Mesothelin-targeting CD40 agonistic multispecific antibody constructs for treatment of solid tumors

Abstract

The present application relates to mesothelin-targeting CD40 agonistic multispecific antibody constructs for the treatment of solid tumors. Specifically, the present application relates to human agonistic CD40 multispecific antibody constructs for use in the treatment of solid tumors by engineering molecules that specifically target the CD40 pathway on tumor-associated APCs without the need for systemic CD40 activation.

Description

[0001] This application is a divisional application of an application filed on December 18, 2020, with application number 202080095225.3, and invention name “CD40 agonistic multispecific antibody construct targeting mesothelin for the treatment of solid tumors”. Technical Field

[0002] The present invention relates to the field of oncology, in particular cancer immunotherapy. The present invention relates to human agonistic CD40 antibodies by engineering molecules that specifically target the CD40 pathway on tumor-associated APCs without the need for systemic CD40 activation. Background Art

[0003] In the rapidly changing field of immunotherapy, treating patients with cancers that do not respond to immune checkpoint inhibitors (such as anti-PD1) represents a major challenge and exciting opportunity. The major determinant of response to PD1 blockade in solid tumors is the extent of tumor-associated T cell infiltration prior to treatment, with patients with poorly infiltrated, “cold” tumors showing minimal clinical benefit. Although clinical data are still emerging, other immunotherapies targeting T cells other than PD1 blockade (such as anti-PD1) are being investigated in solid tumors. Clinical activity of immunotherapy (antibody constructs or CAR-T approaches) may also be limited to "cold" tumors. Combining T-cell-targeted approaches with novel therapies that can enhance T-cell infiltration into solid tumors may be critical to maximizing the number of patients who benefit from immunotherapy.

[0004] CD40 is a member of the TNF receptor (TNFR) superfamily that is preferentially expressed by antigen-presenting cells (APCs), such as dendritic cells, B cells, and macrophages. Interaction with its trimeric ligand on activated T helper cells results in APC activation, which includes upregulation of cytokines / chemokines (such as interleukin 12 [IL-12] and CxCL10), proteins involved in antigen presentation (such as MHC class I and class II ligands), T cell co-stimulatory ligands (such as CD80 and CD86), and a range of other immunomodulatory factors (i.e., adhesion molecules and other TNFRs). These "permitted" APCs can then trigger a cascade of events that lead to a robust adaptive immune response.

[0005] Therapeutics that can activate CD40 signaling have the potential to inflame solid tumors through their ability to enhance the generation of anti-tumor T cells and to enhance the recruitment of T cells directly into tumor lesions. Preclinical studies with anti-CD40 agonists have shown that CD40 triggered by cross-linking antibodies on APCs can replace CD4 T cells to help license APCs and promote the activation and expansion of CD8 effector T cells. In addition, CD40-activated macrophages can also exert direct tumoricidal functions. These anti-CD40 agonist antibodies have been shown to be effective in a variety of syngeneic tumor models alone or in combination with other therapies. Based on these preclinical studies, several CD40 agonistic antibodies are being studied in phase I / II clinical trials in patients with solid tumors. To date, these monoclonal anti-CD40 antibodies have shown some signs of clinical activity, but are often associated with immune-related adverse reactions, such as cytokine release syndrome and evidence of liver damage. These toxicities limit the dose of CD40 agonists that can be delivered, and therefore can lead to insufficient activation of the CD40 pathway in the tumor-associated APC population, thereby negatively affecting the efficacy of this treatment method. Therapeutic agents that can activate CD40 signaling locally in tumor tissues may improve antitumor effects while limiting systemic toxicity.

[0006] To achieve tumor localization, an agonistic multispecific antibody construct was designed that binds CD40 and the tumor-associated antigen (TAA), mesothelin (MSLN). On APCs expressing CD40, the robust agonist activity of this multispecific antibody construct is entirely dependent on the presence of adjacent cells expressing MSLN. The multispecific antibody construct was specifically engineered to lack CD40 agonist activity after binding to CD40 in the absence of cells expressing MSLN. In addition, the introduction of mutations into the IgG Fc domain limits binding to Fc receptors, thereby preventing Fc receptor-mediated CD40 agonist activity. Therefore, the MSLN-dependent CD40 agonist multispecific antibody construct described herein has the potential to drive robust CD40-mediated activation of APCs in a manner that is primarily limited to tumor tissue, thereby minimizing the induction of systemic toxicity. Summary of the invention

[0007] The present invention relates to a multispecific antibody construct comprising:

[0008] (i) a first antibody comprising two light chains and two heavy chains, wherein

[0009] The light chain comprises the first variable region (VL1) and the light chain constant region (CL);

[0010] The heavy chain comprises the first variable region (VH1) and the CH1, hinge, CH2, and CH3 regions;

[0011] The heavy chain comprises at least one amino acid substitution that results in a decrease in the binding affinity of the heavy chain to the human FcγRI receptor (compared to the unsubstituted heavy chain); and

[0012] (ii) a 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 the VH2 are connected via a first peptide linker,

[0013] wherein the scFv is fused at its amino terminus to the carboxyl terminus of each heavy chain via a second peptide linker to form a heavy chain fusion protein; and

[0014] The first antibody specifically binds to and stimulates human CD40 (SEQ ID NO: 1), and the scFv specifically binds to human mesothelin (MSLN) (SEQ ID NO: 2).

[0015] In one embodiment, the multispecific antibody construct specifically agonizes CD40 in an MSLN-dependent manner.

[0016] In one embodiment, the multispecific antibody construct comprises mutations that limit Fc receptor binding and thereby reduce Fc receptor-dependent CD40 agonism.

[0017] In one embodiment, the two light chains are identical and the two heavy chain fusion proteins are identical.

[0018] In one embodiment, the heavy chain comprises an amino acid substitution selected from the group consisting of:

[0019] (i) N297G or N297A;

[0020] (ii) L234A and L235A; and

[0021] (iii) R292C and V302C;

[0022] The amino acid numbering is according to the EU numbering of Kabat.

[0023] In one embodiment, the heavy chain comprises N297G, R292C, and V302C mutations, wherein the amino acid numbering is according to EU numbering as in Kabat.

[0024] In one embodiment, VL1 comprises CDRL1, CDRL2, and CDRL3 selected from the group consisting of:

[0025] SEQ ID NOs: 58, 59, and 60, respectively;

[0026] SEQ ID NOs: 64, 65, and 66, respectively;

[0027] SEQ ID NOs: 70, 71, and 72, respectively;

[0028] SEQ ID NOs: 76, 77, and 78, respectively;

[0029] SEQ ID NOs: 82, 83, and 84, respectively;

[0030] SEQ ID NOs: 88, 89, and 90, respectively;

[0031] SEQ ID NOs: 94, 95, and 96, respectively;

[0032] SEQ ID NOs: 100, 101, and 102, respectively;

[0033] SEQ ID NOs: 106, 107, and 108, respectively;

[0034] SEQ ID NOs: 112, 113, and 114, respectively;

[0035] SEQ ID NOs: 118, 119, and 120, respectively;

[0036] SEQ ID NOs: 124, 125, and 126, respectively; and

[0037] SEQ ID NOs: 130, 131, and 132, respectively;

[0038] VH1 comprises CDRH1, CDRH2, and CDRH3 selected from the group consisting of:

[0039] SEQ ID NOs: 136, 137, and 138, respectively;

[0040] SEQ ID NOs: 142, 143, and 144, respectively;

[0041] SEQ ID NOs: 148, 149, and 150, respectively;

[0042] SEQ ID NOs: 154, 155, and 156, respectively;

[0043] SEQ ID NOs: 160, 161, and 162, respectively;

[0044] SEQ ID NOs: 166, 167, and 168, respectively;

[0045] SEQ ID NOs: 172, 173, and 174, respectively;

[0046] SEQ ID NOs: 178, 179, and 180, respectively;

[0047] SEQ ID NOs: 184, 185, and 186, respectively;

[0048] SEQ ID NOs: 190, 191, and 192, respectively;

[0049] SEQ ID NOs: 196, 197, and 198, respectively;

[0050] SEQ ID NOs: 202, 203, and 204, respectively; and

[0051] SEQ ID NOs: 208, 209, and 210, respectively;

[0052] VL2 comprises CDRL1, CDRL2, and CDRL3 selected from the group consisting of:

[0053] SEQ ID NOs: 230, 231, and 232, respectively;

[0054] SEQ ID NOs: 236, 237, and 238, respectively;

[0055] SEQ ID NOs: 242, 243, and 244, respectively; and

[0056] SEQ ID NOs: 248, 249, and 250, respectively;

[0057] and VH2 comprises CDRH1, CDRH2, and CDRH3 selected from the group consisting of:

[0058] SEQ ID NOs: 254, 255, and 256, respectively;

[0059] SEQ ID NOs: 260, 261, and 262, respectively;

[0060] SEQ ID NOs: 266, 267, and 268, respectively; and

[0061] They are SEQ ID NO: 272, 273, and 274 respectively.

[0062] In one embodiment, 1) VL1 and VH1 are selected from the group consisting of:

[0063] a) a VL1 comprising CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 58, 59, and 60, respectively; and a VH1 comprising CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 136, 137, and 138, respectively;

[0064] b) a VL1 comprising CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 64, 65, and 66, respectively; and a VH1 comprising CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 142, 143, and 144, respectively;

[0065] c) a VL1 comprising CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 70, 71, and 72, respectively; and a VH1 comprising CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 148, 149, and 150, respectively;

[0066] d) a VL1 comprising CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 76, 77, and 6780, respectively; and a VH1 comprising CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 154, 155, and 156, respectively;

[0067] e) a VL1 comprising CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 82, 83, and 84, respectively; and a VH1 comprising CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 160, 161, and 162, respectively;

[0068] f) a VL1 comprising CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 88, 89, and 90, respectively; and a VH1 comprising CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 166, 167, and 168, respectively;

[0069] g) a VL1 comprising CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 94, 95, and 96, respectively; and a VH1 comprising CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 172, 173, and 174, respectively;

[0070] h) a VL1 comprising CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 100, 101, and 102, respectively; and a VH1 comprising CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 178, 179, and 180, respectively;

[0071] i) a VL1 comprising CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 106, 107, and 108, respectively; and a VH1 comprising CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 184, 185, and 186, respectively;

[0072] j) a VL1 comprising CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 112, 113, and 114, respectively; and a VH1 comprising CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 190, 191, and 192, respectively;

[0073] k) a VL1 comprising CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 118, 119, and 120, respectively; and a VH1 comprising CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 196, 197, and 198, respectively;

[0074] l) a VL1 comprising CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 124, 125, and 126, respectively; and a VH1 comprising CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 202, 203, and 204, respectively; and

[0075] m) a VL1 comprising CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 130, 131, and 132, respectively; and a VH1 comprising CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 208, 209, and 210, respectively;

[0076] and

[0077] 2) VL2 and VH2 are selected from the group consisting of:

[0078] a) a VL2 comprising CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 230, 231, and 232, respectively; and a VH2 comprising CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 254, 255, and 256, respectively;

[0079] b) a VL2 comprising CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 236, 237, and 238, respectively; and a VH2 comprising CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 260, 261, and 262, respectively;

[0080] c) a VL2 comprising CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 242, 243, and 244, respectively; and a VH2 comprising CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 266, 267, and 268, respectively; and

[0081] d) a VL2 comprising CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 248, 249, and 250, respectively; and a VH2 comprising CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 272, 273, and 274, respectively.

[0082] In one embodiment, VL1 comprises an amino acid sequence selected from the group consisting of: SEQ ID NO: 5, 9, 13, 17, 21, 25, 29, 33, 37, 41, 45, 49, and 53;

[0083] VH1 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 6, 10, 14, 18, 22, 26, 30, 34, 38, 42, 46, 50, and 54;

[0084] VL2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 213, 217, 221, and 225;

[0085] VH2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 214, 218, 222, and 226.

[0086] In one embodiment,

[0087] 1) VL1 and VH1 are selected from the group consisting of:

[0088] a) VL1 comprising SEQ ID NO: 5 and VH1 comprising SEQ ID NO: 6;

[0089] b) VL1 comprising SEQ ID NO:9 and VH1 comprising SEQ ID NO:10;

[0090] c) a VL1 comprising SEQ ID NO: 13 and a VH1 comprising SEQ ID NO: 14;

[0091] d) VL1 comprising SEQ ID NO: 17 and VH1 comprising SEQ ID NO: 18;

[0092] e) a VL1 comprising SEQ ID NO: 21 and a VH1 comprising SEQ ID NO: 22;

[0093] f) VL1 comprising SEQ ID NO:25 and VH1 comprising SEQ ID NO:26;

[0094] g) VL1 comprising SEQ ID NO:29 and VH1 comprising SEQ ID NO:30;

[0095] h) a VL1 comprising SEQ ID NO: 33 and a VH1 comprising SEQ ID NO: 34;

[0096] i) VL1 comprising SEQ ID NO: 37 and VH1 comprising SEQ ID NO: 38;

[0097] j) VL1 comprising SEQ ID NO:41 and VH1 comprising SEQ ID NO:42;

[0098] k) a VL1 comprising SEQ ID NO:45 and a VH1 comprising SEQ ID NO:46;

[0099] 1) VL1 comprising SEQ ID NO: 49 and VH1 comprising SEQ ID NO: 50; and

[0100] m) a VL1 comprising SEQ ID NO:53 and a VH1 comprising SEQ ID NO:54;

[0101] and

[0102] 2) VL2 and VH2 are selected from the group consisting of:

[0103] a) VL2 comprising SEQ ID NO: 213 and VH2 comprising SEQ ID NO: 214;

[0104] b) VL2 comprising SEQ ID NO: 217 and VH2 comprising SEQ ID NO: 218;

[0105] c) VL2 comprising SEQ ID NO: 221 and VH2 comprising SEQ ID NO: 222; and

[0106] d) VL2 comprising SEQ ID NO:225 and VH2 comprising SEQ ID NO:226.

[0107] In one embodiment, the CL of the light chain is selected from the group consisting of SEQ ID NO:883 and SEQ ID NO:884.

[0108] In one embodiment, 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.

[0109] In one embodiment, the first peptide linker is selected from the group consisting of SEQ ID NOs: 888-893.

[0110] In one embodiment, the second peptide linker is selected from the group consisting of SEQ ID NOs: 887-893.

[0111] In one embodiment, the first peptide linker comprises SEQ ID NO:889 and the second peptide linker comprises SEQ ID NO:887.

[0112] In one embodiment,

[0113] the light chain comprises 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

[0114] The heavy chain fusion protein comprises a sequence selected from the group consisting of SEQ ID NO: 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.

