550] In one aspect, the target binding molecule of the present application is tested for its target binding activity, e.g., by known methods such as ELISA, Western blotting, etc.

[0551] In another aspect, a competition assay can be used to identify a target binding molecule that binds to a scaffold protein that does not compete with other target binding molecules that bind to the scaffold protein of the application. In certain embodiments, such a non- competing first target binding molecule does not bind to the same epitope (e.g., linear or conformational epitope) bound by a second target binding molecule. Detailed exemplary methods for mapping epitopes bound by antibodies are provided in Morris (1996) "Epitope Mapping Protocols" in Methods in Molecular Biology vol. 66 (Humana Press, Totowa, NJ).

[0552] In an exemplary competition assay, immobilized scaffold protein is incubated in a solution comprising a first labeled target binding molecule that binds to the scaffold protein and a second unlabeled target binding molecule that is being tested for its ability to compete with the first target binding molecule for binding to the scaffold protein. The second target binding molecule can be present in a hybridoma supernatant. As a control, immobilized target binding molecule is incubated in a solution comprising 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, excess unbound target binding molecule is 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 relative to the control sample, then the second target binding molecule is indicated to compete 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 relative to the control sample, then the second target binding molecule is indicated not to compete 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).

[0553] Pharmaceutical preparations / pharmaceutical compositions

[0554] Pharmaceutical formulations of the target binding molecule combinations of the present application or the protein complexes of the present application as described herein are prepared by mixing the target binding molecule combinations of the present application or the protein complexes of the present application having the desired degree of purity with one or more optional pharmaceutically-acceptable carriers, in either liquid or solid form, as appropriate. Remington's Pharmaceutical Sciences, 16thEdition, Osol, A. Ed. (1980), describes formulations which are suitable. Pharmaceutical formulations of the present disclosure in either liquid or solid form are prepared by mixing the target binding molecule combinations of the present application or the protein complexes of the present application having the desired degree of purity with one or more optional pharmaceutically-acceptable carriers, in either liquid or solid form, as appropriate. Acceptable carriers, as used herein, are nontoxic to recipients at the dosages and concentrations employed, and include, but are not limited to: buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 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 dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g. Zn-protein complexes); and / or non-ionic surfactants such as polyethylene glycol (PEG). Exemplary pharmaceutically acceptable carriers herein further include an interstitial pharmaceutically-acceptable dispersion agent such as a soluble neutral-active hyaluronidase glycoprotein (sHASEGP), for example, a human soluble PH-20 hyaluronidase glycoprotein, such as rHuPH20 (HYLENEX (R), Baxter International, Inc.). Certain exemplary sHASEGPs and methods of use, including rHuPH20, are described in U.S. Patent Publication Nos. 2005 / 0260186 and 2006 / 0104968. In one aspect, a sHASEGP is combined with one or more additional glycosaminoglycanases, such as a chondroitinase.

[0555] Exemplary lyophilized formulations are described in U.S. Patent No. 6,267,958. Aqueous formulations include those described in U.S. Patent No. 6,171,586 and WO 2006 / 044908, the formulations in the latter including a histidine-acetate buffer.

[0556] The formulations herein can also contain more than one active ingredient selected from the group of active ingredients necessary for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other. Such active ingredients are present in combination in amounts that are effective for the purpose intended.

[0557] The active ingredients can be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsules and poly-(methylmethacrylate) microcapsules, respectively; in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles, and nanocapsules); or in macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

[0558] Sustained-release preparations can be prepared. Suitable examples of sustained-release preparations include semi-permeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, for example, films, or microcapsules.

[0559] Formulations to be used for in vivo administration are typically sterile. Sterility can be readily achieved, for example, by filtration through a sterile filtration membrane.

[0560] Methods of treatment and compositions

[0561] The target binding molecule combination of the application or the protein complex of the application provided herein can be used in a method of treatment.

[0562] In one aspect, the target binding molecule combination of the application or the protein complex of the application is provided for use as a medicament. In a further aspect, the target binding molecule combination of the application or the protein complex of the application is provided for use in the treatment of a cancer or an autoimmune disease. In certain embodiments, a combination of a target binding molecule as defined in [A-1] and another target binding molecule as defined in [A-1] for medical use is provided. In certain embodiments, the target binding molecule combination of the application or the protein complex of the application or a combination of a target binding molecule of the application as defined in [A-1] and another target binding molecule as defined in [A-1] for use in a method of treatment is provided. In certain embodiments, the present application provides the target binding molecule combination of the application or the protein complex of the application or a combination of a target binding molecule of the application as defined in [A-1] and another target binding molecule as defined in [A-1] for use in a method of treating an individual having a cancer or an autoimmune disease, the method comprising administering to the individual an effective amount of the target binding molecule combination of the application or the protein complex of the application or a combination of a target binding molecule of the application as defined in [A-1] and another target binding molecule as defined in [A-1].

[0563] The mechanism of treatment of the target binding molecule combination of the application or the protein complex of the application or the target binding molecule of the application in combination 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 produces the therapeutic effect.

[0564] In a further aspect, the application provides the use of the target binding molecule combination of the application or the protein complex of the application or the target binding molecule of the application as defined in [A-1] in combination with other target binding molecules as defined in [A-1] in the manufacture or preparation of a medicament. In one embodiment, the medicament is for use in treating a cancer or an autoimmune disease. In a further embodiment, the medicament is for use in a method of treating a cancer or an autoimmune disease, wherein the treatment comprises administering to an individual having a cancer or an autoimmune disease an effective amount of the medicament.

[0565] In one embodiment, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent, e.g., as described below.

[0566] In a further aspect, the application provides a pharmaceutical formulation comprising any of the target binding molecule combination of the application or the protein complex of the application or the target binding molecule of the application as defined in [A-1] in combination with other target binding molecules as defined in [A-1] provided herein, e.g., for use in any of the above-described methods of treatment. In one embodiment, the pharmaceutical formulation comprises any of the target binding molecule combination of the application or the protein complex of the application or the target binding molecule of the application as defined in [A-1] in combination 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 of the target binding molecule combination of the application or the protein complex of the application or the target binding molecule of the application as defined in [A-1] in combination with other target binding molecules as defined in [A-1] provided herein, and at least one additional therapeutic agent, e.g., as described below.

[0567] The target binding molecule combination of the application or the protein complex of the application or the target binding molecule of the application as defined in [A-1] in combination with other target binding molecules as defined in [A-1] can be used alone or in combination with other drugs in a therapy. For example, the target binding molecule combination of the application or the protein complex of the application or the target binding molecule of the application as defined in [A-1] in combination with other target binding molecules as defined in [A-1] can be co-administered with at least one additional therapeutic agent.

[0568] Such combination therapies encompass combination administration (where two or more therapeutic agents are administered to a patient simultaneously, or sequentially, in any order where the agents can act independently or synergistically), and separate administration, in which case the administration of the target binding molecule combination of the application or the protein complex of the application or the administration of the target binding molecule combination of the application as defined in [A-1] in combination with other target binding molecules as defined in [A-1] of the application can precede, follow, or be concurrent with the administration of the additional therapeutic agent(s). In one embodiment, the administration of the target binding molecule combination of the application or the protein complex of the application or the administration of the target binding molecule combination of the application as defined in [A-1] in combination with other target binding molecules as defined in [A-1] of the application and the administration of the additional therapeutic agent(s) occur within about one month, or about one, two or three weeks, or about one, two, three, four, five, or six days of each other. The target binding molecule combination of the application or the protein complex of the application or the administration of the target binding molecule combination of the application as defined in [A-1] in combination with other target binding molecules as defined in [A-1] of the application can also be used in combination with radiation therapy.

[0569] The administration of the target binding molecule combination of the application or the protein complex of the application or the administration of the target binding molecule combination of the application as defined in [A-1] in combination with other target binding molecules as defined in [A-1] of the application (and any additional therapeutic agents) can be administered by any suitable means, including parenterally, intrapulmonary, and intranasally, and, if desired, intralesionally for local treatment. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. Dosing can be performed by any suitable route, for example, by injection, such as intravenous or subcutaneous injection, depending in part on whether the administration is short term or chronic. Various administration schedules are contemplated herein, including, but not limited to, single or multiple administrations at various time points, bolus administration, and pulse infusion.

[0570] The target binding molecule combination of the application or the protein complex of the application or the target binding molecule of the application as defined in [A-1] in combination with other target binding molecules as defined in [A-1] will be formulated, dosed, and administered in a fashion consistent with good medical practice. Factors for consideration in this context include the particular disorder being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the part of the body to be treated, the method of administration, the scheduling of administration, and other factors known to medical practitioners. The target binding molecule combination of the application or the protein complex of the application or the target binding molecule of the application as defined in [A-1] in combination with other target binding molecules as defined in [A-1] need not, but can be, administered as part of a combination regimen along with one or more additional therapeutic agent(s) currently used or in development for the prevention or treatment of the disorder, disease or condition. The effective dosage of such other agents depends on the amount of the target binding molecule combination of the application or the protein complex of the application or the target binding molecule of the application as defined in [A-1] in combination with other target binding molecules as defined in [A-1] present in the formulation, the type of disorder or treatment, and other factors discussed above. These are generally used in the same dosages and with administration routes and schedules as used herein or about from 1 to 99% of the dosages used herein are used, or in any dosage and by any route that is empirically / clinically determined to be appropriate.

