Mesoserine-targeted CD40 agonist multispecific antibody construct for the treatment of solid tumors

Abstract

This invention provides a human agonist CD40 multispecific antibody construct for the treatment of solid tumors by designing molecules that specifically target the CD40 pathway or tumor-associated APCs without systemic CD40 activation. [Solution] A multispecific antibody construct is provided, comprising (i) a first antibody having a light chain and a heavy chain having a specific sequence, wherein the first antibody specifically binds to and stimulates human CD40, and (ii) the scFv specifically binds to human mesoserine.

Description

[Technical Field]

[0001] This invention relates to oncology, specifically to the field of cancer immunotherapy. The invention relates to a human agonist CD40 antibody, which involves designing a molecule that specifically targets the CD40 pathway to tumor-associated APCs without systemic CD40 activation. [Background technology]

[0002] Treating cancer patients who do not respond to immune checkpoint inhibitors such as anti-PD1 presents both a major challenge and an exciting opportunity in the rapidly evolving field of immunotherapy. A key determinant of response to PD1 blockers in solid tumors is the degree of tumor-associated T-cell infiltration prior to treatment, with patients with "cold" tumors exhibiting minimal clinical benefit. While clinical data are still lacking, the clinical efficacy of other T-cell targeted immunotherapies besides PD1 blockers, such as solid tumor BiTE® antibody constructs or CAR-T techniques, may also be limited in "cold" tumors. Combining T-cell targeted techniques with a novel class of therapeutics capable of enhancing T-cell infiltration into solid tumors may be crucial in maximizing the number of patients who benefit from immunotherapy.

[0003] CD40 is a member of the TNF receptor (TNFR) superfamily, preferentially expressed by antigen-presenting cells (APCs) such as dendritic cells, B cells, and macrophages. Interaction with its trimer ligand on activated T helper cells results in APC activation, including the upregulation of cytokines / chemokines (such as interleukin-12 [IL-12] and CxCL10), antigen-presenting proteins (such as MHC class I and II ligands), T cell costimulatory ligands (such as CD80 and CD86), and a range of other immunomodulators (i.e., adhesion molecules and other TNFRs). These "licensed" APCs can then activate a cascade of events that trigger the induction of a robust adaptive immune response.

[0004] Therapies that can activate CD40 signaling have the potential to inflame solid tumors by enhancing the generation of antitumor T cells and increasing the direct recruitment of T cells to tumor lesions. Preclinical studies with anti-CD40 agonists suggest that activating CD40 with cross-linked antibodies on APCs can substitute for CD4 T cells, which license APCs and help promote the activation and proliferation of CD8 effector T cells. In addition, CD40-activated macrophages may also exert direct tumor-killing functions. These anti-CD40 agonist antibodies have demonstrated efficacy in multiple isoplastic tumor models, either alone or in combination with other therapies. Based on these preclinical studies, several CD40 agonist antibodies are under investigation in Phase I / II clinical trials in solid tumor patients. To date, these monoclonal anti-CD40 antibodies have shown some signs of clinical efficacy, but are often associated with immune-related adverse effects, such as evidence of cytokine release syndrome and liver injury. These toxicities limit the dose of CD40 agonists that can be delivered, and therefore may negatively impact the efficacy of this therapeutic approach by resulting in insufficient activation of the CD40 pathway in tumor-associated APC populations. Therapeutics that can localize the activation of CD40 signaling in tumor tissue may improve antitumor effects while limiting systemic toxicity.

[0005] To achieve tumor localization, a multispecific agonist antibody construct was designed that binds to both CD40 and the tumor-associated antigen (TAA), mesoserine (MSLN). The robust agonist activity of this multispecific antibody construct against CD40-expressing APCs is entirely dependent on the presence of adjacent MSLN-expressing cells. The multispecific antibody construct was specifically designed to lack CD40 agonist activity upon binding to CD40 in the absence of MSLN-expressing cells. In addition, the introduction of a mutation in the IgG Fc domain restricts binding to the Fc receptor, thereby interfering with Fc receptor-mediated CD40 agonist activity. Therefore, the MSLN-dependent CD40 agonist multispecific antibody construct described herein has the potential to promote robust CD40-mediated activation of APCs in a manner primarily localized to tumor tissue, thereby minimizing the induction of systemic toxicity. [Overview of the project] [Means for solving the problem]

[0006] The present invention (i) A first antibody comprising two light chains and two heavy chains, The light chain comprises a first variable region (VL1) and a light chain constant region (CL); The heavy chain comprises a first heavy chain variable region (VH1) and the CH1, hinge, CH2, and CH3 regions; A first antibody in which the heavy chain contains at least one amino acid substitution resulting in a reduced binding affinity of the heavy chain to the human Fc gamma RI receptor compared to an unsubstituted heavy chain; and (ii) an scFv comprising a second light chain variable region (VL2) and a second heavy chain variable region (VH2) of a second antibody, wherein VL2 and VH2 are linked via a first peptide linker, scFv comprises scFv fused to each carboxyl terminus of the heavy chain via a second peptide linker at its amino terminus, such that a heavy chain fusion protein is formed; and This invention relates to a multispecific antibody construct in which the first antibody specifically binds to and stimulates human CD40 (SEQ ID NO: 1), and scFv specifically binds to human mesoserine (MSLN) (SEQ ID NO: 2).

[0007] In one embodiment, a multispecific antibody construct specifically stimulates CD40 in an MSLN-dependent manner.

[0008] In one embodiment, a multispecific antibody construct includes mutations that restrict Fc receptor binding, thereby reducing Fc receptor-dependent CD40 agonism.

[0009] In one embodiment, the two light chains are identical, and the two heavy chain fusion proteins are identical.

[0010] In one embodiment, the heavy chain is (i) N297G or N297A; (ii) L234A and L235A; and (iii) R292C and V302C Includes amino acid substitutions selected from the group consisting of; The amino acid numbering follows the EU numbering system, which is based on Kabat.

[0011] In one embodiment, the heavy chain contains the N297G, R292C, and V302C mutations, and the amino acid numbering follows the EU numbering system according to Kabat.

[0012] In one embodiment, VL1 is Sequence IDs 58, 59, and 60, respectively; Sequence IDs 64, 65, and 66, respectively; Sequence numbers 70, 71, and 72, respectively; Sequence IDs 76, 77, and 78, respectively; Sequence IDs 82, 83, and 84, respectively; Sequence IDs 88, 89, and 90, respectively; Sequence numbers 94, 95, and 96, respectively; Sequence IDs 100, 101, and 102, respectively; each having SEQ ID NO: 106, 107, and 108; each having SEQ ID NO: 112, 113, and 114; each having SEQ ID NO: 118, 119, and 120; each having SEQ ID NO: 124, 125, and 126; and each having SEQ ID NO: 130, 131, and 132 and comprising CDRL1, CDRL2, and CDRL3 selected from the group consisting of; VH1 is each having SEQ ID NO: 136, 137, and 138; each having SEQ ID NO: 142, 143, and 144; each having SEQ ID NO: 148, 149, and 150; each having SEQ ID NO: 154, 155, and 156; each having SEQ ID NO: 160, 161, and 162; each having SEQ ID NO: 166, 167, and 168; each having SEQ ID NO: 172, 173, and 174; each having SEQ ID NO: 178, 179, and 180; each having SEQ ID NO: 184, 185, and 186; each having SEQ ID NO: 190, 191, and 192; each having SEQ ID NO: 196, 197, and 198; each having SEQ ID NO: 202, 203, and 204; and each having SEQ ID NO: 208, 209, and 210 and comprising CDRH1, CDRH2, and CDRH3 selected from the group consisting of; VL2 is each having SEQ ID NO: 230, 231, and 232; each having SEQ ID NO: 236, 237, and 238; each having SEQ ID NO: 242, 243, and 244; and each having SEQ ID NO: 248, 249, and 250 and comprising CDRL1, CDRL2, and CDRL3 selected from the group consisting of; and VH2 is Sequence IDs 254, 255, and 256, respectively; Sequence IDs 260, 261, and 262, respectively; Sequence IDs 266, 267, and 268, respectively; and Sequence IDs 272, 273, and 274, respectively. It includes CDRH1, CDRH2, and CDRH3 selected from the group consisting of the following.

[0013] In one embodiment, 1) VL1 and VH1 are, a) VL1 containing CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs. 58, 59, and 60, respectively; and VH1 containing CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs. 136, 137, and 138, respectively; b) VL1 containing CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs. 64, 65, and 66, respectively; and VH1 containing CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs. 142, 143, and 144, respectively; c) VL1 containing CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs. 70, 71, and 72, respectively; and VH1 containing CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs. 148, 149, and 150, respectively; d) VL1 containing CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs. 76, 77, and 6780, respectively; and VH1 containing CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs. 154, 155, and 156, respectively; e) VL1 containing CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs. 82, 83, and 84, respectively; and VH1 containing CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs. 160, 161, and 162, respectively; f) VL1 containing CDRL1, CDRL2, and CDRL3 selected from the group consisting of sequence numbers 88, 89, and 90, respectively; and VH1 containing CDRH1, CDRH2, and CDRH3 selected from the group consisting of sequence numbers 166, 167, and 168, respectively; g) VL1 containing CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs. 94, 95, and 96, respectively; and VH1 containing CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs. 172, 173, and 174, respectively; h) VL1 containing CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 100, 101, and 102, respectively; and VH1 containing CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 178, 179, and 180, respectively; i) VL1 containing CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 106, 107, and 108, respectively; and VH1 containing CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 184, 185, and 186, respectively; j) VL1 containing CDRL1, CDRL2, and CDRL3 selected from the group consisting of sequence numbers 112, 113, and 114, respectively; and VH1 containing CDRH1, CDRH2, and CDRH3 selected from the group consisting of sequence numbers 190, 191, and 192, respectively; k) VL1 containing CDRL1, CDRL2, and CDRL3 selected from the group consisting of sequence numbers 118, 119, and 120, respectively; and VH1 containing CDRH1, CDRH2, and CDRH3 selected from the group consisting of sequence numbers 196, 197, and 198, respectively; l) VL1 containing CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 124, 125, and 126, respectively; and VH1 containing CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 202, 203, and 204, respectively; and m) VL1 containing CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 130, 131, and 132, respectively; and VH1 containing CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 208, 209, and 210, respectively. Selected from the group consisting of; and 2) VL2 and VH2 are, a) VL2 containing CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs. 230, 231, and 232, respectively; and VH2 containing CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs. 254, 255, and 256, respectively; b) VL2 containing CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs. 236, 237, and 238, respectively; and VH2 containing CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs. 260, 261, and 262, respectively; c) VL2 containing CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs. 242, 243, and 244, respectively; and VH2 containing CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs. 266, 267, and 268, respectively; and d) VL2 containing CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs. 248, 249, and 250, respectively; and VH2 containing CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs. 272, 273, and 274, respectively. It is selected from the group consisting of the following.

[0014] In one embodiment, VL1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 5, 9, 13, 17, 21, 25, 29, 33, 37, 41, 45, 49, and 53; VH1 contains an amino acid sequence selected from the group consisting of SEQ ID NOs: 6, 10, 14, 18, 22, 26, 30, 34, 38, 42, 46, 50, and 54; VL2 contains an amino acid sequence selected from the group consisting of SEQ ID NOs: 213, 217, 221, and 225; VH2 contains an amino acid sequence selected from the group consisting of SEQ ID NOs: 214, 218, 222, and 226.

[0015] In one embodiment, 1) VL1 and VH1 are, a) VL1 containing Sequence ID 5 and VH1 containing Sequence ID 6; b) VL1 containing SEQ ID NO. 9 and VH1 containing SEQ ID NO. 10; c) VL1 containing SEQ ID NO. 13 and VH1 containing SEQ ID NO. 14; d) VL1 containing SEQ ID NO: 17 and VH1 containing SEQ ID NO: 18; e) VL1 containing Sequence ID 21 and VH1 containing Sequence ID 22; f) VL1 containing sequence number 25 and VH1 containing sequence number 26; g) VL1 containing SEQ ID NO. 29 and VH1 containing SEQ ID NO. 30; h) VL1 containing sequence number 33 and VH1 containing sequence number 34; i) VL1 containing sequence number 37 and VH1 containing sequence number 38; j) VL1 containing sequence number 41 and VH1 containing sequence number 42; k) VL1 containing SEQ ID NO: 45 and VH1 containing SEQ ID NO: 46; l) VL1 containing SEQ ID NO: 49 and VH1 containing SEQ ID NO: 50; and m) VL1 containing SEQ ID NO: 53 and VH1 containing SEQ ID NO: 54 Selected from the group consisting of; and 2) VL2 and VH2 are, a) VL2 containing Sequence ID 213 and VH2 containing Sequence ID 214; b) VL2 containing Sequence ID 217 and VH2 containing Sequence ID 218; c) VL2 containing Sequence ID 221 and VH2 containing Sequence ID 222; and d) VL2 containing SEQ ID NO: 225 and VH2 containing SEQ ID NO: 226 It is selected from the group consisting of the following.

[0016] In one embodiment, the CL of the light chain is selected from the group consisting of SEQ ID NOs: 883 and 884.

[0017] In one embodiment, the heavy chain CH1-hinge-CH2-CH3 is selected from the group consisting of sequence numbers 885 and 886.

[0018] In one embodiment, the first peptide linker is selected from the group consisting of SEQ ID NOs: 888 to 893.

[0019] In one embodiment, the second peptide linker is selected from the group consisting of SEQ ID NOs: 887 to 893.

[0020] In one embodiment, the first peptide linker includes SEQ ID NO: 889, and the second peptide linker includes SEQ ID NO: 887.

[0021] In one embodiment, The light chain contains a sequence selected from the group consisting of SEQ ID NOs: 286, 290, 294, 298, 302, 306, 310, 314, 318, 322, 326, 330, 336, 342, 346, 350, 354, 358, 362, 366, 370, 374, and 378; and The heavy chain fusion protein contains a sequence selected from the group consisting of SEQ ID NOs: 285, 289, 293, 297, 301, 305, 309, 313, 317, 321, 325, 329, 333, 337, 341, 345, 349, 353, 357, 361, 365, 369, 373, and 377.

[0022] In one embodiment, the light chain and heavy chain fusion protein is Sequence IDs 286 and 285, respectively; Sequence IDs 290 and 289, respectively; Sequence IDs 294 and 293, respectively; Sequence IDs 298 and 297, respectively; Sequence IDs 302 and 301, respectively; Sequence IDs 306 and 305, respectively; Sequence IDs 310 and 309, respectively; Sequence IDs 314 and 313, respectively; Sequence IDs 318 and 317, respectively; Sequence IDs 322 and 321, respectively; Sequence IDs 326 and 325, respectively; Sequence IDs 330 and 329, respectively; Sequence IDs 334 and 333, respectively; Sequence IDs 338 and 337, respectively; Sequence IDs 342 and 341, respectively; Sequence IDs 346 and 345, respectively; Sequence IDs 350 and 349, respectively; Sequence IDs 354 and 353, respectively; Sequence IDs 358 and 357, respectively; Sequence IDs 362 and 361, respectively; Sequence IDs 366 and 365, respectively; Sequence IDs 370 and 369, respectively; Sequence ID 374 and Sequence ID 373, respectively; and Sequence IDs 378 and 377, respectively. It contains a polypeptide comprising an amino acid sequence selected from the group consisting of the following.

[0023] In one embodiment, the present invention relates to a polynucleotide encoding the light chain of the antibody construct of the present invention.

[0024] In one embodiment, the present invention relates to a polynucleotide encoding a heavy chain fusion protein of the antibody construct of the present invention.

[0025] In one embodiment, the present invention relates to a vector comprising a polynucleotide encoding the light chain of an antibody construct, a polynucleotide encoding the heavy chain of an antibody construct, or both.

[0026] In one embodiment, the present invention relates to host cells transformed or transfected with polynucleotides encoding the light chain of a vector or antibody construct and polynucleotides encoding the heavy chain of an antibody construct.

[0027] In one embodiment, the present invention relates to a process for generating an antibody construct of the present invention, comprising culturing host cells containing a polynucleotide encoding a light chain and a polynucleotide encoding a heavy chain fusion protein under conditions that enable the expression of the antibody construct, and recovering the antibody construct produced from the culture.

[0028] In one embodiment, the present invention relates to a pharmaceutical composition comprising an antibody construct and carrier, stabilizer, excipient, diluent, solubilizer, surfactant, emulsifier, preservative, or adjuvant according to the present invention.

[0029] In one embodiment, the present invention relates to a method for treating or inducing remission of a solid tumor disease or metastatic cancer disease, comprising the step of administering an effective amount of the antibody construct according to the present invention to a target subject as needed.

[0030] In one embodiment, the solid tumor disease is selected from the group consisting of ovarian cancer, pancreatic cancer, mesothelioma, lung cancer, gastric cancer, and tertiary negative breast cancer or metastatic cancer, all of which originate from any of the aforementioned conditions.

[0031] In one embodiment, the present invention relates to an antibody construct according to the present invention and a kit including, optionally, instructions for use.

[0032] In one aspect, the present invention is (i) A first antibody comprising two light chains and two heavy chains, The light chain comprises a first variable region (VL1) and a light chain constant region (CL); The heavy chain comprises a first heavy chain variable region (VH1) and the CH1, hinge, CH2, and CH3 regions; A first antibody in which the heavy chain contains at least one amino acid substitution resulting in a reduced binding affinity of the heavy chain to the human Fc gamma RI receptor compared to an unsubstituted heavy chain; and (ii) an scFv comprising a second light chain variable region (VL2) and a second heavy chain variable region (VH2) of a second antibody, wherein VL2 and VH2 are linked via a first peptide linker, scFv comprises scFv fused to each carboxyl terminus of the heavy chain via a second peptide linker at its amino terminus, such that a heavy chain fusion protein is formed; and This invention relates to a multispecific antibody construct in which the first antibody specifically binds to human mesoserine (MSLN) (SEQ ID NO: 2), and scFv specifically binds to and stimulates human CD40 (SEQ ID NO: 1).

[0033] In one embodiment, a multispecific antibody construct specifically stimulates CD40 in an MSLN-dependent manner.

[0034] In one embodiment, a multispecific antibody construct includes mutations that restrict Fc receptor binding, thereby reducing Fc receptor-dependent CD40 agonism.

[0035] In one embodiment, the two light chains are identical, and the two heavy chain fusion proteins are identical.

[0036] In one embodiment, the heavy chain is (i) N297G or N297A; (ii) L234A and L235A; and (iii) R292C and V302C Includes amino acid substitutions selected from the group consisting of; The amino acid numbering follows the EU numbering system, which is based on Kabat.

[0037] In one embodiment, the heavy chain contains the N297G, R292C, and V302C mutations, and the amino acid numbering follows the EU numbering system according to Kabat.

[0038] In one embodiment, VL1 is Sequence IDs 230, 231, and 232, respectively; Sequence IDs 236, 237, and 238, respectively; Sequence IDs 242, 243, and 244, respectively; and Sequence IDs 248, 249, and 250, respectively. It includes CDRL1, CDRL2, and CDRL3 selected from the group consisting of; VH1 is Sequence IDs 254, 255, and 256, respectively; Sequence IDs 260, 261, and 262, respectively; Sequence IDs 266, 267, and 268, respectively; and Sequence IDs 272, 273, and 274, respectively. It includes CDRH1, CDRH2, and CDRH3 selected from the group consisting of; VL2 is Sequence IDs 58, 59, and 60, respectively; Sequence IDs 64, 65, and 66, respectively; Sequence numbers 70, 71, and 72, respectively; Sequence IDs 76, 77, and 78, respectively; Sequence IDs 82, 83, and 84, respectively; Sequence IDs 88, 89, and 90, respectively; Sequence numbers 94, 95, and 96, respectively; Sequence IDs 100, 101, and 102, respectively; Sequence IDs 106, 107, and 108, respectively; Sequence numbers 112, 113, and 114, respectively; Sequence IDs 118, 119, and 120, respectively; Sequence IDs 124, 125, and 126, respectively; and Sequence numbers 130, 131, and 132, respectively. It includes CDRL1, CDRL2, and CDRL3 selected from the group consisting of; and VH2 is Sequence numbers 136, 137, and 138, respectively; Sequence IDs 142, 143, and 144, respectively; Sequence IDs 148, 149, and 150, respectively; Sequence IDs 154, 155, and 156, respectively; Sequence IDs 160, 161, and 162, respectively; Sequence IDs 166, 167, and 168, respectively; Sequence IDs 172, 173, and 174, respectively; Sequence IDs 178, 179, and 180, respectively; Sequence IDs 184, 185, and 186, respectively; Sequence numbers 190, 191, and 192, respectively; Sequence IDs 196, 197, and 198, respectively; Sequence IDs 202, 203, and 204, respectively; and Sequence IDs 208, 209, and 210, respectively. It includes CDRH1, CDRH2, and CDRH3 selected from the group consisting of the following.

[0039] In one embodiment, 1) VL1 and VH1 are, a) VL1 containing CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs. 230, 231, and 232, respectively; and VH1 containing CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs. 254, 255, and 256, respectively; b) VL1 containing CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs. 236, 237, and 238, respectively; and VH1 containing CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs. 260, 261, and 262, respectively; c) VL1 containing CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs. 242, 243, and 244, respectively; and VH1 containing CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs. 266, 267, and 268, respectively; and d) VL1 containing CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs. 248, 249, and 250, respectively; and VH1 containing CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs. 272, 273, and 274, respectively. Selected from the group consisting of; and 2) VL2 and VH2 are, a) VL2 containing CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs. 58, 59, and 60, respectively; and VH2 containing CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs. 136, 137, and 138, respectively; b) VL2 containing CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs. 64, 65, and 66, respectively; and VH2 containing CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs. 142, 143, and 144, respectively; c) VL2 containing CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs. 70, 71, and 72, respectively; and VH2 containing CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs. 148, 149, and 150, respectively; d) VL2 containing CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs. 76, 77, and 6780, respectively; and VH2 containing CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs. 154, 155, and 156, respectively; e) VL2 containing CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs. 82, 83, and 84, respectively; and VH2 containing CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs. 160, 161, and 162, respectively; f) VL2 containing CDRL1, CDRL2, and CDRL3 selected from the group consisting of sequence numbers 88, 89, and 90, respectively; and VH2 containing CDRH1, CDRH2, and CDRH3 selected from the group consisting of sequence numbers 166, 167, and 168, respectively; g) VL2 containing CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs. 94, 95, and 96, respectively; and VH2 containing CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs. 172, 173, and 174, respectively; h) VL2 containing CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 100, 101, and 102, respectively; and VH2 containing CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 178, 179, and 180, respectively; i) VL2 containing CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 106, 107, and 108, respectively; and VH2 containing CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 184, 185, and 186, respectively; j) VL2 containing CDRL1, CDRL2, and CDRL3 selected from the group consisting of sequence numbers 112, 113, and 114, respectively; and VH2 containing CDRH1, CDRH2, and CDRH3 selected from the group consisting of sequence numbers 190, 191, and 192, respectively; k) VL2 containing CDRL1, CDRL2, and CDRL3 selected from the group consisting of sequence numbers 118, 119, and 120, respectively; and VH2 containing CDRH1, CDRH2, and CDRH3 selected from the group consisting of sequence numbers 196, 197, and 198, respectively; l) VL2 containing CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 124, 125, and 126, respectively; and VH2 containing CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 202, 203, and 204, respectively; and m) VL2 containing CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 130, 131, and 132, respectively; and VH2 containing CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 208, 209, and 210, respectively. It is selected from the group consisting of the following.

[0040] In one embodiment, VL1 contains an amino acid sequence selected from the group consisting of SEQ ID NOs: 213, 217, 221, and 225; VH1 contains an amino acid sequence selected from the group consisting of SEQ ID NOs: 214, 218, 222, and 226; VL2 contains an amino acid sequence selected from the group consisting of SEQ ID NOs: 5, 9, 13, 17, 21, 25, 29, 33, 37, 41, 45, 49, and 53; and VH2 contains an amino acid sequence selected from the group consisting of SEQ ID NOs: 6, 10, 14, 18, 22, 26, 30, 34, 38, 42, 46, 50, and 54.

[0041] In one embodiment, 1) VL1 and VH1 are, a) VL1 containing sequence number 213 and VH1 containing sequence number 214; b) VL1 containing sequence number 217 and VH1 containing sequence number 218; c) VL1 containing Sequence ID 221 and VH1 containing Sequence ID 222; and d) VL1 containing SEQ ID NO: 225 and VH1 containing SEQ ID NO: 226 Selected from the group consisting of; and 2) VL2 and VH2 are, a) VL2 containing Sequence ID 5 and VH2 containing Sequence ID 6; b) VL2 containing SEQ ID NO. 9 and VH2 containing SEQ ID NO. 10; c) VL2 containing Sequence ID 13 and VH2 containing Sequence ID 14; d) VL2 containing SEQ ID NO: 17 and VH2 containing SEQ ID NO: 18; e) VL2 containing Sequence ID 21 and VH2 containing Sequence ID 22; f) VL2 containing Sequence ID 25 and VH2 containing Sequence ID 26; g) VL2 containing Sequence ID 29 and VH2 containing Sequence ID 30; h) VL2 containing SEQ ID NO. 33 and VH2 containing SEQ ID NO. 34; i) VL2 containing Sequence ID 37 and VH2 containing Sequence ID 38; j) VL2 containing Sequence ID 41 and VH2 containing Sequence ID 42; k) VL2 containing SEQ ID NO: 45 and VH2 containing SEQ ID NO: 46; l) VL2 containing sequence number 49 and VH2 containing sequence number 50; and m) VL2 containing SEQ ID NO: 53 and VH2 containing SEQ ID NO: 54 It is selected from the group consisting of the following.

[0042] In one embodiment, the CL of the light chain is selected from the group consisting of SEQ ID NOs: 883 and 884.

[0043] In one embodiment, the heavy chain CH1-hinge-CH2-CH3 is selected from the group consisting of sequence numbers 885 and 886.

[0044] In one embodiment, the first peptide linker is selected from the group consisting of SEQ ID NOs: 888 to 893.

[0045] In one embodiment, the second peptide linker is selected from the group consisting of SEQ ID NOs: 887 to 893.

[0046] In one embodiment, the first peptide linker includes SEQ ID NO: 889, and the second peptide linker includes SEQ ID NO: 887.

[0047] In one embodiment, The light chain contains a sequence selected from the group consisting of SEQ ID NOs: 382, ​​386, 390, 394, 398, 402, 406, 410, 414, 418, 422, 426, 430, 434, 438, 442, 446, and 450; and The heavy chain fusion protein contains a sequence selected from the group consisting of SEQ ID NOs: 381, 385, 389, 393, 397, 401, 405, 409, 413, 417, 421, 425, 429, 433, 437, 441, 445, and 449.

[0048] In one embodiment, the light chain and heavy chain fusion protein is Sequence IDs 382 and 381, respectively; Sequence IDs 386 and 385, respectively; Sequence IDs 390 and 389, respectively; Sequence IDs 394 and 393, respectively; Sequence IDs 398 and 397, respectively; Sequence IDs 402 and 401, respectively; Sequence IDs 406 and 405, respectively; Sequence IDs 410 and 409, respectively; Sequence IDs 414 and 413, respectively; Sequence IDs 418 and 417, respectively; Sequence IDs 422 and 421, respectively; Sequence IDs 426 and 425, respectively; Sequence IDs 430 and 429, respectively; Sequence IDs 434 and 433, respectively; Sequence IDs 438 and 437, respectively; Sequence IDs 442 and 441, respectively; Sequence IDs 446 and 445, respectively; and Sequence IDs 450 and 449, respectively. It contains a polypeptide comprising an amino acid sequence selected from the group consisting of the following.

[0049] In one aspect, the present invention is a) Two identical heavy chain fusion proteins, each containing a first heavy chain variable region (VH1) and a first CH1 domain (the first CH1 domain is linked to a hinge-CH2-CH3 polypeptide, and the hinge-CH2-CH3 polypeptide is linked to a second heavy chain variable region (VH2), and VH2 is linked to a second CH1 domain; i) The VH1 or first CH1 domain includes at least one amino acid substitution introducing a positively charged amino acid at a residue selected from the group consisting of positions 39, 44, and 183 using EU numbering; and ii) The VH2 or second CH1 domain includes at least one amino acid substitution introducing a negatively charged amino acid with a residue selected from the group consisting of residues corresponding to positions 39, 44, and 183 using EU numbering); and b) A second polypeptide comprising a first light chain (the first light chain comprising a first light chain variable region (VL1) and a first CL region; and the VL1 or the first CL domain comprising at least one amino acid substitution introducing a negatively charged amino acid at a residue selected from the group consisting of positions 38, 100, and 176 using EU numbering); and c) A third polypeptide comprising a second light chain (the second light chain comprising a second light chain variable region (VL2) and a second CL region; and the VL2 or second CL domain comprising at least one amino acid substitution introducing a positively charged amino acid at a residue selected from the group consisting of positions 38, 100, and 176 using EU numbering). A multispecific antibody construct comprising, VH1 and VL1 interact to bind to the first antigen, and VH2 and VL2 interact to bind to the second antigen; Here, The first antigen is human CD40 (SEQ ID NO: 1), and the second antigen is human mesoserine ("MSLN"; SEQ ID NO: 2); or This invention relates to a multispecific antibody construct in which the first antigen is human MSLN (SEQ ID NO: 2) and the second antigen is human CD40 (SEQ ID NO: 1).

[0050] In one aspect, the present invention is a) Two identical heavy chain fusion proteins, each containing a first heavy chain variable region (VH1) and a first CH1 domain (the first CH1 domain is linked to a hinge-CH2-CH3 polypeptide, and the hinge-CH2-CH3 polypeptide is linked to a second heavy chain variable region (VH2), and VH2 is linked to a second CH1 domain; i) The VH1 or first CH1 domain includes at least one amino acid substitution introducing a negatively charged amino acid at a residue selected from the group consisting of positions 39, 44, and 183 using EU numbering; and ii) The VH2 or second CH1 domain includes at least one amino acid substitution introducing a positively charged amino acid at a residue selected from the group consisting of residues corresponding to positions 39, 44, and 183 using EU numbering; and b) A second polypeptide comprising a first light chain (the first light chain comprising a first light chain variable region (VL1) and a first CL region; and the VL1 or the first CL domain comprising at least one amino acid substitution introducing a positively charged amino acid at a residue selected from the group consisting of positions 38, 100, and 176 using EU numbering); and c) A third polypeptide comprising a second light chain (the second light chain comprising a second light chain variable region (VL2) and a second CL region; and the VL2 or the second CL domain comprising at least one amino acid substitution introducing a negatively charged amino acid at a residue selected from the group consisting of positions 38, 100, and 176 using EU numbering). A multispecific antibody construct comprising, VH1 and VL1 interact to bind to the first antigen, and VH2 and VL2 interact to bind to the second antigen; Here, The first antigen is human CD40 (SEQ ID NO: 1), and the second antigen is human mesoserine ("MSLN"; SEQ ID NO: 2); or This invention relates to a multispecific antibody construct in which the first antigen is human MSLN (SEQ ID NO: 2) and the second antigen is human CD40 (SEQ ID NO: 1).

[0051] In one embodiment, the hinge-CH2-CH3 polypeptide is linked to VH2 via a peptide linker.

[0052] In one embodiment, the peptide linker includes a sequence selected from the group consisting of (Gly3Ser)2 (SEQ ID NO: 916), (Gly4Ser)2 (SEQ ID NO: 888), (Gly3Ser)3 (SEQ ID NO: 917), (Gly4Ser)3 (SEQ ID NO: 889), (Gly3Ser)4 (SEQ ID NO: 918), (Gly4Ser)4 (SEQ ID NO: 890), (Gly3Ser)5 (SEQ ID NO: 919), (Gly4Ser)5 (SEQ ID NO: 920), (Gly3Ser)6 (SEQ ID NO: 921), and (Gly4Ser)6 (SEQ ID NO: 922).

[0053] In one embodiment, a) The VH1 or first CH1 domain contains a mutation selected from the group consisting of G39K, G44K, and S183K using EU numbering; b) The VH2 or second CH1 domain contains a mutation selected from the group consisting of G39E, G44E, and S183E using EU numbering; c) The VL1 or first CL domain contains a mutation selected from the group consisting of Q38E, G100E, and S176E using EU numbering; and d) The VL2 or second CL domain contains a mutation selected from the group consisting of Q38K, G100K, and S176K using EU numbering.

[0054] In one embodiment, a) The first CH1 domain contains the S183K mutation using EU numbering; b) The second CH1 domain contains the S183E mutation using EU numbering; c) The first CL domain contains the S176E mutation using EU numbering; and d) The second CL domain contains the S176K mutation, which uses EU numbering.

[0055] In one embodiment, a) VH1 contains the Q39K mutation and the first CH1 domain contains the S183K mutation using EU numbering; b) VH2 contains the Q39E mutation and the second CH1 domain contains the S183E mutation using EU numbering; c) VL1 contains the Q38E mutation and the first CL domain contains the S176E mutation using EU numbering; and d) VL2 contains the Q38K mutation and the second CL domain contains the S176K mutation using EU numbering.

[0056] In one embodiment, a) The first CH1 domain contains the G44K and S183K mutations using EU numbering; b) The second CH1 domain contains the G44E and S183E mutations using EU numbering; c) The first CL domain contains the G100E and S176E mutations using EU numbering; and d) The second CL domain contains the G100K and S176K mutations, which use EU numbering.

[0057] In one embodiment, a) The VH1 or first CH1 domain contains a mutation selected from the group consisting of Q39E, G44E, and S183E using EU numbering; b) The VH2 or second CH1 domain contains a mutation selected from the group consisting of Q39K, G44K, and S183K using EU numbering; c) The VL1 or first CL domain contains a mutation selected from the group consisting of Q38K, G100K, and S176K using EU numbering; and d) The VL2 or second CL domain contains a mutation selected from the group consisting of Q38E, G100E, and S176E using EU numbering.

[0058] In one embodiment, a) The first CH1 domain contains the S183E mutation using EU numbering; b) The second CH1 domain contains the S183K mutation using EU numbering; c) The first CL domain contains the S176K mutation using EU numbering; and d) The second CL domain contains the S176E mutation, which uses EU numbering.

[0059] In one embodiment, a) VH1 contains the Q39E mutation and the first CH1 domain contains the S183E mutation using EU numbering; b) VH2 contains the Q39K mutation and the second CH1 domain contains the S183K mutation using EU numbering; c) VL1 contains the Q38K mutation and the first CL domain contains the S176K mutation using EU numbering; and d) VL2 contains the Q38E mutation and the second CL domain contains the S176E mutation using EU numbering.

[0060] In one embodiment, a) The first CH1 domain contains the G44E and S183E mutations using EU numbering; b) The second CH1 domain contains the G44K and S183K mutations using EU numbering; c) The first CL domain contains the G100K and S176K mutations using EU numbering; and d) The second CL domain contains the G100E and S176E mutations, which use EU numbering.

[0061] In one embodiment, the hinge-CH2-CH3 polypeptide is (i) N297G or N297A; (ii) L234A and L235A; and (iii) R292C and V302C Includes amino acid substitutions selected from the group consisting of; The amino acid numbering follows the EU numbering system, which is based on Kabat.

[0062] In one embodiment, the hinge-CH2-CH3 polypeptide includes N297G, R292C, and V302C mutations, and the amino acid numbering is EU numbering according to Kabat.

[0063] In one embodiment, the first antigen is human CD40 (SEQ ID NO: 1) and the second antigen is human MSLN (SEQ ID NO: 2); and VL1 is Sequence IDs 58, 59, and 60, respectively; Sequence IDs 64, 65, and 66, respectively; Sequence numbers 70, 71, and 72, respectively; Sequence IDs 76, 77, and 78, respectively; Sequence IDs 82, 83, and 84, respectively; Sequence IDs 88, 89, and 90, respectively; Sequence numbers 94, 95, and 96, respectively; Sequence IDs 100, 101, and 102, respectively; Sequence IDs 106, 107, and 108, respectively; Sequence numbers 112, 113, and 114, respectively; Sequence IDs 118, 119, and 120, respectively; Sequence IDs 124, 125, and 126, respectively; and Sequence numbers 130, 131, and 132, respectively. It includes CDRL1, CDRL2, and CDRL3 selected from the group consisting of; VH1 is Sequence numbers 136, 137, and 138, respectively; Sequence IDs 142, 143, and 144, respectively; Sequence IDs 148, 149, and 150, respectively; Sequence IDs 154, 155, and 156, respectively; Sequence IDs 160, 161, and 162, respectively; Sequence IDs 166, 167, and 168, respectively; Sequence IDs 172, 173, and 174, respectively; Sequence IDs 178, 179, and 180, respectively; Sequence IDs 184, 185, and 186, respectively; Sequence numbers 190, 191, and 192, respectively; Sequence IDs 196, 197, and 198, respectively; Sequence IDs 202, 203, and 204, respectively; and Sequence IDs 208, 209, and 210, respectively. It includes CDRH1, CDRH2, and CDRH3 selected from the group consisting of; VL2 is Sequence IDs 230, 231, and 232, respectively; Sequence IDs 236, 237, and 238, respectively; Sequence IDs 242, 243, and 244, respectively; and Sequence IDs 248, 249, and 250, respectively. It includes CDRL1, CDRL2, and CDRL3 selected from the group consisting of; Furthermore, VH2 is Sequence IDs 254, 255, and 256, respectively; Sequence IDs 260, 261, and 262, respectively; Sequence IDs 266, 267, and 268, respectively; and Sequence IDs 272, 273, and 274, respectively. It includes CDRH1, CDRH2, and CDRH3 selected from the group consisting of the following.