[0115] In one embodiment, the light chain and heavy chain fusion proteins comprise polypeptides comprising an amino acid sequence selected from the group consisting of:

[0116] SEQ ID NO:286 and SEQ ID NO:285, respectively;

[0117] SEQ ID NO:290 and SEQ ID NO:289, respectively;

[0118] SEQ ID NO:294 and SEQ ID NO:293, respectively;

[0119] SEQ ID NO:298 and SEQ ID NO:297, respectively;

[0120] SEQ ID NO:302 and SEQ ID NO:301, respectively;

[0121] SEQ ID NO:306 and SEQ ID NO:305, respectively;

[0122] SEQ ID NO:310 and SEQ ID NO:309, respectively;

[0123] SEQ ID NO:314 and SEQ ID NO:313, respectively;

[0124] SEQ ID NO:318 and SEQ ID NO:317, respectively;

[0125] SEQ ID NO:322 and SEQ ID NO:321, respectively;

[0126] SEQ ID NO:326 and SEQ ID NO:325, respectively;

[0127] SEQ ID NO:330 and SEQ ID NO:329, respectively;

[0128] SEQ ID NO:334 and SEQ ID NO:333, respectively;

[0129] SEQ ID NO:338 and SEQ ID NO:337, respectively;

[0130] SEQ ID NO:342 and SEQ ID NO:341, respectively;

[0131] SEQ ID NO:346 and SEQ ID NO:345, respectively;

[0132] SEQ ID NO:350 and SEQ ID NO:349, respectively;

[0133] SEQ ID NO:354 and SEQ ID NO:353, respectively;

[0134] SEQ ID NO:358 and SEQ ID NO:357, respectively;

[0135] SEQ ID NO:362 and SEQ ID NO:361, respectively;

[0136] SEQ ID NO:366 and SEQ ID NO:365, respectively;

[0137] SEQ ID NO:370 and SEQ ID NO:369, respectively;

[0138] SEQ ID NO:374 and SEQ ID NO:373, respectively; and

[0139] They are SEQ ID NO:378 and SEQ ID NO:377 respectively.

[0140] In one aspect, the invention relates to a polynucleotide encoding the light chain of an antibody construct of the invention.

[0141] In one aspect, the invention relates to a polynucleotide encoding a heavy chain fusion protein of an antibody construct of the invention.

[0142] In one aspect, the invention relates to a vector comprising a polynucleotide encoding a light chain of an antibody construct, a polynucleotide encoding a heavy chain of an antibody construct, or both.

[0143] In one aspect, the invention relates to a host cell transformed or transfected with a vector or a polynucleotide encoding a light chain of an antibody construct and a polynucleotide encoding a heavy chain of an antibody construct.

[0144] In one aspect, the present invention relates to a method for producing an antibody construct of the present invention, the method comprising culturing a host cell comprising a polynucleotide encoding a light chain and further comprising a polynucleotide encoding a heavy chain fusion protein under conditions allowing expression of the antibody construct, and recovering the produced antibody construct from the culture.

[0145] In one aspect, the invention relates to a pharmaceutical composition comprising an antibody construct according to the invention and a carrier, stabilizer, excipient, diluent, solubilizer, surfactant, emulsifier, preservative or adjuvant.

[0146] In one aspect, the present invention relates to a method for treating or ameliorating solid tumor disease or metastatic cancer disease, which method comprises administering an effective amount of an antibody construct according to the present invention to a subject in need thereof.

[0147] In one embodiment, the solid tumor disease is selected from the group consisting of ovarian cancer, pancreatic cancer, mesothelioma, lung cancer, gastric cancer, and triple negative breast cancer disease or a metastatic cancer disease derived from any of the foregoing diseases.

[0148] In one aspect, the invention relates to a kit comprising an antibody construct according to the invention and, optionally, instructions for use.

[0149] In one aspect, the present invention relates to a multispecific antibody construct comprising:

[0150] (i) a first antibody comprising two light chains and two heavy chains, wherein

[0151] The light chain comprises the first variable region (VL1) and the light chain constant region (CL);

[0152] The heavy chain comprises the first variable region (VH1) and the CH1, hinge, CH2, and CH3 regions;

[0153] The heavy chain comprises at least one amino acid substitution that results in a decrease in the binding affinity of the heavy chain to the human FcγRI receptor (compared to the unsubstituted heavy chain); and

[0154] (ii) a 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 the VH2 are connected via a first peptide linker,

[0155] wherein the scFv is fused at its amino terminus to the carboxyl terminus of each heavy chain via a second peptide linker to form a heavy chain fusion protein; and

[0156] The first antibody specifically binds to human mesothelin (MSLN) (SEQ ID NO: 2), and the scFv specifically binds to and stimulates human CD40 (SEQ ID NO: 1).

[0157] In one embodiment, the multispecific antibody construct specifically agonizes CD40 in an MSLN-dependent manner.

[0158] In one embodiment, the multispecific antibody construct comprises mutations that limit Fc receptor binding and thereby reduce Fc receptor-dependent CD40 agonism.

[0159] In one embodiment, the two light chains are identical and the two heavy chain fusion proteins are identical.

[0160] In one embodiment, the heavy chain comprises an amino acid substitution selected from the group consisting of:

[0161] (i) N297G or N297A;

[0162] (ii) L234A and L235A; and

[0163] (iii) R292C and V302C;

[0164] The amino acid numbering is according to the EU numbering of Kabat.

[0165] In one embodiment, the heavy chain comprises N297G, R292C, and V302C mutations, wherein the amino acid numbering is according to EU numbering as in Kabat.

[0166] In one embodiment:

[0167] VL1 comprises CDRL1, CDRL2, and CDRL3 selected from the group consisting of:

[0168] SEQ ID NOs: 230, 231, and 232, respectively;

[0169] SEQ ID NOs: 236, 237, and 238, respectively;

[0170] SEQ ID NOs: 242, 243, and 244, respectively; and

[0171] SEQ ID NOs: 248, 249, and 250, respectively;

[0172] VH1 comprises CDRH1, CDRH2, and CDRH3 selected from the group consisting of:

[0173] SEQ ID NOs: 254, 255, and 256, respectively;

[0174] SEQ ID NOs: 260, 261, and 262, respectively;

[0175] SEQ ID NOs: 266, 267, and 268, respectively; and

[0176] SEQ ID NOs: 272, 273, and 274, respectively;

[0177] VL2 comprises CDRL1, CDRL2, and CDRL3 selected from the group consisting of:

[0178] SEQ ID NOs: 58, 59, and 60, respectively;

[0179] SEQ ID NOs: 64, 65, and 66, respectively;

[0180] SEQ ID NOs: 70, 71, and 72, respectively;

[0181] SEQ ID NOs: 76, 77, and 78, respectively;

[0182] SEQ ID NOs: 82, 83, and 84, respectively;

[0183] SEQ ID NOs: 88, 89, and 90, respectively;

[0184] SEQ ID NOs: 94, 95, and 96, respectively;

[0185] SEQ ID NOs: 100, 101, and 102, respectively;

[0186] SEQ ID NOs: 106, 107, and 108, respectively;

[0187] SEQ ID NOs: 112, 113, and 114, respectively;

[0188] SEQ ID NOs: 118, 119, and 120, respectively;

[0189] SEQ ID NOs: 124, 125, and 126, respectively; and

[0190] SEQ ID NOs: 130, 131, and 132, respectively;

[0191] and

[0192] VH2 comprises CDRH1, CDRH2, and CDRH3 selected from the group consisting of:

[0193] SEQ ID NOs: 136, 137, and 138, respectively;

[0194] SEQ ID NOs: 142, 143, and 144, respectively;

[0195] SEQ ID NOs: 148, 149, and 150, respectively;

[0196] SEQ ID NOs: 154, 155, and 156, respectively;

[0197] SEQ ID NOs: 160, 161, and 162, respectively;

[0198] SEQ ID NOs: 166, 167, and 168, respectively;

[0199] SEQ ID NOs: 172, 173, and 174, respectively;

[0200] SEQ ID NOs: 178, 179, and 180, respectively;

[0201] SEQ ID NOs: 184, 185, and 186, respectively;

[0202] SEQ ID NOs: 190, 191, and 192, respectively;

[0203] SEQ ID NOs: 196, 197, and 198, respectively;

[0204] SEQ ID NOs: 202, 203, and 204, respectively; and

[0205] They are SEQ ID NOs: 208, 209, and 210, respectively.

[0206] In one embodiment,

[0207] 1) VL1 and VH1 are selected from the group consisting of:

[0208] a) a VL1 comprising CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 230, 231, and 232, respectively; and a VH1 comprising CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 254, 255, and 256, respectively;

[0209] b) a VL1 comprising CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 236, 237, and 238, respectively; and a VH1 comprising CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 260, 261, and 262, respectively;

[0210] c) a VL1 comprising CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 242, 243, and 244, respectively; and a VH1 comprising CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 266, 267, and 268, respectively; and

[0211] d) a VL1 comprising CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 248, 249, and 250, respectively; and a VH1 comprising CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 272, 273, and 274, respectively;

[0212] and

[0213] 2) VL2 and VH2 are selected from the group consisting of:

[0214] a) a VL2 comprising CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 58, 59, and 60, respectively; and a VH2 comprising CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 136, 137, and 138, respectively;

[0215] b) a VL2 comprising CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 64, 65, and 66, respectively; and a VH2 comprising CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 142, 143, and 144, respectively;

[0216] c) a VL2 comprising CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 70, 71, and 72, respectively; and a VH2 comprising CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 148, 149, and 150, respectively;

[0217] d) a VL2 comprising CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 76, 77, and 6780, respectively; and a VH2 comprising CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 154, 155, and 156, respectively;

[0218] e) a VL2 comprising CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 82, 83, and 84, respectively; and a VH2 comprising CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 160, 161, and 162, respectively;

[0219] f) a VL2 comprising CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 88, 89, and 90, respectively; and a VH2 comprising CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 166, 167, and 168, respectively;

[0220] g) a VL2 comprising CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 94, 95, and 96, respectively; and a VH2 comprising CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 172, 173, and 174, respectively;

[0221] h) a VL2 comprising CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 100, 101, and 102, respectively; and a VH2 comprising CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 178, 179, and 180, respectively;

[0222] i) a VL2 comprising CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 106, 107, and 108, respectively; and a VH2 comprising CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 184, 185, and 186, respectively;

[0223] j) a VL2 comprising CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 112, 113, and 114, respectively; and a VH2 comprising CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 190, 191, and 192, respectively;

[0224] k) a VL2 comprising CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 118, 119, and 120, respectively; and a VH2 comprising CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 196, 197, and 198, respectively;

[0225] 1) a VL2 comprising CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 124, 125, and 126, respectively; and a VH2 comprising CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 202, 203, and 204, respectively; and

[0226] m) a VL2 comprising CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 130, 131, and 132, respectively; and a VH2 comprising CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 208, 209, and 210, respectively.

[0227] In one embodiment:

[0228] VL1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 213, 217, 221, and 225;

[0229] VH1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 214, 218, 222, and 226;

[0230] VL2 comprises an amino acid sequence selected from the group consisting of: SEQ ID NO: 5, 9, 13, 17, 21, 25, 29, 33, 37, 41, 45, 49, and 53; and

[0231] VH2 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 6, 10, 14, 18, 22, 26, 30, 34, 38, 42, 46, 50, and 54.

[0232] In one embodiment:

[0233] 1) VL1 and VH1 are selected from the group consisting of:

[0234] a) VL1 comprising SEQ ID NO: 213 and VH1 comprising SEQ ID NO: 214;

[0235] b) VL1 comprising SEQ ID NO: 217 and VH1 comprising SEQ ID NO: 218;

[0236] c) a VL1 comprising SEQ ID NO: 221 and a VH1 comprising SEQ ID NO: 222; and

[0237] d) VL1 comprising SEQ ID NO: 225 and VH1 comprising SEQ ID NO: 226;

[0238] and

[0239] 2) VL2 and VH2 are selected from the group consisting of:

[0240] a) VL2 comprising SEQ ID NO: 5 and VH2 comprising SEQ ID NO: 6;

[0241] b) VL2 comprising SEQ ID NO: 9 and VH2 comprising SEQ ID NO: 10;

[0242] c) VL2 comprising SEQ ID NO: 13 and VH2 comprising SEQ ID NO: 14;

[0243] d) VL2 comprising SEQ ID NO: 17 and VH2 comprising SEQ ID NO: 18;

[0244] e) VL2 comprising SEQ ID NO: 21 and VH2 comprising SEQ ID NO: 22;

[0245] f) VL2 comprising SEQ ID NO:25 and VH2 comprising SEQ ID NO:26;

[0246] g) VL2 comprising SEQ ID NO:29 and VH2 comprising SEQ ID NO:30;

[0247] h) VL2 comprising SEQ ID NO:33 and VH2 comprising SEQ ID NO:34;

[0248] i) VL2 comprising SEQ ID NO: 37 and VH2 comprising SEQ ID NO: 38;

[0249] j) VL2 comprising SEQ ID NO:41 and VH2 comprising SEQ ID NO:42;

[0250] k) VL2 comprising SEQ ID NO:45 and VH2 comprising SEQ ID NO:46;

[0251] 1) VL2 comprising SEQ ID NO: 49 and VH2 comprising SEQ ID NO: 50; and

[0252] m) VL2 comprising SEQ ID NO:53 and VH2 comprising SEQ ID NO:54.

[0253] In one embodiment, the CL of the light chain is selected from the group consisting of SEQ ID NO:883 and SEQ ID NO:884.

[0254] In one embodiment, 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.

[0255] In one embodiment, the first peptide linker is selected from the group consisting of SEQ ID NOs: 888-893.

[0256] In one embodiment, the second peptide linker is selected from the group consisting of SEQ ID NOs: 887-893.

[0257] In one embodiment, the first peptide linker comprises SEQ ID NO:889 and the second peptide linker comprises SEQ ID NO:887.

[0258] In one embodiment,

[0259] The light chain comprises a sequence selected from the group consisting of: SEQ ID NO: 382, ​​386, 390, 394, 398, 402, 406, 410, 414, 418, 422, 426, 430, 434, 438, 442, 446, and 450; and

[0260] The heavy chain fusion protein comprises a sequence selected from the group consisting of: SEQ ID NO: 381, 385, 389, 393, 397, 401, 405, 409, 413, 417, 421, 425, 429, 433, 437, 441, 445, and 449.

[0261] In one embodiment, the light chain and heavy chain fusion proteins comprise polypeptides comprising an amino acid sequence selected from the group consisting of:

[0262] SEQ ID NO:382 and SEQ ID NO:381, respectively;

[0263] SEQ ID NO:386 and SEQ ID NO:385, respectively;

[0264] SEQ ID NO:390 and SEQ ID NO:389, respectively;

[0265] SEQ ID NO:394 and SEQ ID NO:393, respectively;

[0266] SEQ ID NO:398 and SEQ ID NO:397, respectively;

[0267] SEQ ID NO:402 and SEQ ID NO:401, respectively;

[0268] SEQ ID NO:406 and SEQ ID NO:405, respectively;

[0269] SEQ ID NO:410 and SEQ ID NO:409, respectively;

[0270] SEQ ID NO:414 and SEQ ID NO:413, respectively;

[0271] SEQ ID NO:418 and SEQ ID NO:417, respectively;

[0272] SEQ ID NO:422 and SEQ ID NO:421, respectively;

[0273] SEQ ID NO:426 and SEQ ID NO:425, respectively;

[0274] SEQ ID NO:430 and SEQ ID NO:429, respectively;

[0275] SEQ ID NO:434 and SEQ ID NO:433, respectively;

[0276] SEQ ID NO:438 and SEQ ID NO:437, respectively;

[0277] SEQ ID NO:442 and SEQ ID NO:441, respectively;

[0278] SEQ ID NO:446 and SEQ ID NO:445, respectively; and

[0279] They are SEQ ID NO:450 and SEQ ID NO:449 respectively.

[0280] In one aspect, the invention relates to a polynucleotide encoding the light chain of an antibody construct of the invention.

[0281] In one aspect, the invention relates to a polynucleotide encoding a heavy chain fusion protein of an antibody construct of the invention.

[0282] In one aspect, the invention relates to a host cell comprising a polynucleotide encoding a light chain of an antibody construct of the invention, a polynucleotide encoding a heavy chain fusion protein of an antibody construct, or both.