[0571] For the prevention or treatment of disease, the appropriate dosage of the target binding molecule combination of the application or the protein complex of the application or the target binding molecule of the application as defined in [A-l] in combination with other target binding molecules as defined in [A-l] (when used alone or in combination with one or more other additional therapeutic agents) will depend on the type of disease to be treated, the type of target binding molecule, the severity and course of the disease, whether the target binding molecule combination of the application or the protein complex of the application or the target binding molecule of the application as defined in [A-l] in combination with other target binding molecules as defined in [A-l] is being used for preventive or therapeutic purposes, previous therapy, the patient's clinical history and response to the target binding molecule, and the judgment of the treating physician. The target binding molecule combination of the application or the protein complex of the application or the target binding molecule of the application as defined in [A-l] in combination with other target binding molecules as defined in [A-l] is suitably administered to the patient at one time or over a series of treatments. The initial candidate dosage of the target binding molecule combination of the application or the protein complex of the application or the target binding molecule of the application as defined in [A-l] in combination with other target binding molecules as defined in [A-l] that may be used can be in the range of about 1 μg / kg to 15 mg / kg (e.g., 0.1 mg / kg - 10 mg / kg), depending on the type and severity of the disease, although the dosage range can be from about 0.01 - 100 mg / kg. A typical daily dosage might range from about 1 μg / kg to 100 mg / kg or more, depending on the factors mentioned above. For repeated administrations over several days or longer, depending on the condition, the treatment can be continued as long as required for desired disease symptoms suppression. One exemplary dosage of the target binding molecule combination of the application or the protein complex of the application or the target binding molecule of the application as defined in [A-l] in combination with other target binding molecules as defined in [A-l] will be in the range of about 0.05 mg / kg to about 10 mg / kg. Thus, one or more doses of about 0.5 mg / kg, 2.0 mg / kg, 4.0 mg / kg or 10 mg / kg (or any combination thereof) can be administered to the patient. Such doses can be administered intermittently, e.g. every week or every three weeks (e.g. so that the patient receives from about two to about twenty or, e.g., about six doses of the target binding molecule combination of the application or the protein complex of the application or the target binding molecule of the application as defined in [A-l] in combination with other target binding molecules as defined in [A-l]). The progress of the therapy can be easily monitored by a routine technology and assay.

[0572] Products

[0573] In another aspect of the application, an article of manufacture containing materials useful for the treatment, prevention and / or diagnosis of the disorders described above is provided. The article of manufacture includes a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, IV solution bags, etc. The containers can be formed from a variety of materials such as glass or plastic. The container holds a composition, which is by itself or combined with another composition effective for treating, preventing and / or diagnosing the condition and can have a sterile access port (for example the container can be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). At least one active ingredient in the composition is a target binding molecule combination of the application or a protein complex of the application or a target binding molecule of the application as defined in [A-1] in combination with another target binding molecule as defined in [A-1] of the application. The label or package insert indicates that the composition is used for treating the condition of choice. Moreover, the article of manufacture can further comprise (a) a first container wherein is contained a composition comprising a target binding molecule combination of the application or a protein complex of the application or a target binding molecule of the application as defined in [A-1] in combination with another target binding molecule as defined in [A-1] of the application; and (b) a second container wherein is contained a composition comprising another therapeutic agent or a protein complex of the application. Such article of manufacture of this embodiment of the application can further include a package insert indicating that the compositions can be used to treat the particular condition. Alternatively, or additionally, the article of manufacture can further comprise a second (or third) container comprising a pharmaceutically-acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution and dextrose solution. It can further include other materials from which one of skill in the art would recognize as desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.

[0574] III. Examples

[0575] The following are examples of the methods and compositions of the present application. It is to be understood that various other embodiments can be practiced in light of the general description provided above.

[0576] Example 1: Concepts of the present disclosure

[0577] A schematic representation of a protein complex in the present 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 of the target binding molecules (S1 and S2) are capable of simultaneously, non-competitively binding the scaffold protein in a dual-site fashion. 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), wherein the receptor A and receptor B are capable of associating to form a receptor complex and induce receptor activation and / or signaling activity when in proximity to or associated with each other. The addition of a linker sequence between each binding domain is an option to optimize agonistic activity.

[0578] In the presence of the receptor proteins and in the absence of the scaffold protein, both target binding molecules bind to their respective receptor proteins (receptor A and receptor B) only through the first and third binding domains (R1 and R2), but no signaling activity is induced as the receptor proteins (receptor A and receptor B) are not in proximity to or associated with each other. In the presence of both the receptor receptor proteins and the scaffold protein, the first and third binding domains of the target binding molecules (S1 and S2) bind to the scaffold protein non-competitively and / or in a dual-site fashion, 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 are in proximity to or associated with each other, thereby inducing receptor activation and / or signaling activity. The addition of a linker sequence between each binding domain and a half-life extension domain, such as an Fc domain, is an option to improve functionality.

[0579] As a non-limiting example, the concept of conditional activation is illustrated using two VHH-VHH fusions. Each VHH-VHH fusion comprises a scaffold protein binding domain and a receptor protein binding domain. As a non-limiting example, a SARS-CoV2 receptor binding domain (RBD) or human PDL1 can be the scaffold protein, and IL-2Rbeta, IL-2Rgamma or a Wnt receptor can be the receptor protein.

[0580] VHHs capable of simultaneously binding to RBD in a dual-site fashion or to human PDL1 in a dual-site fashion, and VHHs capable of binding to IL-2Rbeta or IL-2Rgamma are identified. Nb21 and Nb36 are VHHs that each recognize a different epitope of RBD and thus can simultaneously bind to RBD in a dual-site fashion (Nature Communications 2021; 12:4676).

[0581] Figure 1 B shows a possible single molecule format that can induce conditional receptor activation by controlling the distance of the receptors via binding to a scaffold protein. In the presence of the receptor proteins and absence of the scaffold protein, the target binding molecules bind to their respective receptor proteins (Receptor A and Receptor B) only through the first and third binding domains (R1 and R2), but since the receptor proteins (Receptor A and Receptor B) are not in proximity to or associated with each other, only weak signaling activity is induced or no signaling activity is induced. In the presence of both the receptor proteins and the scaffold protein, the first and third binding domains of the target binding molecules (S1 and S2) bind to the scaffold protein bi-site, while the second binding domain binds to the first receptor protein and the fourth binding domain binds to the second receptor protein. Thus, the receptor proteins are in proximity to or associated with each other to form a receptor complex, thereby inducing receptor activation and / or signaling activity. Adding a linker sequence between each binding domain and the half-life extension domain, such as an Fc domain, is an option to improve functionality.

[0582] Example 2: Targeting RBD or PDL1 as scaffold proteins and IL-2Rβ or IL- Preparation of plasmids for 2Rγ-binding VHH-VHH fusions

[0583] Each of the scaffold binding proteins (VHHs capable of binding to RBD or human PDL1) was linked to a VHH capable of binding to human IL-2Rβ or human IL-2Rγ with a signal sequence to construct VHH-VHH fusions (SEQ ID NOs: 1-10). RBD was fused to the transmembrane and intracellular domains of human IL-2Rα with a signal sequence (RBD-IL2RA) (SEQ ID NO: 11) to serve as a scaffold protein. DNA encoding the VHH-VHH fusions, RBD-IL2RA, or human PDL1 (SEQ ID NO: 12) was cloned into a mammalian expression vector.

[0584] Native SARS-CoV2 spike protein, including RBD, is a homotrimeric protein, but RBD-IL2RA lacks the domain to form a trimer and can be expressed as a monomer. Furthermore, while IL2RA (IL-2Rα) is part of the high-affinity IL-2 receptor complex, it does not participate in IL-2R signaling (Cancer Communications 2018; 38:62).

[0585] [Table 1]

[0586] SEQ ID NOs: 1 to 12

[0587]

[0588]

[0589] SEQ ID NOs: 1 and 2 represent a fusion protein of a RBD binding domain (Nb21) and an IL-2Ry binding domain (Vhh2g). 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.

[0590] SEQ ID NOs: 3 and 4 represent a fusion protein of a RBD binding domain (Nb36) and an IL-2Rβ binding domain (Vhh2b). Binding of Nb36 to RBD does not compete with Nb21. In Table 1, the sequence of Vhh2b is underlined and the sequence of Nb36 is double underlined.

[0591] SEQ ID NO: 5 represents a fusion protein of a RBD binding domain (Nb21) and an IL-2Rβ binding domain (Vhh2b). In Table 1, the sequence of Vhh2b is underlined and the sequence of Nb21 is double underlined.

[0592] SEQ ID NO: 7 represents a fusion protein of an IL-2Ry binding domain (Vhh2g) and a PDL1 binding domain (VhhPL1). In Table 1, the sequence of Vhh2g is underlined and the sequence of VhhPL1 is double underlined.

[0593] SEQ ID NO: 8 represents a fusion protein of an IL-2Rβ binding domain (Vhh2b) and a PDL1 binding domain (VhhPL1-12). In Table 1, the sequence of Vhh2b is underlined and the sequence of VhhPL1-12 is double underlined.

[0594] SEQ ID NO: 9 represents a fusion protein of an IL-2Rβ binding domain (Vhh2b) and a PDL1 binding domain (VhhPL1-50). In Table 1, the sequence of Vhh2b is underlined and the sequence of VhhPL1-50 is double underlined.

[0595] SEQ ID NO: 10 represents a fusion protein of an IL-2Rβ binding domain (Vhh2b) and a PDL1 binding domain (VhhPL1). In Table 1, the sequence of Vhh2b is underlined and the sequence of VhhPL1 is double underlined.

[0596] SEQ ID NO: 11 represents a fusion protein of a SARS-CoV2 receptor binding domain (RBD) and the transmembrane and intracellular domains of IL-2Ra (IL-2RA).

[0597] SEQ ID NO: 12 represents human PDL1.

[0598] Example 3: Mixture of VHH-VHH fusions that bind to RBD in a two-site manner and to IL-2 receptor protein Induction of IL-2 receptor signaling by drugs is dependent on RBD expression.

[0599] In this example, the use of RBD as a scaffold protein and IL-2Rβ and IL-2Rγ as conditionally activated receptor proteins demonstrated conditional receptor activation in the presence of a scaffold protein.

[0600] As described in Example 2, VHH-VHH fusions were constructed by linking VHHs binding RBD (Nb21 or Nb36) to VHHs binding human IL-2Rβ (Vhh2b) or human IL-2Rγ (Vhh2g). Nb21 and Nb36 are bi-specific antibodies recognizing 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 database (7S2S and 7S2R).

[0601] Plasmids for VHH-VHH fusions (SEQ ID NO: 1-4) were expressed in HEK-Blue IL-2 cells (InvivoGen, #hkb-il2) by SF Cell Line 4D-Nucleofector X Kit L (Lonza, #V4XC-2024) together with RBD-IL2RA (SEQ ID NO: 11) or PDL1 plasmid (SEQ ID NO: 12). TM Vectors were used as negative controls. Cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin. These transfections were seeded in 96-well plates at 37°C for 3 days in the presence of 5% CO2. After 3 days of incubation, IL-2 receptor complex activation was assessed by measuring the optical density at 620 nm using a Multiskan TM Fermenter (Thermo Scientific, # 1651) using Quanti-Blue solution (InvivoGen, #rep-qbs). Figure 2 A).