[0064] In one embodiment, 1) VL1 and VH1 are, a) VL1 containing CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs. 58, 59, and 60, respectively; and VH1 containing CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs. 136, 137, and 138, respectively; b) VL1 containing CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs. 64, 65, and 66, respectively; and VH1 containing CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs. 142, 143, and 144, respectively; c ) VL1 containing CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs. 70, 71, and 72, respectively; and VH1 containing CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs. 148, 149, and 150, respectively; d) VL1 containing CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs. 76, 77, and 6780, respectively; and VH1 containing CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs. 154, 155, and 156, respectively; e) VL1 containing CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs. 82, 83, and 84, respectively; and VH1 containing CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs. 160, 161, and 162, respectively; f) VL1 containing CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs. 88, 89, and 90, respectively; and VH1 containing CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs. 166, 167, and 168, respectively; g ) VL1 containing CDRL1, CDRL2, and CDRL3 selected from the group consisting of sequence numbers 94, 95, and 96, respectively; and VH1 containing CDRH1, CDRH2, and CDRH3 selected from the group consisting of sequence numbers 172, 173, and 174, respectively; h) VL1 containing CDRL1, CDRL2, and CDRL3 selected from the group consisting of sequence numbers 100, 101, and 102, respectively; and VH1 containing CDRH1, CDRH2, and CDRH3 selected from the group consisting of sequence numbers 178, 179, and 180, respectively; i) VL1 containing CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 106, 107, and 108, respectively; and VH1 containing CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 184, 185, and 186, respectively; j) VL1 containing CDRL1, CDRL2, and CDRL3 selected from the group consisting of sequence numbers 112, 113, and 114, respectively; and VH1 containing CDRH1, CDRH2, and CDRH3 selected from the group consisting of sequence numbers 190, 191, and 192, respectively; k) VL1 containing CDRL1, CDRL2, and CDRL3 selected from the group consisting of sequence numbers 118, 119, and 120, respectively; and VH1 containing CDRH1, CDRH2, and CDRH3 selected from the group consisting of sequence numbers 196, 197, and 198, respectively; l) VL1 containing CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 124, 125, and 126, respectively; and VH1 containing CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 202, 203, and 204, respectively; and m) VL1 containing CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 130, 131, and 132, respectively; and VH1 containing CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 208, 209, and 210, respectively. Selected from the group consisting of; and 2) VL2 and VH2 are, a) VL2 containing CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs. 230, 231, and 232, respectively; and VH2 containing CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs. 254, 255, and 256, respectively; b) VL2 containing CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs. 236, 237, and 238, respectively; and VH2 containing CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs. 260, 261, and 262, respectively; c) VL2 containing CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs. 242, 243, and 244, respectively; and VH2 containing CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs. 266, 267, and 268, respectively; and d) VL2 containing CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs. 248, 249, and 250, respectively; and VH2 containing CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs. 272, 273, and 274, respectively. It is selected from the group consisting of the following.

[0065] In one embodiment, VL1 contains an amino acid sequence selected from the group consisting of SEQ ID NOs: 5, 9, 13, 17, 21, 25, 29, 33, 37, 41, 45, 49, and 53; VH1 contains an amino acid sequence selected from the group consisting of SEQ ID NOs: 6, 10, 14, 18, 22, 26, 30, 34, 38, 42, 46, 50, and 54; VL2 contains an amino acid sequence selected from the group consisting of SEQ ID NOs: 213, 217, 221, and 225; VH2 contains an amino acid sequence selected from the group consisting of SEQ ID NOs: 214, 218, 222, and 226.

[0066] In one embodiment, 1) VL1 and VH1 are, a) VL1 containing Sequence ID 5 and VH1 containing Sequence ID 6; b) VL1 containing SEQ ID NO. 9 and VH1 containing SEQ ID NO. 10; c) VL1 containing SEQ ID NO. 13 and VH1 containing SEQ ID NO. 14; d) VL1 containing SEQ ID NO: 17 and VH1 containing SEQ ID NO: 18; e) VL1 containing Sequence ID 21 and VH1 containing Sequence ID 22; f) VL1 containing sequence number 25 and VH1 containing sequence number 26; g) VL1 containing SEQ ID NO. 29 and VH1 containing SEQ ID NO. 30; h) VL1 containing sequence number 33 and VH1 containing sequence number 34; i) VL1 containing sequence number 37 and VH1 containing sequence number 38; j) VL1 containing sequence number 41 and VH1 containing sequence number 42; k) VL1 containing SEQ ID NO: 45 and VH1 containing SEQ ID NO: 46; l) VL1 containing SEQ ID NO: 49 and VH1 containing SEQ ID NO: 50; and m) VL1 containing SEQ ID NO: 53 and VH1 containing SEQ ID NO: 54 Selected from the group consisting of; and 2) VL2 and VH2 are, a) VL2 containing Sequence ID 213 and VH2 containing Sequence ID 214; b) VL2 containing Sequence ID 217 and VH2 containing Sequence ID 218; c) VL2 containing Sequence ID 221 and VH2 containing Sequence ID 222; and d) VL2 containing SEQ ID NO: 225 and VH2 containing SEQ ID NO: 226 It is selected from the group consisting of the following.

[0067] In one embodiment, the present invention relates to a polynucleotide encoding a first light chain of an antibody construct.

[0068] In one embodiment, the present invention relates to a polynucleotide encoding a second light chain of an antibody construct.

[0069] In one embodiment, the present invention relates to a polynucleotide encoding a heavy chain fusion protein of an antibody construct.

[0070] In one aspect, the present invention is a) Polynucleotide encoding the first light chain of the antibody construct, b) Polynucleotides encoding the second light chain of the antibody construct, c) A polynucleotide encoding the heavy chain fusion protein of the antibody construct, or d) Any combination of a), b), and c) Regarding vectors that include this.

[0071] In one embodiment, the present invention relates to host cells transformed or transfected with a vector or polynucleotide according to the present invention.

[0072] In one embodiment, the present invention relates to a process for generating an antibody construct according to the present invention, comprising culturing a host cell containing a polynucleotide encoding a first light chain, a polynucleotide encoding a second light chain, and a polynucleotide encoding a heavy chain fusion protein under conditions that enable the expression of the antibody construct, and recovering the antibody construct produced from the culture.

[0073] In one embodiment, the present invention relates to a pharmaceutical composition comprising an antibody construct and carrier, stabilizer, excipient, diluent, solubilizer, surfactant, emulsifier, preservative, or adjuvant according to the present invention.

[0074] In one embodiment, the present invention relates to a method for treating or inducing remission of a solid tumor disease or metastatic cancer disease, comprising the step of administering an effective amount of the antibody construct according to the present invention to a target subject as needed.

[0075] In one embodiment, the solid tumor disease is selected from the group consisting of ovarian cancer, pancreatic cancer, mesothelioma, lung cancer, gastric cancer, and tertiary negative breast cancer or metastatic cancer, all of which originate from any of the aforementioned conditions.

[0076] In one embodiment, the present invention relates to an antibody construct according to the present invention and a kit including, optionally, instructions for use.

[0077] In one embodiment, the first antigen is human MSLN (SEQ ID NO: 2) and the second antigen is human CD40 (SEQ ID NO: 1); VL1 is Sequence IDs 230, 231, and 232, respectively; Sequence IDs 236, 237, and 238, respectively; Sequence IDs 242, 243, and 244, respectively; and Sequence IDs 248, 249, and 250, respectively. It includes CDRL1, CDRL2, and CDRL3 selected from the group consisting of; VH1 is Sequence IDs 254, 255, and 256, respectively; Sequence IDs 260, 261, and 262, respectively; Sequence IDs 266, 267, and 268, respectively; and Sequence IDs 272, 273, and 274, respectively. It includes CDRH1, CDRH2, and CDRH3 selected from the group consisting of; VL2 is Sequence IDs 58, 59, and 60, respectively; Sequence IDs 64, 65, and 66, respectively; Sequence numbers 70, 71, and 72, respectively; Sequence IDs 76, 77, and 78, respectively; Sequence IDs 82, 83, and 84, respectively; Sequence IDs 88, 89, and 90, respectively; Sequence numbers 94, 95, and 96, respectively; Sequence IDs 100, 101, and 102, respectively; Sequence IDs 106, 107, and 108, respectively; Sequence numbers 112, 113, and 114, respectively; Sequence IDs 118, 119, and 120, respectively; Sequence IDs 124, 125, and 126, respectively; and Sequence numbers 130, 131, and 132, respectively. It includes CDRL1, CDRL2, and CDRL3 selected from the group consisting of; and VH2 is Sequence numbers 136, 137, and 138, respectively; Sequence IDs 142, 143, and 144, respectively; Sequence IDs 148, 149, and 150, respectively; Sequence IDs 154, 155, and 156, respectively; Sequence IDs 160, 161, and 162, respectively; Sequence IDs 166, 167, and 168, respectively; Sequence IDs 172, 173, and 174, respectively; Sequence IDs 178, 179, and 180, respectively; Sequence IDs 184, 185, and 186, respectively; Sequence numbers 190, 191, and 192, respectively; Sequence IDs 196, 197, and 198, respectively; Sequence IDs 202, 203, and 204, respectively; and Sequence IDs 208, 209, and 210, respectively. It includes CDRH1, CDRH2, and CDRH3 selected from the group consisting of the following.

[0078] In one embodiment, 1) VL1 and VH1 are, a) VL1 containing CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs. 230, 231, and 232, respectively; and VH1 containing CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs. 254, 255, and 256, respectively; b) VL1 containing CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs. 236, 237, and 238, respectively; and VH1 containing CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs. 260, 261, and 262, respectively; c) VL1 containing CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs. 242, 243, and 244, respectively; and VH1 containing CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs. 266, 267, and 268, respectively; and d) VL1 containing CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs. 248, 249, and 250, respectively; and VH1 containing CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs. 272, 273, and 274, respectively. Selected from the group consisting of; and 2) VL2 and VH2 are, a) VL2 containing CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs. 58, 59, and 60, respectively; and VH2 containing CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs. 136, 137, and 138, respectively; b) VL2 containing CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs. 64, 65, and 66, respectively; and VH2 containing CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs. 142, 143, and 144, respectively; c) VL2 containing CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs. 70, 71, and 72, respectively; and VH2 containing CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs. 148, 149, and 150, respectively; d) VL2 containing CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs. 76, 77, and 6780, respectively; and VH2 containing CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs. 154, 155, and 156, respectively. e) VL2 containing CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs. 82, 83, and 84, respectively; and VH2 containing CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs. 160, 161, and 162, respectively; f) VL2 containing CDRL1, CDRL2, and CDRL3 selected from the group consisting of sequence numbers 88, 89, and 90, respectively; and VH2 containing CDRH1, CDRH2, and CDRH3 selected from the group consisting of sequence numbers 166, 167, and 168, respectively; g) VL2 containing CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs. 94, 95, and 96, respectively; and VH2 containing CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs. 172, 173, and 174, respectively; h) VL2 containing CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 100, 101, and 102, respectively; and VH2 containing CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 178, 179, and 180, respectively; i) VL2 containing CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 106, 107, and 108, respectively; and VH2 containing CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 184, 185, and 186, respectively; j) VL2 containing CDRL1, CDRL2, and CDRL3 selected from the group consisting of sequence numbers 112, 113, and 114, respectively; and VH2 containing CDRH1, CDRH2, and CDRH3 selected from the group consisting of sequence numbers 190, 191, and 192, respectively; k) VL2 containing CDRL1, CDRL2, and CDRL3 selected from the group consisting of sequence numbers 118, 119, and 120, respectively; and VH2 containing CDRH1, CDRH2, and CDRH3 selected from the group consisting of sequence numbers 196, 197, and 198, respectively; l) VL2 containing CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 124, 125, and 126, respectively; and VH2 containing CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 202, 203, and 204, respectively; and m) VL2 containing CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 130, 131, and 132, respectively; and VH2 containing CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 208, 209, and 210, respectively. It is selected from the group consisting of the following.

[0079] In one embodiment, VL1 contains an amino acid sequence selected from the group consisting of SEQ ID NOs: 213, 217, 221, and 225; VH1 contains an amino acid sequence selected from the group consisting of SEQ ID NOs: 214, 218, 222, and 226; VL2 contains an amino acid sequence selected from the group consisting of SEQ ID NOs: 5, 9, 13, 17, 21, 25, 29, 33, 37, 41, 45, 49, and 53; and VH2 contains an amino acid sequence selected from the group consisting of SEQ ID NOs: 6, 10, 14, 18, 22, 26, 30, 34, 38, 42, 46, 50, and 54.

[0080] In one embodiment, 1) VL1 and VH1 are, a) VL1 containing sequence number 213 and VH1 containing sequence number 214; b) VL1 containing sequence number 217 and VH1 containing sequence number 218; c) VL1 containing Sequence ID 221 and VH1 containing Sequence ID 222; and d) VL1 containing SEQ ID NO: 225 and VH1 containing SEQ ID NO: 226 Selected from the group consisting of; and 2) VL2 and VH2 are, a) VL2 containing Sequence ID 5 and VH2 containing Sequence ID 6; b) VL2 containing SEQ ID NO. 9 and VH2 containing SEQ ID NO. 10; c) VL2 containing Sequence ID 13 and VH2 containing Sequence ID 14; d) VL2 containing SEQ ID NO: 17 and VH2 containing SEQ ID NO: 18; e) VL2 containing Sequence ID 21 and VH2 containing Sequence ID 22; f) VL2 containing Sequence ID 25 and VH2 containing Sequence ID 26; g) VL2 containing Sequence ID 29 and VH2 containing Sequence ID 30; h) VL2 containing SEQ ID NO. 33 and VH2 containing SEQ ID NO. 34; i) VL2 containing Sequence ID 37 and VH2 containing Sequence ID 38; j) VL2 containing Sequence ID 41 and VH2 containing Sequence ID 42; k) VL2 containing SEQ ID NO: 45 and VH2 containing SEQ ID NO: 46; l) VL2 containing sequence number 49 and VH2 containing sequence number 50; and m) VL2 containing SEQ ID NO: 53 and VH2 containing SEQ ID NO: 54 It is selected from the group consisting of the following.

[0081] In one embodiment, the present invention relates to an antigen-binding protein that specifically binds to and stimulates human CD40 (SEQ ID NO: 1), and comprises a light chain variable region (VL) and a heavy chain variable region (VH). VL is Sequence IDs 58, 59, and 60, respectively; Sequence IDs 64, 65, and 66, respectively; Sequence numbers 70, 71, and 72, respectively; Sequence IDs 76, 77, and 78, respectively; Sequence IDs 82, 83, and 84, respectively; Sequence IDs 88, 89, and 90, respectively; Sequence numbers 94, 95, and 96, respectively; Sequence IDs 100, 101, and 102, respectively; Sequence IDs 106, 107, and 108, respectively; Sequence numbers 112, 113, and 114, respectively; Sequence IDs 118, 119, and 120, respectively; Sequence IDs 124, 125, and 126, respectively; and Sequence numbers 130, 131, and 132, respectively. It includes CDRL1, CDRL2, and CDRL3 selected from the group consisting of; and VH is Sequence numbers 136, 137, and 138, respectively; Sequence IDs 142, 143, and 144, respectively; Sequence IDs 148, 149, and 150, respectively; Sequence IDs 154, 155, and 156, respectively; Sequence IDs 160, 161, and 162, respectively; Sequence IDs 166, 167, and 168, respectively; Sequence IDs 172, 173, and 174, respectively; Sequence IDs 178, 179, and 180, respectively; Sequence IDs 184, 185, and 186, respectively; Sequence numbers 190, 191, and 192, respectively; Sequence IDs 196, 197, and 198, respectively; Sequence IDs 202, 203, and 204, respectively; and Sequence IDs 208, 209, and 210, respectively. It includes CDRH1, CDRH2, and CDRH3 selected from the group consisting of the following.

[0082] In one embodiment, a multispecific antibody construct includes mutations that restrict Fc receptor binding, thereby reducing Fc receptor-dependent CD40 agonism.

[0083] In one embodiment, VL and VH are a) VL containing CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs. 58, 59, and 60, respectively; and VH containing CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs. 136, 137, and 138, respectively; b) VL containing CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs. 64, 65, and 66, respectively; and VH containing CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs. 142, 143, and 144, respectively; c) VL containing CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs. 70, 71, and 72, respectively; and VH containing CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs. 148, 149, and 150, respectively; d) VL containing CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs. 76, 77, and 6780, respectively; and VH containing CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs. 154, 155, and 156, respectively; e) VL containing CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs. 82, 83, and 84, respectively; and VH containing CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs. 160, 161, and 162, respectively; f) VL containing CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs. 88, 89, and 90, respectively; and VH containing CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs. 166, 167, and 168, respectively; g) VL containing CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs. 94, 95, and 96, respectively; and VH containing CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs. 172, 173, and 174, respectively; h) VL containing CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 100, 101, and 102, respectively; and VH containing CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 178, 179, and 180, respectively; i) VL containing CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 106, 107, and 108, respectively; and VH containing CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 184, 185, and 186, respectively; j) VL containing CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 112, 113, and 114, respectively; and VH containing CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 190, 191, and 192, respectively; k) VL containing CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 118, 119, and 120, respectively; and VH containing CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 196, 197, and 198, respectively; l) VL containing CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 124, 125, and 126, respectively; and VH containing CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 202, 203, and 204, respectively; and m) VL containing CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 130, 131, and 132, respectively; and VH containing CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 208, 209, and 210, respectively. It is selected from the group consisting of the following.

[0084] In one embodiment, VL contains an amino acid sequence selected from the group consisting of SEQ ID NOs: 5, 9, 13, 17, 21, 25, 29, 33, 37, 41, 45, 49, and 53; and VH contains an amino acid sequence selected from the group consisting of SEQ ID NOs: 6, 10, 14, 18, 22, 26, 30, 34, 38, 42, 46, 50, and 54.

[0085] In one embodiment, VL and VH are a) VL containing SEQ ID NO. 5 and VH containing SEQ ID NO. 6; b) VL containing SEQ ID NO. 9 and VH containing SEQ ID NO. 10; c) VL containing SEQ ID NO. 13 and VH containing SEQ ID NO. 14; d) VL containing SEQ ID NO: 17 and VH containing SEQ ID NO: 18; e) VL containing Sequence ID 21 and VH containing Sequence ID 22; f) VL containing SEQ ID NO. 25 and VH containing SEQ ID NO. 26; g) VL containing SEQ ID NO. 29 and VH containing SEQ ID NO. 30; h) VL containing SEQ ID NO. 33 and VH containing SEQ ID NO. 34; i) VL containing SEQ ID NO: 37 and VH containing SEQ ID NO: 38; j) VL containing SEQ ID NO: 41 and VH containing SEQ ID NO: 42; k) VL containing SEQ ID NO: 45 and VH containing SEQ ID NO: 46; l) VL containing SEQ ID NO: 49 and VH containing SEQ ID NO: 50; and m) VL containing SEQ ID NO: 53 and VH containing SEQ ID NO: 54 It is selected from the group consisting of the following.

[0086] In one embodiment, the heavy chain is (i) N297G or N297A; (ii) L234A and L235A; and (iii) R292C and V302C Includes amino acid substitutions selected from the group consisting of; The amino acid numbering follows the EU numbering system, which is based on Kabat.

[0087] In one embodiment, the heavy chain contains the N297G, R292C, and V302C mutations, and the amino acid numbering follows the EU numbering system according to Kabat.

[0088] In one embodiment, the antigen-binding protein further comprises a light chain CL polypeptide linked to the VL, the CL polypeptide being selected from the group consisting of SEQ ID NOs: 883 and 884.

[0089] In one embodiment, the antigen-binding protein further comprises a heavy chain CH1-hinge-CH2-CH3 polypeptide, the CH1-hinge-CH2-CH3 polypeptide being selected from the group consisting of SEQ ID NOs. 885 and 886.

[0090] In one embodiment, the antigen-binding protein further includes a second antigen-binding moiety that specifically binds to a second antigen.

[0091] In one embodiment, the second antigen is a tumor-associated antigen (TAA). [Brief explanation of the drawing]

[0092] [Figure 1] This shows the cell-side binding of an anti-MSLN antibody to CHO cells expressing human MSLN. [Figure 2] This shows the activity of both cross-linked and uncross-linked anti-CD40 antibodies in a human B-cell assay. [Figure 3] This shows a bivalent, bispecific antibody format. [Figure 4] This demonstrates the creation of cell lines expressing various levels of MSLN. [Figure 5] This study demonstrates the pharmacokinetic properties and stability of anti-CD40×MSLN bispecific molecules in mice. [Figure 6] This document demonstrates the creation of MC38-human EPCAM and B16F10-human EPCAM expressing cell lines. [Figure 7-1] This molecule exhibits activity as a mouse surrogate anti-CD40 × human EPCAM bispecific molecule. [Figure 7-2] This molecule exhibits activity as a mouse surrogate anti-CD40 × human EPCAM bispecific molecule. [Figure 8] This study demonstrates that a mouse-surrogate anti-CD40 × human EPCAM bispecific molecule is localized to tumors in vivo and exhibits TAA-mediated, cross-linking-dependent activity. [Figure 9] This study demonstrates that a mouse surrogate anti-CD40 × human EPCAM bispecific molecule enhances the CD8+ T cell antitumor response. [Figure 10] This study demonstrates that a mouse-surrogate anti-CD40 × human EPCAM bispecific molecule is localized to tumors in vivo and exhibits TAA-mediated, cross-linking-dependent activity. [Figure 11-1] This study demonstrates that a mouse surrogate anti-CD40 × human EPCAM bispecific molecule does not increase systemic cytokine levels or increase liver inflammation. [Figure 11-2] This study demonstrates that a mouse surrogate anti-CD40 × human EPCAM bispecific molecule does not increase systemic cytokine levels or increase liver inflammation. [Figure 12-1] A murine surrogate anti-CD40×human EpCAM bispecific molecule is shown to induce regression of established MC38-human EpCAM tumors alone and in combination with a PD1 / PDL1 blocking agent. [Figure 12-2] A murine surrogate anti-CD40×human EpCAM bispecific molecule is shown to induce regression of established MC38-human EpCAM tumors alone and in combination with a PD1 / PDL1 blocking agent. [Figure 13] A murine surrogate anti-CD40×human EpCAM bispecific molecule is shown to induce long-term immune memory that protects from tumor rechallenge.

MODE FOR CARRYING OUT THE INVENTION

[0093] Unless otherwise indicated, the term “at least” preceding a series of elements is to be understood as referring to any and all of the elements in the series. One of ordinary skill in the art will recognize or be able to ascertain many equivalents to the specific embodiments of the invention described herein by the mere exercise of routine experimentation. Such equivalents are intended to be encompassed by the present invention.

[0094] The term “and / or” as used anywhere in this specification includes the meanings “and,” “or,” and “any and all combinations of the elements connected by said term.”

[0095] The term “about” or “approximately” as used herein means within ±20%, preferably within ±15%, more preferably within ±10%, and most preferably within ±5% of a given value or range.

[0096] Throughout this specification and the following claims, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", are to be construed as including the stated integer or step, or group of integers or steps, but not as excluding any other integer or step, or group of integers or steps. As used herein, the term "comprise" may be replaced with the term "contain" or "include" or, when used herein, sometimes the term "have".

[0097] As used herein, "consisting of" excludes any element, step, or ingredient not specified in the claim. As used herein, "consisting essentially of" does not exclude materials or steps that do not substantially affect the basic and novel characteristics of the claim.

[0098] In each case herein, any one of the terms "comprise", "consisting essentially of", and "consisting of" may be replaced with any one of the other two terms.

[0099] As used herein, the term “antigen-binding protein” refers to a protein that specifically binds to one or more target antigens. Antigen-binding proteins may include antibodies and their functional fragments. A “functional antibody fragment” is a portion of an antibody that lacks at least some of the amino acids present in the full-length heavy and / or light chain, but is still capable of specifically binding to an antigen. Examples of functional antibody fragments include, but are not limited to, Fab fragments, Fab' fragments, F(ab')2 fragments, Fv fragments, Fd fragments, and complementarity-determining region (CDR) fragments, and may originate from any mammalian source, such as humans, mice, rats, rabbits, or camelids. Functional antibody fragments may compete with intact antibodies for binding to target antigens, and fragments may be produced by modifying intact antibodies (e.g., enzymatic or chemical cleavage) or newly synthesized using recombinant DNA technology or peptide synthesis.

[0100] The term "antibody construct" refers to a molecule whose structure and / or function are based on the structure and / or function of an antibody, such as a full-length or full-length immunoglobulin molecule. Therefore, an antibody construct can bind to its specific target or antigen. Furthermore, the antibody construct according to the present invention includes the minimum structural requirements of the antibody that enable target binding. These minimum requirements can be defined, for example, by the presence of at least three light chain CDRs (i.e., CDR1, CDR2, and CDR3 in the VL region) and / or three heavy chain CDRs (i.e., CDR1, CDR2, and CDR3 in the VH region), preferably all six CDRs. Antibodies on which the construct according to the present invention is based include, for example, monoclonal antibodies, recombinant antibodies, chimeric antibodies, deimmunized antibodies, humanized antibodies, and human antibodies.

[0101] In certain embodiments, the antigen-binding protein of the present invention comprises an antibody. As used herein, the term “antibody” refers to a tetrameric immunoglobulin protein comprising two light-chain polypeptides (each about 25 kDa) and two heavy-chain polypeptides (each about 50–70 kDa). The term “light chain” or “immunoglobulin light chain” refers to a polypeptide comprising a single immunoglobulin light chain variable region (VL) and a single immunoglobulin light chain constant domain (CL) from the amino terminus to the carboxyl terminus. The immunoglobulin light chain constant domain (CL) may be kappa (κ) or lambda (λ). The term “heavy chain” or “immunoglobulin heavy chain” refers to a polypeptide comprising a single immunoglobulin heavy chain variable region (VH), immunoglobulin heavy chain constant domain 1 (CH1), an immunoglobulin hinge region, immunoglobulin heavy chain constant domain 2 (CH2), immunoglobulin heavy chain constant domain 3 (CH3), and optionally, immunoglobulin heavy chain constant domain 4 (CH4) from the amino terminus to the carboxyl terminus. Heavy chains are classified as mu (μ), delta (Δ), gamma (γ), alpha (α), and epsilon (ε), and antibody isotypes are defined as IgM, IgD, IgG, IgA, and IgE, respectively. IgG class antibodies and IgA class antibodies are further divided into subclasses, namely IgG1, IgG2, IgG3, and IgG4, and IgA1 and IgA2, respectively. The heavy chains of IgG, IgA, and IgD antibodies have three domains (CH1, CH2, and CH3), while the heavy chains of IgM and IgE antibodies have four domains (CH1, CH2, CH3, and CH4). The constant domain of the immunoglobulin heavy chain can originate from any immunoglobulin isotype, including its subtypes. The antibody chains are linked via interpolypeptide disulfide bonds between the CL domain and the CH1 domain (i.e., between the light chain and the heavy chain), and between the hinge regions of the antibody heavy chain.

[0102] In human antibodies, CH1 refers to the region containing the amino acid sequence at positions 118 to 215 of the EU index. A highly flexible amino acid region called the "hinge region" exists between CH1 and CH2. CH2 represents the region containing the amino acid sequence at positions 231 to 340 of the EU index, and CH3 represents the region containing the amino acid sequence at positions 341 to 446 of the EU index.

[0103] "CL" represents the constant region of the light chain. In human antibodies, for the κ chain, CL represents the region containing the amino acid sequence from position 108 to 214 of the EU index. In the λ chain, CL represents the region containing the amino acid sequence from position 108 to 215.

[0104] Both the EU index and the AHo numbering scheme (Honegger A. and Plueckthun AJ Mol Biol. 2001 Jun 8:309(3):657-70) in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991) can be used in the present invention. The amino acid positions, complementarity-determining regions (CDRs), and framework regions (FRs) of a given antibody can be identified using either system. For example, the EU heavy chain positions 39, 44, 183, 356, 357, 360, 370, 392, 399, and 409 are identical to the AHo heavy chain positions 46, 51, 230, 484, 485, 491, 501, 528, 535, and 551, respectively.

[0105] In one embodiment, the present invention relates to an antigen-binding protein that specifically binds to and stimulates human CD40 (SEQ ID NO: 1), and comprises a light chain variable region (VL) and a heavy chain variable region (VH). VL is Sequence IDs 58, 59, and 60, respectively; Sequence IDs 64, 65, and 66, respectively; Sequence numbers 70, 71, and 72, respectively; Sequence IDs 76, 77, and 78, respectively; Sequence IDs 82, 83, and 84, respectively; Sequence IDs 88, 89, and 90, respectively; Sequence numbers 94, 95, and 96, respectively; Sequence IDs 100, 101, and 102, respectively; Sequence IDs 106, 107, and 108, respectively; Sequence numbers 112, 113, and 114, respectively; Sequence IDs 118, 119, and 120, respectively; Sequence IDs 124, 125, and 126, respectively; and Sequence numbers 130, 131, and 132, respectively. It includes CDRL1, CDRL2, and CDRL3 selected from the group consisting of; and VH is Sequence numbers 136, 137, and 138, respectively; Sequence IDs 142, 143, and 144, respectively; Sequence IDs 148, 149, and 150, respectively; Sequence IDs 154, 155, and 156, respectively; Sequence IDs 160, 161, and 162, respectively; Sequence IDs 166, 167, and 168, respectively; Sequence IDs 172, 173, and 174, respectively; Sequence IDs 178, 179, and 180, respectively; Sequence IDs 184, 185, and 186, respectively; Sequence numbers 190, 191, and 192, respectively; Sequence IDs 196, 197, and 198, respectively; Sequence IDs 202, 203, and 204, respectively; and Sequence IDs 208, 209, and 210, respectively. It includes CDRH1, CDRH2, and CDRH3 selected from the group consisting of the following.

[0106] In one embodiment, VL and VH are, a) VL comprising CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NO: 58, 59, and 60 respectively; and VH comprising CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NO: 136, 137, and 138 respectively; b) VL comprising CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NO: 64, 65, and 66 respectively; and VH comprising CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NO: 142, 143, and 144 respectively; c) VL comprising CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NO: 70, 71, and 72 respectively; and VH comprising CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NO: 148, 149, and 150 respectively; d) VL comprising CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NO: 76, 77, and 6780 respectively; and VH comprising CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NO: 154, 155, and 156 respectively; e) VL comprising CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NO: 82, 83, and 84 respectively; and VH comprising CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NO: 160, 161, and 162 respectively; f) VL comprising CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NO: 88, 89, and 90 respectively; and VH comprising CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NO: 166, 167, and 168 respectively; g) VL comprising CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NO: 94, 95, and 96 respectively; and VH comprising CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NO: 172, 173, and 174 respectively; h) VL containing CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 100, 101, and 102, respectively; and VH containing CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 178, 179, and 180, respectively; i) VL containing CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 106, 107, and 108, respectively; and VH containing CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 184, 185, and 186, respectively; j) VL containing CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 112, 113, and 114, respectively; and VH containing CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 190, 191, and 192, respectively; k) VL containing CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 118, 119, and 120, respectively; and VH containing CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 196, 197, and 198, respectively; l) VL containing CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 124, 125, and 126, respectively; and VH containing CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 202, 203, and 204, respectively; and m) VL containing CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 130, 131, and 132, respectively; and VH containing CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 208, 209, and 210, respectively. It is selected from the group consisting of the following.

[0107] In one embodiment, VL contains an amino acid sequence selected from the group consisting of SEQ ID NOs: 5, 9, 13, 17, 21, 25, 29, 33, 37, 41, 45, 49, and 53; and VH contains an amino acid sequence selected from the group consisting of SEQ ID NOs: 6, 10, 14, 18, 22, 26, 30, 34, 38, 42, 46, 50, and 54.

[0108] In one embodiment, VL and VH are a) VL containing SEQ ID NO. 5 and VH containing SEQ ID NO. 6; b) VL containing SEQ ID NO. 9 and VH containing SEQ ID NO. 10; c) VL containing SEQ ID NO. 13 and VH containing SEQ ID NO. 14; d) VL containing SEQ ID NO: 17 and VH containing SEQ ID NO: 18; e) VL containing Sequence ID 21 and VH containing Sequence ID 22; f) VL containing SEQ ID NO. 25 and VH containing SEQ ID NO. 26; g) VL containing SEQ ID NO. 29 and VH containing SEQ ID NO. 30; h) VL containing SEQ ID NO. 33 and VH containing SEQ ID NO. 34; i) VL containing SEQ ID NO: 37 and VH containing SEQ ID NO: 38; j) VL containing SEQ ID NO: 41 and VH containing SEQ ID NO: 42; k) VL containing SEQ ID NO: 45 and VH containing SEQ ID NO: 46; l) VL containing SEQ ID NO: 49 and VH containing SEQ ID NO: 50; and m) VL containing SEQ ID NO: 53 and VH containing SEQ ID NO: 54 It is selected from the group consisting of the following.

[0109] In one embodiment, the antigen-binding protein further includes a second antigen-binding moiety that specifically binds to a second antigen.

[0110] In one embodiment, the second antigen is a tumor-associated antigen (TAA).

[0111] The "antibody construct" according to the present invention is a fragment of a full-length antibody such as VH, VHH, VL, (s)dAb, Fv, Fd, Fab, Fab', F(ab')2, or "rIgG" ("half-antibody"). The antibody constructs according to the present invention may also be modified fragments of antibodies, also called antibody variants, such as scFv, di-scFv or bi(s)-scFv, scFv-Fc, scFv-zipper, scFab, Fab2, Fab3, diabody, single-stranded diabody, tandem diabody (Tandab's), tandem di-scFv, tandem tri-scFv, "minibodies" exemplified by structures such as (VH-VL-CH3)2, (scFv-CH3)2, ((scFv)2-CH3+CH3), ((scFv)2-CH3) or (scFv-CH3-scFv)2, multibodies, such as triabodies or tetrabodies, and single-domain antibodies, such as nanobodies or single-variable-domain antibodies containing only one variable domain which may be VHH, VH, or VL, that specifically binds to an antigen or epitope independently of other V regions or domains. Furthermore, the definition of "antibody construct" includes multispecific molecules that incorporate multiple types of antibodies and antibody constructs, such as IgG-linked scFv containing an IgG-linked Fab fragment or IgG-scFv containing IgG-Fab. In one embodiment, scFv linked to an immunoglobulin heavy chain is called a "heavy chain fusion protein." In another embodiment, a VH-CH1 polypeptide linked to an immunoglobulin heavy chain is called a "heavy chain fusion protein."

[0112] A "binding domain," or "antigen-binding domain," may typically include both an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH); however, it does not necessarily have to include both. An Fd fragment, for example, may have two VH regions and often retain the antigen-binding function of a portion of an intact antigen-binding domain. Further examples of antibody fragments, antibody variants, or binding domains include (1) Fab fragments, which are monovalent fragments having VL, VH, CL, and CH1 domains; (2) F(ab')2 fragments, which are bivalent fragments having two Fab fragments linked by disulfide crosslinks in a hinge region; (3) Fd fragments having two VH and CH1 domains; (4) Fv fragments having VL and VH domains in one arm of the antibody; (5) dAb fragments having a VH domain (Ward et al., (1989) Nature 341:544-546); (6) isolated complementarity-determining regions (CDRs); and (7) single-stranded Fv (scFv), the latter of which is preferred (e.g., those derived from scFV libraries). Examples of embodiments of the antibody construct according to the present invention are described, for example, in International Publication No. 00 / 006605, International Publication No. 2005 / 040220, International Publication No. 2008 / 119567, International Publication No. 2010 / 037838, International Publication No. 2013 / 026837, International Publication No. 2013 / 026833, U.S. Patent Application Publication No. 2014 / 0308285, U.S. Patent Application Publication No. 2014 / 0302037, International Publication No. 2014 / 144722, International Publication No. 2014 / 151910, and International Publication No. 2015 / 048272.

[0113] Furthermore, the definition of the term "antibody construct" includes monovalent, divalent, and polyvalent / multivalent constructs, and therefore monospecific constructs that specifically bind to only one antigenic structure, as well as bispecific and multispecific constructs that specifically bind to two or more antigenic structures, e.g., two, three or more, through different binding domains. In addition, the definition of the term "antibody construct" includes molecules consisting of only one polypeptide chain and molecules consisting of two or more polypeptide chains that may be identical (homodimer, homotrimer, or homooligomer) or different (heterodimer, heterotrimer, or heterooligomer). Examples of the antibodies and variants or derivatives identified above are described, in particular, in Harlow and Lane, Antibodies: A Laboratory Manual, CSHL Press (1988) and Using Antibodies: A Laboratory Manual, CSHL Press (1999), Kontermann and Dubel, Antibody Engineering, Springer, 2nd ed. 2010, and Little, Recombinant Antibodies for Immunotherapy, Cambridge University Press 2009.

[0114] The antibody construct of the present invention is preferably an “in vitro antibody construct.” This term refers to an antibody construct as defined above, in which all or part of the variable region (e.g., at least one CDR) is produced by any other method that allows for the selection of non-immune cells, e.g., in vitro phage display, protein chip, or antigen-binding ability to test candidate sequences. Therefore, this term preferably excludes sequences produced solely by genomic rearrangement in animal immune cells. A “recombinant antibody” is an antibody produced using recombinant DNA technology or genetic engineering.

[0115] In one aspect, the present invention is (i) A first antibody comprising two light chains and two heavy chains, The light chain comprises a first variable region (VL1) and a light chain constant region (CL); The heavy chain comprises a first heavy chain variable region (VH1) and the CH1, hinge, CH2, and CH3 regions; A first antibody in which the heavy chain contains at least one amino acid substitution resulting in a reduced binding affinity of the heavy chain to the human Fc gamma RI receptor compared to an unsubstituted heavy chain; and (ii) an scFv comprising a second light chain variable region (VL2) and a second heavy chain variable region (VH2) of a second antibody, wherein VL2 and VH2 are linked via a first peptide linker, scFv comprises scFv fused to each carboxyl terminus of the heavy chain via a second peptide linker at its amino terminus, such that a heavy chain fusion protein is formed; and This invention relates to a multispecific antibody construct in which the first antibody specifically binds to and stimulates human CD40 (SEQ ID NO: 1), and scFv specifically binds to human mesoserine (MSLN) (SEQ ID NO: 2).

[0116] In one embodiment, a multispecific antibody construct specifically stimulates CD40 in an MSLN-dependent manner.

[0117] In one embodiment, a multispecific antibody construct includes mutations that restrict Fc receptor binding, thereby reducing Fc receptor-dependent CD40 agonism.

[0118] In one embodiment, the two light chains are identical, and the two heavy chain fusion proteins are identical.