[0283] In one aspect, the present invention relates to a method for producing an antibody construct of the present invention, comprising culturing a host cell comprising a polynucleotide encoding a light chain and further comprising a polynucleotide encoding a heavy chain fusion protein under conditions allowing expression of the antibody construct, and recovering the produced antibody construct from the culture.

[0284] In one aspect, the invention relates to a pharmaceutical composition comprising an antibody construct of the invention and a carrier, stabilizer, excipient, diluent, solubilizer, surfactant, emulsifier, preservative or adjuvant.

[0285] In one aspect, the present invention relates to a method for treating or ameliorating solid tumor disease or metastatic cancer disease, which method comprises administering an effective amount of the antibody construct of the present invention to a subject in need thereof.

[0286] In one embodiment, the solid tumor disease is selected from the group consisting of ovarian cancer, pancreatic cancer, mesothelioma, lung cancer, gastric cancer, and triple negative breast cancer disease or a metastatic cancer disease derived from any of the foregoing diseases.

[0287] In one aspect, the invention relates to a kit comprising an antibody construct of the invention and optionally instructions for use.

[0288] In one aspect, the present invention relates to a multispecific antibody construct comprising:

[0289] 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 wherein the hinge-CH2-CH3 polypeptide is linked to a second heavy chain variable region (VH2), wherein the VH2 is linked to a second CH1 domain; wherein

[0290] i) the VH1 or first CH1 domain comprises at least one amino acid substitution to introduce a positively charged amino acid at a residue selected from the group consisting of positions 39, 44, and 183, using EU numbering; and

[0291] ii) the VH2 or second CH1 domain comprises at least one amino acid substitution to introduce 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

[0292] 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 wherein the VL1 or the first CL domain comprises at least one amino acid substitution to introduce a negatively charged amino acid at a residue selected from the group consisting of positions 38, 100, and 176, using EU numbering; and

[0293] 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 wherein the VL2 or the second CL domain comprises at least one amino acid substitution to introduce a positively charged amino acid at a residue selected from the group consisting of positions 38, 100, and 176, using EU numbering;

[0294] wherein VH1 and VL1 interact to bind a first antigen and wherein VH2 and VL2 interact to bind a second antigen;

[0295] in:

[0296] The first antigen is human CD40 (SEQ ID NO: 1) and the second antigen is human mesothelin ("MSLN"; SEQ ID NO: 2); or

[0297] The first antigen is human MSLN (SEQ ID NO: 2) and the second antigen is human CD40 (SEQ ID NO: 1).

[0298] In one aspect, the present invention relates to a multispecific antibody construct comprising:

[0299] 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 wherein the hinge-CH2-CH3 polypeptide is linked to a second heavy chain variable region (VH2), wherein the VH2 is linked to a second CH1 domain; wherein

[0300] i) the VH1 or first CH1 domain comprises at least one amino acid substitution to introduce a negatively charged amino acid at a residue selected from the group consisting of positions 39, 44, and 183, using EU numbering; and

[0301] ii) the VH2 or second CH1 domain comprises at least one amino acid substitution to introduce 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

[0302] 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 wherein the VL1 or the first CL domain comprises at least one amino acid substitution to introduce a positively charged amino acid at a residue selected from the group consisting of positions 38, 100, and 176, using EU numbering; and

[0303] 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 wherein the VL2 or the second CL domain comprises at least one amino acid substitution to introduce a negatively charged amino acid at a residue selected from the group consisting of positions 38, 100, and 176, using EU numbering;

[0304] wherein VH1 and VL1 interact to bind a first antigen and wherein VH2 and VL2 interact to bind a second antigen;

[0305] in:

[0306] The first antigen is human CD40 (SEQ ID NO: 1) and the second antigen is human mesothelin ("MSLN"; SEQ ID NO: 2); or

[0307] The first antigen is human MSLN (SEQ ID NO: 2) and the second antigen is human CD40 (SEQ ID NO: 1).

[0308] In one embodiment, the hinge-CH2-CH3 polypeptide is linked to VH2 via a peptide linker.

[0309] In one embodiment, the peptide linker comprises 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).

[0310] In one embodiment,

[0311] a) the VH1 or first CH1 domain comprises a mutation selected from the group consisting of: Q39K, G44K, and S183K, using EU numbering;

[0312] b) the VH2 or second CH1 domain comprises a mutation selected from the group consisting of: Q39E, G44E, and S183E, using EU numbering;

[0313] c) the VL1 or first CL domain comprises a mutation selected from the group consisting of: Q38E, G100E, and S176E, using EU numbering; and

[0314] d) VL2 or the second CL domain comprises a mutation selected from the group consisting of: Q38K, G100K, and S176K, using EU numbering.

[0315] In one embodiment,

[0316] a) the first CH1 domain contains an S183K mutation, using EU numbering;

[0317] b) the second CH1 domain contains the S183E mutation, using EU numbering;

[0318] c) the first CL domain comprises an S176E mutation, using EU numbering; and

[0319] d) The second CL domain contains the S176K mutation, using EU numbering.

[0320] In one embodiment,

[0321] a) VH1 comprises a Q39K mutation and the first CH1 domain comprises a S183K mutation, using EU numbering;

[0322] b) VH2 comprises a Q39E mutation and the second CH1 domain comprises an S183E mutation, using EU numbering;

[0323] c) VL1 comprises a Q38E mutation and the first CL domain comprises an S176E mutation, using EU numbering; and

[0324] d) VL2 comprises a Q38K mutation and the second CL domain comprises a S176K mutation, using EU numbering.

[0325] In one embodiment,

[0326] a) the first CH1 domain contains G44K and S183K mutations, using EU numbering;

[0327] b) the second CH1 domain contains G44E and S183E mutations, using EU numbering;

[0328] c) the first CL domain comprises G100E and S176E mutations, using EU numbering; and

[0329] d) The second CL domain contains G100K and S176K mutations, using EU numbering.

[0330] In one embodiment,

[0331] a) the VH1 or first CH1 domain comprises a mutation selected from the group consisting of: Q39E, G44E, and S183E, using EU numbering;

[0332] b) the VH2 or second CH1 domain comprises a mutation selected from the group consisting of: Q39K, G44K, and S183K, using EU numbering;

[0333] c) the VL1 or first CL domain comprises a mutation selected from the group consisting of: Q38K, G100K, and S176K, using EU numbering; and

[0334] d) VL2 or the second CL domain comprises a mutation selected from the group consisting of: Q38E, G100E, and S176E, using EU numbering.

[0335] In one embodiment,

[0336] a) the first CH1 domain contains the S183E mutation, using EU numbering;

[0337] b) the second CH1 domain contains an S183K mutation, using EU numbering;

[0338] c) the first CL domain comprises an S176K mutation, using EU numbering; and

[0339] d) The second CL domain contains the S176E mutation, using EU numbering.

[0340] In one embodiment,

[0341] a) VH1 comprises a Q39E mutation and the first CH1 domain comprises a S183E mutation, using EU numbering;

[0342] b) VH2 comprises a Q39K mutation and the second CH1 domain comprises a S183K mutation, using EU numbering;

[0343] c) VL1 comprises a Q38K mutation and the first CL domain comprises a S176K mutation, using EU numbering; and

[0344] d) VL2 comprises a Q38E mutation and the second CL domain comprises a S176E mutation, using EU numbering.

[0345] In one embodiment,

[0346] a) the first CH1 domain contains G44E and S183E mutations, using EU numbering;

[0347] b) the second CH1 domain contains G44K and S183K mutations, using EU numbering;

[0348] c) the first CL domain comprises G100K and S176K mutations, using EU numbering; and

[0349] d) The second CL domain contains G100E and S176E mutations, using EU numbering.

[0350] In one embodiment, the hinge-CH2-CH3 polypeptide comprises an amino acid substitution selected from the group consisting of:

[0351] (i) N297G or N297A;

[0352] (ii) L234A and L235A; and

[0353] (iii) R292C and V302C;

[0354] The amino acid numbering is according to the EU numbering of Kabat.

[0355] In one embodiment, the hinge-CH2-CH3 polypeptide comprises N297G, R292C, and V302C mutations, wherein the amino acid numbering is according to EU numbering as in Kabat.

[0356] 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

[0357] VL1 comprises CDRL1, CDRL2, and CDRL3 selected from the group consisting of:

[0358] SEQ ID NOs: 58, 59, and 60, respectively;

[0359] SEQ ID NOs: 64, 65, and 66, respectively;

[0360] SEQ ID NOs: 70, 71, and 72, respectively;

[0361] SEQ ID NOs: 76, 77, and 78, respectively;

[0362] SEQ ID NOs: 82, 83, and 84, respectively;

[0363] SEQ ID NOs: 88, 89, and 90, respectively;

[0364] SEQ ID NOs: 94, 95, and 96, respectively;

[0365] SEQ ID NOs: 100, 101, and 102, respectively;

[0366] SEQ ID NOs: 106, 107, and 108, respectively;

[0367] SEQ ID NOs: 112, 113, and 114, respectively;

[0368] SEQ ID NOs: 118, 119, and 120, respectively;

[0369] SEQ ID NOs: 124, 125, and 126, respectively; and

[0370] SEQ ID NOs: 130, 131, and 132, respectively;

[0371] VH1 comprises CDRH1, CDRH2, and CDRH3 selected from the group consisting of:

[0372] SEQ ID NOs: 136, 137, and 138, respectively;

[0373] SEQ ID NOs: 142, 143, and 144, respectively;

[0374] SEQ ID NOs: 148, 149, and 150, respectively;

[0375] SEQ ID NOs: 154, 155, and 156, respectively;

[0376] SEQ ID NOs: 160, 161, and 162, respectively;

[0377] SEQ ID NOs: 166, 167, and 168, respectively;

[0378] SEQ ID NOs: 172, 173, and 174, respectively;

[0379] SEQ ID NOs: 178, 179, and 180, respectively;

[0380] SEQ ID NOs: 184, 185, and 186, respectively;

[0381] SEQ ID NOs: 190, 191, and 192, respectively;

[0382] SEQ ID NOs: 196, 197, and 198, respectively;

[0383] SEQ ID NOs: 202, 203, and 204, respectively; and

[0384] SEQ ID NOs: 208, 209, and 210, respectively;

[0385] VL2 comprises CDRL1, CDRL2, and CDRL3 selected from the group consisting of:

[0386] SEQ ID NOs: 230, 231, and 232, respectively;

[0387] SEQ ID NOs: 236, 237, and 238, respectively;

[0388] SEQ ID NOs: 242, 243, and 244, respectively; and

[0389] SEQ ID NOs: 248, 249, and 250, respectively;

[0390] and VH2 comprises CDRH1, CDRH2, and CDRH3 selected from the group consisting of:

[0391] SEQ ID NOs: 254, 255, and 256, respectively;

[0392] SEQ ID NOs: 260, 261, and 262, respectively;

[0393] SEQ ID NOs: 266, 267, and 268, respectively; and

[0394] They are SEQ ID NO: 272, 273, and 274 respectively.

[0395] In one embodiment, 1) VL1 and VH1 are selected from the group consisting of:

[0396] a) a VL1 comprising CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 58, 59, and 60, respectively; and a VH1 comprising CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 136, 137, and 138, respectively;

[0397] b) a VL1 comprising CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 64, 65, and 66, respectively; and a VH1 comprising CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 142, 143, and 144, respectively;

[0398] c) a VL1 comprising CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 70, 71, and 72, respectively; and a VH1 comprising CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 148, 149, and 150, respectively;

[0399] d) a VL1 comprising CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 76, 77, and 6780, respectively; and a VH1 comprising CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 154, 155, and 156, respectively;

[0400] e) a VL1 comprising CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 82, 83, and 84, respectively; and a VH1 comprising CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 160, 161, and 162, respectively;

[0401] f) a VL1 comprising CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 88, 89, and 90, respectively; and a VH1 comprising CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 166, 167, and 168, respectively;

[0402] g) a VL1 comprising CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 94, 95, and 96, respectively; and a VH1 comprising CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 172, 173, and 174, respectively;

[0403] h) a VL1 comprising CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 100, 101, and 102, respectively; and a VH1 comprising CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 178, 179, and 180, respectively;

[0404] i) a VL1 comprising CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 106, 107, and 108, respectively; and a VH1 comprising CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 184, 185, and 186, respectively;

[0405] j) a VL1 comprising CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 112, 113, and 114, respectively; and a VH1 comprising CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 190, 191, and 192, respectively;

[0406] k) a VL1 comprising CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 118, 119, and 120, respectively; and a VH1 comprising CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 196, 197, and 198, respectively;

[0407] l) a VL1 comprising CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 124, 125, and 126, respectively; and a VH1 comprising CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 202, 203, and 204, respectively; and

[0408] m) a VL1 comprising CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 130, 131, and 132, respectively; and a VH1 comprising CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 208, 209, and 210, respectively;

[0409] as well as

[0410] 2) VL2 and VH2 are selected from the group consisting of:

[0411] a) a VL2 comprising CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 230, 231, and 232, respectively; and a VH2 comprising CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 254, 255, and 256, respectively;

[0412] b) a VL2 comprising CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 236, 237, and 238, respectively; and a VH2 comprising CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 260, 261, and 262, respectively;

[0413] c) a VL2 comprising CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 242, 243, and 244, respectively; and a VH2 comprising CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 266, 267, and 268, respectively; and

[0414] d) a VL2 comprising CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 248, 249, and 250, respectively; and a VH2 comprising CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 272, 273, and 274, respectively.

[0415] In one embodiment:

[0416] VL1 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 5, 9, 13, 17, 21, 25, 29, 33, 37, 41, 45, 49, and 53;

[0417] VH1 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 6, 10, 14, 18, 22, 26, 30, 34, 38, 42, 46, 50, and 54;

[0418] VL2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 213, 217, 221, and 225;

[0419] VH2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 214, 218, 222, and 226.

[0420] In one embodiment:

[0421] 1) VL1 and VH1 are selected from the group consisting of:

[0422] a) VL1 comprising SEQ ID NO: 5 and VH1 comprising SEQ ID NO: 6;

[0423] b) VL1 comprising SEQ ID NO:9 and VH1 comprising SEQ ID NO:10;

[0424] c) a VL1 comprising SEQ ID NO: 13 and a VH1 comprising SEQ ID NO: 14;

[0425] d) VL1 comprising SEQ ID NO: 17 and VH1 comprising SEQ ID NO: 18;

[0426] e) a VL1 comprising SEQ ID NO: 21 and a VH1 comprising SEQ ID NO: 22;

[0427] f) VL1 comprising SEQ ID NO:25 and VH1 comprising SEQ ID NO:26;

[0428] g) VL1 comprising SEQ ID NO:29 and VH1 comprising SEQ ID NO:30;

[0429] h) a VL1 comprising SEQ ID NO: 33 and a VH1 comprising SEQ ID NO: 34;

[0430] i) VL1 comprising SEQ ID NO: 37 and VH1 comprising SEQ ID NO: 38;

[0431] j) VL1 comprising SEQ ID NO:41 and VH1 comprising SEQ ID NO:42;

[0432] k) a VL1 comprising SEQ ID NO:45 and a VH1 comprising SEQ ID NO:46;

[0433] 1) VL1 comprising SEQ ID NO: 49 and VH1 comprising SEQ ID NO: 50; and

[0434] m) a VL1 comprising SEQ ID NO:53 and a VH1 comprising SEQ ID NO:54;

[0435] and

[0436] 2) VL2 and VH2 are selected from the group consisting of:

[0437] a) VL2 comprising SEQ ID NO: 213 and VH2 comprising SEQ ID NO: 214;

[0438] b) VL2 comprising SEQ ID NO: 217 and VH2 comprising SEQ ID NO: 218;

[0439] c) VL2 comprising SEQ ID NO: 221 and VH2 comprising SEQ ID NO: 222; and

[0440] d) VL2 comprising SEQ ID NO:225 and VH2 comprising SEQ ID NO:226.