[0602] Figure 2A shows the results of IL-2R signal induction. In the presence of RBD-IL2RA, the VHH-VHH fusion was able to activate the IL-2 receptor complex in HEK-Blue IL-2 cells, but not PDL1. This indicates that the induction of IL-2R signal by the mixture of VHH-VHH fusion depends on the expression of the scaffold protein for RBD. Figure 2 B shows a schematic diagram of the experimental system of the present example.

[0603] Example 4: Expression and purification of VHH-VHH fusions that bind to RBD and IL-2 receptor protein.

[0604] The VHH-VHH fusions (SEQ ID NOs: 1, 4, 5, and 6) were transiently expressed using Expi293 cell line (Thermo Fisher, Carlsbad, CA, USA). Antibody purification was performed using protein A affinity chromatography. For the concentration of purified antibody, its absorbance at 280 nm was measured using a spectrophotometer. According to the obtained value, the extinction coefficient calculated by a method such as PACE was used to calculate the antibody concentration (Protein Science 1995; 4: 2411-2423).

[0605] Example 5: A mixture of VHH-VHH fusions targeting the RBD as a scaffold protein to the IL-2 receptor complex Activation depends on two-site binding to RBD.

[0606] To demonstrate that the dual-site binding to the scaffold protein is a condition for IL-2R activation, the effect of a combination of VHH-VHH fusions that can dual-site bind to the scaffold protein was compared with the effect of a combination of VHH-VHH fusions that cannot dual-site bind to the scaffold protein.

[0607] RBD-IL2RA (SEQ ID NO: 11) or PDL1 (SEQ ID NO: 12) were first expressed in HEK-Blue IL-2 cells (InvivoGen, #hkb-il2) using the SF Cell Line 4D-Nucleofector X Kit L (Lonza, #V4XC-2024) by electroporation. 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 different mixtures of recombinant VHH-VHH fusions (SEQ ID NOs: 1, 4, 5, and 6) or recombinant human IL-2 (Peprotech, #200-02) and further incubated for one more day at 37°C in the presence of 5% CO2. On day 2 after electroporation, IL-2 receptor complex activation was assessed by measuring the optical density at 620 nm using a Multiskan TM Read-plate measurements of optical density at 620 nm to assess IL-2 receptor complex activation Figure 3 A and Figure 3 B).

[0608] As Figure 3 A and 3B, the combination of VHH-VHH fusions comprising bi-site RBD antibodies, i.e. Vhh2b-Nb36 and Nb21-Vhh2g, could induce IL-2 receptor signaling in the presence of RBD expression. However, the combination of mono-site RBD antibodies, i.e. Vhh2b-Nb36 and Nb36-Vhh2g or Vhh2b-Nb21 and Nb21-Vhh2g, could not induce IL-2 receptor signaling even in the presence of RBD. This result confirmed that the mixture of VHH-VHH fusions for the activation of IL-2 receptor complex is dependent on bi-site binding to the scaffold protein that is RBD.

[0609] Example 6: A mixture of VHH-VHH fusions that bind to PDL1 and IL-2 receptor proteins binds to the IL-2 receptor complex The induction of signaling is dependent on PDL1 expression.

[0610] The conditional activation of IL-2 receptor complex by the mixture of VHH-VHH fusions binding PDL1 as a target scaffold protein and IL-2 receptor protein as a target receptor protein was demonstrated in HEK-Blue IL-2 cells. Bi-site binding PDL1 VHHs were identified. VhhPL1 is a VHH that can bind PDL1 simultaneously with either VhhPL1-12 or VHHPL1-50 to achieve bi-site binding.

[0611] Plasmids for VHH-VHH fusions binding to PDL1 and human IL-2R beta or human IL-2R gamma (SEQ ID NO: 7-10) were expressed in HEK-Blue IL-2 cells (InvivoGen, #hkb-il2) by SF Cell Line 4D-Nucleofector X Kit L (Lonza #V4XC-2024) together with PDL1 (SEQ ID NO: 12) or RBD-IL2RA plasmid (SEQ ID NO: 11). The combination of VHH-VHH fusions to be tested was the combination of Vhh2g-VhhPL1 with any of Vhh2b-VhhPL1-12, Vhh2b-VhhPL1-50 or Vhh2b-VhhPL1. pmaxGFP TM Vectors were used as negative controls. 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 incubation, IL-2 receptor complex activation was assessed by measuring the optical density at 620 nm using a Multiskan TM Fermenter (Thermo Scientific) with Quanti-Blue solution (InvivoGen, #rep-qbs). Figure 4

[0612] As shown in Figure 4 the mixture of VHH-VHH fusions binding to PDL1 in a dual-site manner and to the IL-2 receptor protein can induce IL-2R signaling under conditions of PDL1 expression. On the other hand, the mixture of single-site VHH-VHH fusions, i.e. Vhh2b-VhhPL1 and Vhh2g-VhhPL1, fails to induce a clear IL-2R signal even in the presence of PDL1. This indicates that the induction of IL-2R signaling by the mixture of VHH-VHH fusions targeting PDL1 and the IL-2 receptor protein is dependent on the expression of PDL1 as a scaffold protein and the dual-site binding to this scaffold protein.

[0613] Example 7: Preparation of plasmids targeting SplitNeo-2 / 15 fused to RBD-binding VHH.

[0614] The concept of this conditional receptor signaling is compared to Split Neo-2 / 15, a split version of the known IL-2 / IL-15 mimetic, which is only active when the two disjoined fragments co-localize at the target cell site (WO2020 / 106708A1).

[0615] ​Split Neo-2 / 15, i.e. Neo2A or Neo2B (WO2020 / 106708 Al) was fused to VHHs (SEQ ID NOs: 13-15) that can bind to RBD with signal sequence and GS linker. DNA encoding these fusion proteins were cloned into mammalian expression vectors.

[0616] [Table 2]

[0617] SEQ ID NOs: 13 to 15

[0618]

[0619] SEQ ID NO: 13 represents a fusion protein of RBD binding domain (Nb21) and Neo2A of Split Neo-2 / 15. In Table 2, the sequence of Nb21 is underlined, and the sequence of Neo2A is double underlined.

[0620] SEQ ID NO: 14 represents a fusion protein of RBD binding domain (Nb21) and Neo2B of Split Neo-2 / 15. In Table 2, the sequence of Nb21 is underlined, and the sequence of Neo2B is double underlined.

[0621] SEQ ID NO: 15 represents a fusion protein of RBD binding domain (Nb36) and Neo2A of Split Neo-2 / 15. In Table 2, the sequence of Nb36 is underlined, and the sequence of Neo2A is double underlined.

[0622] Example 8: The combinations of Vhh2b-Nb36 and Nb21-Vhh2g showed superior selectivity compared to SplitNeo-2 / 15. Selective.

[0623] The mixture of Vhh2b-Nb36 and Nb21-Vhh2g exhibited a selective superiority over the fusion proteins of Split Neo-2 / 15 and RBD binding domain to the activation of IL-2 receptor complex against RBD expression.

[0624] To prevent the accidental formation of Split Neo-2 / 15 heterodimers in the endosomes of the expressing cells, plasmids against Split Neo-2 / 15, being the conjugation of VHHs that can bind to RBD with Neo2A or Neo2B (SEQ ID NO: 13-15), were expressed in HEK-Blue IL-2 cells (InvivoGen, #hkb-il2) by SF Cell Line 4D-Nucleofector X Kit L (Lonza, #V4XC-2024) with RBD-IL2RA (SEQ ID NO: 11) or PDL1 plasmid (SEQ ID NO: 12) respectively. To match the conditions, plasmids of Vhh2b-Nb36 or Nb21-Vhh2g (SEQ ID NO: 1 and 4) were expressed in HEK-Blue IL-2 cells with RBD-IL2RA or PDL1 plasmid respectively. pmaxGFP TM After electroporation, these transfectants were mixed in equal volumes with the different combinations and seeded in 96-well plates at 37°C for 2 days in the presence of 5% CO2. The mixtures of these transfectants were cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin. 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 TM Fermenter (Thermo Fisher Scientific, #155220100) for 30 min at 37°C. The activation of the IL-2 receptor complex was evaluated by measuring the optical density at 620 nm using a Multiskan Figure 5 A and 5B).

[0625] As shown in Figure 5 A, the conjugation of Split-Neo2 / 15 with RBD binding domains showed a limited dependency on RBD expression. On the other hand, the mixture of Vhh2b-Nb36 and Nb21-Vhh2g, which bind to RBD in a dual-site manner, exhibited a complete dependency on RBD expression Figure 5 B). Thus, the concept of conditional receptor signaling has a selectivity over the method Split Neo-2 / 15 known in the art.

[0626] Example 9: Targeting TNFα binding to scaffold protein and IL-2Rβ or Preparation of plasmids for IL-2Rγ-binding VHH-VHH fusions.

[0627] The concept of conditional receptor signaling is further exemplified using TNFα as a scaffold protein and IL-2Rβ or IL-2Rγ as target receptor proteins.

[0628] The scaffold binding protein Vhhtnfa3 was linked to another VHH capable of binding to human IL-2Rβ or human IL-2Rγ with a signal sequence to construct VHH-VHH fusions (SEQ ID NOs: 16-19). DNA encoding these VHH-VHH fusions or human TNFα (SEQ ID NO: 20) were cloned into mammalian expression vectors.

[0629] [Table 3]

[0630] SEQ ID NOs: 16-20

[0631]

[0632] SEQ ID NOs: 16 and 17 represent a fusion protein 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.

[0633] SEQ ID NOs: 18 and 19 represent a fusion protein 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.

[0634] SEQ ID NO: 20 represents human TNFα.

[0635] Example 10: A mixture of VHH-VHH fusions that can bind to the TNFα and IL-2 receptor complex is shown to be effective for IL-2R signaling. The induction of the signal can be induced by binding to a single site of TNFα which is a trimeric antigen.

[0636] Since TNFα is a homotrimeric antigen, the VHH-VHH fusions can each bind to the same epitope on each TNFα monomer (i.e., unit point binding). When the TNFα monomers form a trimer, the scaffold protein binding domains each bind to a TNFα monomer and are in close proximity to each other. Thus, the receptor protein binding domains linked to each scaffold protein binding domain will also be able to bring the receptor proteins in close proximity to each other, allowing for receptor activation and / or induction of signaling activity. Exemplary VHH-VHH fusions of Vhh2b linked to Vhhtnfa3 and Vhh2g linked to Vhhtnfa3 were constructed.