[0119] In one embodiment, VL1 is Sequence IDs 58, 59, and 60, respectively; Sequence IDs 64, 65, and 66, respectively; Sequence numbers 70, 71, and 72, respectively; Sequence IDs 76, 77, and 78, respectively; Sequence IDs 82, 83, and 84, respectively; Sequence IDs 88, 89, and 90, respectively; Sequence numbers 94, 95, and 96, respectively; Sequence IDs 100, 101, and 102, respectively; Sequence IDs 106, 107, and 108, respectively; Sequence numbers 112, 113, and 114, respectively; Sequence IDs 118, 119, and 120, respectively; Sequence IDs 124, 125, and 126, respectively; and Sequence numbers 130, 131, and 132, respectively. It includes CDRL1, CDRL2, and CDRL3 selected from the group consisting of; VH1 is Sequence numbers 136, 137, and 138, respectively; Sequence IDs 142, 143, and 144, respectively; Sequence IDs 148, 149, and 150, respectively; Sequence IDs 154, 155, and 156, respectively; Sequence IDs 160, 161, and 162, respectively; Sequence IDs 166, 167, and 168, respectively; Sequence IDs 172, 173, and 174, respectively; Sequence IDs 178, 179, and 180, respectively; Sequence IDs 184, 185, and 186, respectively; Sequence numbers 190, 191, and 192, respectively; Sequence IDs 196, 197, and 198, respectively; Sequence IDs 202, 203, and 204, respectively; and Sequence IDs 208, 209, and 210, respectively. It includes CDRH1, CDRH2, and CDRH3 selected from the group consisting of; VL2 is Sequence IDs 230, 231, and 232, respectively; Sequence IDs 236, 237, and 238, respectively; Sequence IDs 242, 243, and 244, respectively; and Sequence IDs 248, 249, and 250, respectively; It includes CDRL1, CDRL2, and CDRL3 selected from the group consisting of; Furthermore, VH2 is Sequence IDs 254, 255, and 256, respectively; Sequence IDs 260, 261, and 262, respectively; Sequence IDs 266, 267, and 268, respectively; and Sequence IDs 272, 273, and 274, respectively. It includes CDRH1, CDRH2, and CDRH3 selected from the group consisting of the following.

[0120] In one embodiment, 1) VL1 and VH1 are, a) VL1 containing CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs. 58, 59, and 60, respectively; and VH1 containing CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs. 136, 137, and 138, respectively; b) VL1 containing CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs. 64, 65, and 66, respectively; and VH1 containing CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs. 142, 143, and 144, respectively; c) VL1 containing CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs. 70, 71, and 72, respectively; and VH1 containing CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs. 148, 149, and 150, respectively; d) VL1 containing CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs. 76, 77, and 6780, respectively; and VH1 containing CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs. 154, 155, and 156, respectively; e) VL1 containing CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs. 82, 83, and 84, respectively; and VH1 containing CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs. 160, 161, and 162, respectively; f) VL1 containing CDRL1, CDRL2, and CDRL3 selected from the group consisting of sequence numbers 88, 89, and 90, respectively; and VH1 containing CDRH1, CDRH2, and CDRH3 selected from the group consisting of sequence numbers 166, 167, and 168, respectively; g) VL1 containing CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs. 94, 95, and 96, respectively; and VH1 containing CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs. 172, 173, and 174, respectively; h) VL1 containing CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 100, 101, and 102, respectively; and VH1 containing CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 178, 179, and 180, respectively; i) VL1 containing CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 106, 107, and 108, respectively; and VH1 containing CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 184, 185, and 186, respectively; j) VL1 containing CDRL1, CDRL2, and CDRL3 selected from the group consisting of sequence numbers 112, 113, and 114, respectively; and VH1 containing CDRH1, CDRH2, and CDRH3 selected from the group consisting of sequence numbers 190, 191, and 192, respectively; k) VL1 containing CDRL1, CDRL2, and CDRL3 selected from the group consisting of sequence numbers 118, 119, and 120, respectively; and VH1 containing CDRH1, CDRH2, and CDRH3 selected from the group consisting of sequence numbers 196, 197, and 198, respectively; l) VL1 containing CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 124, 125, and 126, respectively; and VH1 containing CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 202, 203, and 204, respectively; and m) VL1 containing CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 130, 131, and 132, respectively; and VH1 containing CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 208, 209, and 210, respectively. Selected from the group consisting of; and 2) VL2 and VH2 are, a) VL2 containing CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs. 230, 231, and 232, respectively; and VH2 containing CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs. 254, 255, and 256, respectively; b) VL2 containing CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs. 236, 237, and 238, respectively; and VH2 containing CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs. 260, 261, and 262, respectively; c) VL2 containing CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs. 242, 243, and 244, respectively; and VH2 containing CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs. 266, 267, and 268, respectively; and d) VL2 containing CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs. 248, 249, and 250, respectively; and VH2 containing CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs. 272, 273, and 274, respectively. It is selected from the group consisting of the following.

[0121] In one embodiment, VL1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 5, 9, 13, 17, 21, 25, 29, 33, 37, 41, 45, 49, and 53; VH1 contains an amino acid sequence selected from the group consisting of SEQ ID NOs: 6, 10, 14, 18, 22, 26, 30, 34, 38, 42, 46, 50, and 54; VL2 contains an amino acid sequence selected from the group consisting of SEQ ID NOs: 213, 217, 221, and 225; VH2 contains an amino acid sequence selected from the group consisting of SEQ ID NOs: 214, 218, 222, and 226.

[0122] In one embodiment, 1) VL1 and VH1 are, a) VL1 containing Sequence ID 5 and VH1 containing Sequence ID 6; b) VL1 containing SEQ ID NO. 9 and VH1 containing SEQ ID NO. 10; c) VL1 containing SEQ ID NO. 13 and VH1 containing SEQ ID NO. 14; d) VL1 containing SEQ ID NO: 17 and VH1 containing SEQ ID NO: 18; e) VL1 containing Sequence ID 21 and VH1 containing Sequence ID 22; f) VL1 containing sequence number 25 and VH1 containing sequence number 26; g) VL1 containing SEQ ID NO. 29 and VH1 containing SEQ ID NO. 30; h) VL1 containing sequence number 33 and VH1 containing sequence number 34; i) VL1 containing sequence number 37 and VH1 containing sequence number 38; j) VL1 containing sequence number 41 and VH1 containing sequence number 42; k) VL1 containing SEQ ID NO: 45 and VH1 containing SEQ ID NO: 46; l) VL1 containing SEQ ID NO: 49 and VH1 containing SEQ ID NO: 50; and m) VL1 containing SEQ ID NO: 53 and VH1 containing SEQ ID NO: 54 Selected from the group consisting of; and 2) VL2 and VH2 are, a) VL2 containing Sequence ID 213 and VH2 containing Sequence ID 214; b) VL2 containing Sequence ID 217 and VH2 containing Sequence ID 218; c) VL2 containing Sequence ID 221 and VH2 containing Sequence ID 222; and d) VL2 containing SEQ ID NO: 225 and VH2 containing SEQ ID NO: 226 It is selected from the group consisting of the following.

[0123] In one embodiment, The light chain contains a sequence selected from the group consisting of SEQ ID NOs: 286, 290, 294, 298, 302, 306, 310, 314, 318, 322, 326, 330, 336, 342, 346, 350, 354, 358, 362, 366, 370, 374, and 378; and The heavy chain fusion protein contains a sequence selected from the group consisting of SEQ ID NOs: 285, 289, 293, 297, 301, 305, 309, 313, 317, 321, 325, 329, 333, 337, 341, 345, 349, 353, 357, 361, 365, 369, 373, and 377.

[0124] In one embodiment, the light chain and heavy chain fusion protein is Sequence IDs 286 and 285, respectively; Sequence IDs 290 and 289, respectively; Sequence IDs 294 and 293, respectively; Sequence IDs 298 and 297, respectively; Sequence IDs 302 and 301, respectively; Sequence IDs 306 and 305, respectively; Sequence IDs 310 and 309, respectively; Sequence IDs 314 and 313, respectively; Sequence IDs 318 and 317, respectively; Sequence IDs 322 and 321, respectively; Sequence IDs 326 and 325, respectively; Sequence IDs 330 and 329, respectively; Sequence IDs 334 and 333, respectively; Sequence IDs 338 and 337, respectively; Sequence IDs 342 and 341, respectively; Sequence IDs 346 and 345, respectively; Sequence IDs 350 and 349, respectively; Sequence IDs 354 and 353, respectively; Sequence IDs 358 and 357, respectively; Sequence IDs 362 and 361, respectively; Sequence IDs 366 and 365, respectively; Sequence IDs 370 and 369, respectively; Sequence ID 374 and Sequence ID 373, respectively; and Sequence IDs 378 and 377, respectively. It contains a polypeptide comprising an amino acid sequence selected from the group consisting of the following.

[0125] In another embodiment, the present invention is (i) A first antibody comprising two light chains and two heavy chains, The light chain comprises a first variable region (VL1) and a light chain constant region (CL); The heavy chain comprises a first heavy chain variable region (VH1) and the CH1, hinge, CH2, and CH3 regions; A first antibody in which the heavy chain contains at least one amino acid substitution resulting in a reduced binding affinity of the heavy chain to the human Fc gamma RI receptor compared to an unsubstituted heavy chain; and (ii) an scFv comprising a second light chain variable region (VL2) and a second heavy chain variable region (VH2) of a second antibody, wherein VL2 and VH2 are linked via a first peptide linker, scFv comprises scFv fused to each carboxyl terminus of the heavy chain via a second peptide linker at its amino terminus, such that a heavy chain fusion protein is formed; and This invention relates to a multispecific antibody construct in which the first antibody specifically binds to human mesoserine (MSLN) (SEQ ID NO: 2), and scFv specifically binds to and stimulates human CD40 (SEQ ID NO: 1).

[0126] In one embodiment, a multispecific antibody construct specifically stimulates CD40 in an MSLN-dependent manner.

[0127] In one embodiment, a multispecific antibody construct includes mutations that restrict Fc receptor binding, thereby reducing Fc receptor-dependent CD40 agonism.

[0128] In one embodiment, the two light chains are identical, and the two heavy chain fusion proteins are identical.

[0129] In one embodiment, VL1 is Sequence IDs 230, 231, and 232, respectively; Sequence IDs 236, 237, and 238, respectively; Sequence IDs 242, 243, and 244, respectively; and Sequence IDs 248, 249, and 250, respectively. It includes CDRL1, CDRL2, and CDRL3 selected from the group consisting of; VH1 is Sequence IDs 254, 255, and 256, respectively; Sequence IDs 260, 261, and 262, respectively; Sequence IDs 266, 267, and 268, respectively; and Sequence IDs 272, 273, and 274, respectively. It includes CDRH1, CDRH2, and CDRH3 selected from the group consisting of; VL2 is Sequence IDs 58, 59, and 60, respectively; Sequence IDs 64, 65, and 66, respectively; Sequence numbers 70, 71, and 72, respectively; Sequence IDs 76, 77, and 78, respectively; Sequence IDs 82, 83, and 84, respectively; Sequence IDs 88, 89, and 90, respectively; Sequence numbers 94, 95, and 96, respectively; Sequence IDs 100, 101, and 102, respectively; Sequence IDs 106, 107, and 108, respectively; Sequence numbers 112, 113, and 114, respectively; Sequence IDs 118, 119, and 120, respectively; Sequence IDs 124, 125, and 126, respectively; and Sequence numbers 130, 131, and 132, respectively. It includes CDRL1, CDRL2, and CDRL3 selected from the group consisting of; and VH2 is Sequence numbers 136, 137, and 138, respectively; Sequence IDs 142, 143, and 144, respectively; Sequence IDs 148, 149, and 150, respectively; Sequence IDs 154, 155, and 156, respectively; Sequence IDs 160, 161, and 162, respectively; Sequence IDs 166, 167, and 168, respectively; Sequence IDs 172, 173, and 174, respectively; Sequence IDs 178, 179, and 180, respectively; Sequence IDs 184, 185, and 186, respectively; Sequence numbers 190, 191, and 192, respectively; Sequence IDs 196, 197, and 198, respectively; Sequence IDs 202, 203, and 204, respectively; and Sequence IDs 208, 209, and 210, respectively. It includes CDRH1, CDRH2, and CDRH3 selected from the group consisting of the following.

[0130] In one embodiment, 1) VL1 and VH1 are, a) VL1 containing CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs. 230, 231, and 232, respectively; and VH1 containing CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs. 254, 255, and 256, respectively; b) VL1 containing CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs. 236, 237, and 238, respectively; and VH1 containing CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs. 260, 261, and 262, respectively; c) VL1 containing CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs. 242, 243, and 244, respectively; and VH1 containing CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs. 266, 267, and 268, respectively; and d) VL1 containing CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs. 248, 249, and 250, respectively; and VH1 containing CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs. 272, 273, and 274, respectively. Selected from the group consisting of; and 2) VL2 and VH2 are, a) VL2 containing CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs. 58, 59, and 60, respectively; and VH2 containing CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs. 136, 137, and 138, respectively; b) VL2 containing CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs. 64, 65, and 66, respectively; and VH2 containing CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs. 142, 143, and 144, respectively; c) VL2 containing CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs. 70, 71, and 72, respectively; and VH2 containing CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs. 148, 149, and 150, respectively; d) VL2 containing CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs. 76, 77, and 6780, respectively; and VH2 containing CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs. 154, 155, and 156, respectively; e) VL2 containing CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs. 82, 83, and 84, respectively; and VH2 containing CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs. 160, 161, and 162, respectively; f) VL2 containing CDRL1, CDRL2, and CDRL3 selected from the group consisting of sequence numbers 88, 89, and 90, respectively; and VH2 containing CDRH1, CDRH2, and CDRH3 selected from the group consisting of sequence numbers 166, 167, and 168, respectively; g) VL2 containing CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs. 94, 95, and 96, respectively; and VH2 containing CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs. 172, 173, and 174, respectively; h) VL2 containing CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 100, 101, and 102, respectively; and VH2 containing CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 178, 179, and 180, respectively; i) VL2 containing CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 106, 107, and 108, respectively; and VH2 containing CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 184, 185, and 186, respectively; j) VL2 containing CDRL1, CDRL2, and CDRL3 selected from the group consisting of sequence numbers 112, 113, and 114, respectively; and VH2 containing CDRH1, CDRH2, and CDRH3 selected from the group consisting of sequence numbers 190, 191, and 192, respectively; k) VL2 containing CDRL1, CDRL2, and CDRL3 selected from the group consisting of sequence numbers 118, 119, and 120, respectively; and VH2 containing CDRH1, CDRH2, and CDRH3 selected from the group consisting of sequence numbers 196, 197, and 198, respectively; l) VL2 containing CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 124, 125, and 126, respectively; and VH2 containing CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 202, 203, and 204, respectively; and m) VL2 containing CDRL1, CDRL2, and CDRL3 selected from the group consisting of sequence numbers 130, 131, and 132, respectively; and VH2 containing CDRH1, CDRH2, and CDRH3 selected from the group consisting of sequence numbers 208, 209, and 210, respectively.

[0131] In one embodiment, VL1 contains an amino acid sequence selected from the group consisting of SEQ ID NOs: 213, 217, 221, and 225; VH1 contains an amino acid sequence selected from the group consisting of SEQ ID NOs: 214, 218, 222, and 226; VL2 contains an amino acid sequence selected from the group consisting of SEQ ID NOs: 5, 9, 13, 17, 21, 25, 29, 33, 37, 41, 45, 49, and 53; and VH2 contains an amino acid sequence selected from the group consisting of SEQ ID NOs: 6, 10, 14, 18, 22, 26, 30, 34, 38, 42, 46, 50, and 54.

[0132] In one embodiment, 1) VL1 and VH1 are, a) VL1 containing sequence number 213 and VH1 containing sequence number 214; b) VL1 containing sequence number 217 and VH1 containing sequence number 218; c) VL1 containing Sequence ID 221 and VH1 containing Sequence ID 222; and d) VL1 containing SEQ ID NO: 225 and VH1 containing SEQ ID NO: 226 Selected from the group consisting of; and 2) VL2 and VH2 are, a) VL2 containing Sequence ID 5 and VH2 containing Sequence ID 6; b) VL2 containing SEQ ID NO. 9 and VH2 containing SEQ ID NO. 10; c) VL2 containing Sequence ID 13 and VH2 containing Sequence ID 14; d) VL2 containing SEQ ID NO: 17 and VH2 containing SEQ ID NO: 18; e) VL2 containing Sequence ID 21 and VH2 containing Sequence ID 22; f) VL2 containing Sequence ID 25 and VH2 containing Sequence ID 26; g) VL2 containing Sequence ID 29 and VH2 containing Sequence ID 30; h) VL2 containing SEQ ID NO. 33 and VH2 containing SEQ ID NO. 34; i) VL2 containing Sequence ID 37 and VH2 containing Sequence ID 38; j) VL2 containing Sequence ID 41 and VH2 containing Sequence ID 42; k) VL2 containing SEQ ID NO: 45 and VH2 containing SEQ ID NO: 46; l) VL2 containing sequence number 49 and VH2 containing sequence number 50; and m) VL2 containing SEQ ID NO: 53 and VH2 containing SEQ ID NO: 54 It is selected from the group consisting of the following.

[0133] In one embodiment, the CL of the light chain is selected from the group consisting of SEQ ID NOs: 883 and 884.

[0134] In one embodiment, the heavy chain CH1-hinge-CH2-CH3 is selected from the group consisting of sequence numbers 885 and 886.

[0135] In one embodiment, the first peptide linker is selected from the group consisting of SEQ ID NOs: 888 to 893.

[0136] In one embodiment, the second peptide linker is selected from the group consisting of SEQ ID NOs: 887 to 893.

[0137] In one embodiment, the first peptide linker includes SEQ ID NO: 889, and the second peptide linker includes SEQ ID NO: 887.

[0138] In one embodiment, The light chain contains a sequence selected from the group consisting of SEQ ID NOs: 382, ​​386, 390, 394, 398, 402, 406, 410, 414, 418, 422, 426, 430, 434, 438, 442, 446, and 450; and The heavy chain fusion protein contains a sequence selected from the group consisting of SEQ ID NOs: 381, 385, 389, 393, 397, 401, 405, 409, 413, 417, 421, 425, 429, 433, 437, 441, 445, and 449.

[0139] In one embodiment, the light chain and heavy chain fusion protein is Sequence IDs 382 and 381, respectively; Sequence IDs 386 and 385, respectively; Sequence IDs 390 and 389, respectively; Sequence IDs 394 and 393, respectively; Sequence IDs 398 and 397, respectively; Sequence IDs 402 and 401, respectively; Sequence IDs 406 and 405, respectively; Sequence IDs 410 and 409, respectively; Sequence IDs 414 and 413, respectively; Sequence IDs 418 and 417, respectively; Sequence IDs 422 and 421, respectively; Sequence IDs 426 and 425, respectively; Sequence IDs 430 and 429, respectively; Sequence IDs 434 and 433, respectively; Sequence IDs 438 and 437, respectively; Sequence IDs 442 and 441, respectively; Sequence IDs 446 and 445, respectively; and Sequence IDs 450 and 449, respectively. It contains a polypeptide comprising an amino acid sequence selected from the group consisting of the following.

[0140] In one embodiment, the CL of the light chain is selected from the group consisting of SEQ ID NOs: 883 and 884.

[0141] In one embodiment, the heavy chain CH1-hinge-CH2-CH3 is selected from the group consisting of sequence numbers 885 and 886.

[0142] In one embodiment, the first peptide linker is selected from the group consisting of SEQ ID NOs: 888 to 893.

[0143] In one embodiment, the second peptide linker is selected from the group consisting of SEQ ID NOs: 887 to 893.

[0144] In one embodiment, the first peptide linker includes SEQ ID NO: 889, and the second peptide linker includes SEQ ID NO: 887.

[0145] As used herein, the term “monoclonal antibody” (mAb) or monoclonal antibody construct refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., an individual antibody that is identical except for possible naturally occurring mutations and / or post-translational modifications (e.g., isomerization, amidation) that may be present in small amounts. Monoclonal antibodies are highly specific and are induced to a single antigenic site or determinant on an antigen, in contrast to conventional (polyclonal) antibody preparations, which typically contain different antibodies induced to different determinants (or epitopes). In addition to their specificity, monoclonal antibodies are advantageous in that they are synthesized by hybridoma culture and are therefore free from contamination by other immunoglobulins. The modifier “monoclonal” indicates the characteristic of an antibody to be obtained from a substantially homogeneous population of antibodies and should not be interpreted as requiring antibody production by any particular method.

[0146] Any technique that yields antibodies produced by continuous cell line culture can be used to prepare monoclonal antibodies. For example, the monoclonal antibodies used may be produced by the hybridoma method first described by Koehler et al., Nature, 256:495 (1975), or by the recombinant DNA method (see, e.g., U.S. Patent No. 4,816,567). Further examples of techniques for producing human monoclonal antibodies include trioma techniques, human B-cell hybridoma techniques (Kozbor, Immunology Today 4 (1983), 72), and EBV-hybridoma techniques (Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc. (1985), 77-96).

[0147] Next, hybridomas can be screened using standard methods such as enzyme-linked immunosorbent assay (ELISA) and surface plasmon resonance (BIACORE®) analysis to identify one or more hybridomas that produce antibodies that specifically bind to a particular antigen. Any form of the relevant antigen can be used as an immunogen, for example, recombinant antigen, a naturally occurring form, any variant or fragment thereof, and its antigenic peptide. Surface plasmon resonance, as employed in the BIAcore system, can be used to enhance the efficiency of phage antibody binding to epitopes of target antigens such as MSLN or CD40 (Schier, Human Antibodies Hybridomas 7(1996), 97-105; Malmborg, J. Immunol. Methods 183(1995), 7-13).

[0148] Another exemplary method for producing monoclonal antibodies involves screening protein expression libraries, such as phage display or ribosome display libraries. Phage display is described, for example, in Ladner et al., U.S. Patent No. 5,223,409; Smith (1985), Science 228:1315-1317; Clackson et al., Nature, 352:624-628 (1991); and Marks et al., J.Mol.Biol., 222:581-597 (1991).

[0149] In addition to using display libraries, non-human animals, such as rodents (mice, hamsters, rabbits, or rats), can be immunized using relevant antigens. In one embodiment, the non-human animal contains at least a portion of the human immunoglobulin gene. For example, a mouse strain lacking mouse antibody production can be modified using a large fragment of the human Ig (immunoglobulin) locus. Using hybridoma technology, antigen-specific monoclonal antibodies derived from genes with desired specificity can be produced and selected. See, for example, XENOMOUSE®, Green et al. (1994) Nature Genetics 7:13-21, U.S. Patent Application Publication No. 2003-0070185, International Publication No. 96 / 34096 and International Publication No. 96 / 33735.

[0150] Monoclonal antibodies can also be modified after being obtained from non-human animals using recombinant DNA techniques known in the art, such as humanization, deimmunization, and chimerization. Examples of modified antibody constructs include humanized variants of non-human antibodies, "affinity-mature" antibodies (see, e.g., Hawkins et al. J.Mol.Biol. 254, 889-896 (1992) and Lowman et al., Biochemistry 30, 10832-10837 (1991)), and antibody variants with altered effector function (see, e.g., U.S. Patent No. 5,648,260, Kontermann and Dubel (2010) and Little (2009) cited above).

[0151] In immunology, affinity maturation is the process by which B cells produce antibodies with increased affinity for an antigen during an immune response. Repeated exposure to the same antigen causes the host to produce antibodies with progressively increasing affinity. Similar to natural prototypes, in vitro affinity maturation is based on the principles of mutation and selection. In vitro affinity maturation is used without issue to optimize antibodies, antibody constructs, and antibody fragments. Random mutations within the CDR are introduced using radiation, chemical mutagens, or error-prone PCR. In addition, genetic diversity can be increased by chain shuffling. Two or three rounds of mutation and selection using display methods such as phage display typically yield antibody fragments with affinity in the low nanomolar range.

[0152] A preferred type of amino acid substitution variant of an antibody construct involves substitution of one or more hypervariable region residues of the parent antibody (e.g., a humanized antibody or a human antibody). Generally, variants selected for further development will have improved biological properties compared to the parent antibody from which they were generated. A convenient method for generating such substitution variants involves affinity maturation using phage display. Briefly, several hypervariable region sites (e.g., 6-7 sites) are mutated to produce all possible amino acid substitutions at each site. The antibody variants thus generated are presented in a monovalent form from filamentous phage particles as fusions with the M13 gene III product packaged within each particle. The phage-displayed variants are then screened for their biological activity (e.g., binding affinity) as disclosed herein. To identify candidate hypervariable region sites for modification, alanine scanning mutagenesis can be performed to identify hypervariable region residues that significantly contribute to antigen binding. Alternatively, or in addition, it may be beneficial to analyze the crystal structure of the antigen-antibody complex to identify contact sites between the binding domain and, for example, human MSLN. Such contact residues and adjacent residues are candidates for substitution by the techniques detailed herein. After generating such variants, a panel of variants may be screened as described herein, and antibodies exhibiting superior properties in one or more relevant assays may be selected for further development.

[0153] The monoclonal antibodies and antibody constructs of the present invention include, in particular, “chimeric” antibodies (immunoglobulins) in which a portion of the heavy chain and / or light chain originates from a particular species or is identical or homologous to a corresponding sequence in an antibody belonging to a particular class or subclass of antibodies, while the remainder of the chain originates from another species or is identical or homologous to a corresponding sequence in an antibody belonging to another class or subclass of antibodies and fragments of such antibodies, insofar as it exhibits the desired biological activity (U.S. Patent No. 4,816,567; Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)). The chimeric antibodies of interest as used herein include “primatized” antibodies that contain a variable domain antigen-binding sequence and a human constant region sequence derived from a non-human primate (e.g., Old World monkeys, apes, etc.). Various methods for producing chimeric antibodies are described. For example, see Morrison et al., Proc. Natl. Acad. ScL USA 81:6851, 1985; Takeda et al., Nature 314:452, 1985; Cabilly et al., U.S. Patent No. 4,816,567; Boss et al., U.S. Patent No. 4,816,397; Tanaguchi et al., European Patent No. 0171496; European Patent No. 0173494; and British Patent No. 2177096.

[0154] Antibodies, antibody constructs, antibody fragments, or antibody variants can also be modified by specific deletion of human T cell epitopes (a method called "deimmunization") by the methods disclosed in the examples of International Publication No. 98 / 52976 or International Publication No. 00 / 34317. Briefly, the heavy and light chain variable domains of antibodies can be analyzed for peptides that bind to MHC class II. These peptides correspond to potential T cell epitopes (as defined in International Publication No. 98 / 52976 and International Publication No. 00 / 34317). For the detection of potential T cell epitopes, a computer modeling technique called "peptide threading" can be applied, as described in International Publication No. 98 / 52976 and International Publication No. 00 / 34317, and additionally, motifs present in VH and VL sequences can be searched in databases of human MHC class II binding peptides. These motifs bind to any of the 18 major MHC class II DR allotypes and thus become potential T cell epitopes. Detected potential T cell epitopes can be removed by substituting a small number of amino acid residues within the variable domain, or preferably by a single amino acid substitution. Conservative substitutions are typically made. In many, though not all, amino acids common to positions in human germline antibody sequences can be used. Human germline sequences are disclosed, for example, in Tomlinson, et al. (1992), J.Mol.Biol.227:776-798; Cook, GP et al. (1995) Immunol.Today Vol.16(5):237-242; and Tomlinson et al. (1995) EMBO J.14:14:4628-4638. The V BASE catalog provides a comprehensive overview of human immunoglobulin variable region sequences (edited by Tomlinson, LA. et al., MRC Centre for Protein Engineering, Cambridge, UK). These sequences can be used as a source of human sequences, for example, for framework regions and CDRs.For example, the consensus human framework area described in U.S. Patent No. 6,300,064 can also be used.

[0155] A “humanized” antibody, antibody construct, variant, or fragment thereof (e.g., Fv, Fab, Fab', F(ab')2, or other antigen-binding subsequences of an antibody) is an antibody or immunoglobulin that is predominantly human, containing minimal sequences derived from non-human immunoglobulin. In most cases, a humanized antibody is a human immunoglobulin (recipient antibody) in which residues from the recipient’s hypervariable region (also known as the CDR) are replaced with residues from the hypervariable region of a non-human species (e.g., rodents) such as mouse, rat, hamster, or rabbit (donor antibody) that have the desired specificity, affinity, and capability. In some cases, Fv framework region (FR) residues of the human immunoglobulin are replaced with corresponding non-human residues. Furthermore, as used herein, “humanized antibody” may also contain residues not found in either the recipient antibody or the donor antibody. These modifications are made to further refine and optimize the performance of the antibody. A humanized antibody may also contain the immunoglobulin constant region (Fc), usually at least a portion of the constant region of human immunoglobulin. For further details, see Jones et al., Nature, 321:522-525 (1986); Reichmann et al., Nature, 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol., 2:593-596 (1992).

[0156] Humanized antibodies or fragments thereof can be produced by substituting sequences of Fv variable domains not directly involved in antigen binding with equivalent sequences derived from human Fv variable domains. Exemplary methods for producing humanized antibodies or fragments thereof are provided in Morrison (1985) Science 229:1202-1207; Oi et al. (1986) BioTechniques 4:214; and U.S. Patents No. 5,585,089; U.S. Patents No. 5,693,761; U.S. Patents No. 5,693,762; U.S. Patents No. 5,859,205; and U.S. Patents No. 6,407,213. These methods involve isolating, manipulating, and expressing nucleic acid sequences encoding all or part of an immunoglobulin Fv variable domain derived from at least one of the heavy or light chains. Such nucleic acids can be obtained from hybridomas and other sources that produce antibodies against a given target as described above. Next, recombinant DNA encoding a humanized antibody molecule can be cloned into a suitable expression vector.

[0157] Humanized antibodies can be produced using transgenic animals such as mice that express human heavy and light chain genes but cannot express endogenous mouse immunoglobulin heavy and light chain genes. Winter describes exemplary CDR transplantation methods that can be used for the preparation of humanized antibodies described herein (U.S. Patent No. 5,225,539). All of the CDRs of a particular human antibody may be replaced with at least a portion of non-human CDRs, or only a portion of the CDRs may be replaced with non-human CDRs. Only the number of CDRs required for the binding of the humanized antibody to a given antigen needs to be replaced.

[0158] Humanized antibodies can be optimized by introducing conservative substitutions, consensus sequence substitutions, germline substitutions, and / or reverse mutations. Such modified immunoglobulin molecules can be prepared by any of several techniques known in the art (e.g., Teng et al., Proc. Natl. Acad. Sci. USA, 80:7308-7312, 1983; Kozbor et al., Immunology Today, 4:7279, 1983; Olsson et al., Meth. Enzymol., 92:3-16, 1982; and European Patent No. 239400).

[0159] The terms “human antibody,” “human antibody construct,” and “human binding domain” include antibodies, antibody constructs, and binding domains having antibody regions such as variable regions and constant regions or domains that substantially correspond to human germline immunoglobulin sequences known in the art, including those described by Kabat et al. (1991) (cited above). The human antibody, antibody construct, or binding domain of the present invention may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by in vitro random mutagenesis or site-directed mutagenesis, or in vivo somatic mutation) for example, CDR, particularly CDR3. The human antibody, antibody construct, or binding domain may have at least one, two, three, four, five, or more positions substituted with amino acid residues not encoded by human germline immunoglobulin sequences. As used herein, the definitions of human antibody, antibody construct, and binding domain also refer to fully human antibodies that contain only the human sequence of an antibody that has been unartificially and / or genetically modified, which can be obtained by using technologies or systems such as Xenomouse.

[0160] In some embodiments, the antibody construct of the present invention is an “isolated” or “substantially pure” antibody construct. When used to describe the antibody constructs disclosed herein, “isolated” or “substantially pure” means an antibody construct identified, separated and / or recovered from components of its production environment. Preferably, the antibody construct has no or substantially no association with all other components from its production environment. Contaminating components of its production environment, such as components arising from recombinant transfected cells, are typically materials that interfere with diagnostic or therapeutic applications of polypeptides, and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. The antibody construct may, for example, account for at least about 5% by weight or at least about 50% by weight of the total protein in a given sample. Isolated proteins are understood to account for 5% to 99.9% by weight of the total protein content, depending on the environment. Polypeptides can be produced at significantly high concentrations by using an inducible promoter or a high-expression promoter so that polypeptides are produced at high concentration levels. This definition includes the production of antibody constructs in a variety of organisms and / or host cells known in the art. In preferred embodiments, the antibody construct is purified to a degree sufficient to obtain at least 15 residues of the N-terminal or internal amino acid sequence by (1) using a spinning cup sequencer, or to a degree of homogeneity by SDS-PAGE under non-reducible or reducing conditions using Coomassie blue or preferably silver staining. However, the isolated antibody construct is usually prepared by at least one purification step.

[0161] In relation to the present invention, the term "binding domain" refers to a domain that (specifically) binds to / interacts with / recognizes a target molecule (antigen), specifically a given target epitope or target site on MSLN and CD40, respectively. The structure and function of one binding domain (recognition of MSLN), and preferably the other binding domain (recognition of CD40), are based on the structure and / or function of an antibody, e.g., a full-length immunoglobulin molecule or a fully immunoglobulin molecule. According to the present invention, the binding domain is characterized by the presence of three light chain CDRs (i.e., CDRL1, CDRL2, and CDRL3 in the VL region) and / or three heavy chain CDRs (i.e., CDRH1, CDRH2, and CDRH3 in the VH region). The binding domain is envisioned to be prepared or obtained by phage display or library screening, rather than by grafting CDR sequences derived from existing (monoclonal) antibodies onto a scaffold. According to the present invention, the binding domain is in the form of one or more polypeptides. Such polypeptides may comprise a proteinaceous portion and a non-proteinaceous portion (e.g., a chemical linker or a chemical crosslinking agent such as glutaraldehyde). Proteins (including their fragments, preferably biologically active fragments, and peptides, which typically have fewer than 30 amino acids) contain two or more amino acids linked to each other via covalent peptide bonds (resulting in a chain of amino acids). As used herein, the term “polypeptide” usually refers to a group of molecules consisting of more than 30 amino acids. Polypeptides may further form polymers, such as dimers, trimers, and higher-order oligomers, i.e., polymers consisting of two or more polypeptide molecules. The polypeptide molecules forming such dimers, trimers, etc., may be identical or different. The corresponding higher-order structures of such polymers are therefore referred to as homo- or heterodimers, homo- or heterotrimers, etc. An example of a heteropolymer is an antibody molecule, in its naturally occurring form, consisting of two identical polypeptide light chains and two identical polypeptide heavy chains.The terms “peptide,” “polypeptide,” and “protein” also refer to naturally modified peptides / polypeptides / proteins that have undergone post-translational modifications such as glycosylation, acetylation, and phosphorylation. Where used herein, “peptide,” “polypeptide,” or “protein” may also refer to chemically modified substances such as pegylation. Such modifications are well known in the art and are described below herein.

[0162] Preferably, the binding domain that binds to MSLN and / or the binding domain that binds to CD40 is a human binding domain. Antibodies and antibody constructs containing at least one human binding domain avoid some of the problems associated with antibodies or antibody constructs that have non-human variable and / or constant regions, such as those from rodents (e.g., mice, rats, hamsters, or rabbits). The presence of such rodent-derived proteins may result in rapid clearance of the antibody or antibody construct, or may trigger an immune response by the patient to the antibody or antibody construct. To avoid the use of rodent-derived antibodies or antibody constructs, human or fully human antibodies / antibody constructs can be generated by introducing human antibody function into rodents so that the rodents produce fully human antibodies.

[0163] The ability of YAC to clone and reconstruct megabase-sized human loci and to introduce them into mouse germline cells provides a powerful method for elucidating the functional elements of very large or coarsely mapped loci and for generating useful models of human diseases. Furthermore, using such techniques to replace mouse loci with their human equivalents could provide unique insights into the expression and regulation of human gene products during development, their transduction to other systems, and their involvement in disease induction and progression.

[0164] A key practical application of such strategies is the "humanization" of the mouse humoral immune system. Introducing human Ig loci into mice with inactivated endogenous immunoglobulin (Ig) genes provides an opportunity to study the mechanisms underlying programmed antibody expression and construction, as well as their roles in B cell development. Furthermore, such strategies could provide an ideal source for producing fully human monoclonal antibodies (mAbs), a crucial milestone in realizing the potential of antibody therapy in human diseases. Fully human antibodies or antibody constructs are expected to minimize the immunogenicity and allergic reactions inherent in mouse mAbs or mouse-derived mAbs, thereby increasing the efficacy and safety of the administered antibody / antibody construct. The use of fully human antibodies or antibody constructs is expected to offer significant advantages in the treatment of chronic and recurrent human diseases requiring repeated administration of compounds, such as inflammation, autoimmunity, and cancer.

[0165] One approach to achieving this goal involves manipulating mouse strains lacking mouse antibody production using large fragments of the human Ig locus. This is based on the prediction that such mice would produce a broad repertoire of human antibodies without producing mouse antibodies. Large human Ig fragments are thought to retain broad diversity of variable genes as well as appropriate regulation of antibody production and expression. By utilizing mouse mechanisms for antibody diversification and selection, and for the lack of immune tolerance to human proteins, the human antibody repertoire reproduced in these mouse strains should produce high-affinity antibodies against any target antigen, including human antigens. Using hybridoma technology, antigen-specific human mAbs with desired specificity can be easily generated and selected. This general strategy was demonstrated in connection with the generation of the first XenoMouse mouse strain (see Green et al. Nature Genetics 7:13-21 (1994)). This XenoMouse strain was engineered using yeast artificial chromosomes (YACs) containing germline arrangement fragments of 245kb and 190kb sizes, respectively, of the human heavy chain locus and kappa light chain locus, containing the core sequences of the variable and constant regions. These human Ig-containing YACs proved compatible with the mouse strain in terms of both antibody rearrangement and expression, and were capable of replacing inactivated mouse Ig genes. This was demonstrated by their ability to induce B cell development to produce an adult-like human repertoire of fully human antibodies and antigen-specific human mAbs. These results also suggested that the introduction of a large portion of the human Ig locus, containing numerous V genes, additional regulatory elements, and the human Ig constant region, could reproduce a substantially complete repertoire characterized by the human humoral response to infection and immunization. More recently, building upon the work of Green et al., the introduction of megabase-sized germline arrangement YAC fragments of the human heavy chain locus and kappa light chain locus resulted in the introduction of over 80% of the human antibody repertoire. See Mendez et al. Nature Genetics 15:146-156 (1997) and U.S. Patent Application No. 08 / 759,620.