[0441] In one aspect, the invention relates to a polynucleotide encoding a first light chain of an antibody construct.

[0442] In one aspect, the invention relates to a polynucleotide encoding the second light chain of an antibody construct.

[0443] In one aspect, the invention relates to a polynucleotide encoding a heavy chain fusion protein of an antibody construct.

[0444] In one aspect, the present invention relates to a vector comprising:

[0445] a) a polynucleotide encoding the first light chain of the antibody construct,

[0446] b) a polynucleotide encoding the second light chain of the antibody construct,

[0447] c) a polynucleotide encoding a heavy chain fusion protein of an antibody construct, or

[0448] d) Any combination of a), b), and c).

[0449] In one aspect, the invention relates to transformation or transfection of a host cell with a vector or a polynucleotide according to the invention.

[0450] In one aspect, the present invention relates to a method for producing an antibody construct according to the present invention, the method comprising culturing a host cell comprising 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 allowing expression of the antibody construct, and recovering the produced antibody construct from the culture.

[0451] In one aspect, the invention relates to a pharmaceutical composition comprising an antibody construct according to the invention and a carrier, stabilizer, excipient, diluent, solubilizer, surfactant, emulsifier, preservative or adjuvant.

[0452] In one aspect, the present invention relates to a method for treating or ameliorating solid tumor disease or metastatic cancer disease, which method comprises administering an effective amount of an antibody construct according to the present invention to a subject in need thereof.

[0453] In one embodiment, the solid tumor disease is selected from the group consisting of ovarian cancer, pancreatic cancer, mesothelioma, lung cancer, gastric cancer, and triple negative breast cancer disease or a metastatic cancer disease derived from any of the foregoing diseases.

[0454] In one aspect, the invention relates to a kit comprising an antibody construct according to the invention and, optionally, instructions for use.

[0455] In one embodiment, the first antigen is human MSLN (SEQ ID NO: 2) and the second antigen is human CD40 (SEQ ID NO: 1); wherein:

[0456] VL1 comprises CDRL1, CDRL2, and CDRL3 selected from the group consisting of:

[0457] SEQ ID NOs: 230, 231, and 232, respectively;

[0458] SEQ ID NOs: 236, 237, and 238, respectively;

[0459] SEQ ID NOs: 242, 243, and 244, respectively; and

[0460] SEQ ID NOs: 248, 249, and 250, respectively;

[0461] VH1 comprises CDRH1, CDRH2, and CDRH3 selected from the group consisting of:

[0462] SEQ ID NOs: 254, 255, and 256, respectively;

[0463] SEQ ID NOs: 260, 261, and 262, respectively;

[0464] SEQ ID NOs: 266, 267, and 268, respectively; and

[0465] SEQ ID NOs: 272, 273, and 274, respectively;

[0466] VL2 comprises CDRL1, CDRL2, and CDRL3 selected from the group consisting of:

[0467] SEQ ID NOs: 58, 59, and 60, respectively;

[0468] SEQ ID NOs: 64, 65, and 66, respectively;

[0469] SEQ ID NOs: 70, 71, and 72, respectively;

[0470] SEQ ID NOs: 76, 77, and 78, respectively;

[0471] SEQ ID NOs: 82, 83, and 84, respectively;

[0472] SEQ ID NOs: 88, 89, and 90, respectively;

[0473] SEQ ID NOs: 94, 95, and 96, respectively;

[0474] SEQ ID NOs: 100, 101, and 102, respectively;

[0475] SEQ ID NOs: 106, 107, and 108, respectively;

[0476] SEQ ID NOs: 112, 113, and 114, respectively;

[0477] SEQ ID NOs: 118, 119, and 120, respectively;

[0478] SEQ ID NOs: 124, 125, and 126, respectively; and

[0479] SEQ ID NOs: 130, 131, and 132, respectively;

[0480] and

[0481] VH2 comprises CDRH1, CDRH2, and CDRH3 selected from the group consisting of:

[0482] SEQ ID NOs: 136, 137, and 138, respectively;

[0483] SEQ ID NOs: 142, 143, and 144, respectively;

[0484] SEQ ID NOs: 148, 149, and 150, respectively;

[0485] SEQ ID NOs: 154, 155, and 156, respectively;

[0486] SEQ ID NOs: 160, 161, and 162, respectively;

[0487] SEQ ID NOs: 166, 167, and 168, respectively;

[0488] SEQ ID NOs: 172, 173, and 174, respectively;

[0489] SEQ ID NOs: 178, 179, and 180, respectively;

[0490] SEQ ID NOs: 184, 185, and 186, respectively;

[0491] SEQ ID NOs: 190, 191, and 192, respectively;

[0492] SEQ ID NOs: 196, 197, and 198, respectively;

[0493] SEQ ID NOs: 202, 203, and 204, respectively; and

[0494] They are SEQ ID NOs: 208, 209, and 210, respectively.

[0495] In one embodiment,

[0496] 1) VL1 and VH1 are selected from the group consisting of:

[0497] a) a VL1 comprising CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 230, 231, and 232, respectively; and a VH1 comprising CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 254, 255, and 256, respectively;

[0498] b) a VL1 comprising CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 236, 237, and 238, respectively; and a VH1 comprising CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 260, 261, and 262, respectively;

[0499] c) a VL1 comprising CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 242, 243, and 244, respectively; and a VH1 comprising CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 266, 267, and 268, respectively; and

[0500] d) a VL1 comprising CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 248, 249, and 250, respectively; and a VH1 comprising CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 272, 273, and 274, respectively;

[0501] and

[0502] 2) VL2 and VH2 are selected from the group consisting of:

[0503] a) a VL2 comprising CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 58, 59, and 60, respectively; and a VH2 comprising CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 136, 137, and 138, respectively;

[0504] b) a VL2 comprising CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 64, 65, and 66, respectively; and a VH2 comprising CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 142, 143, and 144, respectively;

[0505] c) a VL2 comprising CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 70, 71, and 72, respectively; and a VH2 comprising CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 148, 149, and 150, respectively;

[0506] d) a VL2 comprising CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 76, 77, and 6780, respectively; and a VH2 comprising CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 154, 155, and 156, respectively;

[0507] e) a VL2 comprising CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 82, 83, and 84, respectively; and a VH2 comprising CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 160, 161, and 162, respectively;

[0508] f) a VL2 comprising CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 88, 89, and 90, respectively; and a VH2 comprising CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 166, 167, and 168, respectively;

[0509] g) a VL2 comprising CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 94, 95, and 96, respectively; and a VH2 comprising CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 172, 173, and 174, respectively;

[0510] h) a VL2 comprising CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 100, 101, and 102, respectively; and a VH2 comprising CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 178, 179, and 180, respectively;

[0511] i) a VL2 comprising CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 106, 107, and 108, respectively; and a VH2 comprising CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 184, 185, and 186, respectively;

[0512] j) a VL2 comprising CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 112, 113, and 114, respectively; and a VH2 comprising CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 190, 191, and 192, respectively;

[0513] k) a VL2 comprising CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 118, 119, and 120, respectively; and a VH2 comprising CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 196, 197, and 198, respectively;

[0514] 1) a VL2 comprising CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 124, 125, and 126, respectively; and a VH2 comprising CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 202, 203, and 204, respectively; and

[0515] m) a VL2 comprising CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 130, 131, and 132, respectively; and a VH2 comprising CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 208, 209, and 210, respectively.

[0516] In one embodiment,

[0517] VL1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 213, 217, 221, and 225;

[0518] VH1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 214, 218, 222, and 226;

[0519] VL2 comprises an amino acid sequence selected from the group consisting of: SEQ ID NO: 5, 9, 13, 17, 21, 25, 29, 33, 37, 41, 45, 49, and 53; and

[0520] VH2 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 6, 10, 14, 18, 22, 26, 30, 34, 38, 42, 46, 50, and 54.

[0521] In one embodiment,

[0522] 1) VL1 and VH1 are selected from the group consisting of:

[0523] a) VL1 comprising SEQ ID NO: 213 and VH1 comprising SEQ ID NO: 214;

[0524] b) VL1 comprising SEQ ID NO: 217 and VH1 comprising SEQ ID NO: 218;

[0525] c) a VL1 comprising SEQ ID NO: 221 and a VH1 comprising SEQ ID NO: 222; and

[0526] d) VL1 comprising SEQ ID NO: 225 and VH1 comprising SEQ ID NO: 226;

[0527] and

[0528] 2) VL2 and VH2 are selected from the group consisting of:

[0529] a) VL2 comprising SEQ ID NO: 5 and VH2 comprising SEQ ID NO: 6;

[0530] b) VL2 comprising SEQ ID NO: 9 and VH2 comprising SEQ ID NO: 10;

[0531] c) VL2 comprising SEQ ID NO: 13 and VH2 comprising SEQ ID NO: 14;

[0532] d) VL2 comprising SEQ ID NO: 17 and VH2 comprising SEQ ID NO: 18;

[0533] e) VL2 comprising SEQ ID NO: 21 and VH2 comprising SEQ ID NO: 22;

[0534] f) VL2 comprising SEQ ID NO:25 and VH2 comprising SEQ ID NO:26;

[0535] g) VL2 comprising SEQ ID NO:29 and VH2 comprising SEQ ID NO:30;

[0536] h) VL2 comprising SEQ ID NO:33 and VH2 comprising SEQ ID NO:34;

[0537] i) VL2 comprising SEQ ID NO: 37 and VH2 comprising SEQ ID NO: 38;

[0538] j) VL2 comprising SEQ ID NO:41 and VH2 comprising SEQ ID NO:42;

[0539] k) VL2 comprising SEQ ID NO:45 and VH2 comprising SEQ ID NO:46;

[0540] 1) VL2 comprising SEQ ID NO: 49 and VH2 comprising SEQ ID NO: 50; and

[0541] m) VL2 comprising SEQ ID NO:53 and VH2 comprising SEQ ID NO:54.

[0542] In one aspect, the present invention relates to an antigen binding protein that specifically binds to and stimulates human CD40 (SEQ ID NO: 1), the antigen binding protein comprising a light chain variable region (VL) and a heavy chain variable region (VH), wherein:

[0543] VL comprises a CDRL1, CDRL2, and CDRL3 selected from the group consisting of:

[0544] SEQ ID NOs: 58, 59, and 60, respectively;

[0545] SEQ ID NOs: 64, 65, and 66, respectively;

[0546] SEQ ID NOs: 70, 71, and 72, respectively;

[0547] SEQ ID NOs: 76, 77, and 78, respectively;

[0548] SEQ ID NOs: 82, 83, and 84, respectively;

[0549] SEQ ID NOs: 88, 89, and 90, respectively;

[0550] SEQ ID NOs: 94, 95, and 96, respectively;

[0551] SEQ ID NOs: 100, 101, and 102, respectively;

[0552] SEQ ID NOs: 106, 107, and 108, respectively;

[0553] SEQ ID NOs: 112, 113, and 114, respectively;

[0554] SEQ ID NOs: 118, 119, and 120, respectively;

[0555] SEQ ID NOs: 124, 125, and 126, respectively; and

[0556] SEQ ID NOs: 130, 131, and 132, respectively;

[0557] and

[0558] VH comprises CDRH1, CDRH2, and CDRH3 selected from the group consisting of:

[0559] SEQ ID NOs: 136, 137, and 138, respectively;

[0560] SEQ ID NOs: 142, 143, and 144, respectively;

[0561] SEQ ID NOs: 148, 149, and 150, respectively;

[0562] SEQ ID NOs: 154, 155, and 156, respectively;

[0563] SEQ ID NOs: 160, 161, and 162, respectively;

[0564] SEQ ID NOs: 166, 167, and 168, respectively;

[0565] SEQ ID NOs: 172, 173, and 174, respectively;

[0566] SEQ ID NOs: 178, 179, and 180, respectively;

[0567] SEQ ID NOs: 184, 185, and 186, respectively;

[0568] SEQ ID NOs: 190, 191, and 192, respectively;

[0569] SEQ ID NOs: 196, 197, and 198, respectively;

[0570] SEQ ID NOs: 202, 203, and 204, respectively; and

[0571] They are SEQ ID NOs: 208, 209, and 210, respectively.

[0572] In one embodiment, the multispecific antibody construct comprises mutations that limit Fc receptor binding and thereby reduce Fc receptor-dependent CD40 agonism.

[0573] In one embodiment,

[0574] VL and VH are selected from the group consisting of:

[0575] a) a VL comprising CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 58, 59, and 60, respectively; and a VH comprising CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 136, 137, and 138, respectively;

[0576] b) a VL comprising CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 64, 65, and 66, respectively; and a VH comprising CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 142, 143, and 144, respectively;

[0577] c) a VL comprising CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 70, 71, and 72, respectively; and a VH comprising CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 148, 149, and 150, respectively;

[0578] d) a VL comprising CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 76, 77, and 6780, respectively; and a VH comprising CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 154, 155, and 156, respectively;

[0579] e) a VL comprising CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 82, 83, and 84, respectively; and a VH comprising CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 160, 161, and 162, respectively;

[0580] f) a VL comprising CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 88, 89, and 90, respectively; and a VH comprising CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 166, 167, and 168, respectively;

[0581] g) a VL comprising CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 94, 95, and 96, respectively; and a VH comprising CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 172, 173, and 174, respectively;

[0582] h) a VL comprising CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 100, 101, and 102, respectively; and a VH comprising CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 178, 179, and 180, respectively;

[0583] i) a VL comprising CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 106, 107, and 108, respectively; and a VH comprising CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 184, 185, and 186, respectively;

[0584] j) a VL comprising CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 112, 113, and 114, respectively; and a VH comprising CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 190, 191, and 192, respectively;

[0585] k) a VL comprising CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 118, 119, and 120, respectively; and a VH comprising CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 196, 197, and 198, respectively;

[0586] l) a VL comprising CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 124, 125, and 126, respectively; and a VH comprising CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 202, 203, and 204, respectively; and

[0587] m) a VL comprising CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 130, 131, and 132, respectively; and a VH comprising CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 208, 209, and 210, respectively.

[0588] In one embodiment,

[0589] VL comprises an amino acid sequence selected from the group consisting of: SEQ ID NO: 5, 9, 13, 17, 21, 25, 29, 33, 37, 41, 45, 49, and 53; and

[0590] VH comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 6, 10, 14, 18, 22, 26, 30, 34, 38, 42, 46, 50, and 54.