[0637] Plasmids with VHH-VHH fusions to TNFa and human IL-2Rβ or human IL-2Rγ binding as target scaffold proteins (SEQ ID NOs: 16-19) were expressed in HEK-Blue IL-2 cells (InvivoGen, #hkb-il2) by SF Cell Line 4D-Nucleofector X Kit L (Lonza #V4XC-2024) with TNFa (SEQ ID NO: 20) or PDL1 (SEQ ID NO: 12). pmaxGFP TM Vectors were used as negative controls. 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 for 2 days at 37°C in the presence of 5% CO2. After 2 days of incubation, IL-2 receptor complex activation was evaluated by measuring the optical density at 620 nm using a Multiskan TM Fermenter (Thermo Scientific) with Quanti-Blue solution (InvivoGen, #rep-qbs). Figure 6 A).

[0638] As shown in Figure 6 A, unitary TNFa binding domains targeting the same epitope on the TNFa trimer monomer can induce IL-2 receptor signaling in the presence of TNFa. A schematic of the conditional induction mechanism of IL-2R signaling by a mixture of VHH-VHH fusions that can bind to IL-2Rβ or IL-2Rγ and bind to TNFa in a unitary manner is shown in Figure 6 B.

[0639] Example 11: Expression of VHH-VHH or VHH-scFv fusions binding to RBD and Wnt receptors as LRP or FZD Reach and purify.

[0640] VHHs that can bind to RBD (Nb21 or Nb36) were fused to another receptor protein binding domain that can contain one or more VHHs, or scFv that can bind to human lipoprotein receptor-related protein (LRP) or human frizzled receptor (FZD) with signal sequence (Vhhlrp36 and R2M3, respectively) (SEQ ID NOs: 21-23). DNA encoding these fusion proteins were cloned into mammalian expression vectors. These plasmids were transiently expressed using Expi293 cell line (Thermo Fisher, Carlsbad, CA, USA). Antibody purification was performed using protein A affinity chromatography. For the concentration of purified antibodies, their absorbance at 280 nm was measured using a spectrophotometer. According to the obtained value, the extinction coefficient calculated by methods such as PACE was used to calculate the antibody concentration (Protein Science 1995; 4: 2411-2423).

[0641] [Table 4]

[0642] SEQ ID NOs: 21-23

[0643]

[0644] SEQ ID NO: 21 represents a fusion protein of two LRP binding domains (Vhhlrp36) and RBD binding domain (Nb21). In Table 4, the sequence of Vhhlrp36 is underlined, and the sequence of Nb21 is double underlined.

[0645] SEQ ID NO: 22 represents a fusion protein of two LRP binding domains (Vhhlrp36) and RBD binding domain (Nb36). In Table 4, the sequence of Vhhlrp36 is underlined, and the sequence of Nb36 is double underlined.

[0646] SEQ ID NO: 23 represents a fusion protein of FZD binding domain (R2M3) and RBD binding domain (Nb21). In Table 4, the sequence of R2M3 is underlined, and the sequence of Nb21 is double underlined.

[0647] Example 12: Activation of the Wnt receptor complex by a mixture of VHH-VHH and VHH-scFv fusions depends on the Two-site binding of the RBD.

[0648] The present concept of conditional receptor signaling is further exemplified using RBD as a scaffold protein and Wnt receptors (including LRP and FZD) as conditionally activated receptor proteins. This example also shows that other binding proteins (such as scFv) can also be used as scaffold protein binding domains or receptor protein binding domains.

[0649] RBD-IL2RA (SEQ ID NO: 11) or PDL1 (SEQ ID NO: 12) were first expressed in HEK293 STF cells (ATCC, CRL-3249) using the SF Cell Line 4D-Nucleofector X Kit L (Lonza, #V4XC-2024) by electroporation. These transfectants were seeded in white 96-well plates and incubated overnight at 37°C / 5% CO2 after addition of 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 pg / mL geneticin. On day 1 after electroporation, these cells were treated with different mixtures of VHH-VHH or VHH-scFv fusions (SEQ ID NO: 21-23) that bind to RBD and Wnt receptor proteins (FZD and LRP) and further incubated for one more day at 37°C / 5% CO2. On day 2 after electroporation, Wnt signal activation was assessed by measuring luminescence using Glomax Explorer using the ONE-Glo Luciferase Assay System (Promega, #E6120) Figure 7 A and 7B).

[0650] As Figure 7 shown in Figures Figure 7 A), and in the absence of RBD scaffold proteins Figure 7 B). Thus, activation of Wnt receptor signaling by mixtures of VHH-VHH and VHH-scFv fusions that bind to RBD and Wnt receptor proteins is dependent on RBD expression and bi-site binding to RBD.

[0651] Example 13: Expression and purification of Vhh2b-Nb36 / Nb21-Vhh2gFc fusion protein.

[0652] The concept of conditional receptor activation can also be applied to Figure 1The single molecule format implementation described in B. Two target binding molecules can be linked to form a single molecule, or associate with each other as a protein complex, although there can be one or more domains that extend the half-life of the molecule. In one example, a VHH-VHH fusion can be linked to an Fc domain through knob-in-hole engineering (Protein Engineering 1996; 9:617).

[0653] VHH-VHH fusion Vhh2b-Nb36 (SEQ ID: 4) or Nb21-Vhh2g (SEQ ID: 1) was fused with Fc carrying mutations that promote heterodimerization of Vhh2b-Nb36 and Nb21-Vhh2g. DNA encoding these Fc fusion proteins were cloned into mammalian expression vectors (SEQ ID: 24 and 25). These plasmids were transiently expressed using Expi293 cell line (Thermo Fisher, Carlsbad, CA, USA). Antibody purification was performed using protein A affinity chromatography. For the concentration of purified antibodies, their absorbance at 280 nm was measured using a spectrophotometer. From the obtained value, the extinction coefficient calculated by methods such as PACE was used to calculate the antibody concentration (Protein Science 1995; 4:2411-2423).

[0654] [Table 5]

[0655] SEQ ID NO: 24 and 25

[0656]

[0657] SEQ ID NO: 24 represents the Fc fusion of Vhh2b-Nb36 for making Vhh2b-Nb36 / Nb21-Vhh2g Fc heterodimer. In Table 5, the sequence of Vhh2b-Nb36 is underlined and the sequence of Fc is double underlined.

[0658] SEQ ID NO: 25 represents the Fc fusion of Nb21-Vhh2g for making Vhh2b-Nb36 / Nb21-Vhh2g Fc heterodimer. In Table 5, the sequence of Nb21-Vhh2g is underlined and the sequence of Fc is double underlined.

[0659] Example 14: Compared with the absence of RBD, Vhh2b-Nb36 / Nb21- Vhh2gFc fusion protein can induce stronger IL-2 receptor signaling.

[0660] In HEK-Blue IL-2 cells, the heterodimeric Fc fusion of Vhh2b-Nb36 and Nb21-Vhh2g was more potent in activating the IL-2 receptor complex in the presence of RBD compared to PDL1 expression.

[0661] RBD-IL2RA (SEQ ID NO: 11) or PDL1 (SEQ ID NO: 12) were first expressed in HEK-Blue IL-2 cells (InvivoGen, #hkb-il2) using the SF Cell Line 4D-Nucleofector X Kit L (Lonza, #V4XC-2024) by electroporation. 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: 24 and 25) or recombinant human IL-2 (Peprotech, #200-02) and further incubated for one more day at 37°C in the presence of 5% CO2. On day 2 after electroporation, IL-2 receptor complex activation was assessed by measuring the optical density at 620 nm using Multiskan™ plate reader using Quanti-Blue solution (InvivoGen, #rep-qbs). Figure 8 A and 8B).

[0662] The Vhh2b-Nb36 / Nb21-Vhh2g Fc fusion protein induced a stronger IL-2 receptor complex signal in the presence of RBD compared to the absence of RBD (Figure Figure 8 A). Unlike conventional conjugation of recombinant IL-2 and targeting antibodies (Nature 2022; 610: 161-172), the Vhh2b-Nb36 / Nb21-Vhh2g Fc fusion not only showed a stronger IL-2R signal from lower concentrations, but it also induced a higher maximum STAT5 activation when compared to the presence and absence of RBD. This indicates that the expression of the scaffold protein RBD is a condition for the strong induction of IL-2 receptor complex signal using the Vhh2b-Nb36 / Nb21-Vhh2g Fc fusion protein. On the other hand, direct administration of IL-2R ligand (recombinant human IL-2) did not show any selectivity between RBD-expressing HEK-Blue IL-2 cells and PDL1-expressing HEK-Blue IL-2 cells (Figure Figure 8 B).

[0663] Example 15: VHH-binding to IL2Rα as a scaffold protein and IL-2Rβ and IL-2Rγ as receptor proteins Expression and purification of scFv fusions or IL-2 mutants.

[0664] IL-2 mutant IL2_RETR was fused with His tag sequence (SEQ ID NO: 26). VHH that can bind to human IL-2Ry (Vhh2g) was fused with BT942 scFv (SEQ ID NO: 27) or Dac scFv (SEQ ID NO: 28) as IL-2Ra binding domain. VHH that can bind to human IL-2Ry (Vhh2b) was linked with BT942 scFv (SEQ ID NO: 29). DNA encoding these proteins were cloned into mammalian expression vectors. These plasmids were transiently expressed using Expi293 cell line (ThermoFisher, Carlsbad, CA, USA). Protein purification was performed using Protein A or immobilized metal ion affinity chromatography. For the concentration of purified proteins, their absorbance at 280 nm was measured using spectrophotometer. According to the obtained value, the concentration of antibody was calculated using the extinction coefficient calculated by methods such as PACE (Protein Science 1995; 4: 2411-2423).

[0665] [Table 6]

[0666] SEQ ID NO: 26 to 29

[0667]

[0668] SEQ ID NO: 26 represents IL-2 mutant that lacks binding to IL-2Ry but still binds to IL-2Ra and IL-2Ry.

[0669] SEQ ID NO: 27 represents a fusion protein of IL-2Ry binding domain (Vhh2g) and IL-2Ra binding domain (BT942 scFv). Binding of BT942 scFv to IL-2Ra does not compete with binding of IL2_RETR to IL-2Ra. In Table 6, the sequence of BT942 scFv is underlined, and the sequence of Vhh2g is double underlined.