[0166] The XenoMouse mouse was further developed using U.S. Patent Applications No. 07 / 466,008, No. 07 / 610,515, No. 07 / 919,297, No. 07 / 922,649, No. 08 / 031,801, No. 08 / 112,848, No. 08 / 234,145, No. 08 / 376,279, No. 08 / 430,938, No. 08 / 464,584, No. 08 / 464,582, No. 08 / 463,191, No. 08 / 462,837, and No. 08 / 4 This is further discussed and detailed in Patent Nos. 86,853, 08 / 486,857, 08 / 486,859, 08 / 462,513, 08 / 724,752, and 08 / 759,620; and U.S. Patent Nos. 6,162,963, 6,150,584, 6,114,598, 6,075,181, and 5,939,598, as well as in Japanese Patent Publication Nos. 3068180B2, 3068506B2, and 3068507B2. See also Mendez et al. Nature Genetics 15:146-156 (1997) and Green and Jakobovits J. Exp. Med. 188:483-495 (1998), European Patent No. 0463151B1, International Publication No. 94 / 02602, International Publication No. 96 / 34096, International Publication No. 98 / 24893, International Publication No. 00 / 76310, and International Publication No. 03 / 47336.

[0167] In alternative methods, other companies, including GenPharm International, Inc., utilize the "mini-locus" method. In the mini-locus method, the exogenous Ig locus is mimicked by including fragments (individual genes) from the Ig locus. Thus, one or more VH genes, one or more DH genes, one or more JH genes, a muon constant region, and a second constant region (preferably a gamma constant region) form a construct that is inserted into the animal. This method is based on U.S. Patent No. 5,545,807 to Surani et al., and U.S. Patents No. 5,545,806, 5,625,825, 5,625,126, 5,633,425, 5,661,016, 5,770,429, 5,789,650, 5,814,318, and 5,877,397 to Lonberg and Kay, respectively. The document; U.S. Patent No. 5,874,299; and U.S. Patent No. 6,255,458, U.S. Patent No. 5,591,669 and U.S. Patent No. 6,023,010 against Krimpenfort and Berns, U.S. Patent No. 5,612,205; U.S. Patent No. 5,721,367; and U.S. Patent No. 5,789,215 against Berns et al., and U.S. Patent No. 5,643,763 against Choi and Dunn, and GenPharm This is described in U.S. Patent Applications No. 07 / 574,748, No. 07 / 575,962, No. 07 / 810,279, No. 07 / 853,408, No. 07 / 904,068, No. 07 / 990,860, No. 08 / 053,131, No. 08 / 096,762, No. 08 / 155,301, No. 08 / 161,739, No. 08 / 165,699, and No. 08 / 209,741 of International.See also European Patent No. 0546073B1, International Publication No. 92 / 03918, International Publication No. 92 / 22645, International Publication No. 92 / 22647, International Publication No. 92 / 22670, International Publication No. 93 / 12227, International Publication No. 94 / 00569, International Publication No. 94 / 25585, International Publication No. 96 / 14436, International Publication No. 97 / 13852 and International Publication No. 98 / 24884, and U.S. Patent No. 5,981,175. Furthermore, see Taylor et al. (1992), Chen et al. (1993), Tuaillon et al. (1993), Choi et al. (1993), Lonberg et al. (1994), Taylor et al. (1994), Tuaillon et al. (1995), and Fishwild et al. (1996).

[0168] Kirin also demonstrated the production of human antibodies from mice in which large chromosome fragments or entire chromosomes were introduced by microcell fusion. See European Patent Applications No. 773288 and No. 843961. Xenerex Biosciences is developing a potential human antibody production technology in which SCID mice are reconstituted with human lymphocytes, such as B cells and / or T cells. The mice are then immunized with an antigen and can produce an immune response to that antigen. See U.S. Patents No. 5,476,996; No. 5,698,767; and No. 5,958,765.

[0169] Human-anti-mouse antibody (HAMA) reactions have led the industry to produce chimeric or otherwise humanized antibodies. However, certain human-anti-chimeric antibody (HACA) reactions are expected to be observed, particularly in chronic or multi-dose antibody use. Therefore, to eliminate concerns and / or effects of HAMA or HACA reactions, it is desirable to provide antibody constructs that include human-binding domains to MSLN and CD40.

[0170] The terms "(specifically) bind," "(specifically) recognize," "(specifically) induce," and "(specifically) react" mean, according to the present invention, that the binding domain interacts with or specifically interacts with the target molecule (antigen), in this specification, with a given epitope or a given target site on MSLN and CD40, respectively.

[0171] The term "epitope" refers to a site on an antigen to which a binding domain of an antibody or immunoglobulin, or a derivative, fragment, or variant of an antibody or immunoglobulin, specifically binds. An "epitope" is antigenic, and therefore, the term epitope may also be referred to herein as an "antigenic structure" or "antigenic determinant." Thus, the binding domain is an "antigen interaction site." The binding / interaction is also understood to define "specific recognition."

[0172] An "epitope" can be formed by both consecutive amino acids or discontinuous amino acids that are paralleled by the three-dimensional folding of a protein. A "linear epitope" is an epitope that contains an epitope in which the primary amino acid sequence is recognized. Linear epitopes typically contain at least three or at least four, more commonly at least five, at least six, or at least seven, for example, about eight to about ten amino acids within a specific sequence.

[0173] A "structural epitope," in contrast to a linear epitope, is an epitope where the primary sequence of amino acids containing the epitope is not the sole element defining the recognized epitope (for example, an epitope where the primary sequence of amino acids is not necessarily recognized by the binding domain). Generally, structural epitopes contain more amino acids than linear epitopes. In relation to the recognition of structural epitopes, the binding domain recognizes the three-dimensional structure of an antigen, preferably a peptide or protein, or a fragment thereof (in the context of this invention, the antigenic structure for one of the binding domains is contained within the MSLN protein). For example, when a protein molecule folds to form a three-dimensional structure, specific amino acids and / or polypeptide backbone that form the structural epitope are arranged in parallel, thereby enabling the antibody to recognize that epitope. Methods for determining the three-dimensional structure of an epitope include, but are not limited to, X-ray crystallography, two-dimensional nuclear magnetic resonance (2D-NMR) spectroscopy, and site-directed spin labeling and electron paramagnetic resonance (EPR) spectroscopy.

[0174] The method for epitope mapping is described below. When a region of the human MSLN protein (a sequence of adjacent amino acids) is replaced / substituted with the corresponding region of a non-human and non-primate MSLN antigen (e.g., mouse MSLN, but other possibilities include those from chickens, rats, hamsters, rabbits, etc.), a reduction in binding affinity of the binding domain is expected, unless the binding domain is cross-reactive to the non-human and non-primate MSLN being used. The aforementioned reduction is preferably at least 10%, 20%, 30%, 40%, or 50%; more preferably at least 60%, 70%, or 80%, and most preferably 90%, 95%, or even 100% compared to binding to the corresponding region within the human MSLN protein, with binding to the corresponding region of the human MSLN protein being 100%. The above human MSLN / non-human MSLN chimeras are expected to be expressed in CHO cells. Human MSLN / non-human MSLN chimeras can be fused with the transmembrane and / or cytoplasmic domains of different membrane-bound proteins, such as EpCAM, but such methods were not necessary for the methods described in Examples 1 and 2.

[0175] As an alternative or additional method to epitope mapping, several truncated versions of the extracellular domain of human MSLN may be created to determine specific regions recognized by the binding domain. In these truncated versions, different extracellular MSLN domains / subdomains or regions are deleted stepwise, starting from the N-terminus. These truncated MSLN versions may be expressed in CHO cells. It is also conceivable that truncated MSLN versions may be fused with the transmembrane and / or cytoplasmic domains of different membrane-bound proteins, such as EpCAM. Furthermore, it is conceivable that truncated MSLN versions may contain a signal peptide domain at their N-terminus, e.g., a signal peptide derived from mouse IgG heavy chain signal peptide. Additionally, it is conceivable that truncated MSLN versions may contain a v5 domain at the N-terminus (following the signal peptide) to confirm their precise expression on the cell surface. In truncated MSLN versions that no longer contain the MSLN region recognized by the binding domain, a reduction or loss of binding is expected. The reduction in binding is preferably at least 10%, 20%, 30%, 40%, or 50%; more preferably at least 60%, 70%, or 80%, and most preferably 90%, 95%, or even further 100%, when the binding to the entire human MSLN protein (or its extracellular region or domain) is considered 100%.

[0176] A further method for determining the contribution of specific residues of a target antigen to recognition by an antibody construct or binding domain is alanine scanning, in which each residue to be analyzed is replaced with alanine, for example, by site-directed mutagenesis (see, for example, Morrison KL & Weiss GA. Curr Opin Chem Biol. 2001 Jun;5(3):302-7). The reason alanine is used is that it is not bulky, is chemically inert, and has a methyl functional group that mimics the secondary structure criteria that many other amino acids have. If it is desirable to conserve the size of the mutated residue, bulkier amino acids such as valine or leucine can sometimes be used. Alanine scanning is a well-established technique that has been used for a long time.

[0177] The interaction between the binding domain and the epitope or region containing the epitope means that the binding domain exhibits a measurable affinity for the epitope / region containing the epitope on a specific protein or antigen (herein MSLN and CD40, respectively), and generally does not exhibit significant reactivity to proteins or antigens other than MSLN or CD40. "Measurable affinity" includes bindings having an affinity of about 10 -6 M (KD) or stronger. Preferably, the binding affinity is about 10 -12 ~10 -8 M, 10 -12 ~10 -9 M, 10 -12 ~10 -10 M, 10 -11 ~10 -8 M, preferably about 10 -11 ~10 -9If the value is M, the binding is considered specific. Whether a binding domain specifically reacts with or binds to a target can be easily tested, in particular, by comparing the reaction of the binding domain to a target protein or antigen with the reaction of the binding domain to proteins or antigens other than MSLN or CD40. Preferably, the binding domain of the present invention does not essentially or substantially bind to proteins or antigens other than MSLN or CD40 (for example, one binding domain cannot bind to proteins other than MSLN, and the other binding domain cannot bind to proteins other than CD40).

[0178] The terms "essentially / substantially non-binding" or "unable to bind" mean that the binding domain of the present invention does not bind to proteins or antigens other than MSLN or CD40, that is, if binding to MSLN or CD40 is taken as 100%, it does not exhibit reactivity to proteins or antigens other than MSLN or CD40 of more than 30%, preferably more than 20%, more preferably more than 10%, and particularly preferably more than 9%, 8%, 7%, 6%, or 5%.

[0179] Specific binding is thought to be brought about by specific motifs within the binding domain and the amino acid sequence of the antigen. Therefore, binding occurs as a result of their primary, secondary, and / or tertiary structures, as well as as a result of secondary modifications of said structures. Specific interaction between the antigen interaction site and its specific antigen can lead to simple binding of the site to the antigen. Furthermore, specific interaction between the antigen interaction site and its specific antigen can alternatively or additionally lead to signal initiation, for example, by inducing conformational changes in the antigen or oligomerization of the antigen.

[0180] The Fc region of an antibody interacts with several Fc receptors and ligands to confer a set of important functional capabilities known as effector functions. In the case of IgG, the Fc region includes the CH2 and CH3 groups of the Ig domain. An important family of Fc receptors for IgG isotypes is the Fc gamma receptor (FcγR). These receptors mediate information exchange between antibodies and the cellular arms of the immune system. In humans, this protein family includes FcγRI(CD64), which contains isoforms FcγRIa, FcγRIb, and FcγRIc; FcγRII(CD32), which contains isoforms FcγRIIa (including allotypes H131 and R131), FcγRIIb (including FcγRIIb-1 and FcγRIIb-2), and FcγRIIc; and FcγRIII(CD16), which contains isoforms FcγRIIIa (including allotypes V158 and F158) and FcγRIIIb (including allotypes FcγRIIIb-NA1 and FcγRIIIb-NA2) (Jefferis et al., 2002, Immunol Lett 82:57-65). These receptors typically have an extracellular domain that mediates binding to Fc, a transmembrane domain, and an intracellular domain that can mediate several intracellular signaling phenomena. These receptors are expressed in a variety of immune cells, including monocytes, macrophages, neutrophils, dendritic cells, eosinophils, mast cells, platelets, B cells, large granular lymphocytes, Langerhans cells, natural killer (NK) cells, and γδT cells. The formation of Fc / FcγR complexes recruits these effector cells to the site of the bound antigen, leading to important subsequent immune responses such as intracellular signaling phenomena and the release of inflammatory mediators, B cell activation, endocytosis, phagocytosis, and cytotoxic attack. The ability to mediate cytotoxic and phagocytic effector functions is a potential mechanism by which antibodies destroy targeted cells. The cellular response in which nonspecific cytotoxic cells expressing FcγR recognize bound antibodies on target cells and subsequently cause lysis of the target cells is called antibody-dependent cell-mediated cytotoxicity (ADCC).The cellular response in which nonspecific cytotoxic cells expressing FcγR recognize bound antibodies on target cells, and subsequently trigger phagocytosis of the target cells, is called antibody-dependent cell phagocytosis (ADCP).

[0181] Different IgG subclasses have different affinities to FcγR, with IgG1 and IgG3 typically binding substantially more effectively to different receptors than IgG2 and IgG4. All FcγRs bind to the same region on IgG Fc, but have different affinities: the high-affinity conjugate FcγRI has a Kd of 10⁻⁸ M-1 to IgG1, while the low-affinity receptors FcγRII and FcγRIII generally bind at 10⁻⁶ and 10⁻⁵, respectively.

[0182] As used herein, "Fc gamma receptor" or "FcγR" means any member of the family of proteins that bind to the Fc region of an IgG antibody and are substantially encoded by the FcγR gene. In humans, this family includes, but is not limited to, FcγRI(CD64) including isoforms FcγRIa, FcγRIb, and FcγRIc; FcγRII(CD32) including isoforms FcγRIIa (including allotypes H131 and R131), FcγRIIb (including FcγRIIb-1 and FcγRIIb-2), and FcγRIIc; and FcγRIII(CD16) including isoforms FcγRIIIa (including allotypes V158 and F158) and FcγRIIIb (including allotypes FcγRIIIb-NA1 and FcγRIIIb-NA2) (Jefferis et al., 2002, Immunol Lett 82:57-65), as well as any undiscovered human FcγR or FcγR isoform or allotype. FcγR can originate from any organism, including but not limited to humans, mice, rats, rabbits, and monkeys. Mouse FcγRs include, but are not limited to, FcγRI(CD64), FcγRII(CD32), FcγRIII(CD16), and FcγRIII-2(CD16-2), as well as any undiscovered mouse FcγR or FcγR isoform or allotype.

[0183] Binding to FcγR can be directly determined by determining the affinity of the antibody to FcγR. Alternatively, binding can be indirectly determined by measuring cytokine release in a cell-based assay.

[0184] To facilitate the association of a particular heavy chain with a light chain of its cognate, both the heavy and light chains may contain complementary amino acid substitutions. As used herein, “complementary amino acid substitution” refers to a substitution to a positively charged amino acid in one chain and a corresponding negatively charged amino acid substitution in the other chain. For example, in some embodiments, the heavy chain contains at least one amino acid substitution to introduce a charged amino acid, and the corresponding light chain contains at least one amino acid substitution to introduce a charged amino acid, wherein the charged amino acid introduced into the heavy chain has the opposite charge to the amino acid introduced into the light chain. In certain embodiments, one or more positively charged residues (e.g., lysine, histidine, or arginine) can be introduced into the first light chain (LC1), and one or more negatively charged residues (e.g., aspartic acid or glutamic acid) can be introduced into the heavy chain (HC1) paired at the LC1 / HC1 binding interface. Alternatively, one or more negatively charged residues (e.g., aspartic acid or glutamic acid) can be introduced into the second light chain (LC2), and one or more positively charged residues (e.g., lysine, histidine, or arginine) can be introduced into the heavy chain (HC2) paired at the LC2 / HC2 binding interface. Electrostatic interactions induce LC1 to pair with HC1 and LC2 to pair with HC2 because the oppositely charged residues (polarity) at the interface attract each other. Heavy / light chain pairs with the same charged residue (polarity) at the interface (e.g., LC1 / HC2 and LC2 / HC1) will repel each other, and as a result, the formation of undesirable HC / LC pairs is suppressed.

[0185] In these and other embodiments, the CH1 domain of the heavy chain or the CL domain of the light chain contains an amino acid sequence different from the wild-type IgG amino acid sequence, resulting in one or more positively charged amino acids in the wild-type IgG amino acid sequence being replaced by one or more negatively charged amino acids. Alternatively, the CH1 domain of the heavy chain or the CL domain of the light chain contains an amino acid sequence different from the wild-type IgG amino acid sequence, resulting in one or more negatively charged amino acids in the wild-type IgG amino acid sequence being replaced by one or more positively charged amino acids. In some embodiments, one or more amino acids in the CH1 domain of the first and / or second heavy chain in a multispecific antibody construct at an EU position selected from F126, P127, L128, A141, L145, K147, D148, H168, F170, P171, V173, Q175, S176, S183, V185 and K213 are replaced by charged amino acids. In certain embodiments, the heavy chain residue for substitution with a negatively or positively charged amino acid is S183 (EU numbering system). In some embodiments, S183 is substituted with a positively charged amino acid. In alternative embodiments, S183 is substituted with a negatively charged amino acid. For example, in one embodiment, S183 is substituted with a negatively charged amino acid in the first heavy chain (e.g., S183E), and S183 is substituted with a positively charged amino acid in the second heavy chain (e.g., S183K).

[0186] In embodiments where the light chain is a kappa light chain, one or more amino acids in the CL domain of the first and / or second light chain in the multimeric antibody construct at positions selected from F116, F118, S121, D122, E123, Q124, S131, V133, L135, N137, N138, Q160, S162, T164, S174, and S176 (EU numbering in the kappa light chain) are replaced with charged amino acids. In embodiments where the light chain is a lambda light chain, one or more amino acids in the CL domain of the first and / or second light chain in the multispecific antibody construct at a position selected from T116, F118, S121, E123, E124, K129, T131, V133, L135, S137, E160, T162, S165, Q167, A174, S176, and Y178 (EU numbering in the lambda chain) are replaced with charged amino acids. In some embodiments, the residue for substitution with a negatively or positively charged amino acid is S176 (EU numbering system) in the CL domain of either the kappa or lambda light chain. In certain embodiments, S176 of the CL domain is replaced with a positively charged amino acid. In alternative embodiments, S176 of the CL domain is replaced with a negatively charged amino acid. In one embodiment, S176 is substituted with a positively charged amino acid in the first light chain (e.g., S176K), and S176 is substituted with a negatively charged amino acid in the second light chain (e.g., S176E).

[0187] In addition to or instead of complementary amino acid substitutions in the CH1 and CL domains, the variable regions of the light and heavy chains in a multispecific antibody construct may contain one or more complementary amino acid substitutions to introduce charged amino acids. For example, in some embodiments, the VH region of the heavy chain or the VL region of the light chain of a multispecific antibody construct contains an amino acid sequence different from the wild-type IgG amino acid sequence, resulting in one or more positively charged amino acids in the wild-type IgG amino acid sequence being replaced by one or more negatively charged amino acids. Alternatively, the VH region of the heavy chain or the VL region of the light chain contains an amino acid sequence different from the wild-type IgG amino acid sequence, resulting in one or more negatively charged amino acids in the wild-type IgG amino acid sequence being replaced by one or more positively charged amino acids.

[0188] Examples of V-region interface residues within the VH region (i.e., amino acid residues that mediate the assembly of the VH and VL regions) include EU positions 1, 3, 35, 37, 39, 43, 44, 45, 46, 47, 50, 59, 89, 91, and 93. One or more of these interface residues in the VH region may be substituted with charged (positively or negatively charged) amino acids. In certain embodiments, the amino acid at EU position 39 in the VH region of the first and / or second heavy chain is substituted with a positively charged amino acid, such as lysine. In alternative embodiments, the amino acid at EU position 39 in the VH region of the first and / or second heavy chain is substituted with a negatively charged amino acid, such as glutamic acid. In some embodiments, the amino acid at EU position 39 in the VH region of the first heavy chain is substituted with a negatively charged amino acid (e.g., G39E), and the amino acid at EU position 39 in the VH region of the second heavy chain is substituted with a positively charged amino acid (e.g., G39K). In some embodiments, the amino acid at EU position 44 in the VH region of the first and / or second heavy chain is substituted with a positively charged amino acid, e.g., lysine. In alternative embodiments, the amino acid at EU position 44 in the VH region of the first and / or second heavy chain is substituted with a negatively charged amino acid, e.g., glutamic acid. In certain embodiments, the amino acid at EU position 44 in the VH region of the first heavy chain is substituted with a negatively charged amino acid (e.g., G44E), and the amino acid at EU position 44 in the VH region of the second heavy chain is substituted with a positively charged amino acid (e.g., G44K).

[0189] Examples of V-region interface residues within the VL region (i.e., amino acid residues that mediate the assembly of the VH and VL regions) include EU positions 32, 34, 35, 36, 38, 41, 42, 43, 44, 45, 46, 48, 49, 50, 51, 53, 54, 55, 56, 57, 58, 85, 87, 89, 90, 91, and 100. One or more interface residues in the VL region may be substituted with a charged amino acid, preferably an amino acid having the opposite charge to that introduced into the VH region of the congeneral heavy chain. In some embodiments, the amino acid at EU position 100 in the VL region of the first and / or second light chain is substituted with a positively charged amino acid, such as lysine. In alternative embodiments, the amino acid at EU position 100 in the VL region of the first and / or second light chain is substituted with a negatively charged amino acid, such as glutamic acid. In certain embodiments, the amino acid at EU position 100 in the VL region of the first light chain is substituted with a positively charged amino acid (e.g., G100K), and the amino acid at EU position 100 in the VL region of the second light chain is substituted with a negatively charged amino acid (e.g., G100E).

[0190] Any of the constant domains may be modified to contain one or more of the charge-pair mutations described above, which facilitate the precise assembly of multispecific antibody constructs.

[0191] As used herein, the term “Fc region” refers to the C-terminal region of an immunoglobulin heavy chain that can be produced by papain digestion of an intact antibody. The Fc region of an immunoglobulin generally comprises two constant domains, a CH2 domain and a CH3 domain, and optionally a CH4 domain. In certain embodiments, the Fc region is derived from IgG1, IgG2, IgG3, or IgG4 immunoglobulin. In some embodiments, the Fc region comprises CH2 and CH3 domains derived from human IgG1 or human IgG2 immunoglobulin. The Fc region may retain effector functions such as C1q binding, complement-dependent cell-mediated cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), and phagocytosis. In other embodiments, the Fc region may be modified to reduce or eliminate effector functions, as described further in this specification.

[0192] The heavy chain constant region or Fc region of the multispecific antibody constructs described herein may contain one or more amino acid substitutions that affect the glycosylation and / or effector function of antigen-binding proteins. One function of the Fc region of immunoglobulins is to transmit signals to the immune system when the immunoglobulin binds to its target. This is commonly referred to as “effector function.” The transmission leads to antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP), and / or complement-dependent cell-mediated cytotoxicity (CDC). ADCC and ADCP are mediated through the binding of the Fc region to Fc receptors on the surface of immune system cells. CDC is mediated through the binding of Fc to complement system proteins, such as C1q. In some embodiments, the multispecific antibody constructs of the present invention include one or more amino acid substitutions in the constant region that enhance effector function, such as ADCC activity, CDC activity, ADCP activity, and / or the clearance or half-life of antigen-binding proteins. Examples of amino acid substitutions (EU numbering) that can enhance effector functionality include, but are not limited to, E233L, L234I, L234Y, L235S, G236A, S239D, F243L, F243V, P247I, D280H, K290S, K290E, K290N, K290Y, R292P, E294L, Y296W, S298A, S298 Examples include D, S298V, S298G, S298T, T299A, Y300L, V305I, Q311M, K326A, K326E, K326W, A330S, A330L, A330M, A330F, I332E, D333A, E333S, E333A, K334A, K334V, A339D, A339Q, P396L, or any combination of the above.

[0193] In other embodiments, the multispecific antibody construct of the present invention includes one or more amino acid substitutions in the constant region that reduce effector function. Examples of amino acid substitutions (EU numbering) that can reduce effector function include, but are not limited to, C220S, C226S, C229S, E233P, L234A, L234V, V234A, L234F, L235A, L235E, G237A, P238S, S267E, H268Q, N297A, N297G, V309L, E318A, L328F, A330S, A331S, P331S, or any combination thereof.

[0194] Glycosylation can be a factor in the effector function of antibodies, particularly IgG1 antibodies. Therefore, in some embodiments, the multispecific antibody constructs of the present invention may include one or more amino acid substitutions that affect the level or type of glycosylation of the binding protein. Polypeptide glycosylation is typically either N-linked or O-linked. N-linking refers to the binding of a carbohydrate moiety to the side chain of an asparagine residue. The tripeptide sequences asparagine-X-serine and asparagine-X-threonine (where X is any amino acid other than proline) are recognition sequences for the enzymatic binding of a carbohydrate moiety to the asparagine side chain. Therefore, the presence of either of these tripeptide sequences in a polypeptide creates a potential glycosylation site. O-linked glycosylation refers to the binding of one of the sugars N-acetylgalactosamine, galactose, or xylose to a hydroxyamino acid, most commonly serine or threonine, although 5-hydroxyproline or 5-hydroxylysine may also be used.

[0195] In certain embodiments, glycosylation of the multispecific antibody constructs described herein is increased by adding one or more glycosylation sites, for example, to the Fc region of the binding protein. Addition of glycosylation sites to antigen-binding proteins can be conveniently achieved by modifying the amino acid sequence to include one or more of the above-described tripeptide sequences (in the case of N-linked glycosylation sites). Modification can also be made by adding or substituting one or more serine or threonine residues into the start sequence (in the case of O-linked glycosylation sites). For convenience, the antigen-binding protein amino acid sequence can be modified by changes at the DNA level, particularly by mutating the DNA encoding the target polypeptide at pre-selected base positions so that codons translated to desired amino acids are produced.

[0196] The present invention also encompasses the production of multispecific antibody construct molecules having modified glycosylation structures that result in altered effector activity, such as antigen-binding proteins in which fucosylation is absent or reduced, exhibiting enhanced ADCC activity. Various methods for reducing or eliminating fucosylation are known in the art. For example, ADCC effector activity is mediated by the binding of antibody molecules to the FcγRIII receptor, which has been shown to depend on the carbohydrate structure of N-linked glycosylation at the N297 residue of the CH2 domain. Non-fucosylated antibodies bind to this receptor with high affinity and induce FcγRIII-mediated effector function more efficiently than naturally occurring fucosylated antibodies. For example, recombinant production of non-fucosylated antibodies in CHO cells in which the alpha-1,6-fucosyltransferase enzyme is knocked out results in antibodies with 100-fold increased ADCC activity (see Yamane-Ohnuki et al., Biotechnol Bioeng. 87(5):614-22, 2004). Similar effects can be achieved by reducing the activity of the alpha-1,6-fucosyltransferase enzyme or other enzymes in the fucosylation pathway, for example, by siRNA or antisense RNA treatment, cell line manipulation to knock out the enzyme, or culture using selective glycosylation inhibitors (see Rothman et al., Mol Immunol. 26(12):1113-23, 1989). Some host cell lines, such as Lec13 or the rat hybridoma YB2 / 0 cell line, naturally produce antibodies with lower fucosylation levels (see Shields et al., J Biol Chem. 277(30):26733-40, 2002 and Shinkawa et al., J Biol Chem. 278(5):3466-73, 2003). For example, it has been found that increasing the level of bifurcated glycans by recombinant antibody production in cells overexpressing the GnTIII enzyme also increases ADCC activity (see Umana et al., Nat Biotechnol. 17(2):176-80, 1999).

[0197] In other embodiments, glycosylation of the multispecific antibody constructs described herein is reduced or eliminated by removing one or more glycosylation sites, for example, from the Fc region of the binding protein. N-linked glycosylation of the antigen-binding protein can be reduced or eliminated by amino acid substitutions that eliminate or modify the N-linked glycosylation site. In certain embodiments, the multispecific antibody constructs described herein include mutations at the N297 position (EU numbering), such as N297Q, N297A, or N297G. In certain embodiments, the multispecific antibody constructs described herein include mutations at the L234 and L235 positions (EU numbering), such as L234A and L235A. In a particular embodiment, the multispecific antibody construct of the present invention includes an Fc region derived from a human IgG1 antibody having the N297G mutation. To improve the stability of the molecule containing the N297 mutation, the Fc region of the molecule may be further manipulated. For example, in some embodiments, one or more amino acids in the Fc region are substituted with cysteine ​​to promote disulfide bond formation in the dimeric state. Thus, residues corresponding to V259, A287, R292, V302, L306, V323, or I332 (EU numbering) in the IgG1 Fc region may be substituted with cysteine. In one embodiment, specific pairs of residues are substituted with cysteine ​​so as to preferentially form disulfide bonds with each other, thereby limiting or preventing disulfide bond scrambling. In certain embodiments, such pairs include, but are not limited to, A287C and L306C, V259C and L306C, R292C and V302C, and V323C and I332C. In certain embodiments, the multispecific antibody construct described herein includes an Fc region derived from a human IgG1 antibody having mutations at R292C and V302C. In such embodiments, the Fc region may also include the N297G mutation.

[0198] In one embodiment, the heavy chain is (i) N297G or N297A; (ii) L234A and L235A; and (iii) R292C and V302C Includes amino acid substitutions selected from the group consisting of; The amino acid numbering follows the EU numbering system, which is based on Kabat.

[0199] In one embodiment, the heavy chain contains the N297G, R292C, and V302C mutations, and the amino acid numbering follows the EU numbering system according to Kabat.

[0200] For example, modifications to the multispecific antibody construct of the present invention to increase the serum half-life may also be desirable, such as the incorporation or addition of a salvage receptor-binding epitope (e.g., by mutation in an appropriate region, or by incorporating the epitope into a peptide tag and subsequently fusing it to an antigen-binding protein at either terminal or central position, for example, by DNA or peptide synthesis; see, for example, International Publication No. 96 / 32478), or the addition of molecules such as PEG or other water-soluble polymers, e.g., polysaccharide polymers. The salvage receptor-binding epitope preferably constitutes a region in which any one or more amino acid residues derived from one or two loops of the Fc region are transferred to a similar position in the antigen-binding protein. In one embodiment, three or more residues derived from one or two loops of the Fc region are transferred. In one embodiment, the epitope is taken from the CH2 domain of the Fc region (e.g., the IgG Fc region) and transferred to the CH1, CH3, or VH region, or two or more such regions, of the antigen-binding protein. Alternatively, the epitope may be taken from the CH2 domain of the Fc region and transferred to the CL region, VL region, or both of the antigen-binding protein. For a description of the Fc variant and its interaction with salvage receptors, please refer to the international applications, International Publication Nos. 97 / 34631 and International Publication Nos. 96 / 32478.

[0201] In certain embodiments of the multispecific antibody construct of the present invention, the binding domain located at the amino terminus of the Fc region (i.e., the amino-terminus binding domain) is a Fab fragment fused to the amino terminus of the Fc region via a peptide linker described herein or via an immunoglobulin hinge region. The “immunoglobulin hinge region” refers to the amino acid sequence that links the CH1 and CH2 domains of the immunoglobulin heavy chain. The hinge region of human IgG1 is generally defined as the amino acid sequence from the vicinity of Glu216 or Cys226 to the vicinity of Pro230. The hinge regions of other IgG isotypes may be aligned with the IgG1 sequence by placing the first and last cysteine ​​residues that form the intra-heavy-chain disulfide bond in the same position, and this is determinable to those skilled in the art. In some embodiments, the amino-terminus binding domain is bound to the amino terminus of the Fc region via the human IgG1 hinge region. In other embodiments, the amino-terminus binding domain is bound to the amino terminus of the Fc region via the human IgG2 hinge region. In one embodiment, an amino-terminal binding domain (e.g., a Fab fragment) is fused to the Fc region via the carboxyl terminus of the CH1 region of Fab.

[0202] In some embodiments of the heavy chain fusion protein of the present invention, the binding domain located at the carboxyl terminus of the Fc region (i.e., the carboxyl terminus binding domain) is a Fab fragment. In such embodiments, Fab is fused to the carboxyl terminus of the Fc region (e.g., the carboxyl terminus of the CH3 domain) via a peptide linker through the amino terminus of the VH region of the Fab fragment, or linked in another way. Thus, in one embodiment, Fab is fused to the Fc region via the amino terminus of the VH region of Fab, and the resulting fusion protein includes a CH2 domain, a CH3 domain, a peptide linker, a VH region, and a CH1 region from the N-terminus to the C-terminus.

[0203] The peptide linker that binds the Fc region to the carboxyl-terminal Fab fragment may be any of the peptide linkers described herein. In certain embodiments, the peptide linker that binds the Fc region to the carboxyl-terminal Fab fragment has a length of at least 5 amino acids. In other embodiments, the peptide linker that binds the Fc region to the carboxyl-terminal Fab fragment has a length of at least 8 amino acids. A particularly preferred peptide linker for binding the Fc region to the carboxyl-terminal Fab fragment is (Gly x Ser) n The linker is a glycine-serine linker such as (x=3 or 4, and n=2, 3, 4, 5, or 6) (SEQ ID NO: 923). In one embodiment, the peptide linker that links the Fc region to the carboxyl-terminal Fab fragment is the L10(G4S)2 linker (SEQ ID NO: 888). In another embodiment, the peptide linker that links the Fc region to the carboxyl-terminal Fab fragment is the L9 or G3SG4S linker (SEQ ID NO: 924).

[0204] In some embodiments of the antigen-binding protein of the present invention, where the carboxyl-terminal binding domain is a Fab fragment, the binding domain located at the amino terminus of the Fc region (i.e., the amino-terminal binding domain) is also a Fab fragment. The amino-terminal Fab fragment may be fused to the amino terminus of the Fc region via a peptide linker or immunoglobulin hinge region as described herein. In some embodiments, the amino-terminal Fab fragment is bound to the amino terminus of the Fc region via a human IgG1 hinge region. In other embodiments, the amino-terminal Fab fragment is bound to the amino terminus of the Fc region via a human IgG2 hinge region. In one embodiment, the amino-terminal Fab fragment is fused to the Fc region via the carboxyl terminus of the CH1 region of Fab.

[0205] In some embodiments, the multispecific antibody construct of the present invention comprises a first antibody that specifically binds to a first target, and one polypeptide chain (e.g., heavy chain (VH2-CH1)) of a Fab fragment derived from a second antibody that specifically binds to a second target is fused to the carboxyl terminus of the heavy chain of the first antibody. In such embodiments, the multispecific antibody construct also comprises a polypeptide chain (e.g., light chain (VL2-CL)) containing the other half of the Fab fragment derived from the second antibody. This form is referred to herein as the “IgG-Fab” form, and one embodiment of this type of molecule is schematically shown in Figure 1. Accordingly, in certain embodiments, the present invention comprises a bispecific multivalent antigen-binding protein comprising (i) a light chain derived from a first antibody, (ii) a heavy chain derived from the first antibody (the heavy chain is fused at its carboxyl terminus to a first polypeptide containing the VH-CH1 domain of a second antibody via a peptide linker to form a modified heavy chain), and (iii) a second polypeptide containing the VL-CL domain of a second antibody. When dimerized, the multispecific antibody construct is a homohexamer comprising two modified heavy chains, two light chains, and two polypeptide chains containing the other half of a Fab fragment (Fd fragment) derived from the second antibody. In one embodiment, the first polypeptide fused to the carboxyl terminus of the heavy chain comprises the VH domain and the CH1 domain of the second antibody, and the second polypeptide comprises the VL domain and the CL domain of the second antibody.

[0206] Charge-pair mutations or complementary amino acid substitutions as described herein can be introduced into the Fab region of a first antibody (Fab1) or a second antibody (Fab2) to promote accurate heavy-light pair formation. For example, in some embodiments, the amino acid at EU position 38 of the VL domain in Fab1 is replaced with a negatively charged amino acid (e.g., glutamic acid), and the amino acid at EU position 39 of the VH domain in Fab1 is replaced with a positively charged amino acid (e.g., lysine). In other embodiments, the amino acid at EU position 38 of the VL domain in Fab1 is replaced with a positively charged amino acid (e.g., lysine), and the amino acid at EU position 39 of the VH domain in Fab1 is replaced with a negatively charged amino acid (e.g., glutamic acid). In one particular embodiment, the amino acid at EU position 38 of the VL domain in Fab2 is replaced with a negatively charged amino acid (e.g., glutamic acid), and the amino acid at EU position 39 of the VH domain in Fab2 is replaced with a positively charged amino acid (e.g., lysine). In another embodiment, the amino acid at EU position 38 of the VL domain in Fab2 is replaced with a positively charged amino acid (e.g., lysine), and the amino acid at EU position 39 of the VH domain in Fab2 is replaced with a negatively charged amino acid (e.g., glutamic acid).

[0207] In an embodiment in which the VH-CH1 region (i.e., Fd fragment) derived from the second antibody is fused to the heavy chain of the first antibody, the heavy chain derived from the first antibody contains the S183E mutation (EU numbering), the light chain derived from the first antibody contains the S176K mutation (EU numbering), the light chain derived from the second antibody contains the S176E mutation (EU numbering), and the Fd region derived from the second antibody (fused to the C-terminus of the heavy chain derived from the first antibody) contains the S183K mutation (EU numbering). In other embodiments, the heavy chain derived from the first antibody includes the G44E mutation (EU) and the S183E mutation (EU numbering), the light chain derived from the first antibody includes the G100K mutation (EU) and the S176K mutation (EU numbering), the light chain derived from the second antibody includes the G100E mutation (EU) and the S176E mutation (EU numbering), and the Fd region derived from the second antibody (fused to the C-terminus of the heavy chain derived from the first antibody) includes the G44K mutation (EU) and the S183K mutation (EU numbering). The charges in the above examples can be reversed as long as the charges on the corresponding light or heavy chains are reversed so that the exact heavy / light chain pairs have opposite charges.