[0591] In one embodiment,

[0592] VL and VH are selected from the group consisting of:

[0593] a) a VL comprising SEQ ID NO: 5 and a VH comprising SEQ ID NO: 6;

[0594] b) a VL comprising SEQ ID NO: 9 and a VH comprising SEQ ID NO: 10;

[0595] c) a VL comprising SEQ ID NO: 13 and a VH comprising SEQ ID NO: 14;

[0596] d) a VL comprising SEQ ID NO: 17 and a VH comprising SEQ ID NO: 18;

[0597] e) a VL comprising SEQ ID NO: 21 and a VH comprising SEQ ID NO: 22;

[0598] f) a VL comprising SEQ ID NO: 25 and a VH comprising SEQ ID NO: 26;

[0599] g) a VL comprising SEQ ID NO: 29 and a VH comprising SEQ ID NO: 30;

[0600] h) a VL comprising SEQ ID NO: 33 and a VH comprising SEQ ID NO: 34;

[0601] i) a VL comprising SEQ ID NO: 37 and a VH comprising SEQ ID NO: 38;

[0602] j) a VL comprising SEQ ID NO:41 and a VH comprising SEQ ID NO:42;

[0603] k) a VL comprising SEQ ID NO:45 and a VH comprising SEQ ID NO:46;

[0604] 1) a VL comprising SEQ ID NO: 49 and a VH comprising SEQ ID NO: 50; and

[0605] m) a VL comprising SEQ ID NO:53 and a VH comprising SEQ ID NO:54.

[0606] In one embodiment, the heavy chain comprises an amino acid substitution selected from the group consisting of:

[0607] (i) N297G or N297A;

[0608] (ii) L234A and L235A; and

[0609] (iii) R292C and V302C;

[0610] The amino acid numbering is according to the EU numbering of Kabat.

[0611] In one embodiment, the heavy chain comprises N297G, R292C, and V302C mutations, wherein the amino acid numbering is according to EU numbering as in Kabat.

[0612] In one embodiment, the antigen binding protein further comprises a light chain CL polypeptide linked to VL, wherein the CL polypeptide is selected from the group consisting of SEQ ID NO:883 and SEQ ID NO:884.

[0613] In one embodiment, the antigen binding protein further comprises a heavy chain CH1-hinge-CH2-CH3 polypeptide, wherein the CH1-hinge-CH2-CH3 polypeptide is selected from the group consisting of SEQ ID NO:885 and SEQ ID NO:886.

[0614] In one embodiment, the antigen binding protein further comprises a second antigen binding moiety that specifically binds a second antigen.

[0615] In one embodiment, the second antigen is a tumor associated antigen (TAA). BRIEF DESCRIPTION OF THE DRAWINGS

[0616] Figure 1 Depicted are cellular binding of anti-MSLN antibodies to CHO cells expressing human MSLN.

[0617] Figure 2 The activity of anti-CD40 antibodies with or without cross-linking in a human B cell assay is depicted.

[0618] Figure 3 A bivalent, bispecific antibody format is depicted.

[0619] Figure 4 Depicted is the generation of cell lines expressing different levels of MSLN.

[0620] Figure 5 The pharmacokinetic properties and stability of the anti-CD40xMSLN bispecific molecule in mice were characterized.

[0621] Figure 6 Depicted is the generation of cell lines expressing MC38-human EPCAM and B16F10-human EPCAM.

[0622] Figure 7The activity of the mouse surrogate anti-CD40 x human EPCAM bispecific molecule is depicted.

[0623] Figure 8 It is demonstrated that a mouse surrogate anti-CD40xhuman EPCAM bispecific molecule has tumor-localized, TAA-mediated X-ligation-dependent activity in vivo.

[0624] Fig. 9 Depicted mouse surrogate anti-CD40xhuman EPCAM bispecific molecule enhances CD8+ T cell anti-tumor responses.

[0625] Fig.10 It is demonstrated that a mouse surrogate anti-CD40xhuman EPCAM bispecific molecule has tumor-localized, TAA-mediated X-ligation-dependent activity in vivo.

[0626] Fig.11 It is depicted that mouse surrogate anti-CD40xhuman EPCAM bispecific molecules do not increase systemic cytokine levels or augment liver inflammation.

[0627] Fig.12 Depicted is the mouse surrogate anti-CD40xhuman EPCAM bispecific molecule inducing regression of established MC38-human EPCAM tumors alone and in combination with PD1 / PDL1 blockade.

[0628] Fig.13 Describes that mouse surrogate anti-CD40xhuman EPCAM bispecific molecules induce long-lasting immune memory that protects against tumor rechallenge. DETAILED DESCRIPTION

[0629] Unless otherwise indicated, the term "at least" in front of a series of elements should be understood to refer to each element in the series. Those skilled in the art will recognize, or be able to determine many equivalents of the specific embodiments of the invention described herein using only routine experiments. Such equivalents are intended to be encompassed by the present invention.

[0630] The term "and / or" when used herein includes the meanings of "and", "or" and "all or any other combination of the elements connected by the term".

[0631] As used herein, the term "about" or "approximately" means within ±20%, preferably within ±15%, more preferably within ±10%, and most preferably within ±5% of a given value or range.

[0632] Throughout this specification and the following claims, unless the context requires otherwise, the word "comprise" and variations such as "comprises and comprising" should be understood to imply the inclusion of the stated integers or steps or groups of integers or steps but not the exclusion of any other integers or steps or groups of integers or steps. When used herein, the term "comprises" may be replaced with the terms "containing" or "including", or is sometimes replaced herein with the term "having".

[0633] 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 materially affect the basic and novel characteristics of the claim.

[0634] In each instance herein, any of the terms "comprising," "consisting essentially of," and "consisting of may be replaced with either of the other two terms.

[0635] 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 functional fragments thereof. "Functional antibody fragments" are at least a portion of the amino acids present in the antibody that do not contain the full-length heavy chain and / or light chain, but are still able to specifically bind to the antigen. Functional antibody fragments include, but are not limited to, Fab fragments, Fab' fragments, F(ab')2 fragments, Fv fragments, Fd fragments, and complementary determining region (CDR) fragments, and may be derived from any mammalian source, such as humans, mice, rats, rabbits, or camels. Functional antibody fragments can compete with intact antibodies for binding to target antigens, and these fragments can be obtained by modifying intact antibodies (e.g., enzymatic or chemical cleavage) or synthesizing de novo using recombinant DNA technology or peptide synthesis.

[0636] The term "antibody construct" refers to a molecule wherein the structure and / or function is based on the structure and / or function of an antibody (e.g., full length or complete immunoglobulin molecule). Therefore, the antibody construct can bind to its specific target or antigen. In addition, the antibody construct according to the present invention comprises the minimum structural requirements of the antibody allowing target binding. This minimum requirement 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. The antibody based on the construct according to the present invention includes, for example, monoclonal, recombinant, chimeric, deimmunized, humanized and human antibodies.

[0637] In certain embodiments, the antigen-binding proteins of the present invention include antibodies. As used herein, the term "antibody" refers to a tetrameric immunoglobulin 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) can 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), an immunoglobulin heavy chain constant domain 1 (CH1), an immunoglobulin hinge region, an immunoglobulin heavy chain constant domain 2 (CH2), an immunoglobulin heavy chain constant domain 3 (CH3) and optionally an immunoglobulin heavy chain constant domain 4 (CH4) from the amino terminus to the carboxyl terminus. Heavy chains are classified into mu (μ), delta (Δ), gamma (γ), alpha (α), and epsilon (ε) chains, and they define antibody isotypes as IgM, IgD, IgG, IgA, and IgE, respectively. Antibodies of IgG and IgA categories are further subdivided into several subclasses, namely IgG1, IgG2, IgG3, and IgG4, and IgA1 and IgA2, respectively. The heavy chains in IgG, IgA, and IgD antibodies have three domains (CH1, CH2, and CH3), while the heavy chains in IgM and IgE antibodies have four domains (CH1, CH2, CH3, and CH4). The immunoglobulin heavy chain constant domains can be from any immunoglobulin isotype, including subtypes. The antibody chains are linked together via inter-polypeptide 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 chains.

[0638] In human antibodies, CH1 refers to the region having an amino acid sequence at positions 118-215 of the EU index. A highly flexible amino acid region called a "hinge region" exists between CH1 and CH2. CH2 refers to the region having an amino acid sequence at positions 231 to 340 of the EU index, and CH3 refers to the region having an amino acid sequence at positions 341 to 446 of the EU index.

[0639] "CL" means the constant region of the light chain. In the case of the κ chain of a human antibody, CL means the region having the amino acid sequence at positions 108-214 of the EU index. In the λ chain, CL means the region having the amino acid sequence at positions 108 to 215.

[0640] The EU index (as in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)) and the AHo numbering scheme (Honegger A. and Plückthun AJ Mol Biol. 2001 Jun 8;309(3):657-70) can be used in the present invention. Amino acid positions and complementarity determining regions (CDRs) and framework regions (FRs) of a given antibody can be identified using either system. For example, EU heavy chain positions 39, 44, 183, 356, 357, 360, 370, 392, 399, and 409 are equivalent to AHo heavy chain positions 46, 51, 230, 484, 485, 491, 501, 528, 535, and 551, respectively.

[0641] In one aspect, the present invention relates to an antigen binding protein that specifically binds to and stimulates human CD40 (SEQ ID NO: 1), the antigen binding protein comprising a light chain variable region (VL) and a heavy chain variable region (VH), wherein:

[0642] VL comprises a CDRL1, CDRL2, and CDRL3 selected from the group consisting of:

[0643] SEQ ID NOs: 58, 59, and 60, respectively;

[0644] SEQ ID NOs: 64, 65, and 66, respectively;

[0645] SEQ ID NOs: 70, 71, and 72, respectively;

[0646] SEQ ID NOs: 76, 77, and 78, respectively;

[0647] SEQ ID NOs: 82, 83, and 84, respectively;

[0648] SEQ ID NOs: 88, 89, and 90, respectively;

[0649] SEQ ID NOs: 94, 95, and 96, respectively;

[0650] SEQ ID NOs: 100, 101, and 102, respectively;

[0651] SEQ ID NOs: 106, 107, and 108, respectively;

[0652] SEQ ID NOs: 112, 113, and 114, respectively;

[0653] SEQ ID NOs: 118, 119, and 120, respectively;

[0654] SEQ ID NOs: 124, 125, and 126, respectively; and

[0655] SEQ ID NOs: 130, 131, and 132, respectively;

[0656] and

[0657] VH comprises CDRH1, CDRH2, and CDRH3 selected from the group consisting of:

[0658] SEQ ID NOs: 136, 137, and 138, respectively;

[0659] SEQ ID NOs: 142, 143, and 144, respectively;

[0660] SEQ ID NOs: 148, 149, and 150, respectively;

[0661] SEQ ID NOs: 154, 155, and 156, respectively;

[0662] SEQ ID NOs: 160, 161, and 162, respectively;

[0663] SEQ ID NOs: 166, 167, and 168, respectively;

[0664] SEQ ID NOs: 172, 173, and 174, respectively;

[0665] SEQ ID NOs: 178, 179, and 180, respectively;

[0666] SEQ ID NOs: 184, 185, and 186, respectively;

[0667] SEQ ID NOs: 190, 191, and 192, respectively;

[0668] SEQ ID NOs: 196, 197, and 198, respectively;

[0669] SEQ ID NOs: 202, 203, and 204, respectively; and

[0670] They are SEQ ID NOs: 208, 209, and 210, respectively.

[0671] In one embodiment,

[0672] VL and VH are selected from the group consisting of:

[0673] a) a VL comprising CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 58, 59, and 60, respectively; and a VH comprising CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 136, 137, and 138, respectively;

[0674] b) a VL comprising CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 64, 65, and 66, respectively; and a VH comprising CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 142, 143, and 144, respectively;

[0675] c) a VL comprising CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 70, 71, and 72, respectively; and a VH comprising CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 148, 149, and 150, respectively;

[0676] d) a VL comprising CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 76, 77, and 6780, respectively; and a VH comprising CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 154, 155, and 156, respectively;

[0677] e) a VL comprising CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 82, 83, and 84, respectively; and a VH comprising CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 160, 161, and 162, respectively;

[0678] f) a VL comprising CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 88, 89, and 90, respectively; and a VH comprising CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 166, 167, and 168, respectively;

[0679] g) a VL comprising CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 94, 95, and 96, respectively; and a VH comprising CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 172, 173, and 174, respectively;

[0680] h) a VL comprising CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 100, 101, and 102, respectively; and a VH comprising CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 178, 179, and 180, respectively;

[0681] i) a VL comprising CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 106, 107, and 108, respectively; and a VH comprising CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 184, 185, and 186, respectively;

[0682] j) a VL comprising CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 112, 113, and 114, respectively; and a VH comprising CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 190, 191, and 192, respectively;

[0683] k) a VL comprising CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 118, 119, and 120, respectively; and a VH comprising CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 196, 197, and 198, respectively;

[0684] l) a VL comprising CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 124, 125, and 126, respectively; and a VH comprising CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 202, 203, and 204, respectively; and

[0685] m) a VL comprising CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 130, 131, and 132, respectively; and a VH comprising CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 208, 209, and 210, respectively.

[0686] In one embodiment,

[0687] VL comprises an amino acid sequence selected from the group consisting of: SEQ ID NO: 5, 9, 13, 17, 21, 25, 29, 33, 37, 41, 45, 49, and 53; and

[0688] VH comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 6, 10, 14, 18, 22, 26, 30, 34, 38, 42, 46, 50, and 54.

[0689] In one embodiment,

[0690] VL and VH are selected from the group consisting of:

[0691] a) a VL comprising SEQ ID NO: 5 and a VH comprising SEQ ID NO: 6;

[0692] b) a VL comprising SEQ ID NO: 9 and a VH comprising SEQ ID NO: 10;

[0693] c) a VL comprising SEQ ID NO: 13 and a VH comprising SEQ ID NO: 14;

[0694] d) a VL comprising SEQ ID NO: 17 and a VH comprising SEQ ID NO: 18;

[0695] e) a VL comprising SEQ ID NO: 21 and a VH comprising SEQ ID NO: 22;

[0696] f) a VL comprising SEQ ID NO: 25 and a VH comprising SEQ ID NO: 26;

[0697] g) a VL comprising SEQ ID NO: 29 and a VH comprising SEQ ID NO: 30;

[0698] h) a VL comprising SEQ ID NO: 33 and a VH comprising SEQ ID NO: 34;

[0699] i) a VL comprising SEQ ID NO: 37 and a VH comprising SEQ ID NO: 38;

[0700] j) a VL comprising SEQ ID NO:41 and a VH comprising SEQ ID NO:42;

[0701] k) a VL comprising SEQ ID NO:45 and a VH comprising SEQ ID NO:46;

[0702] 1) a VL comprising SEQ ID NO: 49 and a VH comprising SEQ ID NO: 50; and

[0703] m) a VL comprising SEQ ID NO:53 and a VH comprising SEQ ID NO:54.

[0704] In one embodiment, the antigen binding protein further comprises a second antigen binding moiety that specifically binds a second antigen.

[0705] In one embodiment, the second antigen is a tumor associated antigen (TAA).

[0706] The antibody constructs according to the invention are fragments of a full-length antibody, such as VH, VHH, VL, (s)dAb, Fv, Fd, Fab, Fab', F(ab')2 or "rIgG" ("half antibodies"). The antibody constructs according to the present invention may also include modified fragments of antibodies (also called antibody variants, such as scFv, di-scFv or di(s)-scFv, scFv-Fc, scFv-zipper, scFab, Fab2, Fab3, diabodies, single-chain diabodies, tandem diabodies (Tandab), tandem di-scFv, tandem tri-scFv), "minibodies" (exemplified by the following structures: (VH-VL-CH3)2, (scFv-CH3)2, ((scFv)2-CH3+CH3), ((scFv)2-CH3) or (scFv-CH3-scFv)2), multimeric antibodies such as triabodies or tetrabodies, and single domain antibodies (such as nanobodies or single variable domain antibodies comprising only one variable domain, which domain may be VHH, VH or VL, which specifically binds to an antigen or epitope independently of other V regions or domains). Additionally, included within the definition of "antibody constructs" are multispecific molecules that include various types of antibodies and antibody constructs, such as scFvs linked to IgG or IgG-Fabs, IgG-scFvs that include Fab fragments linked to IgGs. In one embodiment, a scFv linked to an immunoglobulin heavy chain is referred to as a "heavy chain fusion protein." In other embodiments, a VH-CH1 polypeptide linked to an immunoglobulin heavy chain is referred to as a "heavy chain fusion protein."