[0670] SEQ ID NO: 28 represents a fusion protein of IL-2Ry binding domain (Vhh2g) and IL-2Ra binding domain (Dac scFv). Binding of Dac scFv to IL-2Ra competes with binding of IL2_RETR to IL-2Ra. In Table 6, the sequence of Dac scFv is underlined, and the sequence of Vhh2g is double underlined.

[0671] SEQ ID NO: 29 represents a fusion protein of an IL-2Rβ binding domain (Vhh2b) and an IL-2Rα binding domain (BT942 scFv). Binding of BT942 scFv to IL-2Rα does not compete with 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.

[0672] Example 16: In a dual-site manner, it binds to IL-2Rα as the target scaffold protein and IL- Activation of IL-2Rβ and IL-2Rγ by a mixture of IL-2Rβ / γ-binding IL-2 mutants and VHH-scFv fusions is dependent on IL-2Rα binding.

[0673] The use of IL-2Rα as a scaffold protein to bring IL-2Rβ and IL-2Rγ into proximity further exemplifies the concept of conditional receptor signaling. This example also shows that other non-antibody binding proteins can be used as scaffold protein binding domains or receptor protein binding domains. The structure of the quaternary complex of IL-2 with IL-2Rα, IL-2Rβ, and IL-2Rγ is described (Science 2005; 310: 1159-1163) and its structure and binding epitope are shown in the RCSB protein database (2B5I).

[0674] For example, IL2_RETR is a non-antagonistic IL-2 mutant and it binds to IL-2Rα as a scaffold protein and 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 a VHH that binds to IL-2Rγ and a BT942 scFv that binds to IL-2Rα as a scaffold protein. And the binding epitope of BT942 on IL2-Rα is described (Scientific Reports 2021; 11: 22966) and its structure and binding epitope are shown in the RCSB protein database (7F9W). Since binding of BT942 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. A mixture of IL2_RETR and Vhh2g-BT942 scFv together can allow colocalization of IL-2Rα, IL-2Rβ, and IL-2Rγ to induce IL-2R signaling dependent on IL-2Rα binding. A schematic of the IL-2R-dependent activation mechanism of IL-2Rα by a mixture of IL2_RETR and Vhh2g-BT942 scFv is shown in Figure 9 A.

[0675] HEK-Blue IL-2 cells (InvivoGen, #hkb-il2) were seeded in 96-well plates overnight at 37°C in the presence of 5% CO2. Cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin. On day 1 after seeding, cells were treated with or without 5 pg / mL daclizumab (RnD Systems, #MAB9927) for half an hour at 37°C in the presence of 5% CO2. Daclizumab is an anti-IL-2Ra antibody and can inhibit the binding of IL2_RETR to IL-2Ra, daclizumab was used to assess IL-2Ra binding dependent IL-2R activation. A mixture of IL2_RETR (SEQ ID: 26) and Vhh2g-BT942 scFv (SEQ ID: 27) or recombinant human IL-2 (Peprotech, #200-02) was added and further incubated for one more day at 37°C / 5% CO2. On day 2 after seeding, IL-2 receptor complex activation was assessed by measuring the optical density at 620 nm using Multiskan™ plate reader using Quanti-Blue solution (InvivoGen, #rep-qbs). Figure 9 B and 9C).

[0676] As shown in Figure 9 B and 9C, the combination of IL2_RETR and Vhh2g-BT942 scFv targeting IL2Ra as a scaffold protein and IL-2Rb and IL-2Ry as receptor proteins induced IL-2R signaling in HEK-Blue IL-2 cells constitutively expressing IL2Ra. Moreover, this IL-2R signal induction was inhibited by treatment with daclizumab (5 pg / mL), an anti-IL-2Ra neutralizing antibody Figure 9 B). On the other hand, daclizumab treatment did not show a significant effect on IL-2R signaling induced by recombinant IL-2 Figure 9 C). The significant inhibitory effect of daclizumab on IL-2R signaling indicates that the activation of IL-2R by IL2_RETR and Vhh2g-BT942 scFv is dependent on IL-2Ra binding.

[0677] Example 17: Mixture of IL-2 mutants or VHH-VHH fusions targeting IL-2Rα as a scaffold protein Activation of the IL-2 receptor complex depends on dual-site binding to IL-2Rα.

[0678] To demonstrate that two-site binding to a scaffold protein (in this case IL-2Ra) is a condition for IL-2Rb / y activation, the effect of a combination of proteins that can bind to IL-2Ra in two sites is compared to the effect of a combination that cannot bind to IL-2Ra in two sites.

[0679] HEK-Blue IL-2 cells (InvivoGen, #hkb-il2) were seeded in 96-well plates overnight at 37°C in presence of 5% CO2. 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 different mixtures of recombinant IL2_RETR and / or VHH-scFv fusions (SEQ ID NO: 26-29) and incubated at 37°C / 5% CO2 for 3 days. IL-2 receptor complex activation was assessed by using Quanti-Blue solution (InvivoGen, #rep-qbs) by measuring the optical density at 620 nm using a Multiskan™ plate reader (Figure 10).

[0680] Figure 10 shows that the induction of IL-2R signal by mixtures of IL-2 mutants or VHH-ScFV targeting IL-2Ra as a scaffold protein and IL-2Rb / g as receptor proteins is dependent on the dual-site binding to IL-2Ra. In HEK-Blue IL-2 cells constitutively expressing IL-2Ra, the combination of dual-site binding (IL2_RETR and Vhh2g-BT942 scFv or Vhh2b-BT942 scFv and Vhh2g-Dac scFv) induced IL-2R signal, but the combination of unit-site binding (IL2_RETR and Vhh2g-Dac scFv or Vhh2b-BT942 scFv and Vhh2g-BT942 scFv) did not induce IL-2R signal. Dac scFv is a single-chain variable fragment version of daratumumab that competes with IL2_RETR for binding to IL-2Ra, but not with BT942 scFv for binding to IL-2Ra.

[0681] Example 18: VHH-binding to PD1 as a target scaffold protein and IL-2 receptor as a target receptor protein Expression and purification of scFv fusions.

[0682] VHHs that can bind to human IL-2Rβ (Vhh2b or Vhh2b3) or human IL-2Rγ (Vhh2g or Vhh2g6) were linked to human PD1 binding domains that are Nivo scFvLH, Nivo scFvHL, NB01a scFvLH or NB01a scFvHL (SEQ ID NOs: 30-36). DNA encoding these proteins were cloned into mammalian expression vectors. These plasmids were transiently expressed using Expi293 cell line (Thermo Fisher, Carlsbad, CA, USA). Antibody purification was performed using protein A. For the concentration of purified antibodies, their absorbance at 280 nm was measured using a spectrophotometer. According to the obtained value, the extinction coefficient calculated by a method such as PACE was used to calculate the antibody concentration (Protein Science 1995; 4: 2411-2423).

[0683] [Table 7]

[0684] SEQ ID NOs: 30 to 37

[0685]

[0686] SEQ ID NO: 30 represents a fusion protein of IL-2Rγ binding domain (Vhh2g) and PD1 binding domain (Nivo scFvLH). In Table 7, the sequence of Nivo scFvLH is underlined, and the sequence of Vhh2g is double underlined.

[0687] SEQ ID NO: 31 represents a fusion protein of IL-2Rγ binding domain (Vhh2g) and PD1 binding domain (Nivo scFvHL). In Table 7, the sequence of Nivo scFvHL is underlined, and the sequence of Vhh2g is double underlined.

[0688] SEQ ID NO: 32 represents a fusion protein of IL-2Rβ binding domain (Vhh2b) and PD1 binding domain (NB01a scFvLH). In Table 7, the sequence of NB01a scFvLH is underlined, and the sequence of Vhh2b is double underlined.

[0689] SEQ ID NO: 33 represents a fusion protein of IL-2Rβ binding domain (Vhh2b3) and PD1 binding domain (NB01a scFvLH). In Table 7, the sequence of NB01a scFvLH is underlined, and the sequence of Vhh2b3 is double underlined.

[0690] SEQ ID NO:34 represents a fusion protein of an IL-2Ry binding domain (Vhh2g6) and a PD1 binding domain (Nivo scFvHL). In Table 7, the sequence of Nivo scFvHL is underlined and the sequence of Vhh2g6 is double underlined.

[0691] SEQ ID NO:35 represents a fusion protein of an IL-2Ry binding domain (Vhh2g) and a PD1 binding domain (NB01a scFvLH). In Table 7, the sequence of NB01a scFvLH is underlined and the sequence of Vhh2g is double underlined.

[0692] SEQ ID NO:36 represents a fusion protein of an IL-2Ry binding domain (Vhh2g) and a PD1 binding domain (NB01a scFvHL). In Table 7, the sequence of NB01a scFvHL is underlined and the sequence of Vhh2g is double underlined.

[0693] SEQ ID NO:37 represents human PD1.

[0694] Example 19: Recombinant VHH-scFv fusion that binds to PD-1 and IL-2 receptor protein in a dual-site manner The induction of IL-2R signaling by the mixture is dependent on PD1 expression.

[0695] The concept of conditional receptor signaling is further exemplified using PD1 as a scaffold protein and IL-2R and IL-2Ry as receptor proteins for conditional receptor signaling.

[0696] For example, Nivo scFvLH or Nivo scFvHL and NB01a scFvLH are bi-site antibodies recognizing different epitopes of PD1 and can bind to PD1 simultaneously and the epitope on PD1 bound by NB01a has been described (J. Exp. Med 2019; 216: 1525-1541) and its structure and bound epitope is displayed in the RCSB protein database (6HIG). Since the binding of Nivo scFvLH or Nivo scFvHL to PD1 is not in competition with the binding of NB01a scFvLH to PD1, a mixture of these proteins can allow for the co-localization of PD1, IL-2R and IL-2Ry to induce IL-2R signaling dependent on PD1 binding. The epitope on PD1 bound by Nivo has been described (Nature Communications 2017; 08: 14369) and its structure and bound epitope is displayed in the RCSB protein database (5WT9).