[0208] "~corresponding to" means that, when referring to the VH2 and second CH1 domains, the amino acid residues of the VH2 and second CH1 domains are counted from the C-terminus of the first heavy chain when there is no linker. When there is a peptide linker, the amino acid residues of the VH2 and second CH1 domains are counted from the C-terminus of the peptide linker. In either case, amino acid residues are not counted from the N-terminus of the first heavy chain. Rather, with respect to the VH2 and second CH1 domains, counting begins at the first amino acid residue of the VH2 domain. Amino acid residue counting is performed using EU or AHo conventions.

[0209] In certain embodiments, a) VH1 contains the Q39E mutation and the first CH1 domain contains the S183K mutation using EU numbering; b) VH2 contains the Q39K mutation and the second CH1 domain contains the S183E mutation using EU numbering; c) VL1 contains the Q38K mutation and the first CL domain contains the S176E mutation using EU numbering; and d) VL2 contains the Q38E mutation and the second CL domain contains the S176K mutation using EU numbering.

[0210] In a particular embodiment, a) the first CH1 domain includes G44E and S183K mutations using EU numbering; b) the second CH1 domain includes G44K and S183E mutations using EU numbering; c) the first CL domain includes G100K and S176E mutations using EU numbering; and d) the second CL domain includes G100E and S176K mutations using EU numbering.

[0211] In a particular embodiment, a) VH1 contains the Q39K mutation and the first CH1 domain contains the S183E mutation using EU numbering; b) VH2 contains the Q39E mutation and the second CH1 domain contains the S183K mutation using EU numbering; c) VL1 contains the Q38E mutation and the first CL domain contains the S176K mutation using EU numbering; and d) VL2 contains the Q38K mutation and the second CL domain contains the S176E mutation using EU numbering.

[0212] In a particular embodiment, a) the first CH1 domain includes G44K and S183E mutations using EU numbering; b) the second CH1 domain includes G44E and S183K mutations using EU numbering; c) the first CL domain includes G100E and S176K mutations using EU numbering; and d) the second CL domain includes G100K and S176E mutations using EU numbering.

[0213] In certain embodiments, the first heavy chain is fused to VH2 via a peptide linker. In certain embodiments, the peptide linker includes a sequence selected from the group consisting of (Gly3Ser)2 (SEQ ID NO: 916), (Gly4Ser)2 (SEQ ID NO: 888), (Gly3Ser)3 (SEQ ID NO: 917), (Gly4Ser)3 (SEQ ID NO: 889), (Gly3Ser)4 (SEQ ID NO: 918), (Gly4Ser)4 (SEQ ID NO: 890), (Gly3Ser)5 (SEQ ID NO: 919), (Gly4Ser)5 (SEQ ID NO: 920), (Gly3Ser)6 (SEQ ID NO: 921), and (Gly4Ser)6 (SEQ ID NO: 922). These same sequences can also be written as GGGSGGGS (sequence number 916), GGGGSGGGGS (sequence number 888), GGGSGGGSGGGS (sequence number 917), GGGGSGGGGSGGGGGS (sequence number 889), GGGSGGGSGGGSGGGS (sequence number 918), GGGGSGGGGSGGGGSGGGGS (sequence number 890), GGGSGGGSGGGSGGGSGGGS (sequence number 919), GGGGSGGGGSGGGGSGGGGSGGGS (sequence number 920), GGGSGGGSGGGSGGGSGGGSGGGS (sequence number 921), and anGGGGSGGGGSGGGGSGGGGSGGGGGSGGGS (sequence number 922).

[0214] Furthermore, or alternatively, correct heavy-light chain pair formation can be facilitated by exchanging the CH1 and CL domains in the carboxyl-terminal Fab-binding domain. For example, the first polypeptide fused to the carboxyl terminus of the heavy chain may contain the VL and CH1 domains derived from the second antibody, and the second polypeptide may contain the VH and CL domains derived from the second antibody. In another embodiment, the first polypeptide fused to the carboxyl terminus of the heavy chain may contain the VH and CL domains derived from the second antibody, and the second polypeptide may contain the VL and CH1 domains derived from the second antibody.

[0215] In another embodiment, the present invention is a) Two identical heavy chain fusion proteins, each containing a first heavy chain variable region (VH1) and a first CH1 domain (the first CH1 domain is linked to a hinge-CH2-CH3 polypeptide, and the hinge-CH2-CH3 polypeptide is linked to a second heavy chain variable region (VH2), and VH2 is linked to a second CH1 domain; i) The VH1 or first CH1 domain includes at least one amino acid substitution introducing a positively charged amino acid at a residue selected from the group consisting of positions 39, 44, and 183 using EU numbering; and ii) The VH2 or second CH1 domain includes at least one amino acid substitution introducing a negatively charged amino acid with a residue selected from the group consisting of residues corresponding to positions 39, 44, and 183 using EU numbering); and b) A second polypeptide comprising a first light chain (the first light chain comprising a first light chain variable region (VL1) and a first CL region; and the VL1 or the first CL domain comprising at least one amino acid substitution introducing a negatively charged amino acid at a residue selected from the group consisting of positions 38, 100, and 176 using EU numbering); and c) A third polypeptide comprising a second light chain (the second light chain comprising a second light chain variable region (VL2) and a second CL region; and the VL2 or second CL domain comprising at least one amino acid substitution introducing a positively charged amino acid at a residue selected from the group consisting of positions 38, 100, and 176 using EU numbering). A multispecific antibody construct comprising, VH1 and VL1 interact to bind to the first antigen, and VH2 and VL2 interact to bind to the second antigen; Here, The first antigen is human CD40 (SEQ ID NO: 1), and the second antigen is human mesoserine ("MSLN"; SEQ ID NO: 2); or This invention relates to a multispecific antibody construct in which the first antigen is human MSLN (SEQ ID NO: 2) and the second antigen is human CD40 (SEQ ID NO: 1).

[0216] In one aspect, the present invention is a) Two identical heavy chain fusion proteins, each containing a first heavy chain variable region (VH1) and a first CH1 domain (the first CH1 domain is linked to a hinge-CH2-CH3 polypeptide, and the hinge-CH2-CH3 polypeptide is linked to a second heavy chain variable region (VH2), and VH2 is linked to a second CH1 domain; i) The VH1 or first CH1 domain includes at least one amino acid substitution introducing a negatively charged amino acid at a residue selected from the group consisting of positions 39, 44, and 183 using EU numbering; and ii) The VH2 or second CH1 domain includes at least one amino acid substitution introducing a positively charged amino acid at a residue selected from the group consisting of residues corresponding to positions 39, 44, and 183 using EU numbering; and b) A second polypeptide comprising a first light chain (the first light chain comprising a first light chain variable region (VL1) and a first CL region; and the VL1 or the first CL domain comprising at least one amino acid substitution introducing a positively charged amino acid at a residue selected from the group consisting of positions 38, 100, and 176 using EU numbering); and c) A third polypeptide comprising a second light chain (the second light chain comprising a second light chain variable region (VL2) and a second CL region; and the VL2 or the second CL domain comprising at least one amino acid substitution introducing a negatively charged amino acid at a residue selected from the group consisting of positions 38, 100, and 176 using EU numbering). A multispecific antibody construct comprising, VH1 and VL1 interact to bind to the first antigen, and VH2 and VL2 interact to bind to the second antigen; Here, The first antigen is human CD40 (SEQ ID NO: 1), and the second antigen is human mesoserine ("MSLN"; SEQ ID NO: 2); or This invention relates to a multispecific antibody construct in which the first antigen is human MSLN (SEQ ID NO: 2) and the second antigen is human CD40 (SEQ ID NO: 1).

[0217] In one embodiment, the hinge-CH2-CH3 polypeptide is linked to VH2 via a peptide linker.

[0218] In one embodiment, the peptide linker includes a sequence selected from the group consisting of (Gly3Ser)2 (SEQ ID NO: 916), (Gly4Ser)2 (SEQ ID NO: 888), (Gly3Ser)3 (SEQ ID NO: 917), (Gly4Ser)3 (SEQ ID NO: 889), (Gly3Ser)4 (SEQ ID NO: 918), (Gly4Ser)4 (SEQ ID NO: 890), (Gly3Ser)5 (SEQ ID NO: 919), (Gly4Ser)5 (SEQ ID NO: 920), (Gly3Ser)6 (SEQ ID NO: 921), and (Gly4Ser)6 (SEQ ID NO: 922).

[0219] In one embodiment, a) The VH1 or first CH1 domain contains a mutation selected from the group consisting of Q39K, G44K, and S183K using EU numbering; b) The VH2 or second CH1 domain contains a mutation selected from the group consisting of Q39E, G44E, and S183E using EU numbering; c) The VL1 or first CL domain contains a mutation selected from the group consisting of Q38E, G100E, and S176E using EU numbering; and d) The VL2 or second CL domain contains a mutation selected from the group consisting of Q38K, G100K, and S176K using EU numbering.

[0220] In one embodiment, a) The first CH1 domain contains the S183K mutation using EU numbering; b) The second CH1 domain contains the S183E mutation using EU numbering; c) The first CL domain contains the S176E mutation using EU numbering; and d) The second CL domain contains the S176K mutation, which uses EU numbering.

[0221] In one embodiment, a) VH1 contains the Q39K mutation and the first CH1 domain contains the S183K mutation using EU numbering; b) VH2 contains the Q39E mutation and the second CH1 domain contains the S183E mutation using EU numbering; c) VL1 contains the Q38E mutation and the first CL domain contains the S176E mutation using EU numbering; and d) VL2 contains the Q38K mutation and the second CL domain contains the S176K mutation using EU numbering.

[0222] In one embodiment, a) The first CH1 domain contains the G44K and S183K mutations using EU numbering; b) The second CH1 domain contains the G44E and S183E mutations using EU numbering; c) The first CL domain contains the G100E and S176E mutations using EU numbering; and d) The second CL domain contains the G100K and S176K mutations, which use EU numbering.

[0223] In one embodiment, a) The VH1 or first CH1 domain contains a mutation selected from the group consisting of Q39E, G44E, and S183E using EU numbering; b) The VH2 or second CH1 domain contains a mutation selected from the group consisting of Q39K, G44K, and S183K using EU numbering; c) The VL1 or first CL domain contains a mutation selected from the group consisting of Q38K, G100K, and S176K using EU numbering; and d) The VL2 or second CL domain contains a mutation selected from the group consisting of Q38E, G100E, and S176E using EU numbering.

[0224] In one embodiment, a) The first CH1 domain contains the S183E mutation using EU numbering; b) The second CH1 domain contains the S183K mutation using EU numbering; c) The first CL domain contains the S176K mutation using EU numbering; and d) The second CL domain contains the S176E mutation, which uses EU numbering.

[0225] In one embodiment, a) VH1 contains the Q39E mutation and the first CH1 domain contains the S183E mutation using EU numbering; b) VH2 contains the Q39K mutation and the second CH1 domain contains the S183K mutation using EU numbering; c) VL1 contains the Q38K mutation and the first CL domain contains the S176K mutation using EU numbering; and d) VL2 contains the Q38E mutation and the second CL domain contains the S176E mutation using EU numbering.

[0226] In one embodiment, a) The first CH1 domain contains the G44E and S183E mutations using EU numbering; b) The second CH1 domain contains the G44K and S183K mutations using EU numbering; c) The first CL domain contains the G100K and S176K mutations using EU numbering; and d) The second CL domain contains the G100E and S176E mutations, which use EU numbering.

[0227] In one embodiment, the hinge-CH2-CH3 polypeptide is (i) N297G or N297A; (ii) L234A and L235A; and (iii) R292C and V302C Includes amino acid substitutions selected from the group consisting of; The amino acid numbering follows the EU numbering system, which is based on Kabat.

[0228] In one embodiment, the hinge-CH2-CH3 polypeptide includes N297G, R292C, and V302C mutations, and the amino acid numbering is EU numbering according to Kabat.

[0229] In one embodiment, the first antigen is human CD40 (SEQ ID NO: 1) and the second antigen is human MSLN (SEQ ID NO: 2); and VL1 is Sequence IDs 58, 59, and 60, respectively; Sequence IDs 64, 65, and 66, respectively; Sequence numbers 70, 71, and 72, respectively; Sequence IDs 76, 77, and 78, respectively; Sequence IDs 82, 83, and 84, respectively; Sequence IDs 88, 89, and 90, respectively; Sequence numbers 94, 95, and 96, respectively; Sequence IDs 100, 101, and 102, respectively; Sequence IDs 106, 107, and 108, respectively; Sequence numbers 112, 113, and 114, respectively; Sequence IDs 118, 119, and 120, respectively; Sequence IDs 124, 125, and 126, respectively; and Sequence numbers 130, 131, and 132, respectively. It includes CDRL1, CDRL2, and CDRL3 selected from the group consisting of; VH1 is Sequence numbers 136, 137, and 138, respectively; Sequence IDs 142, 143, and 144, respectively; Sequence IDs 148, 149, and 150, respectively; Sequence IDs 154, 155, and 156, respectively; Sequence IDs 160, 161, and 162, respectively; Sequence IDs 166, 167, and 168, respectively; Sequence IDs 172, 173, and 174, respectively; Sequence IDs 178, 179, and 180, respectively; Sequence IDs 184, 185, and 186, respectively; Sequence numbers 190, 191, and 192, respectively; Sequence IDs 196, 197, and 198, respectively; Sequence IDs 202, 203, and 204, respectively; and Sequence IDs 208, 209, and 210, respectively. It includes CDRH1, CDRH2, and CDRH3 selected from the group consisting of; VL2 is Sequence IDs 230, 231, and 232, respectively; Sequence IDs 236, 237, and 238, respectively; Sequence IDs 242, 243, and 244, respectively; and Sequence IDs 248, 249, and 250, respectively. It includes CDRL1, CDRL2, and CDRL3 selected from the group consisting of; Furthermore, VH2 is Sequence IDs 254, 255, and 256, respectively; Sequence IDs 260, 261, and 262, respectively; Sequence IDs 266, 267, and 268, respectively; and Sequence IDs 272, 273, and 274, respectively. It includes CDRH1, CDRH2, and CDRH3 selected from the group consisting of the following.

[0230] In one embodiment, 1) VL1 and VH1 are, a) VL1 containing CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs. 58, 59, and 60, respectively; and VH1 containing CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs. 136, 137, and 138, respectively; b) VL1 containing CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs. 64, 65, and 66, respectively; and VH1 containing CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs. 142, 143, and 144, respectively; c ) VL1 containing CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs. 70, 71, and 72, respectively; and VH1 containing CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs. 148, 149, and 150, respectively; d) VL1 containing CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs. 76, 77, and 6780, respectively; and VH1 containing CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs. 154, 155, and 156, respectively; e) VL1 containing CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs. 82, 83, and 84, respectively; and VH1 containing CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs. 160, 161, and 162, respectively; f) VL1 containing CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs. 88, 89, and 90, respectively; and VH1 containing CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs. 166, 167, and 168, respectively; g ) VL1 containing CDRL1, CDRL2, and CDRL3 selected from the group consisting of sequence numbers 94, 95, and 96, respectively; and VH1 containing CDRH1, CDRH2, and CDRH3 selected from the group consisting of sequence numbers 172, 173, and 174, respectively; h) VL1 containing CDRL1, CDRL2, and CDRL3 selected from the group consisting of sequence numbers 100, 101, and 102, respectively; and VH1 containing CDRH1, CDRH2, and CDRH3 selected from the group consisting of sequence numbers 178, 179, and 180, respectively; i) VL1 containing CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 106, 107, and 108, respectively; and VH1 containing CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 184, 185, and 186, respectively; j) VL1 containing CDRL1, CDRL2, and CDRL3 selected from the group consisting of sequence numbers 112, 113, and 114, respectively; and VH1 containing CDRH1, CDRH2, and CDRH3 selected from the group consisting of sequence numbers 190, 191, and 192, respectively; k) VL1 containing CDRL1, CDRL2, and CDRL3 selected from the group consisting of sequence numbers 118, 119, and 120, respectively; and VH1 containing CDRH1, CDRH2, and CDRH3 selected from the group consisting of sequence numbers 196, 197, and 198, respectively; l) VL1 containing CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 124, 125, and 126, respectively; and VH1 containing CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 202, 203, and 204, respectively; and m) VL1 containing CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 130, 131, and 132, respectively; and VH1 containing CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 208, 209, and 210, respectively. Selected from the group consisting of; and 2) VL2 and VH2 are, a) VL2 containing CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs. 230, 231, and 232, respectively; and VH2 containing CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs. 254, 255, and 256, respectively; b) VL2 containing CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs. 236, 237, and 238, respectively; and VH2 containing CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs. 260, 261, and 262, respectively; c) VL2 containing CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs. 242, 243, and 244, respectively; and VH2 containing CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs. 266, 267, and 268, respectively; and d) VL2 containing CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs. 248, 249, and 250, respectively; and VH2 containing CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs. 272, 273, and 274, respectively. It is selected from the group consisting of the following.

[0231] In one embodiment, VL1 contains an amino acid sequence selected from the group consisting of SEQ ID NOs: 5, 9, 13, 17, 21, 25, 29, 33, 37, 41, 45, 49, and 53; VH1 contains an amino acid sequence selected from the group consisting of SEQ ID NOs: 6, 10, 14, 18, 22, 26, 30, 34, 38, 42, 46, 50, and 54; VL2 contains an amino acid sequence selected from the group consisting of SEQ ID NOs: 213, 217, 221, and 225; VH2 contains an amino acid sequence selected from the group consisting of SEQ ID NOs: 214, 218, 222, and 226.

[0232] In one embodiment, 1) VL1 and VH1 are, a) VL1 containing Sequence ID 5 and VH1 containing Sequence ID 6; b) VL1 containing SEQ ID NO. 9 and VH1 containing SEQ ID NO. 10; c) VL1 containing SEQ ID NO. 13 and VH1 containing SEQ ID NO. 14; d) VL1 containing SEQ ID NO: 17 and VH1 containing SEQ ID NO: 18; e) VL1 containing Sequence ID 21 and VH1 containing Sequence ID 22; f) VL1 containing sequence number 25 and VH1 containing sequence number 26; g) VL1 containing SEQ ID NO. 29 and VH1 containing SEQ ID NO. 30; h) VL1 containing sequence number 33 and VH1 containing sequence number 34; i) VL1 containing sequence number 37 and VH1 containing sequence number 38; j) VL1 containing sequence number 41 and VH1 containing sequence number 42; k) VL1 containing SEQ ID NO: 45 and VH1 containing SEQ ID NO: 46; l) VL1 containing SEQ ID NO: 49 and VH1 containing SEQ ID NO: 50; and m) VL1 containing SEQ ID NO: 53 and VH1 containing SEQ ID NO: 54 Selected from the group consisting of; and 2) VL2 and VH2 are, a) VL2 containing Sequence ID 213 and VH2 containing Sequence ID 214; b) VL2 containing Sequence ID 217 and VH2 containing Sequence ID 218; c) VL2 containing Sequence ID 221 and VH2 containing Sequence ID 222; and d) VL2 containing SEQ ID NO: 225 and VH2 containing SEQ ID NO: 226 It is selected from the group consisting of the following.

[0233] In one embodiment, the first antigen is human MSLN (SEQ ID NO: 2) and the second antigen is human CD40 (SEQ ID NO: 1); VL1 is Sequence IDs 230, 231, and 232, respectively; Sequence IDs 236, 237, and 238, respectively; Sequence IDs 242, 243, and 244, respectively; and Sequence IDs 248, 249, and 250, respectively. It includes CDRL1, CDRL2, and CDRL3 selected from the group consisting of; VH1 is Sequence IDs 254, 255, and 256, respectively; Sequence IDs 260, 261, and 262, respectively; Sequence IDs 266, 267, and 268, respectively; and Sequence IDs 272, 273, and 274, respectively. It includes CDRH1, CDRH2, and CDRH3 selected from the group consisting of; VL2 is Sequence IDs 58, 59, and 60, respectively; Sequence IDs 64, 65, and 66, respectively; Sequence numbers 70, 71, and 72, respectively; Sequence IDs 76, 77, and 78, respectively; Sequence IDs 82, 83, and 84, respectively; Sequence IDs 88, 89, and 90, respectively; Sequence numbers 94, 95, and 96, respectively; Sequence IDs 100, 101, and 102, respectively; Sequence IDs 106, 107, and 108, respectively; Sequence numbers 112, 113, and 114, respectively; Sequence IDs 118, 119, and 120, respectively; Sequence IDs 124, 125, and 126, respectively; and Sequence numbers 130, 131, and 132, respectively. It includes CDRL1, CDRL2, and CDRL3 selected from the group consisting of; and VH2 is Sequence numbers 136, 137, and 138, respectively; Sequence IDs 142, 143, and 144, respectively; Sequence IDs 148, 149, and 150, respectively; Sequence IDs 154, 155, and 156, respectively; Sequence IDs 160, 161, and 162, respectively; Sequence IDs 166, 167, and 168, respectively; Sequence IDs 172, 173, and 174, respectively; Sequence IDs 178, 179, and 180, respectively; Sequence IDs 184, 185, and 186, respectively; Sequence numbers 190, 191, and 192, respectively; Sequence IDs 196, 197, and 198, respectively; Sequence IDs 202, 203, and 204, respectively; and Sequence IDs 208, 209, and 210, respectively. It includes CDRH1, CDRH2, and CDRH3 selected from the group consisting of the following.

[0234] In one embodiment, 1) VL1 and VH1 are, a) VL1 containing CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs. 230, 231, and 232, respectively; and VH1 containing CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs. 254, 255, and 256, respectively; b) VL1 containing CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs. 236, 237, and 238, respectively; and VH1 containing CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs. 260, 261, and 262, respectively; c) VL1 containing CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs. 242, 243, and 244, respectively; and VH1 containing CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs. 266, 267, and 268, respectively; and d) VL1 containing CDRL1, CDRL2, and CDRL3 selected from the group consisting of sequence numbers 248, 249, and 250, respectively; and VH1 containing CDRH1, CDRH2, and CDRH3 selected from the group consisting of sequence numbers 272, 273, and 274, respectively; and 2) VL2 and VH2 are, a) VL2 containing CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs. 58, 59, and 60, respectively; and VH2 containing CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs. 136, 137, and 138, respectively; b) VL2 containing CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs. 64, 65, and 66, respectively; and VH2 containing CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs. 142, 143, and 144, respectively; c) VL2 containing CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs. 70, 71, and 72, respectively; and VH2 containing CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs. 148, 149, and 150, respectively; d) VL2 containing CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs. 76, 77, and 6780, respectively; and VH2 containing CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs. 154, 155, and 156, respectively; e) VL2 containing CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs. 82, 83, and 84, respectively; and VH2 containing CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs. 160, 161, and 162, respectively; f) VL2 containing CDRL1, CDRL2, and CDRL3 selected from the group consisting of sequence numbers 88, 89, and 90, respectively; and VH2 containing CDRH1, CDRH2, and CDRH3 selected from the group consisting of sequence numbers 166, 167, and 168, respectively; g) VL2 containing CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs. 94, 95, and 96, respectively; and VH2 containing CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs. 172, 173, and 174, respectively; h) VL2 containing CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 100, 101, and 102, respectively; and VH2 containing CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 178, 179, and 180, respectively; i) VL2 containing CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 106, 107, and 108, respectively; and VH2 containing CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 184, 185, and 186, respectively; j) VL2 containing CDRL1, CDRL2, and CDRL3 selected from the group consisting of sequence numbers 112, 113, and 114, respectively; and VH2 containing CDRH1, CDRH2, and CDRH3 selected from the group consisting of sequence numbers 190, 191, and 192, respectively; k) VL2 containing CDRL1, CDRL2, and CDRL3 selected from the group consisting of sequence numbers 118, 119, and 120, respectively; and VH2 containing CDRH1, CDRH2, and CDRH3 selected from the group consisting of sequence numbers 196, 197, and 198, respectively; l) VL2 containing CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 124, 125, and 126, respectively; and VH2 containing CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 202, 203, and 204, respectively; and m) VL2 containing CDRL1, CDRL2, and CDRL3 selected from the group consisting of sequence numbers 130, 131, and 132, respectively; and VH2 containing CDRH1, CDRH2, and CDRH3 selected from the group consisting of sequence numbers 208, 209, and 210, respectively.

[0235] In one embodiment, VL1 contains an amino acid sequence selected from the group consisting of SEQ ID NOs: 213, 217, 221, and 225; VH1 contains an amino acid sequence selected from the group consisting of SEQ ID NOs: 214, 218, 222, and 226; VL2 contains an amino acid sequence selected from the group consisting of SEQ ID NOs: 5, 9, 13, 17, 21, 25, 29, 33, 37, 41, 45, 49, and 53; and VH2 contains an amino acid sequence selected from the group consisting of SEQ ID NOs: 6, 10, 14, 18, 22, 26, 30, 34, 38, 42, 46, 50, and 54.

[0236] In one embodiment, 1) VL1 and VH1 are, a) VL1 containing sequence number 213 and VH1 containing sequence number 214; b) VL1 containing sequence number 217 and VH1 containing sequence number 218; c) VL1 containing Sequence ID 221 and VH1 containing Sequence ID 222; and d) VL1 containing SEQ ID NO: 225 and VH1 containing SEQ ID NO: 226 Selected from the group consisting of; and 2) VL2 and VH2 are, a) VL2 containing Sequence ID 5 and VH2 containing Sequence ID 6; b) VL2 containing SEQ ID NO. 9 and VH2 containing SEQ ID NO. 10; c) VL2 containing Sequence ID 13 and VH2 containing Sequence ID 14; d) VL2 containing SEQ ID NO: 17 and VH2 containing SEQ ID NO: 18; e) VL2 containing Sequence ID 21 and VH2 containing Sequence ID 22; f) VL2 containing Sequence ID 25 and VH2 containing Sequence ID 26; g) VL2 containing Sequence ID 29 and VH2 containing Sequence ID 30; h) VL2 containing SEQ ID NO. 33 and VH2 containing SEQ ID NO. 34; i) VL2 containing Sequence ID 37 and VH2 containing Sequence ID 38; j) VL2 containing Sequence ID 41 and VH2 containing Sequence ID 42; k) VL2 containing SEQ ID NO: 45 and VH2 containing SEQ ID NO: 46; l) VL2 containing sequence number 49 and VH2 containing sequence number 50; and m) VL2 containing SEQ ID NO: 53 and VH2 containing SEQ ID NO: 54 It is selected from the group consisting of the following.

[0237] The present invention comprises one or more isolated nucleic acids encoding the multispecific antibody constructs and their components described herein. The nucleic acid molecules of the present invention include DNA and RNA in both single-stranded and double-stranded forms, as well as corresponding complementary sequences. DNA includes, for example, cDNA, genomic DNA, chemically synthesized DNA, PCR-amplified DNA, and combinations thereof. The nucleic acid molecules of the present invention include full-length genes or cDNA molecules and combinations of their fragments. In one embodiment, the nucleic acids of the present invention are derived from human sources, but the present invention also includes those derived from non-human species.

[0238] In this specification, "isolated nucleic acid," as used interchangeably with "isolated polynucleotide," refers to a nucleic acid isolated from a neighboring gene sequence present in the genome of the organism from which the nucleic acid was isolated, in the case of a nucleic acid isolated from a naturally occurring source. For example, in the case of a nucleic acid synthesized enzymatically or chemically from a template, such as a PCR product, cDNA molecule, or oligonucleotide, the nucleic acid obtained from such a process is understood to be a nucleic acid existing on its own. An isolated nucleic acid molecule refers to a nucleic acid molecule in the form of a distinct fragment, or a nucleic acid molecule as a component of a larger nucleic acid construct. In one embodiment, the nucleic acid is substantially free of contaminating endogenous material. The nucleic acid molecule is derived from DNA or RNA that has been isolated at least once in a substantially pure form and in an amount or concentration that allows for the identification, manipulation, and recovery of its constituent nucleotide sequences by standard biochemical methods (e.g., as outlined in Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1989)). Such sequences are typically provided and / or constructed in the form of an open reading frame that is not interrupted by internal untranslated sequences or introns, which are present within eukaryotic genes. The sequence of untranslated DNA may be located on the 5' or 3' side of the open reading frame, in which case this does not interfere with the manipulation or expression of the coding region. Unless otherwise specified, the left end of any single-stranded polynucleotide sequence described herein is the 5' end, and the left direction of a double-stranded polynucleotide sequence is called the 5' direction. The direction of production of a nascent RNA transcript from 5' to 3' is called the transcription direction, the sequence region on the DNA strand having the same sequence as the RNA transcript on the 5' side of the 5' end of the RNA transcript is called the “upstream sequence”, and the sequence region on the DNA strand having the same sequence as the RNA transcript on the 3' side of the 3' end of the RNA transcript is called the “downstream sequence”.

[0239] In one embodiment, the present invention relates to a polynucleotide encoding the light chain of the antibody construct of the present invention.

[0240] In one embodiment, the present invention relates to a polynucleotide encoding a heavy chain fusion protein of the antibody construct of the present invention.

[0241] In one embodiment, the present invention relates to a vector comprising a polynucleotide encoding the light chain of an antibody construct, a polynucleotide encoding the heavy chain of an antibody construct, or both.

[0242] In one embodiment, the present invention relates to host cells transformed or transfected with polynucleotides encoding the light chain of a vector or antibody construct and polynucleotides encoding the heavy chain of an antibody construct.

[0243] In one embodiment, the present invention relates to a process for generating an antibody construct of the present invention, comprising culturing host cells containing a polynucleotide encoding a light chain and a polynucleotide encoding a heavy chain fusion protein under conditions that enable the expression of the antibody construct, and recovering the antibody construct produced from the culture.

[0244] The antigen-binding protein variants described herein can be prepared by generating variant-encoding DNA using site-directed mutagenesis of nucleotides in the polypeptide-encoding DNA, by cassette or PCR mutagenesis, or by other techniques well known in the art, and then expressing the recombinant DNA in cell culture as outlined herein. However, antigen-binding proteins, including variant CDRs having up to approximately 100–150 residues, can be prepared by in vitro synthesis using established techniques. Variants typically exhibit qualitative biological activity similar to that of their native analogs, e.g., binding to antigens. Such variants include, for example, deletions and / or insertions and / or substitutions of residues in the amino acid sequence of the antigen-binding protein. Any combination of deletions, insertions, and substitutions is performed to arrive at the final construct, provided that the final construct retains the desired properties. Amino acid changes may also alter the post-translational processes of the antigen-binding protein, such as changing the number or location of glycosylation sites. In certain embodiments, antigen-binding protein variants are prepared with the aim of modifying amino acid residues directly involved in epitope binding. In other embodiments, for the purposes described herein, modification of residues not directly involved in epitope binding, or residues not involved in epitope binding at all, is desirable. Mutagenesis in either the CDR region and / or the framework region is attempted. Those skilled in the art can use analysis of covariance techniques to design useful modifications to the amino acid sequence of antigen-binding proteins.For example, see Choulier, et al., Proteins 41:475-484, 2000; Demarest et al., J.Mol.Biol.335:41-48, 2004; Hugo et al., Protein Engineering 16(5):381-86, 2003; Aurora et al., U.S. Patent Application Publication No. 2008 / 0318207A1; Glaser et al., U.S. Patent Application Publication No. 2009 / 0048122A1; Urech et al., International Publication No. 2008 / 110348A1; Borras et al., International Publication No. 2009 / 000099A2. Such modifications, determined by analysis of covariance, can improve the potency, pharmacokinetic, pharmacodynamic and / or manufacturability properties of antigen-binding proteins.

[0245] Nucleic acid sequences of the present invention. As will be understood by those skilled in the art, due to the degeneracy of the genetic code, a very large number of nucleic acids may be produced, all of which encode the CDR of the present invention (as well as the heavy and light chains or other components of the antigen-binding proteins described herein). Therefore, once a particular amino acid sequence is identified, those skilled in the art will be able to produce any number of different nucleic acids by simply modifying the sequence of one or more codons in a manner that does not alter the amino acid sequence of the encoded protein.

[0246] The present invention also includes a vector comprising one or more nucleic acids encoding one or more components of the multispecific antibody construct of the present invention (e.g., a variable region, a light chain, a heavy chain, a modified heavy chain, and an Fd fragment). The term “vector” refers to any molecule or entity (e.g., a nucleic acid, a plasmid, a bacteriophage, or a virus) used to transfer protein-coding information into a host cell. Examples of vectors include, but are not limited to, plasmids, viral vectors, non-episomal mammalian vectors, and expression vectors, e.g., recombinant expression vectors. The terms “expression vector” or “expression construct” as used herein refer to a recombinant DNA molecule containing a desired coding sequence and appropriate nucleic acid regulatory sequences necessary for the expression of a coding sequence operably ligated in a particular host cell. An expression vector may, but is not limited to, sequences that affect or control transcription, translation, and, if introns are present, sequences that affect the RNA splicing of the coding region operably ligated to them. Nucleic acid sequences necessary for expression in prokaryotes include promoters, optionally operator sequences, ribosome binding sites, and possibly other sequences. Eukaryotic cells are known to utilize promoters, enhancers, and termination and polyadenylation signals. Secretory signal peptide sequences can also be optionally encoded by expression vectors and operably ligated to the desired coding sequence, thereby causing recombinant host cells to secrete the expressed polypeptide, which, if desired, allows for easier isolation of the polypeptide from the cell. For example, in some embodiments, the signal peptide sequence may be added / fused to the amino terminus of any of the polypeptide sequences of the present invention. In a particular embodiment, a signal peptide having the amino acid sequence MDMRVPAQLLGLLLLWLRGARC (SEQ ID NO: 894) is fused to the amino terminus of any of the polypeptide sequences of the present invention. In other embodiments, a signal peptide having the amino acid sequence MAWALLLLTLLTQGTGSWA (SEQ ID NO: 895) is fused to the amino terminus of any of the polypeptide sequences of the present invention.In yet another embodiment, a signal peptide having the amino acid sequence MTCSPLLLTLLIHCTGSWA (SEQ ID NO: 896) is fused to the amino terminus of any of the polypeptide sequences of the present invention. Other suitable signal peptide sequences that can be fused to the amino terminus of the polypeptide sequences described herein include MEAPAQLLFLLLLWLPDTTG (SEQ ID NO: 897), MEWTWRVLFLVAAATGAHS (SEQ ID NO: 898), METPAQLLFLLLLWLPDTTG (SEQ ID NO: 899), METPAQLLFLLLLWLPDTTG (SEQ ID NO: 900), MKHLWFFLLLVAAPRWVLS (SEQ ID NO: 901), and MEWSWVFLFFLSVTTGVHS (SEQ ID NO: 902). Other signal peptides are known to those skilled in the art and can be fused to any of the polypeptide chains of the present invention, for example, to promote or optimize their expression in specific host cells.

[0247] Typically, the expression vector used in host cells to produce the bispecific antigen protein of the present invention will contain a sequence for plasmid maintenance, as well as a sequence for cloning and expressing exogenous nucleotide sequences encoding components of the multispecific antibody construct. Such sequences, collectively referred to as “flanking sequences” in certain embodiments, will typically include the following nucleotide sequences: a promoter, one or more enhancer sequences, an origin of replication, a transcription termination sequence, a complete intron sequence containing donor and acceptor splice sites, a sequence encoding a leader sequence for polypeptide secretion, a ribosome binding site, a polyadenylation sequence, a polylinker region for inserting the nucleic acid encoding the polypeptide to be expressed, and one or more selection marker elements. Each of these sequences is described below.

[0248] Optionally, the vector may contain a “tag” coding sequence, i.e., an oligonucleotide molecule located at the 5' or 3' end of the polypeptide coding sequence, which encodes polyHis (e.g., hexaHis (SEQ ID NO: 925)), FLAG, HA (hemagglutinin influenza virus), myc, or another “tag” molecule for which a commercially available antibody exists. This tag is typically fused to the polypeptide during polypeptide expression and can function as a means for affinity purification or detection of the polypeptide from host cells. Affinity purification can be achieved, for example, by column chromatography using an antibody against the tag as an affinity matrix. Optionally, the tag can then be removed from the purified polypeptide by various means, such as using a specific cleavage peptidase.

[0249] Flanking sequences may be homogeneous (i.e., derived from the same species and / or strain as the host cell), heterogeneous (i.e., derived from a species other than the host cell species or strain), hybrid (i.e., a combination of flanking sequences from two or more sources), synthetic, or native. Therefore, the source of a flanking sequence may be any prokaryote or eukaryote, any vertebrate or invertebrate, or any plant, provided that the flanking sequence is functional in the host cellular mechanism and can be activated by the host cellular mechanism.

[0250] Flanking sequences useful in the vectors of the present invention may be obtained by any of several methods well known in the art. Typically, flanking sequences useful herein will have been previously identified by mapping and / or restriction endonuclease digestion and can therefore be isolated from a suitable tissue source using a suitable restriction endonuclease. In some cases, the entire nucleotide sequence of the flanking sequence may be known. Here, the flanking sequence may be synthesized using methods routinely used in nucleic acid synthesis or cloning.

[0251] Whether all or only a portion of the flanking sequence is known, the flanking sequence may be obtained by polymerase chain reaction (PCR) and / or by screening a genomic library with suitable probes such as oligonucleotides and / or flanking sequence fragments derived from the same or a different species. If the flanking sequence is unknown, a DNA fragment containing the flanking sequence can be isolated from a larger DNA fragment that may, for example, contain a coding sequence or even one or more other genes. Isolation can be achieved by generating a suitable DNA fragment by restriction endonuclease digestion, followed by isolation using agarose gel purification, Qiagen® column chromatography (Chatsworth, CA), or other methods known to those skilled in the art. The selection of suitable enzymes for achieving this purpose will be readily apparent to those skilled in the art.

[0252] The origin of replication is typically a component of commercially available prokaryotic expression vectors, and this origin assists in the amplification of the vector within host cells. If the selected vector does not contain an origin of replication site, it may be chemically synthesized based on a known sequence and ligated into the vector. For example, the origin of replication derived from plasmid pBR322 (New England Biolabs, Beverly, MA) is suitable for most Gram-negative bacteria, and various viral origins (e.g., SV40, polyoma, adenovirus, varicella-stomatitis virus (VSV), or papillomavirus, e.g., HPV or BPV) are useful for cloning vectors in mammalian cells. In general, the origin of replication component is not necessary for mammalian expression vectors (for example, the SV40 origin is often used only because it also contains the initial viral promoter).