[0707] A "binding domain" or "antigen binding domain" typically can include an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH); however, it does not necessarily include both. For example, a Fd fragment has two VH regions and generally retains some antigen binding functions of a complete antigen binding domain. Additional examples of antibody fragments, antibody variants, or binding domain formats include (1) a Fab fragment, a monovalent fragment having VL, VH, CL, and CH1 domains; (2) a F(ab')2 fragment, a bivalent fragment having two Fab fragments connected by a disulfide bridge at the hinge region; (3) a Fd fragment having two VH and CH1 domains; (4) an Fv fragment having the VL and VH domains of a single arm of an antibody; (5) a dAb fragment having a VH domain (Ward et al., (1989) Nature 341:544-546); (6) isolated complementarity determining regions (CDRs), and (7) single-chain Fv (scFv), the latter being preferred (e.g., derived from an scFV library). Examples of embodiments of antibody constructs according to the invention are e.g. described in WO 00 / 006605, WO 2005 / 040220, WO 2008 / 1 19567, WO 2010 / 037838, WO 2013 / 026837, WO 2013 / 026833, US 2014 / 0308285, US 2014 / 0302037, WO 2014 / 144722, WO 2014 / 151910, and WO 2015 / 048272.

[0708] In addition, the definition of the term "antibody construct" includes monovalent, divalent and multivalent (polyvalent / multivalent) constructs, and thus includes monospecific constructs that specifically bind to only one antigenic structure, and bispecific and multispecific constructs that specifically bind to more than one (e.g., two, three or more) antigenic structures through a unique binding domain. In addition, the definition of the term "antibody construct" includes molecules consisting of only one polypeptide chain and molecules consisting of more than one polypeptide chain, which chains can be the same (homodimers, homotrimers or homooligomers) or different (heterodimers, heterotrimers or heterooligomers). Examples of the antibodies identified above and variants or derivatives thereof are described inter alia 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 edition 2010 and Little, Recombinant Antibodies for Immunotherapy, Cambridge University Press 2009.

[0709] The antibody constructs of the present invention are preferably "in vitro generated antibody constructs". This term refers to an antibody construct according to the above definition, wherein all or part of the variable region (e.g., at least one CDR) is generated in a non-immune cell selection, such as in vitro phage display, protein chip or any other method that can test the ability of candidate sequences to bind to antigens. Therefore, the term preferably excludes sequences generated only by genomic rearrangement in the immune cells of an animal. "Recombinant antibodies" are antibodies made by using recombinant DNA technology or genetic engineering.

[0710] In one aspect, the present invention relates to a multispecific antibody construct comprising:

[0711] (i) a first antibody comprising two light chains and two heavy chains, wherein

[0712] The light chain comprises the first variable region (VL1) and the light chain constant region (CL);

[0713] The heavy chain comprises the first variable region (VH1) and the CH1, hinge, CH2, and CH3 regions;

[0714] The heavy chain comprises at least one amino acid substitution that results in a decrease in the binding affinity of the heavy chain to the human FcγRI receptor (compared to the unsubstituted heavy chain); and

[0715] (ii) a 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 the VH2 are connected via a first peptide linker,

[0716] wherein the scFv is fused at its amino terminus to the carboxyl terminus of each heavy chain via a second peptide linker to form a heavy chain fusion protein; and

[0717] The first antibody specifically binds to and stimulates human CD40 (SEQ ID NO: 1), and the scFv specifically binds to human mesothelin (MSLN) (SEQ ID NO: 2).

[0718] In one embodiment, the multispecific antibody construct specifically agonizes CD40 in an MSLN-dependent manner.

[0719] In one embodiment, the multispecific antibody construct comprises mutations that limit Fc receptor binding and thereby reduce Fc receptor-dependent CD40 agonism.

[0720] In one embodiment, the two light chains are identical and the two heavy chain fusion proteins are identical.

[0721] In one embodiment, VL1 comprises CDRL1, CDRL2, and CDRL3 selected from the group consisting of:

[0722] SEQ ID NOs: 58, 59, and 60, respectively;

[0723] SEQ ID NOs: 64, 65, and 66, respectively;

[0724] SEQ ID NOs: 70, 71, and 72, respectively;

[0725] SEQ ID NOs: 76, 77, and 78, respectively;

[0726] SEQ ID NOs: 82, 83, and 84, respectively;

[0727] SEQ ID NOs: 88, 89, and 90, respectively;

[0728] SEQ ID NOs: 94, 95, and 96, respectively;

[0729] SEQ ID NOs: 100, 101, and 102, respectively;

[0730] SEQ ID NOs: 106, 107, and 108, respectively;

[0731] SEQ ID NOs: 112, 113, and 114, respectively;

[0732] SEQ ID NOs: 118, 119, and 120, respectively;

[0733] are SEQ ID NOs: 124, 125, and 126, respectively; and

[0734] SEQ ID NOs: 130, 131, and 132, respectively;

[0735] VH1 comprises CDRH1, CDRH2, and CDRH3 selected from the group consisting of:

[0736] SEQ ID NOs: 136, 137, and 138, respectively;

[0737] SEQ ID NOs: 142, 143, and 144, respectively;

[0738] SEQ ID NOs: 148, 149, and 150, respectively;

[0739] SEQ ID NOs: 154, 155, and 156, respectively;

[0740] SEQ ID NOs: 160, 161, and 162, respectively;

[0741] SEQ ID NOs: 166, 167, and 168, respectively;

[0742] SEQ ID NOs: 172, 173, and 174, respectively;

[0743] SEQ ID NOs: 178, 179, and 180, respectively;

[0744] SEQ ID NOs: 184, 185, and 186, respectively;

[0745] SEQ ID NOs: 190, 191, and 192, respectively;

[0746] SEQ ID NOs: 196, 197, and 198, respectively;

[0747] are SEQ ID NOs: 202, 203, and 204, respectively; and

[0748] SEQ ID NOs: 208, 209, and 210, respectively;

[0749] VL2 comprises CDRL1, CDRL2, and CDRL3 selected from the group consisting of:

[0750] SEQ ID NOs: 230, 231, and 232, respectively;

[0751] SEQ ID NOs: 236, 237, and 238, respectively;

[0752] are SEQ ID NOs: 242, 243, and 244, respectively; and

[0753] SEQ ID NOs: 248, 249, and 250, respectively;

[0754] and VH2 comprises CDRH1, CDRH2, and CDRH3 selected from the group consisting of:

[0755] SEQ ID NOs: 254, 255, and 256, respectively;

[0756] SEQ ID NOs: 260, 261, and 262, respectively;

[0757] are SEQ ID NOs: 266, 267, and 268, respectively; and

[0758] They are SEQ ID NO: 272, 273, and 274 respectively.

[0759] In one embodiment, 1) VL1 and VH1 are selected from the group consisting of:

[0760] a) a VL1 comprising CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 58, 59, and 60, respectively; and a VH1 comprising CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 136, 137, and 138, respectively;

[0761] b) a VL1 comprising CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 64, 65, and 66, respectively; and a VH1 comprising CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 142, 143, and 144, respectively;

[0762] c) a VL1 comprising CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 70, 71, and 72, respectively; and a VH1 comprising CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 148, 149, and 150, respectively;

[0763] d) a VL1 comprising CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 76, 77, and 6780, respectively; and a VH1 comprising CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 154, 155, and 156, respectively;

[0764] e) a VL1 comprising CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 82, 83, and 84, respectively; and a VH1 comprising CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 160, 161, and 162, respectively;

[0765] f) a VL1 comprising CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 88, 89, and 90, respectively; and a VH1 comprising CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 166, 167, and 168, respectively;

[0766] g) a VL1 comprising CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 94, 95, and 96, respectively; and a VH1 comprising CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 172, 173, and 174, respectively;

[0767] h) a VL1 comprising CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 100, 101, and 102, respectively; and a VH1 comprising CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 178, 179, and 180, respectively;

[0768] i) a VL1 comprising CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 106, 107, and 108, respectively; and a VH1 comprising CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 184, 185, and 186, respectively;

[0769] j) a VL1 comprising CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 112, 113, and 114, respectively; and a VH1 comprising CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 190, 191, and 192, respectively;

[0770] k) a VL1 comprising CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 118, 119, and 120, respectively; and a VH1 comprising CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 196, 197, and 198, respectively;

[0771] l) a VL1 comprising CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 124, 125, and 126, respectively; and a VH1 comprising CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 202, 203, and 204, respectively; and

[0772] m) a VL1 comprising CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 130, 131, and 132, respectively; and a VH1 comprising CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 208, 209, and 210, respectively;

[0773] and

[0774] 2) VL2 and VH2 are selected from the group consisting of:

[0775] a) a VL2 comprising CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 230, 231, and 232, respectively; and a VH2 comprising CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 254, 255, and 256, respectively;

[0776] b) a VL2 comprising CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 236, 237, and 238, respectively; and a VH2 comprising CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 260, 261, and 262, respectively;

[0777] c) a VL2 comprising CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 242, 243, and 244, respectively; and a VH2 comprising CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 266, 267, and 268, respectively; and

[0778] d) a VL2 comprising CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 248, 249, and 250, respectively; and a VH2 comprising CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 272, 273, and 274, respectively.

[0779] In one embodiment, VL1 comprises an amino acid sequence selected from the group consisting of: SEQ ID NO: 5, 9, 13, 17, 21, 25, 29, 33, 37, 41, 45, 49, and 53;

[0780] VH1 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 6, 10, 14, 18, 22, 26, 30, 34, 38, 42, 46, 50, and 54;

[0781] VL2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 213, 217, 221, and 225;

[0782] VH2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 214, 218, 222, and 226.

[0783] In one embodiment,

[0784] 1) VL1 and VH1 are selected from the group consisting of:

[0785] a) VL1 comprising SEQ ID NO: 5 and VH1 comprising SEQ ID NO: 6;

[0786] b) VL1 comprising SEQ ID NO:9 and VH1 comprising SEQ ID NO:10;

[0787] c) a VL1 comprising SEQ ID NO: 13 and a VH1 comprising SEQ ID NO: 14;

[0788] d) VL1 comprising SEQ ID NO: 17 and VH1 comprising SEQ ID NO: 18;

[0789] e) a VL1 comprising SEQ ID NO: 21 and a VH1 comprising SEQ ID NO: 22;

[0790] f) VL1 comprising SEQ ID NO:25 and VH1 comprising SEQ ID NO:26;

[0791] g) VL1 comprising SEQ ID NO:29 and VH1 comprising SEQ ID NO:30;

[0792] h) a VL1 comprising SEQ ID NO: 33 and a VH1 comprising SEQ ID NO: 34;

[0793] i) VL1 comprising SEQ ID NO: 37 and VH1 comprising SEQ ID NO: 38;

[0794] j) VL1 comprising SEQ ID NO:41 and VH1 comprising SEQ ID NO:42;

[0795] k) a VL1 comprising SEQ ID NO:45 and a VH1 comprising SEQ ID NO:46;

[0796] 1) VL1 comprising SEQ ID NO: 49 and VH1 comprising SEQ ID NO: 50; and

[0797] m) a VL1 comprising SEQ ID NO:53 and a VH1 comprising SEQ ID NO:54;

[0798] and

[0799] 2) VL2 and VH2 are selected from the group consisting of:

[0800] a) VL2 comprising SEQ ID NO: 213 and VH2 comprising SEQ ID NO: 214;

[0801] b) VL2 comprising SEQ ID NO: 217 and VH2 comprising SEQ ID NO: 218;

[0802] c) VL2 comprising SEQ ID NO: 221 and VH2 comprising SEQ ID NO: 222; and

[0803] d) VL2 comprising SEQ ID NO:225 and VH2 comprising SEQ ID NO:226.

[0804] In one embodiment,

[0805] the light chain comprises 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

[0806] The heavy chain fusion protein comprises a sequence selected from the group consisting of SEQ ID NO: 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.

[0807] In one embodiment, the light chain and heavy chain fusion proteins comprise polypeptides comprising an amino acid sequence selected from the group consisting of:

[0808] SEQ ID NO:286 and SEQ ID NO:285, respectively;

[0809] SEQ ID NO:290 and SEQ ID NO:289, respectively;

[0810] SEQ ID NO:294 and SEQ ID NO:293, respectively;

[0811] SEQ ID NO:298 and SEQ ID NO:297, respectively;

[0812] SEQ ID NO:302 and SEQ ID NO:301, respectively;

[0813] SEQ ID NO:306 and SEQ ID NO:305, respectively;

[0814] SEQ ID NO:310 and SEQ ID NO:309, respectively;

[0815] SEQ ID NO:314 and SEQ ID NO:313, respectively;

[0816] SEQ ID NO:318 and SEQ ID NO:317, respectively;

[0817] SEQ ID NO:322 and SEQ ID NO:321, respectively;

[0818] SEQ ID NO:326 and SEQ ID NO:325, respectively;

[0819] SEQ ID NO:330 and SEQ ID NO:329, respectively;

[0820] SEQ ID NO:334 and SEQ ID NO:333, respectively;

[0821] SEQ ID NO:338 and SEQ ID NO:337, respectively;

[0822] SEQ ID NO:342 and SEQ ID NO:341, respectively;

[0823] SEQ ID NO:346 and SEQ ID NO:345, respectively;

[0824] SEQ ID NO:350 and SEQ ID NO:349, respectively;

[0825] SEQ ID NO:354 and SEQ ID NO:353, respectively;

[0826] SEQ ID NO:358 and SEQ ID NO:357, respectively;

[0827] SEQ ID NO:362 and SEQ ID NO:361, respectively;

[0828] SEQ ID NO:366 and SEQ ID NO:365, respectively;

[0829] SEQ ID NO:370 and SEQ ID NO:369, respectively;

[0830] SEQ ID NO:374 and SEQ ID NO:373, respectively; and

[0831] They are SEQ ID NO:378 and SEQ ID NO:377 respectively.

[0832] In another aspect, the present invention relates to a multispecific antibody construct comprising:

[0833] (i) a first antibody comprising two light chains and two heavy chains, wherein

[0834] The light chain comprises the first variable region (VL1) and the light chain constant region (CL);

[0835] The heavy chain comprises the first variable region (VH1) and the CH1, hinge, CH2, and CH3 regions;

[0836] The heavy chain comprises at least one amino acid substitution that results in a decrease in the binding affinity of the heavy chain to the human FcγRI receptor (compared to the unsubstituted heavy chain); and

[0837] (ii) a 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 the VH2 are connected via a first peptide linker,

[0838] wherein the scFv is fused at its amino terminus to the carboxyl terminus of each heavy chain via a second peptide linker to form a heavy chain fusion protein; and

[0839] The first antibody specifically binds to human mesothelin (MSLN) (SEQ ID NO: 2), and the scFv specifically binds to and stimulates human CD40 (SEQ ID NO: 1).

[0840] In one embodiment, the multispecific antibody construct specifically agonizes CD40 in an MSLN-dependent manner.