[0697] Human PD1 (SEQ ID NO: 37) or PDL1 (SEQ ID NO: 12) were first expressed in HEK-Blue IL-2 cells (InvivoGen, #hkb-il2) using the SF Cell Line 4D-Nucleofector X Kit L (Lonza, #V4XC-2024) by electroporation. 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 different mixtures of recombinant VHH-scFv fusions (SEQ ID NO: 30-33) or recombinant human IL-2 (Peprotech, #200-02) and further incubated for one more day at 37°C / 5% CO2. On day 2 after electroporation, IL-2 receptor complex activation was assessed by measuring the optical density at 620 nm using Multiskan™ plate reader using Quanti-Blue solution (InvivoGen, #rep-qbs) (Figures 11A and 1 IB).

[0698] As shown in Figures 11A and 1 IB, mixtures of VHH-scFv fusions that bind to PD1 as a scaffold protein and to IL-2Rβ or IL-2Rγ as target receptor proteins in a dual-site manner can induce IL-2R signaling under conditions of PD1 scaffold protein expression. In the absence of the scaffold protein PD1, IL-2R signaling is not induced.

[0699] Example 20: A mixture of VHH-VHH fusions targeting PD1 as a scaffold protein against the IL-2 receptor complex Activation is dependent on dual-site binding to PD1.

[0700] To demonstrate that dual-site binding to a scaffold protein (in this case PD1) is a condition for IL-2Rβ / γ activation, the effect of a combination of VHH-VHH fusions that can bind to PD1 in a dual-site was compared to the effect of a combination of VHH-VHH fusions that cannot bind to PD1 in a dual-site.

[0701] Human PD1 (SEQ ID NO: 37) was first expressed in HEK-Blue IL-2 cells (InvivoGen, #hkb-il2) using the SF Cell Line 4D-Nucleofector X Kit L (Lonza, #V4XC-2024) by electroporation. After electroporation, these transfectants were seeded in a 96-well plate 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 different mixtures of recombinant VHH-scFv fusions (SEQ ID NO: 30, 32-36) and further incubated for one more day at 37°C / 5% CO2. On day 2 after electroporation, IL-2 receptor complex activation was assessed by measuring the optical density at 620 nm using Multiskan™ plate reader with Quanti-Blue solution (InvivoGen, #rep-qbs) (Figure 12).

[0702] As shown in Figure 12, the induction of IL-2R signaling by mixtures of VHH-scFv fusions binding to PD1 as a scaffold protein and to IL-2Rβ or IL-2Rγ as a target receptor protein depends on the two-site binding to PD1. In PD1 -expressing HEK-Blue IL-2 cells, IL-2R signaling was induced using Nivo scFvHL or Nivo scFvLH and NB01a scFvHL and NB01a scFvLH as two-site PD1 binding combinations of PD1 binding domains, but not using NB01a scFvHL or NB01a scFvLH alone as one-site binding combinations of PD1 binding domains.

[0703] Example 21: Expression and purification of anti-PD1 antibodies (Abs)

[0704] DNA encoding heavy (Hch) and light chains (Lch) of anti-PD1 antibodies (SEQ ID NO: 38-59) were cloned into mammalian expression vectors. For the preparation of purified anti-PD1 Abs, the Expi293 cell line (Thermo Fisher, Carlsbad, CA, USA) was used for transient expression of the respective Hch and Lch expression plasmids. The Hch and Lch pairs for each antibody are described in Table 8. Protein purification was performed using protein A. For the concentration of the purified proteins, the absorbance at 280 nm was measured using a spectrophotometer. From the obtained values, the extinction coefficient calculated by methods such as PACE was used to calculate the antibody concentration (Protein Science 1995; 4: 2411-2423).

[0705] [Table 8]

[0706]

[0707]

[0708]

[0709] Example 22: Binding activity of anti-PD1 Ab

[0710] The binding activity of anti-PD1 Abs was assessed by flow cytometry. Anti-PD1 Abs (10 pg / mL) and human PD1-expressing cell line (NFAT-luc2 / PD1 Jurkat cell line, Promega, #CS187102) were incubated in staining buffer (Biolegend, #420201) for 20 minutes. Unbound Abs were removed by washing twice with staining buffer. PD1-bound human IgG Abs were detected by goat F(ab')2 anti-human IgG, mouse ads-PE (Southern Biotech, #2043-09). Data acquisition was performed on LSRFortessa X-20 (Becton Dickinson) (Figure 13).

[0711] As shown in Figure 13, all anti-PD1 Abs showed clear binding to human PD1-expressing cell line.

[0712] Example 23: Neutralizing activity of anti-PD1 Ab

[0713] The neutralization activity of anti-PD1 Abs was assessed by PD1 / PD-L1 blockade assay system (Promega, #CS187109). PD-L1+CHOK1 cells (Promega, #CS187108) were seeded in 384-well white plates at 37°C overnight in the presence of 5% CO2. Cells were cultured in RPMI1640 containing 5% fetal bovine serum and 1% penicillin-streptomycin. On day 1 after seeding, anti-PD1 Abs (final concentration of 5 pg / mL) and PD1+Jurkat cells (NFAT-luc2 / PD1 Jurkat cell line, Promega, #CS187102) were added to PD-L1+CHOK1 seeded wells and further incubated for 6 hours. Nivolumab (Selleck, #A2002, final concentration of 50 pg / mL) was used as a positive control. After 6 hours, TCR activation was detected by Bio-Glo luciferase assay system (Promega, #G7940) and GloMax Multi-Detection Plate Reader (Promega, #GM3500) (Figure 14).

[0714] As shown in FIG. 14, PDA0041, PDA0070, PDA0107, PDA0138, GY5, and GY14 exhibited PD1 blocking activity, but PDA0044, PDA0129, PDC0037, PDC0053, and PDE0171 did not exhibit PD1 blocking activity.

[0715] Example 24: Binds to PD1 as a target scaffold protein and to IL-2Rβ and IL-2Rγ as target receptor proteins Expression and purification of the combined VHH-IgG fusion protein.

[0716] Anti-IL-2Rγ VHH or anti-IL-2Rβ VHH was fused to Lch (SEQ ID NOs: 60-65, 71-76) of neutralizing anti-PD1 IgG Ab or Hch (SEQ ID NOs: 66-70, 77-81) of non-neutralizing anti-PD1 IgG Ab through a linker. In addition, anti-IL-2Rβ VHH or IL-2Rγ VHH was fused to anti-PD1 scFv (SEQ ID NOs: 82, 83) through a linker. DNA encoding these proteins was cloned into a mammalian expression vector. To prepare purified Ab, Expi293 cell line (Thermo Fisher, Carlsbad, CA, USA) was used for transient expression of the corresponding Hch and Lch expression plasmids. The Hch and Lch pairs of each antibody are described in Table 9. Protein purification was performed using protein A. For the concentration of the purified protein, the absorbance at 280 nm thereof was measured using a spectrophotometer. According to the obtained value, the extinction coefficient calculated by a method such as PACE was used to calculate the antibody concentration (Protein Science 1995; 4: 2411-2423).

[0717] [Table 9]

[0718]

[0719]

[0720]

[0721]

[0722]

[0723]

[0724]

[0725]

[0726]

[0727]

[0728] Example 25: Combinations of neutralizing and non-neutralizing anti-PD1 Abs fused with anti-IL-2Rβ or IL-2Rγ VHHs in PD1+ Efficiently induces IL-2 receptor activation in HEK-Blue IL-2 cells.

[0729] Human PD1 (SEQ ID NO: 37) or PDL1 (SEQ ID NO: 12) were first expressed in HEK-Blue IL-2 cells (InvivoGen, #hkb-il2) using the SF Cell Line 4D-Nucleofector X Kit L (Lonza, #V4XC-2024) by electroporation. 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. One day after electroporation, these cells were re-treated with different mixtures of neutralizing anti-PD1 Ab fused to anti-IL-2Ry VHH (final concentration of 12.5 nM) and non-neutralizing anti-PD1 Ab fused to anti-IL-2Ry VHH (final concentration of 12.5 nM) for one more day at 37°C / 5% CO2. Recombinant human IL-2 (Peprotech, #200-02) was used as a positive control. Two days after electroporation, IL-2 receptor complex activation was assessed by measuring the optical density at 620 nm using Quanti-Blue solution (InvivoGen, #rep-qbs) (Figures 15A and 15B).

[0730] As shown in Figures 15A and 15B, mixtures of neutralizing and non-neutralizing anti-PD1 Ab with IL2Ry or IL2Ry binding domains (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 demonstrate that it is possible to identify combinations that can demonstrate PD1 -dependent IL-2R agonist activity by binding neutralizing and non-neutralizing anti-PD1 Ab. This is an effective method to screen non-competitive or bivalent binders to build the target binding molecule combinations or protein complexes of the present application.

[0731] Example 26: Combinations of neutralizing anti-PD1Ab and non-neutralizing anti-PD1Ab fused to anti-IL-2Rβ or IL-2Rγ VHH Induced IL-2 receptor activation in PD1+ primary T cells but not in PD1-negative NK92 cells.

[0732] EasySep TMHuman CD4+ T cells isolation kit (STEMCELL, #17952) was used to isolate human CD4+ T cells from human PBMC (STEMCELL, #70025.2). After isolation, CD4+ T cells were treated with plates coated with anti-CD3 Ab (Biolegend, #317347, 5 pg / mL) and anti-CD28 Ab (Biolegend, #302943, 5 pg / mL) and recombinant human IL-2 (Peprotech, #200-02) for 3 days at 37°C / 5% CO2. CTS Optimizer T cell expansion SFM (GIBCO, #A1048501) containing penicillin / streptomycin (GIBCO, #15140122), GlutaMax (GIBCO, #35050061) and 10% fetal bovine serum was used for culture medium. After 3 days of culture, CD4+ T cells were harvested and washed twice with culture medium and left overnight with culture medium only. After resting, T cells were stimulated with a mixture of Vhh2g-PDA0041 and Vhh2b-PDE0171, Vhh2g-GY5 and Vhh2b-PDA0129, or Vhh2b-NivoscFv and Vhh2g4-NB01a scFv for 30 min at 37°C. Recombinant human IL-2 (Peprotech, #200-02) was used as a positive control and NK92 cells (ATCC, #CRL-2407) as PD1 negative IL-2 responsive cells. These cells were immediately fixed with CytoFix (BD Biosciences, #554655) for 20 min at room temperature. Cells were washed once with staining buffer (Biolegend, #420201) and subsequently freeze permeabilization buffer (BD Biosciences, #558050) was added to the samples and incubated on ice for 30 min. Cells were washed twice with staining buffer and then stained with Alexa Fluor (registered trademark) 647 Mouse Anti-Stat5 (pY694) (BD Biosciences, #562076) for at least 60 min at room temperature. Cells were washed once with staining buffer before analysis. Data acquisition was performed on a LSRFortessa X-20 (Becton Dickinson) (Figures 16A and 16B).