[0253] Transcription termination sequences are typically located 3' to the end of the polypeptide coding region and function to terminate transcription. In prokaryotic cells, the transcription termination sequence is usually a GC-rich fragment followed by a polyT sequence. This sequence can be readily cloned from libraries, or even purchased commercially as part of a vector, but it can also be readily synthesized using known nucleic acid synthesis methods.

[0254] Selection marker genes encode proteins necessary for the survival and proliferation of host cells grown in a selective culture medium. Typical selection marker genes encode proteins that (a) confer resistance to antibiotics or other toxins, such as ampicillin, tetracycline, or kanamycin, to prokaryotic host cells; (b) compensate for deficiencies in the cellular nutritional requirements; or (c) supply essential nutrients unavailable from complex or standard media. Specific selection markers include kanamycin resistance genes, ampicillin resistance genes, and tetracycline resistance genes. Advantageously, neomycin resistance genes can be used for selection in both prokaryotic and eukaryotic host cells.

[0255] Other select genes may be used to amplify the genes that will be expressed. Amplification is the process by which genes required for the production of proteins important for proliferation or cell survival are repeated in tandem within the chromosomes of successive generations of recombinant cells. Examples of suitable select markers for mammalian cells include the dihydrofolate reductase (DHFR) and promoter resthymidine kinase genes. Mammalian cell transformants are placed under selective pressure, in which only these transformants are adapted to survive by the select gene present in the vector. Selective pressure is imposed by culturing the transformed cells under conditions in which the concentration of the selector in the medium is successively increased, thereby leading to amplification of both the selectable gene and the DNA encoding another gene, such as one or more components of the multispecific antibody construct described herein. As a result, large quantities of polypeptides are synthesized from the amplified DNA.

[0256] The ribosome binding site is typically required for mRNA translation initiation and is characterized by a Shine-Dalgarno sequence (prokaryotes) or a Kozak sequence (eukaryotes). This element is typically located at the 3' end of the promoter and at the 5' end of the coding sequence of the polypeptide to be expressed. In certain embodiments, one or more coding regions may be operably ligated to an internal ribosome binding site (IRES), enabling translation of two open reading frames from a single RNA transcript.

[0257] In some cases, such as when glycosylation is desired in eukaryotic host cell expression systems, various pre-sequences or pro-sequences can be manipulated to improve glycosylation or yield. For example, the peptidase cleavage site of a particular signal peptide can be modified, or a pro-sequence can be added, which can also affect glycosylation. The final protein product may have one or more additional amino acids at position -1 (relative to the first amino acid of the mature protein) that are associated with expression and may not have been completely removed. For example, the final protein product may have one or two amino acid residues found at the peptidase cleavage site, attached to the amino terminus. Alternatively, if the enzyme cleaves at such a region within the mature polypeptide, the use of certain enzymatic cleavage sites may result in a slightly cleaved form of the desired polypeptide.

[0258] The expression vectors and cloning vectors of the present invention typically contain a promoter that is recognized by a host organism and operably ligated to a molecule encoding a polypeptide. The term "operably ligated," as used herein, refers to the ligation of two or more nucleic acid sequences such that a nucleic acid molecule is produced that can direct the transcription of a given gene and / or the synthesis of a desired protein molecule. For example, a regulatory sequence in a vector "operably ligated" to a protein-coding sequence is ligated to the protein-coding sequence such that the expression of the protein-coding sequence occurs under conditions compatible with the transcriptional activity of the regulatory sequence. More specifically, a promoter and / or enhancer sequence (including any combination of cis-acting transcriptional regulatory elements) is operably ligated to a coding sequence if it stimulates or modulates the transcription of the coding sequence in a suitable host cell or other expression system.

[0259] A promoter is a non-transcriptional sequence located upstream (i.e., at the 5' end) of the start codon of a structural gene (generally within approximately 100–1000 bp) and controls the transcription of that structural gene. Conventionally, promoters are grouped into two classes: inductive promoters and constitutive promoters. Inductive promoters initiate an increase in the level of transcription from DNA under their control in response to any change in culture conditions, such as the presence or absence of nutrients or changes in temperature. Constitutive promoters, on the other hand, transcribe the genes to which they are operably ligated uniformly, i.e., with little or no control over gene expression. A large number of promoters recognized by various potential host cells are well known. A suitable promoter is operably ligated to the DNA encoding, for example, the heavy chain, light chain, modified heavy chain, or other components of the multispecific antibody construct of the present invention by removing the promoter from the source DNA by restriction enzyme digestion and inserting the desired promoter sequence into a vector.

[0260] Promoterians suitable for use in yeast hosts are also well known in the art. Advantageously, yeast enhancers are used in conjunction with yeast promoters. Promoterians suitable for use in mammalian host cells are well known and include, but are not limited to, those derived from the genomes of viruses such as polyomaviruses, fowlpox virus, adenoviruses (such as adenovirus type 2), bovine papillomavirus, aerovirus, cytomegalovirus, retroviruses, hepatitis B virus, and simian virus 40 (SV40). Other suitable mammalian promoters include heterozoan promoters, such as heat shock promoters and actin promoters.

[0261] Further potential promoters include the SV40 early promoter (Benoist and Chambon, 1981, Nature 290:304-310); the CMV promoter (Thornsen et al., 1984, Proc. Natl. Acad. USA 81:659-663); the promoter included in the long terminal repeat at the 3' end of Roussarcoma virus (Yamamoto et al., 1980, Cell 22:787-797); the herpa thymidine kinase promoter (Wagner et al., 1981, Proc. Natl. Acad. Sci. USA 78:1444-1445); promoters and regulatory sequences derived from the metallothionein gene (Prinster et al., 1982, Nature 296:39-42); and prokaryotic promoters such as the beta-lactamase promoter (Villa-Kamaroff et al.) Examples include, but are not limited to, the tac promoter (DeBoer et al., 1978, Proc. Natl. Acad. Sci. USA 75:3727-3731) or the tac promoter (DeBoer et al., 1983, Proc. Natl. Acad. Sci. USA 80:21-25). The following animal transcriptional regulatory regions, which exhibit tissue specificity and are utilized in transgenic animals, are also included: the elastase I gene regulatory region active in pancreatic acinar cells (Swift et al., 1984, Cell 38:639-646; Ornitz et al., 1986, Cold Spring Harbor Symp. Quant. Biol. 50:399-409; MacDonald, 1987, Hepatology 7:425-515); the insulin gene regulatory region active in pancreatic β-cells (Hanahan, 1985, Nature 315:115-122); and the immunoglobulin gene regulatory region active in lymphocytes (Grosschedl et al., 1984, Cell 38:647-658; Adames et al., 1985, Nature 318:533-538; Alexander et al., 1987, Mol. Cell. Biol.7:1436-1444); Mouse mammary tumor virus regulatory region active in testes, mammary glands, lymphocytes and mast cells (Leder et al., 1986, Cell 45:485-495); Albumin gene regulatory region active in the liver (Pinkert et al., 1987, Genes and Devel. 1:268-276); α-fetoprotein gene regulatory region active in the liver (Krumlauf et al., 1985, Mol.Cell.Biol. 5:1639-1648; Hammer et al., 1987, Science 253:53-58); α1-antitrypsin gene regulatory region active in the liver (Kelsey et al., 1987, Genes and Devel. 1:161-171); β-globin gene regulatory region active in bone marrow cells (Mogram et al., 1985, Nature 315:338-340; Kollias et al., 1986, Cell 46:89-94); the myelin basic protein gene regulatory region active in oligodendrocyte cells in the brain (Readhead et al., 1987, Cell 48:703-712); the myosin light chain 2 gene regulatory region active in skeletal muscle (Sani, 1985, Nature 314:283-286); and the gonadotropin-releasing hormone gene regulatory region active in the hypothalamus (Mason et al., 1986, Science 234:1372-1378).

[0262] Enhancer sequences may be inserted into vectors to increase the transcription of DNA encoding components of a multispecific antibody construct (e.g., light chain, heavy chain, modified heavy chain, Fd fragment) by higher eukaryotes. Enhancers are typically cis-acting elements of DNA, about 10–300 bp in length, that act on promoters to increase transcription. Enhancers are relatively independent of direction and position and can be found at both the 5' and 3' ends of the transcription unit. Several enhancer sequences are known to be available from mammalian genes (e.g., globin, elastase, albumin, alpha-fetoprotein, and insulin). However, typically, viral enhancers are used. The SV40 enhancer, cytomegalovirus initial promoter enhancer, polyoma enhancer, and adenovirus enhancer, known in this technique, are exemplary enhancing elements for eukaryotic promoter activation. The enhancer may be located either 5' or 3' of the coding sequence in the vector, but is typically located 5' from the promoter. A sequence encoding a suitable native or heterologous signal sequence (leader sequence or signal peptide) can be incorporated into the expression vector to promote extracellular antibody secretion. The choice of signal peptide or leader depends on the type of host cell from which the antibody will be produced, and heterologous signal sequences may replace native signal sequences. Examples of signal peptides are listed above. Other signal peptides that function in mammalian host cells include the interleukin-7 (IL-7) signal sequence described in U.S. Patent No. 4,965,195; the interleukin-2 receptor signal sequence described in Cosman et al., 1984, Nature 312:768; the interleukin-4 receptor signal peptide described in European Patent No. 0367 566; the type I interleukin-1 receptor signal peptide described in U.S. Patent No. 4,968,607; and the type II interleukin-1 receptor signal peptide described in European Patent No. 0460 846.

[0263] The expression vector provided may be constructed from a starting vector, such as a commercially available vector. Such a vector may or may not contain all of the desired flanking sequences. If one or more of the flanking sequences described herein are not initially present in the vector, they may be obtained individually and ligated into the vector. Methods used to obtain each of the flanking sequences are well known to those skilled in the art. The expression vector can be introduced into a host cell to produce a protein containing a fusion protein encoded by the nucleic acids described herein.

[0264] In certain embodiments, nucleic acids encoding different components of the multispecific antibody construct of the present invention may be inserted into the same expression vector. For example, the nucleic acid encoding the light chain of the anti-first target antigen may be cloned into the same vector as the nucleic acid encoding the heavy chain of the anti-first target antigen. In such embodiments, the two nucleic acids may be separated under the control of a single promoter by an intrasequence ribosome entry site (IRES) so that the light and heavy chains are expressed from the same mRNA transcript. Alternatively, the two nucleic acids may be under the control of two distinct promoters so that the light and heavy chains are expressed from two distinct mRNA transcripts. In some embodiments, the nucleic acids encoding the light and heavy chains of the anti-first target antigen are cloned into one expression vector, and the nucleic acids encoding the light and heavy chains of the anti-second target antigen are cloned into a second expression vector.

[0265] Similarly, for an IgG-Fab multispecific antibody construct, the nucleic acids encoding each of the three components may be cloned into the same expression vector. In some embodiments, the nucleic acid encoding the light chain of the IgG-Fab molecule and the nucleic acid encoding the second polypeptide (containing the other half of the C-terminal Fab domain) are cloned into one expression vector, while the nucleic acid encoding the modified heavy chain (a fusion protein containing the heavy chain and half of the Fab domain) is cloned into a second expression vector. In certain embodiments, all components of the multispecific antibody construct described herein are expressed from the same host cell population. For example, even if one or more components are cloned into separate expression vectors, the host cell is simultaneously transfected with both expression vectors, resulting in a single cell producing all components of the multispecific antibody construct.

[0266] After a vector is constructed and one or more nucleic acid molecules encoding components of the multispecific antibody construct described herein are inserted into appropriate sites on the vector, the completed vector can be inserted into a host cell suitable for amplification and / or polypeptide expression. Thus, the present invention encompasses isolated host cells containing one or more expression vectors encoding components of the multispecific antibody construct. The term “host cell,” as used herein, means a cell that is transformed with nucleic acid, or can be transformed, and thereby expresses the gene of interest. This term includes offspring of parental cells, regardless of whether the morphology or genetic structure of the offspring is identical to that of the original parental cell, as long as the gene of interest is present. In one embodiment, a host cell containing an isolated nucleic acid of the present invention operably ligated to at least one expression control sequence (e.g., a promoter or enhancer) is a “recombinant host cell.”

[0267] Transformation of selected host cells with an antigen-binding protein expression vector can be achieved by well-known methods, including transfection, infection, calcium phosphate coprecipitation, electroporation, microinjection, lipofection, DEAE-dextran-mediated transfection, or other known techniques. The chosen method will, to some extent, depend on the type of host cell used. These methods and other suitable methods are well known to those skilled in the art and are described, for example, Sambrook et al., 2001, cited above.

[0268] When cultured under appropriate conditions, host cells synthesize antigen-binding proteins, which can then be harvested from the culture medium (if the host cell secretes it into the medium) or directly from the host cell producing it (if it is not secreted). The selection of a suitable host cell will depend on various factors, including the desired expression level, polypeptide modifications desirable or required for activity (such as glycosylation or phosphorylation), and the ease of folding into a biologically active molecule.

[0269] Exemplary host cells include prokaryotes, yeasts, or higher eukaryotic cells. Prokaryotic host cells include eubacteria such as Gram-negative or Gram-positive microorganisms, such as Enterobacteriaceae, for example Escherichia, for example E. coli, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, for example Salmonella typhimurium, and Serratia, for example Serratia marcescens. This includes the genera *Marcescens*, *Shigella*, and *Bacillus*, such as *B. subtilis* and *B. licheniformis*, *Pseudomonas*, and *Streptomyces*. Eukaryotic microorganisms, such as filamentous fungi or yeasts, are suitable cloning or expression hosts for recombinant polypeptides. *Saccharomyces cerevisiae*, or common baker's yeast, is the most commonly used lower eukaryotic host microorganism.However, genera such as Pichia (e.g., P. pastoris), Schizosaccharomyces pombe, Kluyveromyces, Yarrowia, Candida, Trichoderma reesia, Neurospora crassa, and Schwanniomyces (e.g., Schwanniomyces occidentalis) are not included. Aspergillus (occidentalis), as well as filamentous fungi, such as the genera Neurospora, Penicillium, Tolypocladium, and Aspergillus, several other genera, species, and strains such as A. nidulans and A. niger are generally available and useful here.

[0270] Host cells for the expression of glycosylated antigen-binding proteins can be derived from multicellular organisms. Examples of invertebrate cells include plant cells and insect cells. Numerous baculovirus strains and variants, as well as corresponding insect-acceptable host cells derived from hosts such as the fall armyworm (Spodoptera frugiperda) (caterpillar), Aedes aegypti (mosquito), Aedes albopictus (mosquito), Drosophila melanogaster (fruit fly), and silkworm (Bombyx mori), have been identified. Various virus strains for transfection of such cells, e.g., the L-1 variant of Autographa californica NPV and the Bm-5 strain of silkworm (Bombyx mori) NPV, are publicly available.

[0271] Vertebrate host cells are also suitable hosts, and recombinant production of antigen-binding proteins from such cells is a common practice. Mammalian cell lines available as hosts for expression include, but are not limited to, immortalized cell lines available from the American Type Culture Collection (ATCC), such as, but are not limited to, Chinese hamster ovary (CHO) cells, e.g., CHOK1 cells (ATCC CCL61), DXB-11, DG-44 and Chinese hamster ovary cell / -DHFR (CHO, Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216, 1980); monkey kidney CV1 cell line transformed with SV40 (COS-7, ATCC CRL 1651); human embryonic kidney cell line (293 cells or 293 cells subcloned for growth in suspension culture, (Graham et al., J. Gen Virol. 36:59, 1977)); baby hamster kidney cells (BHK, ATCC CCL 10); Mouse Sertoli cells (TM4, Mather, Biol. Reprod. 23:243-251, 1980); Monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); Human cervical cancer cells (HELA, ATCC CCL 2); Canine kidney cells (MDCK, ATCC CCL 34); Buffalo rat liver cells (BRL 3A, ATCC CRL 1442); Human lung cells (W138, ATCC CCL 75); Human hepatocellular carcinoma cells (Hep G2, HB 8065); Mouse mammary cancer cells (MMT 060562, ATCC CCL 51); TRI cells (Mather et al., Annals NY Acad. Sci. 383:44-68, 1982); MRC 5 cells or FS4 cells; Mammalian myeloma cells and many other cell lines are also mentioned. In certain embodiments, cell lines may be selected by determining which cell lines have a high expression level of the multispecific antibody construct of the present invention and produce it constitutively. In other embodiments, cell lines derived from B cell lines that do not produce antibodies of their own but have the ability to produce and secrete heterologous antibodies may be selected.In some embodiments, CHO cells are host cells for expressing the multispecific antibody construct of the present invention.

[0272] For the production of multispecific antibody constructs, host cells are transformed or transfected with the nucleic acids or vectors described above and cultured in a conventional nutrient medium modified to be suitable for promoter induction, transformant selection, or amplification of genes encoding a desired sequence. In addition, novel vectors and transfected cell lines having multiple copies of transcription units separated by a selection marker are particularly useful for the expression of antigen-binding proteins. Accordingly, the present invention also provides a method for preparing a multispecific antibody construct as described herein, comprising culturing host cells containing one or more expression vectors as described herein in a culture medium under conditions that enable the expression of a multispecific antibody construct encoded by the one or more expression vectors; and recovering the multispecific antibody construct from the culture medium.

[0273] The host cells used to produce the antigen-binding protein of the present invention can be cultured in a variety of media. Commercial media such as Ham F10 (Sigma), Minimum Essential Medium (MEM, Sigma), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle Medium (DMEM, Sigma) are suitable for culturing host cells. Furthermore, any of the media described in Ham et al., Meth. Enz. 58:44, 1979; Barnes et al., Anal. Biochem. 102:255, 1980; U.S. Patent No. 4,767,704; U.S. Patent No. 4,657,866; U.S. Patent No. 4,927,762; U.S. Patent No. 4,560,655; or U.S. Patent No. 5,122,469; International Publication No. 90103430; International Publication No. 87 / 00195; or U.S. Reissue Patent No. 30,985 can be used as the culture medium for host cells. Any of these media may be supplemented as needed with hormones and / or other growth factors (such as insulin, transferrin, or epidermal growth factor), salts (such as sodium chloride, calcium, magnesium, and phosphates), buffers (such as HEPES), nucleotides (such as adenosine and thymidine), antibiotics (such as gentamicin®), trace elements (usually defined as inorganic compounds present at final concentrations in the micromolar range), and glucose or equivalent energy sources. Any other necessary nutritional supplements may also be included in appropriate concentrations known to those skilled in the art. Culture conditions such as temperature and pH are those previously used with host cells selected for expression and will be obvious to those skilled in the art.

[0274] When host cells are cultured, multispecific antibody constructs can be produced intracellularly, in the perimembranous space, or directly secreted into the culture medium. If the antigen-binding protein is produced intracellularly, the first step is to remove particulate fragments, i.e., host cells or lysed fragments, for example, by centrifugation or ultrafiltration. The bispecific antigen-binding protein can be purified using, for example, hydroxyapatite chromatography, cation or anion exchange chromatography, or affinity chromatography using the antigen of interest or protein A or protein G as the affinity ligand. Protein A can be used to purify proteins containing polypeptides based on human γ1, γ2, or γ4 heavy chains (Lindmark et al., J.Immunol.Meth.62:1-13, 1983). Protein G is recommended for all mouse isotypes and human γ3 (Guss et al., EMBO J.5:15671575, 1986). The matrix to which the affinity ligand binds is most often agarose, but other matrices are also available. Mechanically stable matrices such as controlled pore glass or poly(styrenedivinyl)benzene allow for faster flow rates and shorter processing times than those achievable with agarose. If the protein contains a CH3 domain, Bakerbond ABX® resin (JTBaker, Phillipsburg, NJ) is useful for purification. Depending on the specific multispecific antibody construct to be recovered, other techniques for protein purification such as ethanol precipitation, reverse-phase HPLC, chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation are also possible.

[0275] The multispecific antibody constructs of the present invention are useful for detecting target antigens in biological samples and for identifying cells or tissues that express target antigens. The multispecific antibody constructs described herein may be used for diagnostic purposes to detect, diagnose, or monitor diseases and / or conditions associated with target antigens. Furthermore, methods are provided for detecting the presence of target antigens in a sample using classical immunohistochemical methods known to those skilled in the art (e.g., Tijssen, 1993, Practice and Theory of Enzyme Immunoassays, Vol 15 (Eds RHBurdon and PH van Knippenberg, Elsevier, Amsterdam); Zola, 1987, Monoclonal Antibodies: A Manual of Techniques, pp. 147-158 (CRC Press, Inc.); Jalkanen et al., 1985, J. Cell. Biol. 101: 976-985; Jalkanen et al., 1987, J. Cell Biol. 105: 3087-3096). Detection of any target can be performed in vivo or in vitro.

[0276] One embodiment provides a multispecific antibody construct of the present invention or a multispecific antibody construct produced according to the process of the present invention for use in the prevention, treatment, or improvement of tumors or cancerous or metastatic cancerous diseases.

[0277] According to a preferred embodiment of the present invention, the tumor or cancerous disease is a solid tumor disease.

[0278] The formulations described herein are useful as pharmaceutical compositions for treating, improving and / or preventing the pathological medical conditions described herein in patients in need thereof. The term “treatment” refers to both therapeutic treatment and preventive or deterrent measures. Treatment includes the application or administration of formulations to the body, isolated tissues or cells of a patient having a disease / disorder, symptoms of a disease / disorder, or predisposition to a disease / disorder, with the aim of curing, resolving, alleviating, mitigating, altering, correcting, improving, reversing, or influencing the disease, symptoms of a disease or predisposition to a disease.

[0279] As used herein, the term “improvement” means any improvement in the disease state of a patient having a tumor or cancer or metastatic cancer as described herein, by administration of the multispecific antibody construct according to the present invention to a target requiring such construct. Such improvement may also be considered as slowing or halting the progression of the patient’s tumor or cancer or metastatic cancer. As used herein, the term “prevention” means avoiding the onset or recurrence of a patient having a tumor or cancer or metastatic cancer as described herein, by administration of the multispecific antibody construct according to the present invention to a target requiring such construct.

[0280] The term “disease” refers to any condition that would benefit from treatment with the multispecific antibody constructs or pharmaceutical compositions described herein. This includes chronic and acute disorders or diseases, including pathological conditions that make mammals susceptible to the disease of interest. A “neoplasm” is an abnormal growth of tissue, which is not necessarily but usually forms a mass. When it forms a mass, it is generally called a “tumor.” Neoplasms or tumors can be benign, occult malignant (precancerous), or malignant. Malignant neoplasms are generally called cancers. They can usually invade and destroy surrounding tissues and form metastases, that is, they spread to other parts, tissues, or organs of the body. Thus, the term “metastatic cancer” includes metastases to other tissues or organs other than those of the primary tumor. Lymphomas and leukemias are lymphoid neoplasms. For the purposes of this invention, they are also included in the terms “tumor” or “cancer.”

[0281] In preferred embodiments of the present invention, the tumor or cancerous disease is a solid tumor disease, and the metastatic cancerous disease may originate from any of the above.

[0282] Preferred tumors or cancerous diseases according to the present invention are selected from the group consisting of breast cancer, carcinoid cancer, cervical cancer, colorectal cancer, endometrial cancer, gastric cancer, head and neck cancer, mesothelioma, liver cancer, lung cancer, ovarian cancer, pancreatic cancer, prostate cancer, skin cancer, kidney cancer, and gastric cancer. More preferably, tumors or cancerous diseases that are solid tumor diseases may be selected from the group consisting of ovarian cancer, pancreatic cancer, mesothelioma, lung cancer, gastric cancer, and 3-negative breast cancer. Metastatic cancerous diseases may originate from any of the above.

[0283] The present invention also provides a method for the treatment or improvement of a tumor or cancerous disease or metastatic cancerous disease, comprising the step of administering the multispecific antibody construct of the present invention or a multispecific antibody construct produced according to the process of the present invention to a subject in need thereof.

[0284] The terms “in need” or “in need of treatment” include subjects who already have the disorder and subjects for whom the disorder will be prevented. “In need” or “patient” includes human and other mammalian subjects receiving either preventive or therapeutic treatment.

[0285] The multispecific antibody constructs of the present invention are generally designed to suit specific routes and methods of administration, specific doses and frequencies of administration, and specific treatments for specific diseases, particularly in terms of bioavailability and persistence. The materials of the composition are preferably formulated at concentrations acceptable at the administration site.

[0286] Therefore, formulations and compositions can be designed in accordance with the present invention to be delivered by any preferred route of administration. In relation to the present invention, the route of administration is: • Local routes (e.g., on the skin, by inhalation, nose, eyes, auricle / ear, vagina, mucous membranes); • Intestinal pathways (e.g., oral, gastrointestinal, sublingual, sublabial, buccal, rectal); and This includes, but is not limited to, parenteral routes (e.g., intravenous, intraarterial, intraosseous, intramuscular, intracerebral, intraventricular, epidural, intramedullary, subcutaneous, intraperitoneal, extraamniotic, intraarticular, intracardiac, intradermal, intrafocal, intrauterine, intrabladder, intravitreous, percutaneous, intranasal, transmucosal, synovial bursa, intraluminal).

[0287] The pharmaceutical composition and multispecific antibody construct of the present invention are particularly useful for parenteral administration, such as subcutaneous or intravenous delivery, by injection, such as bolus injection, or by infusion, such as continuous infusion. The pharmaceutical composition may be administered using a medical device. Examples of medical devices for administering pharmaceutical compositions are described in U.S. Patent Nos. 4,475,196; 4,439,196; 4,447,224; 4,447,233; 4,486,194; 4,487,603; 4,596,556; 4,790,824; 4,941,880; 5,064,413; 5,312,335; 5,312,335; 5,383,851; and 5,399,163.

[0288] In particular, the present invention enables the uninterrupted administration of a preferred composition. As a non-limiting example, uninterrupted or substantially uninterrupted, i.e., continuous administration can be achieved by a patient-worn miniature pump system for regulating the inflow of the therapeutic agent into the patient's body. A pharmaceutical composition comprising the multispecific antibody construct of the present invention can be administered by using such a pump system. Such pump systems are generally known in the art and typically rely on the periodic replacement of a cartridge containing the therapeutic agent to be injected. When a cartridge is replaced in such a pump system, a temporary interruption may occur in the inflow of the therapeutic agent into the patient's body, which is otherwise uninterrupted. Even in such cases, the administration stage before and after cartridge replacement will still be considered within the meaning of the pharmaceutical means and methods of the present invention, which constitute the “uninterrupted administration” of such therapeutic agent.

[0289] The multispecific antibody construct of the present invention may be administered intravenously or subcutaneously by a fluid delivery device or a small pump system comprising a fluid delivery mechanism for delivering fluid from a reservoir and a drive mechanism for driving the delivery mechanism. A pump system for subcutaneous administration may include a needle or cannula for penetrating the patient's skin and delivering the preferred composition into the patient's body. The pump system can be directly fixed or attached to the patient's skin, whether vein, artery, or blood vessel, to allow direct contact between the pump system and the patient's skin. This pump system can be attached to the patient's skin for 24 hours to several days. There may also be small pump systems with a small reservoir volume. In non-limiting examples, the reservoir volume for the preferred pharmaceutical composition to be administered may be 0.1 to 50 ml.

[0290] Continuous administration can also be percutaneous, using patches that are applied to the skin and replaced from time to time. Those skilled in the art are aware of patch systems for drug delivery suitable for this purpose. It should be noted that percutaneous administration is particularly suitable for uninterrupted administration, for example, because a new second patch can be applied to the skin surface directly adjacent to the first used patch, immediately before the first used patch is removed, and the replacement of the first used patch can be completed at the same time. There are no problems with inflow interruption or battery failure.

[0291] If the pharmaceutical composition is lyophilized, the lyophilized material is first reconstituted with an appropriate liquid before administration. The lyophilized material can be reconstituted with, for example, bacteriostatic water for injection (BWFI), physiological saline, phosphate-buffered saline (PBS), or the same formulation in which the protein was present before lyophilization.

[0292] The compositions of the present invention can be administered to subjects at a suitable dose, which can be determined by dose-escalation studies, for example, by administering the interspecies-specific multispecific antibody constructs of the present invention described herein to primates other than chimpanzees, such as macaques, in increasing doses. As described herein, the interspecies-specific multispecific antibody constructs of the present invention have the advantage of being usable in the same form in preclinical studies in primates other than chimpanzees and being usable as drugs in humans. The administration plan will be determined by the attending physician based on clinical factors. As is well known in the medical field, the dosage for a given patient depends on many factors, including the patient's size, body surface area, age, the individual compounds administered, sex, time and route of administration, overall health, and other drugs being administered simultaneously.

[0293] The term “effective dose” or “effective dosage” is defined as the amount sufficient to achieve, or at least partially achieve, the desired effect. The term “therapeutic effective dose” is defined as the amount sufficient to cure, or at least partially suppress, the disease and its complications in a patient who already has the disease. The amount or dose that is effective for this use will depend on the condition being treated (indication), the multispecific antibody construct being delivered, the nature and purpose of the treatment, the severity of the disease, prior treatment, the patient’s medical history and responsiveness to the drug, the route of administration, body size (weight, body surface area, or organ size) and / or the patient’s condition (age and overall health), as well as the patient’s overall immune system status. The appropriate dose may be adjusted at the discretion of the attending physician so that it can be administered to the patient in a single dose or in multiple doses, and to obtain the optimal therapeutic effect.

[0294] A therapeutically effective amount of the multispecific antibody construct of the present invention preferably reduces the severity of disease symptoms, increases the frequency or duration of disease-free periods, or prevents functional impairment or disability resulting from the suffering of the disease. With regard to the treatment of MSLN-expressing tumors, a therapeutically effective amount of the multispecific antibody construct of the present invention, for example, an anti-MSLN / anti-CD40 multispecific antibody construct, preferably inhibits cell proliferation or tumor growth by at least about 20%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% compared to untreated patients. The ability of a compound to inhibit tumor growth can be evaluated in animal models that serve as predictors of efficacy in human tumors.

[0295] The pharmaceutical composition may be administered as a single treatment or, as necessary, in combination with additional therapies such as anticancer therapy, for example, other proteinaceous and non-proteinaceous drugs. These drugs may be administered simultaneously with the composition comprising the multispecific antibody construct of the present invention as defined herein, or separately at predetermined time intervals and doses before or after the administration of the multispecific antibody construct.

[0296] As used herein, the term “effective and non-toxic dose” refers to an acceptable dose of the multispecific antibody construct of the present invention that is sufficient to result in a drastic reduction of diseased cells, tumor removal, tumor regression, or disease stabilization without causing or essentially causing serious toxic effects. Such an effective and non-toxic dose may be determined, for example, by dose escalation studies described in the Art, and the dose should be less than the dose that induces serious adverse events (dose-limiting toxicity, DLT).

[0297] As used herein, the term “toxicity” refers to the toxic effects of a drug that manifest as adverse events or serious adverse events. These adverse events may refer to systemic drug intolerance and / or local intolerance after administration. Toxicity may also include teratogenic or carcinogenic effects caused by the drug.

[0298] As used herein, the terms “safety,” “in vivo safety,” or “tolerability” are defined as the administration of a drug that does not induce serious adverse events immediately after administration (local tolerance) and during longer periods of drug use. “Safety,” “in vivo safety,” or “tolerability” can be assessed periodically, for example, during treatment and follow-up periods. Measurements include clinical assessments, e.g., screening for organ findings and abnormal clinical laboratory values. Clinical assessments may be performed, and deviations from normal findings may be recorded / coded according to NCI-CTC and / or MedDRA standards. Organ findings may include, for example, criteria such as allergy / immunology, blood / bone marrow, cardiac arrhythmias, and coagulation, as shown in the Common Terminology Criteria for adverse events v3.0 (CTCAE). Laboratory parameters that may be tested include, for example, hematology, clinical chemistry, coagulation profiles, and urinalysis, as well as tests of other body fluids, e.g., serum, plasma, lymph, or cerebrospinal fluid. Therefore, safety can be evaluated by measuring examination parameters and recording adverse events, for example, through physical examination, imaging techniques (i.e., ultrasound, X-ray, CT scan, magnetic resonance imaging (MRI), other measurements using technical devices (i.e., electrocardiogram), and vital signs. For example, in the use and methods according to the present invention, adverse events in primates other than chimpanzees may be tested by histopathological and / or histochemical methods. The above terms are also referenced, for example, in the Preclinical safety evaluation of biotechnology-derived pharmaceuticals S6; ICH Harmonised Tripartite Guideline; ICH Steering Committee meeting of July 16, 1997.

[0299] In further embodiments, the present invention provides a kit comprising the multispecific antibody construct of the present invention, the multispecific antibody construct produced by the method of the present invention, the polynucleotide of the present invention, the vector of the present invention, and / or the host cell of the present invention.

[0300] In relation to the present invention, the term "kit" means two or more components packaged together in a container, vessel, or other, where one of the components corresponds to the multispecific antibody construct, pharmaceutical composition, vector, or host cell of the present invention. Thus, a kit can be described as a set of products and / or equipment sufficient to achieve a particular purpose, which can be sold individually.

[0301] The kit may include one or more containers of any suitable shape, size, and material (preferably waterproof, e.g., plastic or glass) containing a dose of the multispecific antibody construct or pharmaceutical composition of the present invention suitable for administration (see above). The kit may further include instructions for use (e.g., in the form of a leaflet or instruction manual), means for administering the multispecific antibody construct of the present invention, means for reconstituting the multispecific antibody construct of the present invention, e.g., syringes, pumps, infusors, etc., and / or means for diluting the multispecific antibody construct of the present invention.

[0302] The present invention also provides kits for single-dose units. The kits of the present invention may also include a first container containing a dried / lyophilized multispecific antibody construct and a second container containing an aqueous formulation. In certain embodiments of the present invention, kits are provided that include single-chamber and multi-chamber pre-filled syringes (e.g., liquid syringes and rio-syringes). [Examples]

[0303] Production and characterization of anti-mesoserine antibodies Anti-mesoselin antibody production. Fully human antibodies against human mesoserine were produced as previously described (U.S. Patent Application Publication No. 20170029502A1) or by immunizing XENOMOUSE® transgenic mice (U.S. Patents No. 6,114,598, 6,162,963, 6,833,268, 7,049,426, 7,064,244, incorporated herein by reference as a whole; Green et al., 1994, Nature Genetics 7:13-21; Mendez et al., 1997, Nature Genetics 15:146-156; Green and Jakobovitis, 1998, J.Ex.Med, 188:483-495; Kellerman and Green, Current Opinion in Biotechnology). (13,593-597, 2002). Animals derived from XMG4-K and XMG4-KL XENOMOUSE® strains were used for these immunizations. The animals were alternately immunized with soluble human mesoserine-His and cynomolgus monkey mesoserine-His. Animals with intrinsic titers to the highest antigen-specific serum directed towards human mesoserine and cynomolgus monkey mesoserine were used for hybridoma production (Kohler and Milstein, 1975). Pooled lymphocytes derived from the spleen and / or inflow region lymph nodes (from each harvest) were isolated from lymphoid tissue by grinding in a suitable medium (e.g., Dulbecco's Modified Eagle Medium (DMEM); Invitrogen, Carlsbad, CA). B cells were selected and / or proliferated using standard methods and fused with suitable fusion partners using techniques known in the art. Next, hybridoma supernatants containing binding to human mesoserine and cynomolgus monkey mesoserine were selected for further characterization.

[0304] Sequencing of anti-mesoserine antibodies. For antibodies derived from XENOMOUSE®, RNA (all or mRNA) was purified from wells containing anti-mesoserine antibody-producing hybridoma cells using the Qiagen RNeasy mini or Invitrogen mRNA catcher plus kit. Using the purified RNA, cDNA synthesis was performed by reverse transcription, followed by amplification of the antibody heavy chain and light chain variable region (V) genes by polymerase chain reaction (RT-PCR). Fully human antibody gamma heavy chains were obtained using the Qiagen 1-step reverse transcriptase PCR kit (Qiagen). Fully human kappa light chains were obtained using the Qiagen 1-step reverse transcriptase PCR kit (Qiagen). Amino acid sequences were predicted from the corresponding nucleic acid sequences by bioinformatics. Next, the obtained amino acid sequences were analyzed to determine the germline sequence origin of the antibodies and identify differences from the germline sequences. The amino acid sequences corresponding to the complementarity-determining regions (CDRs) of the sequenced antibodies were aligned, and these alignments were used to group clones by similarity.

[0305] Antibody production. The selected and sequenced anti-mesoserine antibody heavy and light chains were subcloned into mammalian expression vectors to generate individual antibodies, which were then purified. All antibodies were reformatted with the human IgG1SEFL2(REF) heavy chain sequence.

[0306] Binding of antibodies to human and cynomolgus monkey mesoserine. The binding of anti-mesoserine antibodies to human mesoserine was confirmed by flow cytometry of CHO cells engineered to express human mesoserine (huMSLN-CHO). Human MSLN-transfected CHO cells were incubated with various concentrations of purified anti-mesoserine antibody, washed, and labeled with a secondary antibody conjugated with human IgG-specific fluorescence. The cells were then analyzed by flow cytometry. The percentage of fluorescence-positive cells was plotted against antibody concentration (Figure 1), and EC 50The binding affinity of anti-human mesoserine to the soluble forms of recombinant human and cynomolgus monkey (cyno) mesoserine was also measured by the Octet assay (Table 1), which quantifies the binding rate (Kon), dissociation rate (Kdis), and equilibrium binding constant (KD). These data demonstrated the binding of all anti-MSLN antibodies to both human and cynomolgus monkey mesoserine.

[0307] [Table 1]

[0308] Production and characterization of anti-CD40 agonist antibodies Production of anti-CD40 antibodies. Fully human antibodies against human CD40 were produced by immunizing XENOMOUSE® transgenic mice (U.S. Patent Nos. 6,114,598; 6,162,963; 6,833,268; 7,049,426; 7,064,244; Green et al., 1994, Nature Genetics 7:13-21; Mendez et al., 1997, Nature Genetics 15:146-156; Green and Jakobovitis, 1998, J.Ex.Med, 188:483-495; Kellerman and Green, Current Opinion in Biotechnology 13,593-597, 2002). Animals derived from XMG4-K and XMG4-KL XENOMOUSE® strains were used for this immunization. Multiple immunogens and immunization pathways were used to generate anti-human CD40 immune responses. For immunization with soluble recombinant protein, mice were alternately immunized with soluble human CD40-Fc and cynomolgus monkey CD40-Fc. For cell-based immunization, CHO-S cells were transiently transfected with either wild-type human CD40 or cynomolgus monkey CD40 as the immunogen source. Animals were immunized with either of these transiently transfected CHO cells. Animals with intrinsic titers to the highest antigen-specific serum directed towards human CD40 and cynomolgus monkey CD40 were used for hybridoma production (Kohler and Milstein, 1975). Pooled lymphocytes derived from the spleen and / or influx region lymph nodes (from each harvest) were isolated from lymphoid tissue by grinding in a suitable medium (e.g., Dulbecco's Modified Eagle Medium (DMEM); Invitrogen, Carlsbad, CA). B cells were selected and / or proliferated using standard methods and fused with suitable fusion partners using techniques known in the art. Hybridoma supernatants with binding to human and cynomolgus monkey CD40 were identified.