[0841] In one embodiment, the multispecific antibody construct comprises mutations that limit Fc receptor binding and thereby reduce Fc receptor-dependent CD40 agonism.

[0842] In one embodiment, the two light chains are identical and the two heavy chain fusion proteins are identical.

[0843] In one embodiment:

[0844] VL1 comprises CDRL1, CDRL2, and CDRL3 selected from the group consisting of:

[0845] SEQ ID NOs: 230, 231, and 232, respectively;

[0846] SEQ ID NOs: 236, 237, and 238, respectively;

[0847] are SEQ ID NOs: 242, 243, and 244, respectively; and

[0848] SEQ ID NOs: 248, 249, and 250, respectively;

[0849] VH1 comprises CDRH1, CDRH2, and CDRH3 selected from the group consisting of:

[0850] SEQ ID NOs: 254, 255, and 256, respectively;

[0851] SEQ ID NOs: 260, 261, and 262, respectively;

[0852] SEQ ID NOs: 266, 267, and 268, respectively; and

[0853] SEQ ID NOs: 272, 273, and 274, respectively;

[0854] VL2 comprises CDRL1, CDRL2, and CDRL3 selected from the group consisting of:

[0855] SEQ ID NOs: 58, 59, and 60, respectively;

[0856] SEQ ID NOs: 64, 65, and 66, respectively;

[0857] SEQ ID NOs: 70, 71, and 72, respectively;

[0858] SEQ ID NOs: 76, 77, and 78, respectively;

[0859] SEQ ID NOs: 82, 83, and 84, respectively;

[0860] SEQ ID NOs: 88, 89, and 90, respectively;

[0861] SEQ ID NOs: 94, 95, and 96, respectively;

[0862] SEQ ID NOs: 100, 101, and 102, respectively;

[0863] SEQ ID NOs: 106, 107, and 108, respectively;

[0864] SEQ ID NOs: 112, 113, and 114, respectively;

[0865] SEQ ID NOs: 118, 119, and 120, respectively;

[0866] SEQ ID NOs: 124, 125, and 126, respectively; and

[0867] SEQ ID NOs: 130, 131, and 132, respectively;

[0868] and

[0869] VH2 comprises CDRH1, CDRH2, and CDRH3 selected from the group consisting of:

[0870] SEQ ID NOs: 136, 137, and 138, respectively;

[0871] SEQ ID NOs: 142, 143, and 144, respectively;

[0872] SEQ ID NOs: 148, 149, and 150, respectively;

[0873] SEQ ID NOs: 154, 155, and 156, respectively;

[0874] SEQ ID NOs: 160, 161, and 162, respectively;

[0875] SEQ ID NOs: 166, 167, and 168, respectively;

[0876] SEQ ID NOs: 172, 173, and 174, respectively;

[0877] SEQ ID NOs: 178, 179, and 180, respectively;

[0878] SEQ ID NOs: 184, 185, and 186, respectively;

[0879] SEQ ID NOs: 190, 191, and 192, respectively;

[0880] SEQ ID NOs: 196, 197, and 198, respectively;

[0881] SEQ ID NOs: 202, 203, and 204, respectively; and

[0882] They are SEQ ID NOs: 208, 209, and 210, respectively.

[0883] In one embodiment,

[0884] 1) VL1 and VH1 are selected from the group consisting of:

[0885] a) a VL1 comprising CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 230, 231, and 232, respectively; and a VH1 comprising CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 254, 255, and 256, respectively;

[0886] b) a VL1 comprising CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 236, 237, and 238, respectively; and a VH1 comprising CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 260, 261, and 262, respectively;

[0887] c) a VL1 comprising CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 242, 243, and 244, respectively; and a VH1 comprising CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 266, 267, and 268, respectively; and

[0888] d) a VL1 comprising CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 248, 249, and 250, respectively; and a VH1 comprising CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 272, 273, and 274, respectively;

[0889] and

[0890] 2) VL2 and VH2 are selected from the group consisting of:

[0891] a) a VL2 comprising CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 58, 59, and 60, respectively; and a VH2 comprising CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 136, 137, and 138, respectively;

[0892] b) a VL2 comprising CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 64, 65, and 66, respectively; and a VH2 comprising CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 142, 143, and 144, respectively;

[0893] c) a VL2 comprising CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 70, 71, and 72, respectively; and a VH2 comprising CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 148, 149, and 150, respectively;

[0894] d) a VL2 comprising CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 76, 77, and 6780, respectively; and a VH2 comprising CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 154, 155, and 156, respectively;

[0895] e) a VL2 comprising CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 82, 83, and 84, respectively; and a VH2 comprising CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 160, 161, and 162, respectively;

[0896] f) a VL2 comprising CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 88, 89, and 90, respectively; and a VH2 comprising CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 166, 167, and 168, respectively;

[0897] g) a VL2 comprising CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 94, 95, and 96, respectively; and a VH2 comprising CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 172, 173, and 174, respectively;

[0898] h) a VL2 comprising CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 100, 101, and 102, respectively; and a VH2 comprising CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 178, 179, and 180, respectively;

[0899] i) a VL2 comprising CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 106, 107, and 108, respectively; and a VH2 comprising CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 184, 185, and 186, respectively;

[0900] j) a VL2 comprising CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 112, 113, and 114, respectively; and a VH2 comprising CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 190, 191, and 192, respectively;

[0901] k) a VL2 comprising CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 118, 119, and 120, respectively; and a VH2 comprising CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 196, 197, and 198, respectively;

[0902] 1) a VL2 comprising CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 124, 125, and 126, respectively; and a VH2 comprising CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 202, 203, and 204, respectively; and

[0903] m) a VL2 comprising CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 130, 131, and 132, respectively; and a VH2 comprising CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 208, 209, and 210, respectively.

[0904] In one embodiment:

[0905] VL1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 213, 217, 221, and 225;

[0906] VH1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 214, 218, 222, and 226;

[0907] VL2 comprises an amino acid sequence selected from the group consisting of: SEQ ID NO: 5, 9, 13, 17, 21, 25, 29, 33, 37, 41, 45, 49, and 53; and

[0908] VH2 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 6, 10, 14, 18, 22, 26, 30, 34, 38, 42, 46, 50, and 54.

[0909] In one embodiment:

[0910] 1) VL1 and VH1 are selected from the group consisting of:

[0911] a) VL1 comprising SEQ ID NO: 213 and VH1 comprising SEQ ID NO: 214;

[0912] b) VL1 comprising SEQ ID NO: 217 and VH1 comprising SEQ ID NO: 218;

[0913] c) a VL1 comprising SEQ ID NO: 221 and a VH1 comprising SEQ ID NO: 222; and

[0914] d) VL1 comprising SEQ ID NO: 225 and VH1 comprising SEQ ID NO: 226;

[0915] and

[0916] 2) VL2 and VH2 are selected from the group consisting of:

[0917] a) VL2 comprising SEQ ID NO: 5 and VH2 comprising SEQ ID NO: 6;

[0918] b) VL2 comprising SEQ ID NO: 9 and VH2 comprising SEQ ID NO: 10;

[0919] c) VL2 comprising SEQ ID NO: 13 and VH2 comprising SEQ ID NO: 14;

[0920] d) VL2 comprising SEQ ID NO: 17 and VH2 comprising SEQ ID NO: 18;

[0921] e) VL2 comprising SEQ ID NO: 21 and VH2 comprising SEQ ID NO: 22;

[0922] f) VL2 comprising SEQ ID NO:25 and VH2 comprising SEQ ID NO:26;

[0923] g) VL2 comprising SEQ ID NO:29 and VH2 comprising SEQ ID NO:30;

[0924] h) VL2 comprising SEQ ID NO:33 and VH2 comprising SEQ ID NO:34;

[0925] i) VL2 comprising SEQ ID NO: 37 and VH2 comprising SEQ ID NO: 38;

[0926] j) VL2 comprising SEQ ID NO:41 and VH2 comprising SEQ ID NO:42;

[0927] k) VL2 comprising SEQ ID NO:45 and VH2 comprising SEQ ID NO:46;

[0928] 1) VL2 comprising SEQ ID NO: 49 and VH2 comprising SEQ ID NO: 50; and

[0929] m) VL2 comprising SEQ ID NO:53 and VH2 comprising SEQ ID NO:54.

[0930] In one embodiment, the CL of the light chain is selected from the group consisting of SEQ ID NO:883 and SEQ ID NO:884.

[0931] In one embodiment, 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.

[0932] In one embodiment, the first peptide linker is selected from the group consisting of SEQ ID NOs: 888-893.

[0933] In one embodiment, the second peptide linker is selected from the group consisting of SEQ ID NOs: 887-893.

[0934] In one embodiment, the first peptide linker comprises SEQ ID NO:889 and the second peptide linker comprises SEQ ID NO:887.

[0935] In one embodiment,

[0936] The light chain comprises a sequence selected from the group consisting of: SEQ ID NO: 382, ​​386, 390, 394, 398, 402, 406, 410, 414, 418, 422, 426, 430, 434, 438, 442, 446, and 450; and

[0937] The heavy chain fusion protein comprises a sequence selected from the group consisting of: SEQ ID NO: 381, 385, 389, 393, 397, 401, 405, 409, 413, 417, 421, 425, 429, 433, 437, 441, 445, and 449.

[0938] In one embodiment, the light chain and heavy chain fusion proteins comprise polypeptides comprising an amino acid sequence selected from the group consisting of:

[0939] SEQ ID NO:382 and SEQ ID NO:381, respectively;

[0940] SEQ ID NO:386 and SEQ ID NO:385, respectively;

[0941] SEQ ID NO:390 and SEQ ID NO:389, respectively;

[0942] SEQ ID NO:394 and SEQ ID NO:393, respectively;

[0943] SEQ ID NO:398 and SEQ ID NO:397, respectively;

[0944] SEQ ID NO:402 and SEQ ID NO:401, respectively;

[0945] SEQ ID NO:406 and SEQ ID NO:405, respectively;

[0946] SEQ ID NO:410 and SEQ ID NO:409, respectively;

[0947] SEQ ID NO:414 and SEQ ID NO:413, respectively;

[0948] SEQ ID NO:418 and SEQ ID NO:417, respectively;

[0949] SEQ ID NO:422 and SEQ ID NO:421, respectively;

[0950] SEQ ID NO:426 and SEQ ID NO:425, respectively;

[0951] SEQ ID NO:430 and SEQ ID NO:429, respectively;

[0952] SEQ ID NO:434 and SEQ ID NO:433, respectively;

[0953] SEQ ID NO:438 and SEQ ID NO:437, respectively;

[0954] SEQ ID NO:442 and SEQ ID NO:441, respectively;

[0955] SEQ ID NO:446 and SEQ ID NO:445, respectively; and

[0956] They are SEQ ID NO:450 and SEQ ID NO:449 respectively.

[0957] In one embodiment, the CL of the light chain is selected from the group consisting of SEQ ID NO:883 and SEQ ID NO:884.

[0958] In one embodiment, 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.

[0959] In one embodiment, the first peptide linker is selected from the group consisting of SEQ ID NOs: 888-893.

[0960] In one embodiment, the second peptide linker is selected from the group consisting of SEQ ID NOs: 887-893.

[0961] In one embodiment, the first peptide linker comprises SEQ ID NO:889 and the second peptide linker comprises SEQ ID NO:887.

[0962] As used herein, the term "monoclonal antibody" (mAb) or monoclonal antibody construct refers to an antibody obtained from a substantially homogeneous antibody population, i.e., the individual antibodies constituting the population are 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, directed against a single antigenic site or determinant on an antigen, in contrast to conventional (polyclonal) antibody preparations, which typically include different antibodies directed against different determinants (or epitopes). In addition to their specificity, monoclonal antibodies have the advantage that they are synthesized by hybridoma culture and are therefore not contaminated by other immunoglobulins. The modifier "monoclonal" indicates that the antibody is characterized by being obtained from a substantially homogeneous antibody population and should not be considered as requiring the production of the antibody by any particular method.

[0963] In order to prepare monoclonal antibodies, any technology providing antibodies produced by continuous cell line culture can be used. For example, the monoclonal antibody to be used can be prepared by the hybridoma method first described by Koehler et al., Nature [Nature], 256:495 (1975), or can be prepared by recombinant DNA method (see, e.g., U.S. Patent No. 4,816,567). Examples of other technologies for producing human monoclonal antibodies include trioma technology, human B cell hybridoma technology (Kozbor, Immunology Today [Today's Immunology] 4 (1983), 72) and EBV-hybridoma technology (Cole et al., Monoclonal Antibodies and Cancer Therapy [monoclonal antibodies and cancer therapy], Alan R. Liss Company (1985), 77-96).

[0964] Standard methods such as enzyme-linked immunosorbent assay (ELISA) and surface plasmon resonance (BIACORE TM ) analysis to screen hybridomas 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, such as a recombinant antigen, a naturally occurring form, any variant or fragment thereof, and antigenic peptides thereof. Surface plasmon resonance, as used in the BIAcore system, can be used to increase the efficiency of phage antibodies that bind to epitopes of target antigens (MSLN or CD40) (Schier, Human Antibodies Hybridomas 7 (1996), 97-105; Malmborg, J. Immunol. Methods 183 (1995), 7-13).

[0965] Another exemplary method for preparing monoclonal antibodies includes screening protein expression libraries, such as phage display or ribosome display libraries. Phage display is described, for example, in Ladner et al., U.S. Pat. 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).

[0966] In addition to using display libraries, the relevant antigens can be used to immunize non-human animals, such as rodents (such as mice, hamsters, rabbits or rats). In one embodiment, the non-human animal includes at least a portion of a human immunoglobulin gene. For example, a large fragment of a human Ig (immunoglobulin) locus can be used to engineer a mouse strain that is defective in mouse antibody production. By using hybridoma technology, antigen-specific monoclonal antibodies derived from genes with desired specificity can be produced and selected. See, for example, XENOMOUSE TM , Green et al. (1994) Nature Genetics 7: 13-21, US 2003-0070185, WO 96 / 34096 and WO 96 / 33735.

[0967] Monoclonal antibodies can also be obtained from non-human animals and then modified using recombinant DNA techniques known in the art, such as humanization, deimmunization, chimerization, etc. Examples of modified antibody constructs include humanized variants of non-human antibodies, "affinity matured" 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 mutants with altered effector function(s) (see, e.g., U.S. Pat. No. 5,648,260, Kontermann and Dubel (2010), cited above, and Little (2009), cited above).

[0968] In immunology, affinity maturation is a process by which B cells produce antibodies with increased affinity for antigens during an immune response. By repeated exposure to the same antigen, the host will produce antibodies with successively greater affinity. Similar to the natural prototype, in vitro affinity maturation is based on the principles of mutation and selection. In vitro affinity maturation has been successfully used to optimize antibodies, antibody constructs, and antibody fragments. Random mutations within CDRs 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 mutations and selection using display methods such as phage display typically produce antibody fragments with low nanomolar range affinities.

[0969] The amino acid substitution variation of the preferred type of antibody construct includes replacing one or more hypervariable region residues of the parent antibody (e.g., humanized or human antibody). Generally, one or more obtained variants selected for further development will have improved biological properties relative to the parent antibody that generates them. A convenient way to generate such substitution variants includes affinity maturation using phage display. In short, 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 produced are displayed as fusions of the gene III product of M13 packaged in each particle from filamentous phage particles in a monovalent manner. Then the biological activity (e.g., binding affinity) of the variants displayed by phage as disclosed herein is screened. In order to identify candidate hypervariable region sites for modification, alanine scanning mutagenesis can be performed to identify hypervariable region residues that contribute significantly to antigen binding. Alternatively or in addition, it may be advantageous to analyze the crystal structure of the antigen-antibody complex to identify the contact points between the binding domain and, for example, human MSLN. According to the technology detailed herein, such contact residues and adjacent residues are candidates for substitution. Once such variants are generated, the panel of variants is subjected to screening as described herein, and antibodies with superior properties in one or more relevant assays can be selected for further development.