[0733] As shown in Figures 16A and 16B, mixtures of Vhh2g-PDA0041 and Vhh2b-PDE0171, Vhh2g-GY5 and Vhh2b-PDA0129, or Vhh2b-Nivo scFv and Vhh2g4-NB01a scFv induced significant STAT5 activation in activated human CD4+ T cells that highly express PD1, but did not induce significant STAT5 activation in NK92 cells that are negative for PD1 expression. These data indicate that the combination of neutralizing anti-PD1 Abs and non-neutralizing anti-PD1 Abs fused to anti-IL-2Rß or IL-2Ry binding domains can induce PD1-dependent IL-2 receptor activation not only in reporter cells, but also in primary T cells.

[0734] Example 27: VHHs binding to PD1 as scaffold protein and to IL-2R beta and IL-2R gamma as receptor proteins Expression and purification of IgG fusion proteins.

[0735] Anti-IL-2Rß VHHs (i.e., Vhh2b1, Vhh2b3, or Vhh2b4) were fused to the Hch of PDE0171 (SEQ ID NOs: 84-86) and anti-IL-2Ry VHHs (i.e., Vhh2g1, Vhh2g2, Vhh2g4, Vhh2g6, Vhh2g8, or Vhh2g10) were fused to the Lch of PDA0041 (SEQ ID NOs: 87-92) via linkers. These anti-IL-2Rß / y VHHs are described in WO2022 / 032006A2, WO2022 / 031884A2, or Cell (2022; 185: 1414-1430.e19). DNA encoding these proteins were cloned into mammalian expression vectors. For the preparation of purified Abs, Expi293 cell line (Thermo Fisher, Carlsbad, CA, USA) was used for transient expression of the corresponding Hch and Lch expression plasmids. The Hch and Lch pairs for each antibody are described in Table 10. Protein purification was performed using protein A. For the concentration of the purified proteins, the absorbance at 280 nm was measured using a spectrophotometer. According to the obtained values, the extinction coefficient calculated by methods such as PACE was used to calculate the antibody concentration (Protein Science 1995; 4: 2411-2423).

[0736] [Table 10]

[0737]

[0738]

[0739] Example 28: Mixtures of anti-PD1 Abs (PDA0041) fused to various IL-2R gamma binding domains and anti-PD1 Abs (PDE00171) fused to various IL-2R beta binding domains induce IL-2 receptor activation in PD1+HEK-Blue IL-2 cells. VHHs binding to PD1 as scaffold protein and to IL-2R beta and IL-2R gamma as receptor proteins Expression and purification of scFv-IgG or VHH-IgG fusion proteins.

[0740] Human PD1 (SEQ ID NO: 37) or PDL1 (SEQ ID NO: 12) were first expressed in HEK-Blue IL-2 cells (InvivoGen, #hkb-il2) using the SF Cell Line 4D-Nucleofector X Kit L (Lonza, #V4XC-2024) by electroporation. 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 different mixtures of anti-PD1 Ab (PDA0041) fused to various IL-2Ry binding domains (Vhh2g, Vhh2gl, Vhh2g2, Vhh2g4, Vhh2g6, Vhh2g8, or Vhh2glO) and anti-PD1 Ab (PDE0171) fused to various IL-2Ry binding domains (Vhh2b, Vhh2bl, Vhh2b3, or Vhh2b4), and further incubated for one more day at 37°C / 5% CO2. Each Ab concentration was 12.5 nM. Recombinant human IL-2 (Peprotech, #200-02) was used as a positive control. On day 3 after electroporation, IL-2 receptor complex activation was assessed by measuring the optical density at 620 nm using Quanti-Blue solution (InvivoGen, #rep-qbs) (Figures 17A and 17B).

[0741] As shown in Figures 17A and 17B, mixtures of anti-PD1 Ab (PDA0041) fused to various IL-2Ry binding domains and anti-PD1 Ab (PDEA00171) fused to various IL-2Ry binding domains induced IL-2 receptor activation in PD1+HEK-Blue IL-2 cells, but not in the absence of PD1 expression. These data indicate that various IL-2Ry / g binding domains can be used to induce PD1-dependent IL-2 receptor activation.

[0742] Example 29: Mixtures of Vhhpc13-AM3 and PDA0129 scFv-AL1, 2, 3, 4 or 5 binding to PD1 as target scaffold protein and to IL-2R beta or IL-2R gamma as target receptor proteins induce IL-2R activation in HEK-Blue IL-2 cells. Expression and purification of scFv-VHH fusion proteins binding to CD25 as target scaffold protein and to IL-2R beta and IL-2R gamma as target receptor proteins.

[0743] Anti-PD1 VHH (Vhhpc13) was fused to Hch of anti-IL2Ry (AM3) (SEQ ID NO: 93) and anti-PD1 scFv (PDA0129 scFv) was fused to Hch of anti-IL2Rp (AL1, AL2, AL3, AL4 or AL5) (SEQ ID NO: 94) through linkers. These Hch fusion proteins and corresponding Lch (SEQ ID NO: 95-100) were cloned into mammalian expression vectors. These anti-IL-2Rp or anti-IL-2Ry antibodies are reported on WO2023 / 139293A1. To prepare purified Abs, Expi293 cell line (Thermo Fisher, Carlsbad, CA, USA) was used to transiently express the corresponding Hch and Lch expression plasmids. The Hch and Lch pairs for each antibody are described in Table 11. Protein A was used for protein purification. For the concentration of the purified proteins, the absorbance at 280 nm was measured using a spectrophotometer. From the obtained values, the extinction coefficient calculated by methods such as PACE was used to calculate the antibody concentration (Protein Science 1995; 4: 2411-2423).

[0744] [Table 11]

[0745]

[0746] Example 30: Mixtures of neutralizing anti-CD25 scFv with IL-2R beta / gamma binding domains and non-neutralizing anti-CD25 scFv with IL-2R beta / gamma binding domains induce IL-2 receptor activation in activated primary CD4+T cells. Expression and purification of VHH-scFv fusion proteins binding to PDL1 as target scaffold protein and to CSF2RA or CSF2RB as target receptor proteins. Mixtures of VHH-scFv fusion proteins binding to PDL1 as scaffold protein and to CSF2RA or CSF2RB as target receptor proteins induce CSF2RA / B activation in HEK-Blue GM-CSF cells.

[0747] Human PD1 (SEQ ID NO: 37) or PDL1 (SEQ ID NO: 12) were first expressed in HEK-Blue IL-2 cells (InvivoGen, #hkb-il2) using the SF Cell Line 4D-Nucleofector X Kit L (Lonza, #V4XC-2024) by electroporation. 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 different mixtures of fusion proteins binding to PD1 as scaffold protein and IL-2Rβ or IL-2Rγ as receptor protein (final concentration of 25 nM) and further incubated for one more day at 37°C / 5% CO2. Recombinant human IL-2 (Peprotech, #200-02) was used as positive control. On day 2 after electroporation, IL-2 receptor complex activation was assessed by measuring the optical density at 620 nm using Quanti-Blue solution (InvivoGen, #rep-qbs) (Figures 18A and 18B).

[0748] As shown in Figures 18A and 18B, mixtures of Vhhpc13-AM3 and 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 not only anti-IL-2Rβ / γ VHHs, but also other binding domains such as Fabs, can be used to induce PD1-dependent IL-2 receptor activation.

[0749] Example 31: Expression and purification of VHH-Fab fusion proteins binding to RBD as target scaffold protein and to cMET as target receptor protein. Activation of cMET receptor by mixtures of VHH-Fab fusions binding to RBD and cMET is dependent on dual site binding to RBD.

[0750] Anti-CD25 scFv was fused with VHH that can bind to human IL-2Rγ (Vhh2g) or VHH that can bind to IL-2Rβ (Vhh2b) (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, and Gen scFv (US 8,961,968 B2) or Dac scFv (Biomedicines 2019; 7: 18) were used as neutralizing anti-CD25 antibodies. DNA encoding these proteins was cloned into a mammalian expression vector. 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. For the concentration of the purified protein, the absorbance at 280 nm thereof was measured using a spectrophotometer. According to the obtained value, the extinction coefficient calculated by a method such as PACE was used to calculate the antibody concentration (Protein Science 1995; 4: 2411-2423).

[0751] [Table 12]

[0752]

[0753]

[0754] Example 32: Expression and purification of bispecific antibodies binding to MUC1 or CEA as target scaffold protein and to FZD or LRP as target receptor protein. Mixtures of bispecific Abs binding to MUC1 and Wnt receptors (FZD or LRP) induce Wnt receptor activation in the presence of MUC1 expression.

[0755] EasySep TMHuman CD4+ T cells isolation kit (STEMCELL, #17952) was used to isolate human CD4+ T cells from human PBMC (STEMCELL, #70025.2). After isolation, CD4+ T cells were treated with plates coated with anti-CD3 Ab (Biolegend, #317347, 5 pg / mL) and anti-CD28 Ab (Biolegend, #302943, 5 pg / mL) and recombinant human IL-2 (Peprotech, #200-02) for 3 days at 37°C / 5% CO2. CTS Optimizer T cell expansion SFM (GIBCO, #A1048501) containing penicillin / streptomycin (GIBCO, #15140122), GlutaMax (GIBCO, #35050061) and 10% fetal bovine serum was used as culture medium. After 3 days of culture, CD4+ T cells were harvested and washed twice with culture medium and left overnight in culture medium conditions. After overnight, T cells were stimulated with a mix of VHH-scFv fusion proteins binding to CD25 as target scaffold protein and to either IL-2Rß or IL-2Ry as target receptor protein (final concentration of 25 nM) for 25 minutes at 37°C. After stimulation, these cells were immediately fixed with CytoFix (BD Biosciences, #554655) for 20 minutes at room temperature. Cells were washed once with staining buffer (Biolegend, #420201) and subsequently freeze 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 AlexaFluor (registered trademark) 647 mouse anti-Stat5 (pY694) (BD Biosciences, #562076) was added and incubated for at least 60 minutes at room temperature. Cells were washed once with staining buffer before analysis. Data acquisition was performed on a LSRFortessa X-20 (Becton Dickinson) (Figure 19).