[0309] Antibody production. Using selected hybridoma supernatants that exhibited binding to human and cynomolgus monkey CD40, purified anti-CD40 antibodies were generated using techniques known in the art (Table 2).

[0310] [Table 2]

[0311] Anti-CD40 antibodies that bind to human and cynomolgus monkey CD40. The binding affinity of anti-human CD40 antibodies to human and cynomolgus monkey CD40 was also measured by the Octet assay (Table 3), which quantifies the binding rate (Kon), dissociation rate (Kdis), and equilibrium binding constant (KD). Cross-reactivity of anti-CD40 antibodies to relevant TNF receptor superfamily (TNFRSF) members was assessed by transient transfection of human TNFR1, TNFR2, TNFR10, or TNFR14 on HEK293 cells using expression vectors encoding these genes. Binding of specified anti-human CD40 antibodies to these transfected cells was determined by flow cytometry. Minimal binding of anti-CD40 to other TNFRSF members was demonstrated by assessing geometric mean fluorescence intensity compared to control cells (Table 4). Cross-reactivity of anti-CD40 antibodies to cynomolgus monkey and mouse CD40 was assessed by transient transfection of cynomolgus monkey or mouse CD40 on HEK293 cells using expression vectors encoding these genes. The binding of a specified anti-human CD40 antibody to cells overexpressing cynomolgus monkey and mouse CD40 was determined by flow cytometry. By evaluating the geometric mean fluorescence intensity compared to control cells, we demonstrated robust binding of anti-CD40 to cynomolgus monkey (cyno) CD40 but no binding to mouse CD40 (Table 5). Together, these data demonstrate strong and equivalent binding of the anti-CD40 antibody to human and cynomolgus monkey CD40, but minimal binding to relevant TNFRSF members or mouse CD40.

[0312] [Table 3]

[0313] [Table 4]

[0314] [Table 5]

[0315] Crosslinking-dependent activation of CD40 by anti-CD40 antibodies. Stimulation of the CD40 receptor on human B cells results in activation and proliferation. Purified primary human B cells were seeded in 384-well assay plates and treated with IL-4 / IL-21 as a comitogen, various concentrations of anti-CD40 antibody, and either in the presence (Figure 2A) or absence (Figure 2B) of protein G. A single molecule of protein G can bind to multiple IgG molecules and therefore crosslink antibodies in solution. Cells were incubated for 5 days, and cell proliferation was tested using CellTiter-Glo. Since all anti-CD40 antibodies tested were IgG4, an unrelated IgG4 isotype antibody was included as a negative control. EC50 and percentage of maximum activity were calculated (Table 6). All anti-CD40 antibodies induced robust proliferation of B cells in the presence of protein G but exhibited minimal activity in the absence of protein G, indicating that anti-CD40 antibodies alone that bind to CD40 are insufficient to stimulate CD40, and that additional crosslinking of antibodies is required.

[0316] [Table 6]

[0317] The effect of anti-CD40 antibodies on the binding of human CD40 ligand to human CD40. To evaluate the ability of anti-CD40 antibodies to block the interaction between CD40 and its ligand, a flow cytometry-based ligand binding assay was performed to measure the binding of fluorescently labeled soluble human CD40L to human CD40 overexpressed on 293T cells by transient transfection with an expression vector encoding this gene. The percentage of ligand binding inhibition was calculated as [(CD40L binding gMFI in the absence of anti-CD40) - (CD40L binding gMFI in the presence of anti-CD40)] / (CD40L binding gMFI in the absence of anti-CD40). gMFI: geometric mean of fluorescence intensity. All anti-CD40 antibodies showed minimal effect on the ability of CD40L to bind to the CD40 receptor (Table 7).

[0318] [Table 7]

[0319] The effect of anti-human CD40 antibodies on CD40L-induced B cell activation. Several antibodies specific to TNFRSF members have been shown to enhance receptor activation by their normal ligands by potentially clustering the receptor and lowering the threshold for ligand-induced activation. To evaluate the ability of anti-CD40 antibodies to enhance CD40 ligand activity, purified primary human B cells were treated with various concentrations of recombinant human CD40L in or without a specified CD40 antibody at 1 ug / ml. Cells were incubated for 5 days, and cell proliferation was tested using CellTiter-Glo. None of the anti-CD40 antibodies induced significant changes in EC50 of CD40 ligands that stimulate human B cell proliferation (Table 8).

[0320] [Table 8]

[0321] Sequencing of anti-CD40 antibodies. RNA (all or mRNA) was purified from wells containing anti-mesoserine antibody-producing hybridoma cells using the Qiagen RNeasy mini or Invitrogen mRNA catcher plus kit. The purified RNA was used to amplify the antibody heavy chain and light chain variable region (V) genes by reverse transcription of cDNA synthesis, followed by polymerase chain reaction (RT-PCR). The fully human antibody gamma heavy chain was obtained using the Qiagen 1-step reverse transcriptase PCR kit (Qiagen). The fully human kappa light chain was obtained using the Qiagen 1-step reverse transcriptase PCR kit (Qiagen). The amino acid sequences were predicted by bioinformatics from the corresponding nucleic acid sequences. Next, the obtained amino acid sequences were analyzed to determine the germline sequence origin of the antibody and identify differences from the germline sequence. The amino acid sequences corresponding to the complementarity-determining regions (CDRs) of the sequenced antibodies were aligned, and these alignments were used to group clones by similarity.

[0322] Optimization of anti-CD40 antibody sequences. The selected anti-CD40 antibodies were manipulated using standard recombinant DNA techniques to remove potentially undesirable sequences in the antibody heavy and light chain variable regions. These antibodies were produced by overexpression in cell lines and purified using techniques known in the art. The purified antibodies were then evaluated in human B cell proliferation assays, both with and without protein G crosslinking, as described above. 50 The percentage of maximum activity was calculated (Table 9), which indicates that many of the manipulated antibody variants maintained similar crosslink-dependent agonist activity to their parent antibody sequences.

[0323] [Table 9]

[0324] Preparation and Characterization of Mesoserine-Targeted CD40 Agonist Bivalent Bispecific Antibodies Production of bispecific antibodies. To produce mesoserine-dependent CD40 agonists, bivalent bispecific antibodies capable of binding to both human mesoserine and human CD40 were prepared in either IgG-Fab format (Figure 3A) or IgG-scFv format (Figure 3B) using the variable region binding domains of the above-mentioned anti-mesoserine and anti-CD40 antibodies.

[0325] Preparation and evaluation of mesoserine × CD40 bispecific agonist antibodies in IgG-Fab format. A series of mesoserine × CD40 bispecific antibodies were prepared in IgG-Fab format (Table 10), and their binding to human and cynomolgus monkey CD40 and human and cynomolgus monkey mesoserine in soluble forms was evaluated using the Octet assay (Table 11). Binding rate (K on ), dissociation rate (K dis ), and equilibrium coupling constant (K D The EC50 values ​​for CD86 upregulation and markers of CD40-mediated B cell activation were calculated. The majority of the bispecific antibodies showed high affinity binding to both human and cynomolgus monkey CD40 and mesoserine. Next, these bispecific antibodies were evaluated for their ability to induce mesoserine-inducible activation of human B cells. Human mesoserine-expressing CHO cells were seeded in 96-well plates. The following day, various concentrations of mesoserine × CD40 IgG-Fab antibodies were added to the wells along with purified human B cells, and the plates were incubated for a further 48 hours. Upregulation of CD86 and markers of CD40-mediated B cell activation were quantified on B cells by flow cytometry. EC50 values ​​for CD86 upregulation were calculated, demonstrating that the majority of the mesoserine × CD40 IgG-Fab bispecific antibodies enabled B cell activation in the presence of human mesoserine-expressing CHO cells (Table 12).

[0326] [Table 10]

[0327] [Table 11]

[0328] [Table 12]

[0329] [Table 13]

[0330] Preparation and evaluation of mesoserine × CD40 bispecific agonist antibodies in IgG-scFv format. A series of mesoserine × CD40 bispecific antibodies were prepared in IgG-scFv form (Table 13), and their binding to human and cynomolgus monkey CD40 and human and cynomolgus monkey mesoserine in soluble forms was evaluated using the Octet assay (Table 14). Binding rate (Kon), dissociation rate (Kdis), and equilibrium binding constant (KD) were calculated. The majority of the bispecific antibodies showed high affinity binding to both human and cynomolgus monkey CD40 and mesoserine. Next, these bispecific antibodies were evaluated for their ability to induce mesoserine-inducible activation of human B cells. Human mesoserine-expressing CHO cells were seeded in 96-well plates. The following day, various concentrations of mesoserine × CD40 IgG-scFv antibody were added to the wells along with purified human B cells, and the plates were incubated for a further 48 hours. Markers for CD86 upregulation and CD40-mediated B cell activation were quantified on B cells by flow cytometry. EC50 values ​​for CD86 upregulation were calculated, demonstrating that the majority of mesoserine × CD40 IgG-scFv bispecific antibodies enabled B cell activation in the presence of human mesoserine-expressing CHO cells (Table 15).

[0331] [Table 14]

[0332] [Table 15]

[0333] [Table 16]

[0334] [Table 17]

[0335] [Table 18]

[0336] Sequence optimization of mesoserine × CD40 bispecific agonist antibodies in IgG-scFv format. The selected mesoserine × CD40 bispecific IgG-scFv antibodies were manipulated using standard recombinant DNA techniques to remove potentially undesirable sequences in the antibody heavy and light chain variable regions. These antibodies were produced by overexpression in cell lines and purified using techniques known in the art. Next, these bispecific antibodies were evaluated for their ability to induce mesoserine-inducible activation of human B cells. Human mesoserine-expressing CHO cells were seeded in 96-well plates. The following day, various concentrations of mesoserine × CD40 IgG-scFv sequence variant antibodies were added to the wells along with purified human B cells, and the plates were incubated for a further 48 hours. Upregulation of CD86, a marker of CD40-mediated B cell activation, was quantified on B cells by flow cytometry. EC50 values ​​were calculated for upregulation of CD86, demonstrating that the majority of mesoserine × CD40 IgG-scFv sequence variant bispecific antibodies retain their ability to induce B cell activation in the presence of human mesoserine-expressing CHO cells (Table 16).

[0337] [Table 19]

[0338] Effects of various levels of mesoserine on the activity of selected mesoserine × CD40 bispecific antibodies in human B cell functional assays. MC38 cells were engineered to express varying levels of human mesoserine, ranging from low (MC38.MSLN1) to high (MC38.MSLN5). Since MC38 cells are mouse-derived, they do not express human mesoserine (Figure 4). Parental MC38 cells and MC38 cells expressing varying levels of human mesoserine were seeded in 96-well plates and cultured overnight. The following day, mesoserine × CD40 bispecific antibodies were added to the cells at varying concentrations. Next, isolated human B cells were added to the wells, and the plates were incubated for a further 48 hours. Upregulation of CD86 on B cells was assessed by flow cytometry and used as a measure of B cell activation. EC50 and maximum activity (Emax) values ​​were calculated, demonstrating that the mesoserine × CD40 bispecific antibody was able to induce B cell activation across all levels of MSLN expression (Table 17). Cell lines expressing lower levels of mesoserine induced lower maximal activity of the mesoserine × CD40 bispecific antibody, as measured by the geometric mean of the fluorescence intensity of CD86 upregulation on B cells. The mesoserine × CD40 bispecific antibody was inactive in the presence of parental MC38 cells, consistent with the mesoserine-dependent activity of these molecules against CD40 agonist activity.

[0339] [Table 20]

[0340] [Table 21]

[0341] Effects of various levels of mesoserine on the activity of selected mesoserine × CD40 bispecific antibodies in functional assays of monocyte-derived dendritic cells. MC38 cells engineered to express varying levels of human mesoserine (Figure 4) or parental MC38 cells were seeded in 96-well plates and cultured overnight. The following day, mesoserine × CD40 bispecific antibody was added to the cells at varying concentrations. Dendritic cells derived from monocytes differentiated from human monocytes using GMCSF and IL4 were added to the wells, and the plates were incubated for a further 48 hours. The supernatant was collected from each well, and the concentration of IL12p40 secreted from the dendritic cells was quantified using an ELISA kit. EC50 and maximum activity (Emax) values ​​were calculated, demonstrating that the mesoserine × CD40 bispecific antibody was able to induce dendritic cell activation across both high and low levels of MSLN expression (Table 17). Cell lines expressing lower levels of mesoserine induced lower maximum activity of the mesoserine × CD40 bispecific antibody. Mesoserine × CD40 bispecific antibodies showed no activity in the presence of parental MC38 cells, consistent with the mesoserine-dependent activity of these molecules against CD40 agonist activity.

[0342] Activity of selected mesoserine × CD40 bispecific antibodies in a cynomolgus monkey B cell functional assay using cynomolgus monkey mesoserine-expressing CHO cells. CHO cells engineered to express cynomolgus monkey mesoserine were added to 96-well plates and cultured overnight. The following day, selected mesoserine × CD40 bispecific antibodies were added to the cells at various concentrations. Next, isolated cynomolgus monkey B cells were added to the wells, and the plates were incubated for a further 48 hours. Upregulation of CD23 on cynomolgus monkey B cells was assessed by flow cytometry and used as a measure of CD40-induced B cell activation. EC50 was calculated, demonstrating that the mesoserine × CD40 bispecific antibody can induce cynomolgus monkey B cell activation in the presence of cynomolgus monkey mesoserine (Table 18).

[0343] [Table 22]

[0344] Pharmacokinetics and stability of mesoserine × CD40 bispecific antibody in mice. To characterize the stability of mesoserine × CD40 bispecific antibodies in vivo, selected mesoserine × CD40 bispecific molecules were injected into mice at a dose of 1 mg / kg. Animals were bled at various time points after injection, and serum was isolated. Using immunoassays, the concentration of mesoserine × CD40 bispecific antibodies in serum was quantified by capturing with human CD40 and detecting with anti-human IgG, or capturing with human mesoserine and detecting with anti-human IgG. The combination of these two assays provides the ability to detect molecular degradation or instability. The CD40 or mesoserine capture assay yielded comparable results, suggesting that the molecules remained unchanged for 14 days in vivo (Figure 5). All molecules except one had similar pharmacokinetic properties.

[0345] Immunostimulatory and antitumor activity of tumor-targeted CD40 agonist bispecific antibodies in mouse models Production of mouse-surrogate human EPCAM-dependent CD40 agonist bispecific antibodies and human EPCAM-expressing mouse tumor cell lines. Using an anti-mouse CD40 agonist antibody (clone FGK45) and anti-human EPCAM antibodies (clones 4-7), a surrogate CD40 bispecific antibody (anti-muCD40 × huEPCAM bispecific antibody) in the IgG-scFv form possessing the mouse IgG1 N297G Fc domain was constructed. The bispecific antibody contains anti-CD40 at the N-terminus of the molecule and anti-EPCAM as scFv at the C-terminus of the molecule. Human EPCAM was overexpressed in mouse MC38 colon cancer cell lines and mouse B16F10 melanoma cell lines by transduction with retroviruses encoding both human EPCAM and human truncated nerve growth factor receptor (NGFR) reporter genes (Figure 6). Human EpCAM-MS38 or human EpCAM-B16 tumor cells were inoculated into the right flank of the graft at a rate of 3e5 cells per graft and grown for 24 days or 11 days, respectively. Tumors were measured twice weekly with digital calipers to demonstrate that tumor cell lines expressing human EPCAM maintained their ability to grow and form tumors in mice. Flow cytometry of tumors collected from mice and subjected to enzymatic dissociation demonstrated that human EPCAM expression was maintained on the surface of tumor cells during in vivo tumorigenesis (Figure 6).

[0346] Human EPCAM-dependent agonist activity of EPCAM×CD40 agonist antibody on mouse B cells. Human EpCAM-overexpressing MC38 cells (huEpCAM-MC38) or parental MC38 cells were seeded in 96 plates and cultured overnight. The following day, anti-muCD40×huEPCAM bispecific antibody, parental anti-CD40 antibody (FGK54), or isotype control antibody were added to the cells at various concentrations. Next, isolated mouse B cells were added to the wells, and the plates were incubated for a further 48 hours. Upregulation of CD86 on B cells was assessed by flow cytometry and used as a measure of B cell activation. EC50 values ​​were calculated, demonstrating that the anti-muCD40×huEPCAM bispecific antibody was able to induce mouse B cell activation (Figure 7). The anti-muCD40×huEPCAM bispecific antibody demonstrated robust agonist activity dependent on the presence of human EPCAM and showed dramatically higher efficacy in activating B cells compared to the FGK45 anti-CD40 monoclonal antibody.

[0347] In vivo immunostimulatory activity of anti-muCD40 × huEPCAM bispecific antibody in MC38 tumor model To evaluate the effect of a tumor-targeted CD40 agonist bispecific antibody on in vivo immune cell activation, human EpCAM-expressing MC38 tumor cells were inoculated into the right flank of mice at a rate of 3e5 cells per graft and grown for 8–10 days. Mice were then randomized by tumor volume (60–100 m3) and treated with intraperitoneal administration of the specified antibody at a dose of 5 mg / kg. Tumors, tumor-inflowing lymph nodes (dLNs), and non-inflowing lymph nodes (ndLNs) were collected 24–48 hours post-treatment, and single-cell suspensions were prepared by enzymatic digestion of the tissues. Flow cytometry analysis was performed to stain for surface immune cell lineage markers and activation markers, as well as intracellular cytokines, to determine cell proportions and phenotypes (Figure 8). As expected, CD40 agonist antibody (anti-CD40) treatment activated dendritic cells (DCs) located in the tumor and peripheral tissues, including dLNs and ndLNs. In contrast, only the anti-muCD40×huEPCAM bispecific antibody activated tumor-infiltrating DCs in human EPCAM-expressing MC38 tumors, but did not activate DCs in peripheral tissues (dLN and ndLN) (Figure 8A). In addition, the anti-muCD40×huEPCAM bispecific antibody increased the ratio of CD8 T cells to regulatory T cells and interferon-gamma (IFNg)-producing CD4+ T cells in tumors (Figure 8B). Total T cells were also isolated from the spleen of huEpCAM-expressing MC38 tumor-bearing mice treated with the specified antibody. Bone marrow-derived dendritic cells (BMDCs) were generated from naive C57BL / 6 bone marrow cells by incubation with recombinant GM-CSF and IL4 for 7 days. BMDCs were instantaneously applied overnight to the specified tumor cells and subsequently co-cultured with isolated T cells for 18 hours. Using the ELISPOT assay, we measured IFNg production by antigen-specific T cells and demonstrated that the anti-muCD40 × huEPCAM bispecific antibody significantly increased T cells specific to both human EPCAM-expressing MC38 and the parental MC38 antigen (Figure 9). In summary, these data demonstrate that tumor-targeted CD40 agonist bispecific antibodies can activate bone marrow populations in tumors such as DCs in a tumor-associated antigen-dependent manner, resulting in enhanced antitumor T cell responses.

[0348] In vivo immunostimulatory activity of anti-muCD40 × huEPCAM bispecific antibody in a B16F10 tumor model To evaluate the effect of tumor-targeted CD40 agonist bispecific antibodies on immune cell activation in additional tumor models, vector-controlled or human EpCAM-expressing B16F10 tumor cells were inoculated into the right flank of mice at a rate of 3e5 cells per graft and grown for 8–10 days. Mice were then randomized by tumor volume (60–100 m3) and treated with intraperitoneal administration of the specified antibody at a dose of 5 mg / kg. Tumors, tumor dLNs, and ndLNs were collected 24–48 hours post-treatment, and single-cell suspensions were prepared by enzymatic digestion of the tissue. Flow cytometry analysis was performed to stain for surface immune cell lineage markers and activation markers, as well as intracellular cytokines, to determine cell proportions and phenotypes (Figure 10). As expected, the CD40 agonist antibody (anti-CD40) treats DCs located in peripheral tissues, including tumors, dLNs, and ndLNs in both parental huEpCAM-negative B16F10 tumors and huEpCAM-expressing B16F10 tumors. In contrast, only the anti-muCD40×huEPCAM bispecific antibody activated tumor-infiltrating DCs in huEpCAM-expressing B16F10 tumors, but not in parental huEpCAM-B16F10 tumors. The anti-muCD40×huEPCAM bispecific antibody did not activate DCs in dLNs or ndLNs, regardless of whether these mice had human EPCAM-expressing or non-expressing B16F10 tumors (Figure 10A). In addition, the anti-muCD40×huEPCAM bispecific antibody increased IFNg-producing effector CD4+ and CD8+ T cells in human EPCAM-expressing B16F10 tumors, but not in parental B16F10 tumors (Figure 10B). These data provide further evidence that tumor-targeted CD40 agonist bispecific antibodies can enhance the anti-tumor T cell response by activating tumor-infiltrating immune cells in a tumor-associated antigen-dependent manner.

[0349] The anti-muCD40 × huEPCAM bispecific antibody does not induce systemic serum cytokine upregulation or liver injury in mice.CD40 agonist antibodies exhibited dose-limiting toxicity in clinical trials, including liver damage and elevated serum cytokine levels, likely due to systemic immune cell activation. To compare systemic immune cell activation and liver damage between untargeted CD40 agonist antibodies and tumor-targeted CD40 agonist bispecific antibodies, mice carrying established human EPCAM-expressing MC38 tumors (above) were treated with a nonspecific isotype control antibody, a mouse CD40 agonist antibody (anti-CD40), a CD40 antibody with a mutation in the Fc domain that reduces Fc receptor binding to make it an inadequate agonist (anti-CD40 N297G), or an anti-muCD40×huEPCAM bispecific antibody (anti-CD40×huEPCAM) and serum, and harvested 24–48 hours later. The concentrations of selected cytokines were measured using a mouse cytokine / chemokine multiplex assay kit. Anti-CD40 antibodies increased the concentrations of several inflammatory cytokines in mouse serum, consistent with systemic immune cell activation. In contrast, neither anti-CD40 N297G nor anti-muCD40×huEPCAM bispecific antibody induced an increase in serum cytokine concentration (Figure 11A). Similar results were observed in the B16F10 tumor model, where, in contrast to untargeted anti-CD40 antibodies, anti-muCD40×huEPCAM bispecific antibody did not induce an increase in serum cytokine concentration in either mice carrying vector-controlled B16F10 tumors or mice carrying human EPCAM-expressing B16F10 tumors (Figure 11B). Liver tissue from mice carrying human EPCAM-expressing MC38 tumors and treated with anti-CD40 agonist antibody or anti-muCD40×huEPCAM bispecific antibody was also fixed in 10% neutral buffered formalin, embedded in paraffin, thinned, and stained with hematoxylin and eosin for histological analysis. Anti-CD40 agonist antibodies induced multifocal mononuclear cell infiltration in the liver, and these livers contained images of single-cell necrosis, whereas livers from mice treated with anti-muCD40 × huEPCAM bispecific antibodies showed no discernible histopathological changes (Figure 11C).In summary, these data indicate that tumor-targeted CD40 agonist antibodies can induce local immune cell activation in tumors without causing systemic cytokine production or liver damage associated with anti-CD40 toxicity.

[0350] Antitumor effect of human EPCAM × mouse CD40 bispecific antibody in mouse tumor model To evaluate the efficacy of tumor-targeted CD40 agonist antibodies against immune-mediated inhibition of tumor growth and the potential of this therapeutic approach in combination with immune checkpoint inhibitor PD1 blockers, mice carrying human EPCAM...

Claims

1. A multispecific antibody construct, (i) A first antibody comprising two light chains and two heavy chains, The light chain comprises a first variable region (VL1) and a light chain steady region (CL); The heavy chain comprises a first heavy chain variable region (VH1) and regions CH1, hinge, CH2, and CH3; A first antibody wherein the heavy chain comprises at least one amino acid substitution that results in a reduction in the binding affinity of the heavy chain to the human Fc gamma RI receptor compared to an unsubstituted heavy chain; and (ii) an scFv comprising a second light chain variable region (VL2) and a second heavy chain variable region (VH2) of a second antibody, wherein the VL2 and VH2 are linked via a first peptide linker, The scFv comprises an scFv fused to each carboxyl terminus of the heavy chain via a second peptide linker at its amino terminus, such that a heavy chain fusion protein is formed; and A multispecific antibody construct in which the first antibody specifically binds to and stimulates human CD40 (SEQ ID NO: 1), and the scFv specifically binds to human mesoserine (MSLN) (SEQ ID NO: 2).

2. The antibody construct according to claim 1, wherein the two light chains are identical and the two heavy chain fusion proteins are identical.

3. The aforementioned heavy chain, (i) N297G or N297A; (ii) L234A and L235A; and (iii) R292C and V302C It includes amino acid substitutions selected from the group consisting of; The antibody construct according to claim 1 or 2, wherein the numbering of the amino acids is EU numbering according to Kabat.

4. The antibody construct according to claim 3, wherein the heavy chain comprises N297G, R292C, and V302C mutations, and the numbering of the amino acids is EU numbering according to Kabat.

5. The aforementioned VL1 is Sequence IDs 58, 59, and 60, respectively; Sequence IDs 64, 65, and 66, respectively; Sequence IDs 70, 71, and 72, respectively; Sequence IDs 76, 77, and 78, respectively; Sequence IDs 82, 83, and 84, respectively; Sequence IDs 88, 89, and 90, respectively; Sequence IDs 94, 95, and 96, respectively; Sequence IDs 100, 101, and 102, respectively; Sequence IDs 106, 107, and 108, respectively; Sequence IDs 112, 113, and 114, respectively; Sequence IDs 118, 119, and 120, respectively; Sequence IDs 124, 125, and 126, respectively; and Sequence IDs 130, 131, and 132, respectively. It includes CDRL1, CDRL2, and CDRL3 selected from the group consisting of; The aforementioned VH1 is Sequence IDs 136, 137, and 138, respectively; Sequence IDs 142, 143, and 144, respectively; Sequence IDs 148, 149, and 150, respectively; Sequence IDs 154, 155, and 156, respectively; Sequence IDs 160, 161, and 162, respectively; Sequence IDs 166, 167, and 168, respectively; Sequence IDs 172, 173, and 174, respectively; Sequence IDs 178, 179, and 180, respectively; Sequence IDs 184, 185, and 186, respectively; Sequence numbers 190, 191, and 192, respectively; Sequence IDs 196, 197, and 198, respectively; Sequence IDs 202, 203, and 204, respectively; and Sequence IDs 208, 209, and 210, respectively. It includes CDRH1, CDRH2, and CDRH3 selected from the group consisting of; The aforementioned VL2 is Sequence IDs 230, 231, and 232, respectively; Sequence IDs 236, 237, and 238, respectively; Sequence IDs 242, 243, and 244, respectively; and Sequence IDs 248, 249, and 250, respectively. It includes CDRL1, CDRL2, and CDRL3 selected from the group consisting of; Furthermore, the VH2 is Sequence IDs 254, 255, and 256, respectively; Sequence numbers 260, 261, and 262, respectively; Sequence IDs 266, 267, and 268, respectively; and Sequence IDs 272, 273, and 274, respectively. An antibody construct according to any one of claims 1 to 4, comprising CDRH1, CDRH2, and CDRH3 selected from the group consisting of the following.

6. 1) The VL1 and VH1 are, a) VL1 containing CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs. 58, 59, and 60, respectively; and VH1 containing CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs. 136, 137, and 138, respectively; b) VL1 containing CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 64, 65, and 66, respectively; and VH1 containing CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 142, 143, and 144, respectively; c) VL1 comprising CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs. 70, 71, and 72, respectively; and VH1 comprising CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs. 148, 149, and 150, respectively; d) VL1 comprising CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 76, 77, and 6780, respectively; and VH1 comprising CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 154, 155, and 156, respectively; e) VL1 containing CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 82, 83, and 84, respectively; and VH1 containing CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 160, 161, and 162, respectively; f) VL1 containing CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 88, 89, and 90, respectively; and VH1 containing CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 166, 167, and 168, respectively; g) VL1 containing CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 94, 95, and 96, respectively; and VH1 containing CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 172, 173, and 174, respectively; h) VL1 containing CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 100, 101, and 102, respectively; and VH1 containing CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 178, 179, and 180, respectively; i) VL1 containing CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 106, 107, and 108, respectively; and VH1 containing CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 184, 185, and 186, respectively; j) VL1 containing CDRL1, CDRL2, and CDRL3 selected from the group consisting of sequence numbers 112, 113, and 114, respectively; and VH1 containing CDRH1, CDRH2, and CDRH3 selected from the group consisting of sequence numbers 190, 191, and 192, respectively; k) VL1 containing CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 118, 119, and 120, respectively; and VH1 containing CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 196, 197, and 198, respectively; l) VL1 containing CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 124, 125, and 126, respectively; and VH1 containing CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 202, 203, and 204, respectively; and m) VL1 containing CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 130, 131, and 132, respectively; and VH1 containing CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 208, 209, and 210, respectively. Selected from the group consisting of; and 2) The VL2 and VH2 are, a) VL2 containing CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 230, 231, and 232, respectively; and VH2 containing CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 254, 255, and 256, respectively; b) VL2 containing CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 236, 237, and 238, respectively; and VH2 containing CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 260, 261, and 262, respectively; c) VL2 containing CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 242, 243, and 244, respectively; and VH2 containing CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 266, 267, and 268, respectively; and d) VL2 containing CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 248, 249, and 250, respectively; and VH2 containing CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 272, 273, and 274, respectively. An antibody construct according to any one of claims 1 to 5, selected from the group consisting of the following.

7. The VL1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 5, 9, 13, 17, 21, 25, 29, 33, 37, 41, 45, 49, and 53; The VH1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 6, 10, 14, 18, 22, 26, 30, 34, 38, 42, 46, 50, and 54; The VL2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 213, 217, 221, and 225; The antibody construct according to any one of claims 1 to 6, wherein the VH2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 214, 218, 222, and 226.

8. 1) The VL1 and VH1 are, a) VL1 containing SEQ ID NO: 5 and VH1 containing SEQ ID NO: 6; b) VL1 containing SEQ ID NO: 9 and VH1 containing SEQ ID NO: 10; c) VL1 containing sequence number 13 and VH1 containing sequence number 14; d) VL1 containing SEQ ID NO: 17 and VH1 containing SEQ ID NO: 18; e) VL1 containing sequence number 21 and VH1 containing sequence number 22; f) VL1 containing sequence number 25 and VH1 containing sequence number 26; g) VL1 containing SEQ ID NO: 29 and VH1 containing SEQ ID NO: 30; h) VL1 containing sequence number 33 and VH1 containing sequence number 34; i) VL1 containing Sequence ID 37 and VH1 containing Sequence ID 38; j) VL1 containing sequence number 41 and VH1 containing sequence number 42; k) VL1 containing sequence number 45 and VH1 containing sequence number 46; l) VL1 containing sequence number 49 and VH1 containing sequence number 50; and m) VL1 containing sequence number 53 and VH1 containing sequence number 54 Selected from the group consisting of; and 2) The VL2 and VH2 are, a) VL2 containing sequence number 213 and VH2 containing sequence number 214; b) VL2 containing Sequence ID 217 and VH2 containing Sequence ID 218; c) VL2 containing sequence number 221 and VH2 containing sequence number 222; and d) VL2 containing SEQ ID NO: 225 and VH2 containing SEQ ID NO: 226 An antibody construct according to any one of claims 1 to 7, selected from the group consisting of the following.

9. The antibody construct according to any one of claims 1 to 8, wherein the CL of the light chain is selected from the group consisting of SEQ ID NO: 883 and SEQ ID NO:

884.

10. The antibody construct according to any one of claims 1 to 9, wherein the CH1-hinge-CH2-CH3 of the heavy chain is selected from the group consisting of SEQ ID NO: 885 and SEQ ID NO:

886.

11. The antibody construct according to any one of claims 1 to 10, wherein the first peptide linker is selected from the group consisting of SEQ ID NOs: 888 to 893.

12. The antibody construct according to any one of claims 1 to 11, wherein the second peptide linker is selected from the group consisting of SEQ ID NOs: 887 to 893.

13. The antibody construct according to any one of claims 1 to 12, wherein the first peptide linker comprises SEQ ID NO: 889 and the second peptide linker comprises SEQ ID NO:

887.

14. The light chain contains a sequence selected from the group consisting of sequence numbers 286, 290, 294, 298, 302, 306, 310, 314, 318, 322, 326, 330, 336, 342, 346, 350, 354, 358, 362, 366, 370, 374, and 378; and The antibody construct according to any one of claims 1 to 13, wherein the heavy chain fusion protein comprises a sequence selected from the group consisting of SEQ ID NOs: 285, 289, 293, 297, 301, 305, 309, 313, 317, 321, 325, 329, 333, 337, 341, 345, 349, 353, 357, 361, 365, 369, 373, and 377.

15. The light chain and the heavy chain fusion protein Sequence ID 286 and Sequence ID 285, respectively; Sequence IDs 290 and 289, respectively; Sequence IDs 294 and 293, respectively; Sequence IDs 298 and 297, respectively; Sequence ID 302 and Sequence ID 301, respectively; Sequence ID 306 and Sequence ID 305, respectively; Sequence IDs 310 and 309, respectively; Sequence ID 314 and Sequence ID 313, respectively; Sequence ID 318 and Sequence ID 317, respectively; Sequence ID 322 and Sequence ID 321, respectively; Sequence ID 326 and Sequence ID 325, respectively; Sequence IDs 330 and 329, respectively; Sequence ID 334 and Sequence ID 333, respectively; Sequence ID 338 and Sequence ID 337, respectively; Sequence ID 342 and Sequence ID 341, respectively; Sequence ID 346 and Sequence ID 345, respectively; Sequence IDs 350 and 349, respectively; Sequence ID 354 and Sequence ID 353, respectively; Sequence ID 358 and Sequence ID 357, respectively; Sequence ID 362 and Sequence ID 361, respectively; Sequence ID 366 and Sequence ID 365, respectively; Sequence IDs 370 and 369, respectively; Sequence ID 374 and Sequence ID 373, respectively; and Sequence ID 378 and Sequence ID 377, respectively. An antibody construct according to any one of claims 1 to 14, comprising a polypeptide having an amino acid sequence selected from the group consisting of the following.

16. A polynucleotide encoding the light chain of the antibody construct according to any one of claims 1 to 15.

17. A polynucleotide encoding the heavy chain fusion protein of the antibody construct according to any one of claims 1 to 16.

18. A vector comprising the polynucleotide described in claim 16, the polynucleotide described in claim 17, or both.

19. Host cells transformed or transfected with the polynucleotides described in claim 16 and the polynucleotides described in claim 17.

20. A process for generating an antibody construct according to any one of claims 1 to 15, comprising culturing a host cell comprising a polynucleotide encoding the light chain and a polynucleotide encoding the heavy chain fusion protein under conditions that enable the expression of the antibody construct, and recovering the antibody construct generated from the culture.

21. A pharmaceutical composition comprising an antibody construct according to any one of claims 1 to 15, and a carrier, stabilizer, excipient, diluent, solubilizer, surfactant, emulsifier, preservative, or adjuvant.

22. A method for treating or inducing remission of a solid tumor disease or metastatic cancer, comprising the step of administering an effective amount of an antibody construct according to any one of claims 1 to 15 to a subject as needed.

23. The method according to claim 22, wherein the solid tumor disease is selected from the group consisting of ovarian cancer, pancreatic cancer, mesothelioma, lung cancer, gastric cancer, and tertiary negative breast cancer or metastatic cancer, which originate from any of the above.

24. A kit comprising an antibody construct according to any one of claims 1 to 15, and optionally, instructions for use.

25. A multispecific antibody construct, (i) A first antibody comprising two light chains and two heavy chains, The light chain comprises a first variable region (VL1) and a light chain steady region (CL); The heavy chain comprises a first heavy chain variable region (VH1) and regions CH1, hinge, CH2, and CH3; A first antibody wherein the heavy chain comprises at least one amino acid substitution that results in a reduction in the binding affinity of the heavy chain to the human Fc gamma RI receptor compared to an unsubstituted heavy chain; and (ii) an scFv comprising a second light chain variable region (VL2) and a second heavy chain variable region (VH2) of a second antibody, wherein the VL2 and VH2 are linked via a first peptide linker, The scFv comprises an scFv fused to each carboxyl terminus of the heavy chain via a second peptide linker at its amino terminus, such that a heavy chain fusion protein is formed; and A multispecific antibody construct in which the first antibody specifically binds to human mesoserine (MSLN) (SEQ ID NO: 2), and the scFv specifically binds to and stimulates human CD40 (SEQ ID NO: 1).

26. The antibody construct according to claim 25, wherein the two light chains are identical and the two heavy chain fusion proteins are identical.

27. The aforementioned heavy chain, (i) N297G or N297A; (ii) L234A and L235A; and (iii) R292C and V302C It includes amino acid substitutions selected from the group consisting of; The antibody construct according to any one of claims 25 to 26, wherein the numbering of the amino acids is EU numbering according to Kabat.

28. The antibody construct according to claim 27, wherein the heavy chain comprises N297G, R292C, and V302C mutations, and the numbering of the amino acids is EU numbering according to Kabat.