[0756] As shown in Figure 19, mixtures of Vhh2g-Gen scFv HL / LH and Vhh2b-RG6292 scFv, Vhh2b-BT942 scFv, or Vhh2b-7G7 / B6 scFv, which are combinations of neutralizing and non-neutralizing anti-CD25 scFv, induce significant STAT5 activation in activated CD4+ T cells. In addition, mixtures of Vhh2g-BT942 scFv HL / LH and Vhh2b-Gen scFv or Vhh2b-Dac scFv, which are also combinations of neutralizing and non-neutralizing anti-CD25 scFv, induce significant STAT5 activation in activated CD4+ T cells. Again, this indicates that identifying combinations of neutralizing and non-neutralizing Abs to a scaffold protein is an effective approach to screen for non-competitive or dual-site binding agents to construct the target binding molecule combinations or protein complexes of the application.

[0757] Example 33: Mixtures of bispecific Abs binding to CEA and Wnt receptors induce Wnt receptor activation in the presence of CEA. Expression and purification of VHH-VHH fusion proteins binding to CD8 as scaffold protein and to IL-2R beta and IL-2R gamma as receptor proteins

[0758] VhhPL1, which is an anti-PDL1 VHH, was fused to anti-CSF2RA scFv (116.08 scFv, 116.18 scFv) (SEQ ID NOs: 110-113), and VhhPL1-1, which is another anti-PDL1 VHH, was fused to anti-CSF2RB scFv (131.16 scFv, 131.B2 scFv) (SEQ ID NOs: 114-117). These anti-CSF2RA or anti-CSF2RB Abs are reported in WO2023 / 027177A1. DNA encoding these proteins was cloned into mammalian expression vectors. These plasmids were transiently expressed using Expi293 cell line (Thermo Fisher, Carlsbad, CA, USA). Protein purification was performed using protein A or immobilized metal ion affinity chromatography. For the concentration of purified proteins, their absorbance at 280 nm was measured using a spectrophotometer. According to the obtained value, the extinction coefficient calculated by a method such as PACE was used to calculate the antibody concentration (Protein Science 1995; 4: 2411-2423).

[0759] [Table 13]

[0760]

[0761]

[0762] Example 34: Combination of VHH-VHH fusion proteins targeting CD8 as target scaffold protein and IL-2R beta / gamma as target receptor proteins induces IL-2 receptor activation in CD8+T cells but not in CD4+T cells. ​

[0763] Human PDL1 (SEQ ID NO: 12) or RBD-IL2RA (SEQ ID NO: 11) were first expressed in HEK-Blue GM-CSF cells (InvivoGen, #hkb-gmcsfr) using the SF Cell Line 4D-Nucleofector X Kit L (Lonza, #V4XC-2024) by electroporation. 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. One day after electroporation, these cells were treated with different mixtures of VHH-scFv fusion proteins that can bind to PDL1 (VHH) and CSF2RA (scFv) (SEQ ID NO: 110-113) and VHH-scFv fusion proteins that can bind to PDL1 (VHH) and CSF2RB (scFv) (SEQ ID NO: 114-117) for one more day at 37°C / 5% CO2. The final concentration of each Ab was 10 nM. Two days after electroporation, the activation of GM-CSF receptor (for CSF2RA and CSF2RB) was assessed by measuring the optical density at 620 nm using Quanti-Blue solution (InvivoGen, #rep-qbs) (FIGS. 20A and 20B).

[0764] As shown in FIGS. 20A and 20B, mixtures of VHH-scFv fusion proteins that bind to PDL1 as the target scaffold protein and 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 the scaffold-dependent mechanism can not only activate the IL-2 receptor, but also the GM-CSF receptor or other cytokine receptors.

[0765] Example 35: ​ ​

[0766] VHHs that can bind to SARS-CoV2 RBD (Nb21 or Nb36) were fused to Lch of anti-human cMET Fab (SEQ ID NOs: 119, 120). DNA encoding these VHH-Lch fusion proteins and Hch of anti-human cMET Fab (SEQ ID NO: 118) were cloned into mammalian expression vectors. Anti-cMET Fab is reported in Protein Data Bank (PDB: 6I04). To prepare purified Abs, Expi293 cell line (Thermo Fisher, Carlsbad, CA, USA) was used for transient expression of the corresponding Hch and Lch expression plasmids. The Hch and Lch pairs for each antibody are described in Table 14. Antibody purification was performed using Protein A or immobilized metal ion affinity chromatography. For the concentration of purified antibodies, the absorbance at 280 nm was measured using a spectrophotometer. According to the obtained value, the extinction coefficient calculated by methods such as PACE was used to calculate the antibody concentration (Protein Science 1995; 4: 2411-2423).

[0767] [Table 14]

[0768]

[0769] Example 36: ​ ​

[0770] 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 onto 96-well plates and stimulated for 2 days with VHH-Fab fusion proteins (Nb21-Met6I04 Fab, Nb36-Met6I04 Fab) binding to RBD as a target scaffold protein and cMET as a target receptor protein in the presence of recombinant SARS-CoV-2 spike RBD protein (R&D systems, #10523-CV-100, final concentration of 640 pM). Recombinant human HGF protein (R&D systems, #294-HGN / CF) was used as a positive control. Two days after stimulation, culture supernatants were collected and IL-11 induced by cMET activation was detected using human IL-11 DuoSet ELISA kit (R&D systems, #DY218) (Figures 21A, 21B, and 21C).

[0771] As shown in Figures 21A, 21B and 21C, mixtures of VHH-Fab fusion proteins of anti-RBD and anti-cMET Fabs that bind to RBD in a two-site manner induced significant cMET activation in the presence of recombinant SARS-CoV-2 spike RBD protein (Figure 21A), but did not induce significant cMET activation in the case of unit site Abs (Figures 21B and 21C). These data suggest that the scaffold-dependent mechanism can not only activate IL-2 receptor, but also other cytokine receptors.

[0772] Example 37: ​ ​

[0773] DNA encoding Hch and Lch of R2M3-R2M3 (anti-FZD Ab, SEQ ID NOs: 121, 123), Hch and Lch of AR20.5 (anti-MUC1 Ab, SEQ ID NOs: 124, 127), and Hch and Lch of CEA.Mab3 (anti-CEA antibody, SEQ ID NOs: 126, 128) were cloned into mammalian expression vectors. In addition, DNA encoding Vhhlrp36-Vhhlrp36 (anti-LRP Ab, SEQ ID NO: 122), CEA.VHH (anti-CEA Ab, SEQ ID NO: 125) were cloned into mammalian expression vectors. For the preparation of purified Abs, Expi293 cell line (Thermo Fisher, Carlsbad, CA, USA) was used for transient expression of the corresponding Hch and Lch expression plasmids. The Hch and Lch pairs for each antibody are described in Table 15. From these purified Abs, bispecific antibodies against FZD and MUC1 (R2M3-R2M3 / / AR20.5) or FZD and CEA (R2M3-R2M3 / / CEA.VHH, R2M3-R2M3 / / CEA.MAb3) were prepared by using Fab arm exchange technique (as described in WO2015 / 046467). In addition, bispecific antibodies against LRP and MUC1 (Vhhlrp36-Vhhlrp36 / / AR20.5) or LRP and CEA (Vhhlrp36-Vhhlrp36 / / CEA.VHH, Vhhlrp36-Vhhlrp36 / / CEA.MAb3) were prepared by using Fab arm exchange technique. For the concentration of purified antibodies, the absorbance at 280 nm was measured using a spectrophotometer. According to the obtained value, the extinction coefficient calculated by a method such as PACE was used to calculate the antibody concentration (Protein Science 1995; 4: 2411-2423).

[0774] [Table 15]

[0775]

[0776]

[0777] Example 38: ​ ​

[0778] MUC1 (SEQ ID NO: 129) or RBD-IL2RA (SEQ ID NO: 11) were first expressed in HEK293 STF cells (ATCC, #CRL-3249) using the SF Cell Line 4D-Nucleofector X Kit L (Lonza, #V4XC-2024) by electroporation. These transfections were seeded in white 384-well plates and incubated overnight at 37°C / 5% CO2 after addition of 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 pg / mL geneticin. On day 1 after electroporatio...

Claims

1. A target binding molecule combination 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 non-competitively binding to the scaffold protein, and The first receptor protein and the second receptor protein are each receptor subunits capable of associating to form a receptor complex.

2. A protein complex 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 non-competitively binding to the scaffold protein, The first receptor protein and the second receptor protein are each receptor subunits capable of associating to form a receptor complex.

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

4. The target binding molecule combination or protein complex according to any one of claims 1 to 3, wherein the first binding domain and the third binding domain are each capable of binding to a subunit of the scaffold protein, and the subunits are capable of associating to form the scaffold protein.

5. The target binding molecule combination or protein complex according to any one of claims 1 to 4, wherein the scaffold protein is selected from the group consisting of 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, T IM3, 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, Nectine-4, EGFRvIII, IL13RA2, TMEFF2, Muc16, EpCAM, FcRH2, AFP, PSA, amyloid β, MBP, and ASGPR.

6. The target binding molecule combination or protein complex according to any one of claims 1 to 5, wherein the first receptor protein and the second receptor protein are each independently selected from the group consisting of 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, IL18 RAP, ST2, IL17RA, IL17RC, 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, ACVR1C, ACVR1, AMHR2, BMPR1A, BMPR1B, BMPR2, TRA, TRB, CD 3E, CD16, TREM2, FGFR1, FGFR2, FGFR3, FGFR4, Fzd1, Fzd2, Fzd3, Fzd4, Fzd5, Fzd6, Fzd7, Fzd8 Fzd9, Fzd10, LRP5, LRP6, and LGR5. 7 . The target binding molecule combination or 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. The target binding molecule combination 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, affibody, cytokine, ligand or split cytokine.

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

10. A vector or vectors comprising a nucleic acid molecule or nucleic acid molecules according to claim 9.

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

12. A method for producing a target binding molecule combination or protein complex according to any one of claims 1 to 8, the method comprising the following steps: (iii) culturing the host cell or cells according to claim 11 under conditions suitable for protein expression; (iv) optionally lysing the host cell; and (iii) isolating the target binding molecule assembly or protein complex.

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

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

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

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