29. The aforementioned VL1 is Sequence IDs 230, 231, and 232, respectively; Sequence IDs 236, 237, and 238, respectively; Sequence IDs 242, 243, and 244, respectively; and Sequence IDs 248, 249, and 250, respectively. It includes CDRL1, CDRL2, and CDRL3 selected from the group consisting of; The aforementioned VH1 is Sequence IDs 254, 255, and 256, respectively; Sequence numbers 260, 261, and 262, respectively; Sequence IDs 266, 267, and 268, respectively; and Sequence IDs 272, 273, and 274, respectively. It includes CDRH1, CDRH2, and CDRH3 selected from the group consisting of; The aforementioned VL2 is Sequence IDs 58, 59, and 60, respectively; Sequence IDs 64, 65, and 66, respectively; Sequence IDs 70, 71, and 72, respectively; Sequence IDs 76, 77, and 78, respectively; Sequence IDs 82, 83, and 84, respectively; Sequence IDs 88, 89, and 90, respectively; Sequence IDs 94, 95, and 96, respectively; Sequence IDs 100, 101, and 102, respectively; Sequence IDs 106, 107, and 108, respectively; Sequence IDs 112, 113, and 114, respectively; Sequence IDs 118, 119, and 120, respectively; Sequence IDs 124, 125, and 126, respectively; and Sequence IDs 130, 131, and 132, respectively. It includes CDRL1, CDRL2, and CDRL3 selected from the group consisting of; and The aforementioned VH2 is Sequence IDs 136, 137, and 138, respectively; Sequence IDs 142, 143, and 144, respectively; Sequence IDs 148, 149, and 150, respectively; Sequence IDs 154, 155, and 156, respectively; Sequence IDs 160, 161, and 162, respectively; Sequence IDs 166, 167, and 168, respectively; Sequence IDs 172, 173, and 174, respectively; Sequence IDs 178, 179, and 180, respectively; Sequence IDs 184, 185, and 186, respectively; Sequence numbers 190, 191, and 192, respectively; Sequence IDs 196, 197, and 198, respectively; Sequence IDs 202, 203, and 204, respectively; and Sequence IDs 208, 209, and 210, respectively. An antibody construct according to any one of claims 25 to 28, comprising CDRH1, CDRH2, and CDRH3 selected from the group consisting of the above.

30. 1) The VL1 and VH1 are, a) VL1 containing CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 230, 231, and 232, respectively; and VH1 containing CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 254, 255, and 256, respectively; b) VL1 comprising CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 236, 237, and 238, respectively; and VH1 comprising CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 260, 261, and 262, respectively; c) VL1 containing CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 242, 243, and 244, respectively; and VH1 containing CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 266, 267, and 268, respectively; and d) VL1 containing CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 248, 249, and 250, respectively; and VH1 containing CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 272, 273, and 274, respectively. Selected from the group consisting of; and 2) The VL2 and VH2 are, a) VL2 containing CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs. 58, 59, and 60, respectively; and VH2 containing CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs. 136, 137, and 138, respectively; b) VL2 containing CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 64, 65, and 66, respectively; and VH2 containing CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 142, 143, and 144, respectively; c) VL2 containing CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs. 70, 71, and 72, respectively; and VH2 containing CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs. 148, 149, and 150, respectively; d) VL2 containing CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 76, 77, and 6780, respectively; and VH2 containing CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 154, 155, and 156, respectively; e) VL2 containing CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs. 82, 83, and 84, respectively; and VH2 containing CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs. 160, 161, and 162, respectively; f) VL2 containing CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 88, 89, and 90, respectively; and VH2 containing CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 166, 167, and 168, respectively; g) VL2 containing CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 94, 95, and 96, respectively; and VH2 containing CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 172, 173, and 174, respectively; h) VL2 containing CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 100, 101, and 102, respectively; and VH2 containing CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 178, 179, and 180, respectively; i) VL2 containing CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 106, 107, and 108, respectively; and VH2 containing CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 184, 185, and 186, respectively; j) VL2 containing CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 112, 113, and 114, respectively; and VH2 containing CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 190, 191, and 192, respectively; k) VL2 containing CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 118, 119, and 120, respectively; and VH2 containing CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 196, 197, and 198, respectively; l) VL2 containing CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 124, 125, and 126, respectively; and VH2 containing CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 202, 203, and 204, respectively; and m) VL2 containing CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 130, 131, and 132, respectively; and VH2 containing CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 208, 209, and 210, respectively. An antibody construct according to any one of claims 25 to 29, selected from the group consisting of the following.

31. The VL1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 213, 217, 221, and 225; The VH1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 214, 218, 222, and 226; The VL2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 5, 9, 13, 17, 21, 25, 29, 33, 37, 41, 45, 49, and 53; and The antibody construct according to any one of claims 25 to 30, wherein the VH2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 6, 10, 14, 18, 22, 26, 30, 34, 38, 42, 46, 50, and 54.

32. 1) The VL1 and VH1 are, a) VL1 containing sequence number 213 and VH1 containing sequence number 214; b) VL1 containing sequence number 217 and VH1 containing sequence number 218; c) VL1 containing SEQ ID NO: 221 and VH1 containing SEQ ID NO: 222; and d) VL1 containing SEQ ID NO: 225 and VH1 containing SEQ ID NO: 226 Selected from the group consisting of; and 2) The VL2 and VH2 are, a) VL2 containing Sequence ID 5 and VH2 containing Sequence ID 6; b) VL2 containing SEQ ID NO: 9 and VH2 containing SEQ ID NO: 10; c) VL2 containing SEQ ID NO: 13 and VH2 containing SEQ ID NO: 14; d) VL2 containing SEQ ID NO: 17 and VH2 containing SEQ ID NO: 18; e) VL2 containing sequence number 21 and VH2 containing sequence number 22; f) VL2 containing sequence number 25 and VH2 containing sequence number 26; g) VL2 containing SEQ ID NO: 29 and VH2 containing SEQ ID NO: 30; h) VL2 containing sequence number 33 and VH2 containing sequence number 34; i) VL2 containing Sequence ID 37 and VH2 containing Sequence ID 38; j) VL2 containing sequence number 41 and VH2 containing sequence number 42; k) VL2 containing sequence number 45 and VH2 containing sequence number 46; l) VL2 containing Sequence ID 49 and VH2 containing Sequence ID 50; and m) VL2 containing sequence number 53 and VH2 containing sequence number 54 An antibody construct according to any one of claims 25 to 31, selected from the group consisting of the following.

33. The antibody construct according to any one of claims 25 to 32, wherein the CL of the light chain is selected from the group consisting of SEQ ID NO: 883 and SEQ ID NO:

884.

34. The antibody construct according to any one of claims 25 to 33, wherein the CH1-hinge-CH2-CH3 of the heavy chain is selected from the group consisting of SEQ ID NO: 885 and SEQ ID NO:

886.

35. The antibody construct according to any one of claims 25 to 34, wherein the first peptide linker is selected from the group consisting of SEQ ID NOs: 888 to 893.

36. The antibody construct according to any one of claims 25 to 35, wherein the second peptide linker is selected from the group consisting of SEQ ID NOs: 887 to 893.

37. The antibody construct according to any one of claims 25 to 36, wherein the first peptide linker comprises SEQ ID NO: 889 and the second peptide linker comprises SEQ ID NO:

887.

38. The light chain comprises a sequence selected from the group consisting of sequence numbers 382, ​​386, 390, 394, 398, 402, 406, 410, 414, 418, 422, 426, 430, 434, 438, 442, 446, and 450; and The antibody construct according to any one of claims 25 to 37, wherein the heavy chain fusion protein comprises a sequence selected from the group consisting of SEQ ID NOs: 381, 385, 389, 393, 397, 401, 405, 409, 413, 417, 421, 425, 429, 433, 437, 441, 445, and 449.

39. The light chain and the heavy chain fusion protein Sequence ID 382 and Sequence ID 381, respectively; Sequence ID 386 and Sequence ID 385, respectively; Sequence IDs 390 and 389, respectively; Sequence ID 394 and Sequence ID 393, respectively; Sequence ID 398 and Sequence ID 397, respectively; Sequence ID 402 and Sequence ID 401, respectively; Sequence ID 406 and Sequence ID 405, respectively; Sequence IDs 410 and 409, respectively; Sequence ID 414 and Sequence ID 413, respectively; Sequence ID 418 and Sequence ID 417, respectively; Sequence ID 422 and Sequence ID 421, respectively; Sequence ID 426 and Sequence ID 425, respectively; Sequence IDs 430 and 429, respectively; Sequence ID 434 and Sequence ID 433, respectively; Sequence ID 438 and Sequence ID 437, respectively; Sequence ID 442 and Sequence ID 441, respectively; Sequence ID 446 and Sequence ID 445, respectively; and Sequence IDs 450 and 449, respectively. The antibody construct according to any one of claims 25 to 38, comprising a polypeptide having an amino acid sequence selected from the group consisting of the following.

40. A polynucleotide encoding the light chain of an antibody construct according to any one of claims 25 to 39.

41. A polynucleotide encoding the heavy chain fusion protein of the antibody construct according to any one of claims 25 to 39.

42. A vector comprising the polynucleotide described in claim 40, the polynucleotide described in claim 41, or both.

43. Host cells transformed or transfected with the polynucleotide described in claim 40 and the polynucleotide described in claim 42.

44. A process for generating an antibody construct according to any one of claims 25 to 39, comprising culturing a host cell comprising a polynucleotide encoding the light chain and a polynucleotide encoding the heavy chain fusion protein under conditions that enable the expression of the antibody construct, and recovering the antibody construct generated from the culture.

45. A pharmaceutical composition comprising an antibody construct according to any one of claims 25 to 39, and a carrier, stabilizer, excipient, diluent, solubilizer, surfactant, emulsifier, preservative, or adjuvant.

46. A method for treating or inducing remission of a solid tumor disease or metastatic cancer, comprising the step of administering an effective amount of an antibody construct according to any one of claims 25 to 39 to a subject as needed.

47. The method according to claim 46, wherein the solid tumor disease is selected from the group consisting of ovarian cancer, pancreatic cancer, mesothelioma, lung cancer, gastric cancer, and trinegative breast cancer or metastatic cancer, which originate from any of the above.

48. A kit comprising an antibody construct according to any one of claims 25 to 39, and optionally an instruction manual for use.

49. A multispecific antibody construct, a) Two identical heavy chain fusion proteins, each comprising a first heavy chain variable region (VH1) and a first CH1 domain, wherein the first CH1 domain is linked to a hinge-CH2-CH3 polypeptide, and the hinge-CH2-CH3 polypeptide is linked to a second heavy chain variable region (VH2), and the VH2 is linked to a second CH1 domain; i) The VH1 or first CH1 domain includes at least one amino acid substitution introducing a positively charged amino acid at a residue selected from the group consisting of positions 39, 44, and 183 using EU numbering; and ii) A heavy chain fusion protein wherein the VH2 or second CH1 domain includes at least one amino acid substitution introducing a negatively charged amino acid at a residue selected from the group consisting of residues corresponding to positions 39, 44, and 183 using EU numbering; and b) A second polypeptide comprising a first light chain, wherein the first light chain comprises a first light chain variable region (VL1) and a first CL region; and the VL1 or the first CL domain comprises at least one amino acid substitution introducing a negatively charged amino acid at a residue selected from the group consisting of positions 38, 100, and 176 using EU numbering; and c) A third polypeptide comprising a second light chain, wherein the second light chain comprises a second light chain variable region (VL2) and a second CL region; and the VL2 or the second CL domain comprises at least one amino acid substitution introducing a positively charged amino acid at a residue selected from the group consisting of positions 38, 100, and 176 using EU numbering. A multispecific antibody construct comprising, The VH1 and VL1 interact to bind to the first antigen, and the VH2 and VL2 interact to bind to the second antigen; Here, The first antigen is human CD40 (SEQ ID NO: 1), and the second antigen is human mesoserine ("MSLN"; SEQ ID NO: 2); or A multispecific antibody construct in which the first antigen is human MSLN (SEQ ID NO: 2) and the second antigen is human CD40 (SEQ ID NO: 1).

50. A multispecific antibody construct, a) Two identical heavy chain fusion proteins, each comprising a first heavy chain variable region (VH1) and a first CH1 domain, wherein the first CH1 domain is linked to a hinge-CH2-CH3 polypeptide, and the hinge-CH2-CH3 polypeptide is linked to a second heavy chain variable region (VH2), and the VH2 is linked to a second CH1 domain; i) The VH1 or first CH1 domain includes at least one amino acid substitution introducing a negatively charged amino acid at a residue selected from the group consisting of positions 39, 44, and 183 using EU numbering; and ii) A heavy chain fusion protein wherein the VH2 or second CH1 domain includes at least one amino acid substitution introducing a positively charged amino acid at a residue selected from the group consisting of residues corresponding to positions 39, 44, and 183 using EU numbering; and b) A second polypeptide comprising a first light chain, wherein the first light chain comprises a first light chain variable region (VL1) and a first CL region; and the VL1 or the first CL domain comprises at least one amino acid substitution introducing a positively charged amino acid at a residue selected from the group consisting of positions 38, 100, and 176 using EU numbering; and c) A third polypeptide comprising a second light chain, wherein the second light chain comprises a second light chain variable region (VL2) and a second CL region; and the VL2 or the second CL domain comprises at least one amino acid substitution introducing a negatively charged amino acid at a residue selected from the group consisting of positions 38, 100, and 176 using EU numbering. A multispecific antibody construct comprising, The VH1 and VL1 interact to bind to the first antigen, and the VH2 and VL2 interact to bind to the second antigen; Here, The first antigen is human CD40 (SEQ ID NO: 1), and the second antigen is human mesoserine ("MSLN"; SEQ ID NO: 2); or A multispecific antibody construct in which the first antigen is human MSLN (SEQ ID NO: 2) and the second antigen is human CD40 (SEQ ID NO: 1).

51. The antibody construct according to any one of claims 49 or 50, wherein the hinge-CH2-CH3 polypeptide is linked to the VH2 via a peptide linker.

52. The peptide linker is (Gly ３ Ser) ２ (SEQ ID NO: 916), (Gly ４ Ser) ２ (SEQ ID NO: 888), (Gly ３ Ser) ３ (SEQ ID NO: 917), (Gly ４ Ser) ３ (SEQ ID NO: 889), (Gly ３ Ser) ４ (SEQ ID NO: 918), (Gly ４ Ser) ４ (SEQ ID NO: 890), (Gly ３ Ser) ５ (SEQ ID NO: 919), (Gly ４ Ser) ５ (SEQ ID NO: 920), (Gly ３ Ser) ６ (SEQ ID NO: 921), and (Gly ４ Ser) ６ (SEQ ID NO: 922), the antibody construct according to claim 51, comprising a sequence selected from the group consisting of.

53. a) The VH1 or first CH1 domain contains a mutation selected from the group consisting of Q39K, G44K, and S183K using EU numbering; b) The VH2 or second CH1 domain contains a mutation selected from the group consisting of Q39E, G44E, and S183E using EU numbering; c) The VL1 or first CL domain contains a mutation selected from the group consisting of Q38E, G100E, and S176E using EU numbering; and d) The antigen-binding protein according to claim 49, wherein the VL2 or second CL domain contains a mutation selected from the group consisting of Q38K, G100K, and S176K using EU numbering.

54. a) The first CH1 domain contains the S183K mutation using EU numbering; b) The second CH1 domain contains the S183E mutation using EU numbering; c) The first CL domain contains the S176E mutation using EU numbering; and d) The antigen-binding protein according to claim 53, wherein the second CL domain contains an S176K mutation using EU numbering.

55. a) The VH1 contains the Q39K mutation and the first CH1 domain contains the S183K mutation using EU numbering; b) The VH2 contains the Q39E mutation and the second CH1 domain contains the S183E mutation using EU numbering; c) The VL1 contains the Q38E mutation and the first CL domain contains the S176E mutation using EU numbering; and d) The antigen-binding protein according to claim 53, wherein the VL2 contains the Q38K mutation and the second CL domain contains the S176K mutation using EU numbering.

56. a) The first CH1 domain contains G44K and S183K mutations using EU numbering; b) The second CH1 domain contains G44E and S183E mutations using EU numbering; c) The first CL domain contains G100E and S176E mutations using EU numbering; and d) The antigen-binding protein according to claim 53, wherein the second CL domain includes G100K and S176K mutations using EU numbering.

57. a) The VH1 or first CH1 domain contains a mutation selected from the group consisting of Q39E, G44E, and S183E using EU numbering; b) The VH2 or second CH1 domain contains a mutation selected from the group consisting of Q39K, G44K, and S183K using EU numbering; c) The VL1 or first CL domain contains a mutation selected from the group consisting of Q38K, G100K, and S176K using EU numbering; and d) The antigen-binding protein according to claim 50, wherein the VL2 or second CL domain contains a mutation selected from the group consisting of Q38E, G100E, and S176E using EU numbering.

58. a) The first CH1 domain contains the S183E mutation using EU numbering; b) The second CH1 domain contains the S183K mutation using EU numbering; c) The first CL domain contains the S176K mutation using EU numbering; and d) The antigen-binding protein according to claim 57, wherein the second CL domain contains the S176E mutation using EU numbering.

59. a) The VH1 contains the Q39E mutation and the first CH1 domain contains the S183E mutation using EU numbering; b) The VH2 contains the Q39K mutation and the second CH1 domain contains the S183K mutation using EU numbering; c) The VL1 contains the Q38K mutation and the first CL domain contains the S176K mutation using EU numbering; and d) The antigen-binding protein according to claim 57, wherein the VL2 contains the Q38E mutation and the second CL domain contains the S176E mutation using EU numbering.

60. a) The first CH1 domain contains G44E and S183E mutations using EU numbering; b) The second CH1 domain contains G44K and S183K mutations using EU numbering; c) The first CL domain contains G100K and S176K mutations using EU numbering; and d) The antigen-binding protein according to claim 57, wherein the second CL domain includes G100E and S176E mutations using EU numbering.

61. The aforementioned hinge-CH2-CH3 polypeptide (i) N297G or N297A; (ii) L234A and L235A; and (iii) R292C and V302C It includes amino acid substitutions selected from the group consisting of; The antibody construct according to any one of claims 49 to 50, wherein the numbering of the amino acids is EU numbering according to Kabat.

62. The antibody construct according to claim 61, wherein the hinge-CH2-CH3 polypeptide comprises N297G, R292C, and V302C mutations, and the amino acid numbering is EU numbering according to Kabat.

63. The first antigen is human CD40 (SEQ ID NO: 1) and the second antigen is human MSLN (SEQ ID NO: 2); and The aforementioned VL1 is Sequence IDs 58, 59, and 60, respectively; Sequence IDs 64, 65, and 66, respectively; Sequence IDs 70, 71, and 72, respectively; Sequence IDs 76, 77, and 78, respectively; Sequence IDs 82, 83, and 84, respectively; Sequence IDs 88, 89, and 90, respectively; Sequence IDs 94, 95, and 96, respectively; Sequence IDs 100, 101, and 102, respectively; Sequence IDs 106, 107, and 108, respectively; Sequence IDs 112, 113, and 114, respectively; Sequence IDs 118, 119, and 120, respectively; Sequence IDs 124, 125, and 126, respectively; and Sequence IDs 130, 131, and 132, respectively. It includes CDRL1, CDRL2, and CDRL3 selected from the group consisting of; The aforementioned VH1 is Sequence IDs 136, 137, and 138, respectively; Sequence IDs 142, 143, and 144, respectively; Sequence IDs 148, 149, and 150, respectively; Sequence IDs 154, 155, and 156, respectively; Sequence IDs 160, 161, and 162, respectively; Sequence IDs 166, 167, and 168, respectively; Sequence IDs 172, 173, and 174, respectively; Sequence IDs 178, 179, and 180, respectively; Sequence IDs 184, 185, and 186, respectively; Sequence numbers 190, 191, and 192, respectively; Sequence IDs 196, 197, and 198, respectively; Sequence IDs 202, 203, and 204, respectively; and Sequence IDs 208, 209, and 210, respectively. It includes CDRH1, CDRH2, and CDRH3 selected from the group consisting of; The aforementioned VL2 is Sequence IDs 230, 231, and 232, respectively; Sequence IDs 236, 237, and 238, respectively; Sequence IDs 242, 243, and 244, respectively; and Sequence IDs 248, 249, and 250, respectively; It includes CDRL1, CDRL2, and CDRL3 selected from the group consisting of; Furthermore, the VH2 is Sequence IDs 254, 255, and 256, respectively; Sequence numbers 260, 261, and 262, respectively; Sequence IDs 266, 267, and 268, respectively; and Sequence IDs 272, 273, and 274, respectively. An antibody construct according to any one of claims 49 to 62, comprising CDRH1, CDRH2, and CDRH3 selected from the group consisting of the above.

64. 1) The VL1 and VH1 are, a) VL1 containing CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs. 58, 59, and 60, respectively; and VH1 containing CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs. 136, 137, and 138, respectively; b) VL1 containing CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 64, 65, and 66, respectively; and VH1 containing CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 142, 143, and 144, respectively; c) VL1 comprising CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs. 70, 71, and 72, respectively; and VH1 comprising CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs. 148, 149, and 150, respectively; d) VL1 comprising CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 76, 77, and 6780, respectively; and VH1 comprising CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 154, 155, and 156, respectively; e) VL1 containing CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 82, 83, and 84, respectively; and VH1 containing CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 160, 161, and 162, respectively; f) VL1 containing CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 88, 89, and 90, respectively; and VH1 containing CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 166, 167, and 168, respectively; g) VL1 containing CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 94, 95, and 96, respectively; and VH1 containing CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 172, 173, and 174, respectively; h) VL1 containing CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 100, 101, and 102, respectively; and VH1 containing CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 178, 179, and 180, respectively; i) VL1 containing CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 106, 107, and 108, respectively; and VH1 containing CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 184, 185, and 186, respectively; j) VL1 containing CDRL1, CDRL2, and CDRL3 selected from the group consisting of sequence numbers 112, 113, and 114, respectively; and VH1 containing CDRH1, CDRH2, and CDRH3 selected from the group consisting of sequence numbers 190, 191, and 192, respectively; k) VL1 containing CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 118, 119, and 120, respectively; and VH1 containing CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 196, 197, and 198, respectively; l) VL1 containing CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 124, 125, and 126, respectively; and VH1 containing CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 202, 203, and 204, respectively; and m) VL1 containing CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 130, 131, and 132, respectively; and VH1 containing CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 208, 209, and 210, respectively. Selected from the group consisting of; and 2) The VL2 and VH2 are, a) VL2 containing CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 230, 231, and 232, respectively; and VH2 containing CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 254, 255, and 256, respectively; b) VL2 containing CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 236, 237, and 238, respectively; and VH2 containing CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 260, 261, and 262, respectively; c) VL2 containing CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 242, 243, and 244, respectively; and VH2 containing CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 266, 267, and 268, respectively; and d) VL2 containing CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 248, 249, and 250, respectively; and VH2 containing CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 272, 273, and 274, respectively. An antibody construct according to claim 63, selected from the group consisting of the following.

65. The VL1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 5, 9, 13, 17, 21, 25, 29, 33, 37, 41, 45, 49, and 53; The VH1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 6, 10, 14, 18, 22, 26, 30, 34, 38, 42, 46, 50, and 54; The VL2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 213, 217, 221, and 225; The antibody construct according to claim 63, wherein the VH2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 214, 218, 222, and 226.

66. 1) The VL1 and VH1 are, a) VL1 containing SEQ ID NO: 5 and VH1 containing SEQ ID NO: 6; b) VL1 containing SEQ ID NO: 9 and VH1 containing SEQ ID NO: 10; c) VL1 containing sequence number 13 and VH1 containing sequence number 14; d) VL1 containing SEQ ID NO: 17 and VH1 containing SEQ ID NO: 18; e) VL1 containing sequence number 21 and VH1 containing sequence number 22; f) VL1 containing sequence number 25 and VH1 containing sequence number 26; g) VL1 containing SEQ ID NO: 29 and VH1 containing SEQ ID NO: 30; h) VL1 containing sequence number 33 and VH1 containing sequence number 34; i) VL1 containing Sequence ID 37 and VH1 containing Sequence ID 38; j) VL1 containing sequence number 41 and VH1 containing sequence number 42; k) VL1 containing sequence number 45 and VH1 containing sequence number 46; l) VL1 containing sequence number 49 and VH1 containing sequence number 50; and m) VL1 containing sequence number 53 and VH1 containing sequence number 54 Selected from the group consisting of; and 2) The VL2 and VH2 are, a) VL2 containing sequence number 213 and VH2 containing sequence number 214; b) VL2 containing Sequence ID 217 and VH2 containing Sequence ID 218; c) VL2 containing sequence number 221 and VH2 containing sequence number 222; and d) VL2 containing SEQ ID NO: 225 and VH2 containing SEQ ID NO: 226 An antibody construct according to claim 65, selected from the group consisting of the following.

67. A polynucleotide encoding the first light chain of an antibody construct according to any one of claims 49 to 66 and 76 to 80.

68. A polynucleotide encoding the second light chain of an antibody construct according to any one of claims 49 to 66 and 76 to 80.

69. A polynucleotide encoding the heavy chain fusion protein of the antibody construct according to any one of claims 49 to 66 and 76 to 80.

70. a) The polynucleotide according to claim 67, b) The polynucleotide according to claim 68, c) The polynucleotide described in claim 69, or d) Any combination of a), b), and c) A vector containing this.

71. A host cell transformed or transfected with the polynucleotide described in claim 67, the polynucleotide described in claim 68, and the polynucleotide described in claim 69.

72. A process for generating an antibody construct according to any one of claims 49 to 66 and 76 to 80, comprising culturing a host cell comprising a polynucleotide encoding the first light chain, a polynucleotide encoding the second light chain, and a polynucleotide encoding the heavy chain fusion protein under conditions that enable the expression of the antibody construct, and recovering the antibody construct generated from the culture.

73. A pharmaceutical composition comprising an antibody construct according to any one of claims 49 to 66 and 76 to 80, and a carrier, stabilizer, excipient, diluent, solubilizer, surfactant, emulsifier, preservative, or adjuvant.

74. A method for treating or inducing remission of a solid tumor disease or metastatic cancer, comprising the step of administering an effective amount of an antibody construct according to any one of claims 49 to 66 and 76 to 80 to a subject as needed.

75. The method according to claim 74, wherein the solid tumor disease is selected from the group consisting of ovarian cancer, pancreatic cancer, mesothelioma, lung cancer, gastric cancer, and tertiary negative breast cancer or metastatic cancer, which originate from any of the above.

76. A kit comprising an antibody construct according to any one of claims 49-66 and 76-80, and optionally, instructions for use.

77. The first antigen is human MSLN (SEQ ID NO: 2) and the second antigen is human CD40 (SEQ ID NO: 1); The aforementioned VL1 is Sequence IDs 230, 231, and 232, respectively; Sequence IDs 236, 237, and 238, respectively; Sequence IDs 242, 243, and 244, respectively; and Sequence IDs 248, 249, and 250, respectively. It includes CDRL1, CDRL2, and CDRL3 selected from the group consisting of; The aforementioned VH1 is Sequence IDs 254, 255, and 256, respectively; Sequence numbers 260, 261, and 262, respectively; Sequence IDs 266, 267, and 268, respectively; and Sequence IDs 272, 273, and 274, respectively. It includes CDRH1, CDRH2, and CDRH3 selected from the group consisting of; The aforementioned VL2 is Sequence IDs 58, 59, and 60, respectively; Sequence IDs 64, 65, and 66, respectively; Sequence IDs 70, 71, and 72, respectively; Sequence IDs 76, 77, and 78, respectively; Sequence IDs 82, 83, and 84, respectively; Sequence IDs 88, 89, and 90, respectively; Sequence IDs 94, 95, and 96, respectively; Sequence IDs 100, 101, and 102, respectively; Sequence IDs 106, 107, and 108, respectively; Sequence IDs 112, 113, and 114, respectively; Sequence IDs 118, 119, and 120, respectively; Sequence IDs 124, 125, and 126, respectively; and Sequence IDs 130, 131, and 132, respectively. It includes CDRL1, CDRL2, and CDRL3 selected from the group consisting of; and The aforementioned VH2 is Sequence IDs 136, 137, and 138, respectively; Sequence IDs 142, 143, and 144, respectively; Sequence IDs 148, 149, and 150, respectively; Sequence IDs 154, 155, and 156, respectively; Sequence IDs 160, 161, and 162, respectively; Sequence IDs 166, 167, and 168, respectively; Sequence IDs 172, 173, and 174, respectively; Sequence IDs 178, 179, and 180, respectively; Sequence IDs 184, 185, and 186, respectively; Sequence numbers 190, 191, and 192, respectively; Sequence IDs 196, 197, and 198, respectively; Sequence IDs 202, 203, and 204, respectively; and Sequence IDs 208, 209, and 210, respectively. An antibody construct according to any one of claims 49 to 62, comprising CDRH1, CDRH2, and CDRH3 selected from the group consisting of the above.

78. 1) The VL1 and VH1 are, a) VL1 containing CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 230, 231, and 232, respectively; and VH1 containing CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 254, 255, and 256, respectively; b) VL1 comprising CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 236, 237, and 238, respectively; and VH1 comprising CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 260, 261, and 262, respectively; c) VL1 containing CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 242, 243, and 244, respectively; and VH1 containing CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 266, 267, and 268, respectively; and d) VL1 containing CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 248, 249, and 250, respectively; and VH1 containing CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 272, 273, and 274, respectively. Selected from the group consisting of; and 2) The VL2 and VH2 are, a) VL2 containing CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs. 58, 59, and 60, respectively; and VH2 containing CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs. 136, 137, and 138, respectively; b) VL2 containing CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 64, 65, and 66, respectively; and VH2 containing CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 142, 143, and 144, respectively; c) VL2 containing CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs. 70, 71, and 72, respectively; and VH2 containing CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs. 148, 149, and 150, respectively; d) VL2 containing CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 76, 77, and 6780, respectively; and VH2 containing CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 154, 155, and 156, respectively. e) VL2 containing CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs. 82, 83, and 84, respectively; and VH2 containing CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs. 160, 161, and 162, respectively; f) VL2 containing CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 88, 89, and 90, respectively; and VH2 containing CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 166, 167, and 168, respectively; g) VL2 containing CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 94, 95, and 96, respectively; and VH2 containing CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 172, 173, and 174, respectively; h) VL2 containing CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 100, 101, and 102, respectively; and VH2 containing CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 178, 179, and 180, respectively; i) VL2 containing CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 106, 107, and 108, respectively; and VH2 containing CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 184, 185, and 186, respectively; j) VL2 containing CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 112, 113, and 114, respectively; and VH2 containing CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 190, 191, and 192, respectively; k) VL2 containing CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 118, 119, and 120, respectively; and VH2 containing CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 196, 197, and 198, respectively; l) VL2 containing CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 124, 125, and 126, respectively; and VH2 containing CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 202, 203, and 204, respectively; and m) VL2 containing CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 130, 131, and 132, respectively; and VH2 containing CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 208, 209, and 210, respectively. An antibody construct according to claim 77, selected from the group consisting of the following.

79. The VL1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 213, 217, 221, and 225; The VH1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 214, 218, 222, and 226; The VL2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 5, 9, 13, 17, 21, 25, 29, 33, 37, 41, 45, 49, and 53; and The antibody construct according to claim 77, wherein the VH2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 6, 10, 14, 18, 22, 26, 30, 34, 38, 42, 46, 50, and 54.

80. 1) The VL1 and VH1 are, a) VL1 containing sequence number 213 and VH1 containing sequence number 214; b) VL1 containing sequence number 217 and VH1 containing sequence number 218; c) VL1 containing SEQ ID NO: 221 and VH1 containing SEQ ID NO: 222; and d) VL1 containing SEQ ID NO: 225 and VH1 containing SEQ ID NO: 226 Selected from the group consisting of; and 2) The VL2 and VH2 are, a) VL2 containing Sequence ID 5 and VH2 containing Sequence ID 6; b) VL2 containing SEQ ID NO: 9 and VH2 containing SEQ ID NO: 10; c) VL2 containing SEQ ID NO: 13 and VH2 containing SEQ ID NO: 14; d) VL2 containing SEQ ID NO: 17 and VH2 containing SEQ ID NO: 18; e) VL2 containing sequence number 21 and VH2 containing sequence number 22; f) VL2 containing sequence number 25 and VH2 containing sequence number 26; g) VL2 containing SEQ ID NO: 29 and VH2 containing SEQ ID NO: 30; h) VL2 containing sequence number 33 and VH2 containing sequence number 34; i) VL2 containing Sequence ID 37 and VH2 containing Sequence ID 38; j) VL2 containing sequence number 41 and VH2 containing sequence number 42; k) VL2 containing sequence number 45 and VH2 containing sequence number 46; l) VL2 containing Sequence ID 49 and VH2 containing Sequence ID 50; and m) VL2 containing sequence number 53 and VH2 containing sequence number 54 An antibody construct according to claim 79, selected from the group consisting of the following.

81. An antigen-binding protein that specifically binds to and stimulates human CD40 (SEQ ID NO: 1), comprising a light chain variable region (VL) and a heavy chain variable region (VH), The aforementioned VL is Sequence IDs 58, 59, and 60, respectively; Sequence IDs 64, 65, and 66, respectively; Sequence IDs 70, 71, and 72, respectively; Sequence IDs 76, 77, and 78, respectively; Sequence IDs 82, 83, and 84, respectively; Sequence IDs 88, 89, and 90, respectively; Sequence IDs 94, 95, and 96, respectively; Sequence IDs 100, 101, and 102, respectively; Sequence IDs 106, 107, and 108, respectively; Sequence IDs 112, 113, and 114, respectively; Sequence IDs 118, 119, and 120, respectively; Sequence IDs 124, 125, and 126, respectively; and Sequence IDs 130, 131, and 132, respectively. It includes CDRL1, CDRL2, and CDRL3 selected from the group consisting of; and The aforementioned VH is, Sequence IDs 136, 137, and 138, respectively; Sequence IDs 142, 143, and 144, respectively; Sequence IDs 148, 149, and 150, respectively; Sequence IDs 154, 155, and 156, respectively; Sequence IDs 160, 161, and 162, respectively; Sequence IDs 166, 167, and 168, respectively; Sequence IDs 172, 173, and 174, respectively; Sequence IDs 178, 179, and 180, respectively; Sequence IDs 184, 185, and 186, respectively; Sequence numbers 190, 191, and 192, respectively; Sequence IDs 196, 197, and 198, respectively; Sequence IDs 202, 203, and 204, respectively; and Sequence IDs 208, 209, and 210, respectively. Antigen-binding proteins comprising CDRH1, CDRH2, and CDRH3 selected from the group consisting of the following.

82. The aforementioned VL and VH are a) VL containing CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs. 58, 59, and 60, respectively; and VH containing CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs. 136, 137, and 138, respectively; b) VL containing CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 64, 65, and 66, respectively; and VH containing CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 142, 143, and 144, respectively; c) VL containing CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs. 70, 71, and 72, respectively; and VH containing CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs. 148, 149, and 150, respectively; d) VL containing CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 76, 77, and 6780, respectively; and VH containing CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 154, 155, and 156, respectively; e) VL containing CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs. 82, 83, and 84, respectively; and VH containing CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs. 160, 161, and 162, respectively; f) VL containing CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 88, 89, and 90, respectively; and VH containing CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 166, 167, and 168, respectively; g) VL containing CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 94, 95, and 96, respectively; and VH containing CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 172, 173, and 174, respectively; h) VL containing CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 100, 101, and 102, respectively; and VH containing CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 178, 179, and 180, respectively; i) VL containing CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 106, 107, and 108, respectively; and VH containing CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 184, 185, and 186, respectively; j) VL containing CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 112, 113, and 114, respectively; and VH containing CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 190, 191, and 192, respectively; k) VL containing CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 118, 119, and 120, respectively; and VH containing CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 196, 197, and 198, respectively; l) VL containing CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 124, 125, and 126, respectively; and VH containing CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 202, 203, and 204, respectively; and m) VL containing CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 130, 131, and 132, respectively; and VH containing CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 208, 209, and 210, respectively. An antibody construct according to claim 81, selected from the group consisting of the following.

83. The VL comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 5, 9, 13, 17, 21, 25, 29, 33, 37, 41, 45, 49, and 53; and The antibody construct according to claim 81, wherein the VH comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 6, 10, 14, 18, 22, 26, 30, 34, 38, 42, 46, 50, and 54.

84. The aforementioned VL2 and VH2 are a) VL containing SEQ ID NO: 5 and VH containing SEQ ID NO: 6; b) VL containing SEQ ID NO: 9 and VH containing SEQ ID NO: 10; c) VL containing sequence number 13 and VH containing sequence number 14; d) VL containing SEQ ID NO: 17 and VH containing SEQ ID NO: 18; e) VL containing SEQ ID NO: 21 and VH containing SEQ ID NO: 22; f) VL containing sequence number 25 and VH containing sequence number 26; g) VL containing SEQ ID NO: 29 and VH containing SEQ ID NO: 30; h) VL containing SEQ ID NO: 33 and VH containing SEQ ID NO: 34; i) VL containing Sequence ID 37 and VH containing Sequence ID 38; j) VL containing sequence number 41 and VH containing sequence number 42; k) VL containing SEQ ID NO: 45 and VH containing SEQ ID NO: 46; l) VL containing sequence number 49 and VH containing sequence number 50; and m) VL containing sequence number 53 and VH containing sequence number 54 An antibody construct according to claim 83, selected from the group consisting of the following.

85. The aforementioned heavy chain, (i) N297G or N297A; (ii) L234A and L235A; and (iii) R292C and V302C It includes amino acid substitutions selected from the group consisting of; The antibody construct according to any one of claims 81 to 84, wherein the numbering of the amino acids is EU numbering according to Kabat.

86. The antibody construct according to claim 85, wherein the heavy chain comprises N297G, R292C, and V302C mutations, and the numbering of the amino acids is EU numbering according to Kabat.

87. The antibody construct according to any one of claims 81 to 86, wherein the antigen-binding protein further comprises a light chain CL polypeptide linked to the VL, and the CL polypeptide is selected from the group consisting of SEQ ID NO: 883 and SEQ ID NO:

884.

88. The antibody construct according to any one of claims 81 to 87, wherein the antigen-binding protein further comprises a heavy chain CH1-hinge-CH2-CH3 polypeptide, and the CH1-hinge-CH2-CH3 polypeptide is selected from the group consisting of SEQ ID NO: 885 and SEQ ID NO:

886.

89. The antibody construct according to any one of claims 81 to 88, wherein the antigen-binding protein further comprises a second antigen-binding moiety that specifically binds to a second antigen.

90. The antibody construct according to claim 89, wherein the second antigen is a tumor-associated antigen (TAA).

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