[0970] The monoclonal antibodies and antibody constructs of the present invention particularly include "chimeric" antibodies (immunoglobulins) in which a portion of the heavy and / or light chains is identical or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, and the remainder of one or more chains is identical or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, as long as they exhibit the desired biological activity (U.S. Pat. No. 4,816,567; Morrison et al., Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences of the United States], 81: 6851-6855 (1984)). Chimeric antibodies of interest herein include "primitized" antibodies, which contain variable domain antigen-binding sequences derived from non-human primates (e.g., Old World Monkey, ape, etc.) and human constant region sequences. A variety of methods for preparing chimeric antibodies have been described. See, e.g., Morrison et al., Proc. Natl. Acad. ScL USA 81:6851, 1985; Takeda et al., Nature 314:452, 1985; Cabilly et al., U.S. Pat. No. 4,816,567; Boss et al., U.S. Pat. No. 4,816,397; Tanaguchi et al., EP 0171496; EP 0173494; and GB 2177096.

[0971] Antibodies, antibody constructs, antibody fragments or antibody variants can also be modified by specifically deleting human T cell epitopes (called "deimmunization" methods) by methods disclosed in, for example, WO 98 / 52976 or WO 00 / 34317. In short, the heavy and light chain variable domains of antibodies can be analyzed for peptides bound to class II MHC; these peptides represent potential T cell epitopes (as defined in WO 98 / 52976 and WO 00 / 34317). In order to detect potential T cell epitopes, a computer modeling method called "peptide threading" can be applied, and in addition, for motifs present in VH and VL sequences, databases of human MHC class II binding peptides can be searched, as described in WO 98 / 52976 and WO 00 / 34317. These motifs are combined with any of 18 major MHC class II DR allotypes, and therefore constitute potential T cell epitopes. The detected potential T cell epitopes can be eliminated by replacing a small amount of amino acid residues in the variable domain, or preferably by replacing a single amino acid. Typically, conservative substitutions are made. Generally, but not exclusively, 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 16(5): 237-242; and Tomlinson et al. (1995) EMBO J. 14: 14: 4628-4638. The VBASE directory provides a comprehensive directory 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. Common human framework regions can also be used, for example as described in U.S. Pat. No. 6,300,064.

[0972] "Humanized" antibodies, antibody constructs, variants or fragments thereof (such as Fv, Fab, Fab', F(ab')2 or other antigen-binding subsequences of antibodies) are antibodies or immunoglobulins of most human sequences, which contain one or more minimal sequences derived from non-human immunoglobulins. In most cases, humanized antibodies are human immunoglobulins (receptor antibodies), in which the residues from the hypervariable regions (also known as CDR) of the receptor are replaced by residues from the hypervariable regions (donor antibodies) of non-human (e.g., rodent) species (such as mice, rats, hamsters or rabbits) with the desired specificity, affinity and capacity. In some cases, the Fv framework region (FR) residues of human immunoglobulins are replaced by corresponding non-human residues. In addition, "humanized antibodies" as used herein may also be included in residues not found in either the receptor antibody or the donor antibody. These modifications are performed to further improve and optimize antibody performance. Humanized antibodies may also include at least a portion of an immunoglobulin constant region (Fc), which is typically a human immunoglobulin. For more 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).

[0973] Humanized antibodies or fragments thereof can be generated by replacing sequences of Fv variable domains that are not directly involved in antigen binding with equivalent sequences from human Fv variable domains. Exemplary methods for producing humanized antibodies or fragments thereof are provided by: Morrison (1985) Science [Science] 229: 1202-1207; Oi et al. (1986) BioTechniques [Biotechnology] 4: 214; and US 5,585,089; US 5,693,761; US ​​5,693,762; US 5,859,205; and US 6,407,213. Those methods include separating, manipulating and expressing nucleic acid sequences encoding all or part of the immunoglobulin Fv variable domains from at least one of the heavy chain or light chain. Such nucleic acids can be obtained from hybridomas and from other sources by producing antibodies against a predetermined target as described above. The recombinant DNA encoding the humanized antibody molecule can then be cloned into a suitable expression vector.

[0974] Humanized antibodies can also be produced using transgenic animals such as mice that express human heavy chain and light chain genes but cannot express endogenous mouse immunoglobulin heavy chain and light chain genes. Winter describes an exemplary CDR transplantation method (U.S. Patent number 5,225,539) that can be used to prepare humanized antibodies as herein described. All CDRs of specific human antibodies can be replaced with at least a portion of non-human CDRs, or only some CDRs can be replaced with non-human CDRs. Only need to substitute humanized antibodies and predetermined antigen in combination with required CDR quantity.

[0975] Humanized antibodies can be optimized by introducing conservative substitutions, consensus sequence substitutions, germline substitutions and / or back mutations. Such altered immunoglobulin molecules can be prepared by any of several techniques known in the art (e.g., Teng et al., Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences of the United States], 80:7308-7312, 1983; Kozbor et al., Immunology Today [Today's Immunology], 4:7279, 1983; Olsson et al., Meth. Enzymol. [Enzymology Methods], 92:3-16, 1982; and EP 239 400).

[0976] The terms "human antibody", "human antibody construct" and "human binding domain" include antibodies, antibody constructs and binding domains having antibody regions, such as variable and constant regions or domains substantially corresponding to human germline immunoglobulin sequences known in the art, including, for example, those described by Kabat et al. (1991) (cited above). The human antibody, antibody construct or binding domain of the present invention may include, for example, amino acid residues in CDR and particularly CDR3 that are not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutations in vivo). The human antibody, antibody construct or binding domain may have at least one, two, three, four, five or more positions replaced by amino acid residues that are not encoded by human germline immunoglobulin sequences. However, the definitions of human antibodies, antibody constructs and binding domains as used herein also encompass "fully human antibodies", which only include non-artificial and / or genetically altered human antibody sequences, such as those that can be derived using techniques such as Xenomouse or systems.

[0977] In some embodiments, the antibody construct of the present invention is an "isolated" or "substantially pure" antibody construct. When used to describe the antibody construct disclosed herein, "isolated" or "substantially pure" means that the antibody construct has been identified, separated and / or recovered from the components of its production environment. Preferably, the antibody construct does not contain all other components from its production environment or is not substantially associated with all other components from its production environment. The contaminating components of its production environment (such as those produced by recombinant transfected cells) are substances that usually interfere with the diagnostic or therapeutic use of the polypeptide, and the substances may include enzymes, hormones and other proteins or non-protein solutes. By weight, the antibody construct can, for example, account for at least about 5% or at least about 50% of the total protein in a given sample. It should be understood that, by weight, the isolated protein can account for 5% to 99.9% of the total protein content, depending on the situation. It is possible to prepare a polypeptide at an increased concentration level by using an inducible promoter or a high expression promoter to prepare a significantly higher concentration of polypeptide. This definition includes producing antibody constructs in a variety of organisms and / or host cells known in the art. In preferred embodiments, the antibody construct is (1) purified to a degree sufficient to obtain at least 15 N-terminal or internal amino acid sequence residues by use of a spinning cup sequenator, or (2) purified to homogeneity by SDS-PAGE under non-reducing or reducing conditions using Coomassie blue or, preferably, silver stain. Typically, however, an isolated antibody construct is prepared by at least one purification step.

[0978] The term "binding domain" in relation to the present invention characterizes a domain that (specifically) binds / interacts / recognizes a given target epitope or a given target side on a target molecule (antigen) (here: MSLN and CD40, respectively). The structure and function of one binding domain (recognizing MSLN) and preferably also the structure and / or function of the other binding domain (recognizing CD40) are based on the structure and / or function of an antibody (e.g. a full-length or complete 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 of the VL region) and three heavy chain CDRs (i.e. CDRH1, CDRH2 and CDRH3 of the VH region). It is envisaged that the binding domain is generated or obtainable by phage display or library screening methods, rather than being generated or obtained by transplanting CDR sequences from pre-existing (monoclonal) antibodies into a scaffold. According to the present invention, the binding domain is in the form of one or more polypeptides. Such polypeptides may include protein parts and non-protein parts (e.g., chemical linkers or chemical cross-linking agents, such as glutaraldehyde). Proteins (including fragments thereof, preferably biologically active fragments, and peptides generally having less than 30 amino acids) contain two or more amino acids coupled to each other via covalent peptide bonds (producing amino acid chains). As used herein, the term "polypeptide" describes a group of molecules that are generally composed of more than 30 amino acids. Polypeptides can further form multimers, such as dimers, trimers, and higher-order oligomers, i.e., composed of more than one polypeptide molecule. The polypeptide molecules that form such dimers, trimers, etc. may be the same or different. Therefore, the corresponding higher-order structures of such multimers are referred to as homodimers or heterodimers, homotrimers or heterotrimers, etc. An example of a heteromultimer is an antibody molecule, which in its naturally occurring form consists of two identical light polypeptide chains and two identical heavy polypeptide chains. The terms "peptide", "polypeptide" and "protein" also refer to naturally modified peptides / polypeptides / proteins, wherein the modification is achieved, for example, by post-translational modifications such as glycosylation, acetylation, phosphorylation, etc. When referred to herein, a "peptide", "polypeptide" or "protein" may also be chemically modified, such as pegylated. Such modifications are well known in the art and are described below.

[0979] 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 comprising at least one human binding domain avoid some of the problems associated with antibodies or antibody constructs having variable and / or constant regions from non-human, such as rodents (e.g., mice, rats, hamsters, or rabbits). The presence of such rodent-derived proteins can result in rapid clearance of the antibody or antibody construct, or can result in the patient generating an immune response to the antibody or antibody construct. In order to avoid the use of rodent-derived antibodies or antibody constructs, human or fully human antibodies / antibody constructs can be produced by introducing human antibody functions into rodents so that the rodents produce fully human antibodies.

[0980] The ability to clone and reconstruct megabase-sized human loci in YACs and introduce them into the mouse germline provides a powerful approach for elucidating the functional components of very large or poorly mapped loci and for generating useful human disease models. In addition, using this technology to replace mouse loci with their human equivalents can provide unique insights into the expression and regulation of human gene products during development, their communication with other systems, and their involvement in disease induction and progression.

[0981] The important practical application of this strategy is the "humanization" of the mouse humoral immune system. The human immunoglobulin (Ig) locus is introduced into the mouse in which the endogenous Ig gene has been inactivated to provide the opportunity for studying the programmed expression of antibodies and the mechanism of assembly and their effects in B cell development. In addition, this strategy can provide an ideal source for the generation of fully human monoclonal antibodies (mAbs)-this is an important milestone that helps to realize the prospects of antibody therapy in human diseases. It is expected that fully human antibodies or antibody constructs will minimize the immunogenicity and allergic responses inherent to mouse or mouse-derived mAbs, and thus increase the efficacy and safety of the antibodies / antibody constructs used. It can be expected that the use of fully human antibodies or antibody constructs provides significant advantages in the treatment of chronic and recurrent human diseases such as inflammation, autoimmunity and cancer that require repeated compound administration.

[0982] One method to achieve this goal is to engineer a mouse strain that is defective in mouse antibody production with a large fragment of the human Ig locus, and it is expected that such mice will produce a large amount of human antibodies in the absence of mouse antibodies. Large human Ig fragments will retain large variable gene diversity and appropriate regulation of antibody production and expression. By utilizing the antibody diversification and selection mechanism of mice and lacking immune tolerance to human proteins, the regenerated human antibody library in these mouse strains should produce high-affinity antibodies for any antigen of interest (including human antigens). By using hybridoma technology, antigen-specific human mAbs with desired specificity can be easily produced and selected. This general strategy is related to the first generation XenoMouse mouse strain (see Green et al. Nature Genetics [Natural Genetics] 7: 13-21 (1994)). XenoMouse strains are engineered with yeast artificial chromosomes (YACs), which contain germline configuration fragments of 245kb and 190kb sizes of human heavy chain loci and κ light chain loci, respectively, and these fragments contain core variable regions and constant region sequences. It is proved that YAC containing human Ig is compatible with the mouse system regarding the rearrangement and expression of antibodies, and can replace the inactivated mouse Ig gene. This is proved by its ability to induce B cell development, produce an adult-like human repertoire of fully human antibodies, and produce antigen-specific human mAbs. These results also show that the introduction of a larger portion of the human Ig locus containing more V genes, additional regulatory elements, and human Ig constant regions may substantially summarize the complete library with the characteristics of human humoral responses of infected and immune people. The work of Green et al. has recently been extended to the introduction of a human antibody library greater than about 80% by introducing a megabase-sized germline configuration YAC fragment of a human heavy chain locus and a kappa light chain locus, respectively. See Mendez et al. Nature Genetics [Natural Genetics] 15: 146-156 (1997) and U.S. Patent Application Serial No. 08 / 759,620.

[0983] The generation of XenoMouse mice is further discussed and described in the following: U.S. Patent Applications Serial Nos. 07 / 466,008, 07 / 610,515, 07 / 919,297, 07 / 922,649, 08 / 031,801, 08 / 112,848, 08 / 234,145, 08 / 376,279, 08 / 430,938, 08 / 464,584, 08 / 464,649. 4,582, serial number 08 / 463,191, serial number 08 / 462,837, serial number 08 / 486,853, serial number 08 / 486,857, serial number 08 / 486,859, serial number 08 / 462,513, serial number 08 / 724,752, and serial number 08 / 759,620; and U.S. Patent Nos. 6,162,963; 6,150,584; 6,114,598; 6,075,181 and 5,939,598 and Japanese Patent Nos. 3 068 180 B2, 3 068 506 B2, and 3 068 507 B2. See also Mendez et al. Nature Genetics 15:146-156 (1997) and Green and Jakobovits J. Exp. Med. 188:483-495 (1998), EP 0463 151 B1, WO 94 / 02602, WO 96 / 34096, WO 98 / 24893, WO 00 / 76310 and WO 03 / 47336.

[0984] In an alternative approach, other companies (including GenPharm International) have utilized a "minilocus" approach. In the minilocus approach, an 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 mu constant region, and a second constant region (preferably a gamma constant region) form a construct for insertion into an animal. This method is described in: U.S. Pat. Nos. 5,545,807 and 5,545,806 to Surani et al.; 5,625,825; 5,625,126; 5,633,425; 5,661,016; 5,770,429; 5,789,650; 5,814,318; 5,877,397; 5,874,299; and 6,255,458 (each to Lonberg and Kay), U.S. Pat. Nos. 5,591,669 and 6,023.010 to Krimpenfort and Berns, U.S. Pat. No. 5,612,205 to Berns et al.; ,721,367; and 5,789,215, and U.S. Patent No. 5,643,763 to Choi and Dunn, and GenPharm International U.S. Patent Application Serial Nos. 07 / 574,748, 07 / 575,962, 07 / 810,279, 07 / 853,408, 07 / 904,068, 07 / 990,860, 08 / 053,131, 08 / 096,762, 08 / 155,301, 08 / 161,739, 08 / 165,699, 08 / 209,741. See also EP 0 546 073 B1, WO 92 / 03918, WO 92 / 22645, WO 92 / 22647, WO 92 / 22670, WO 93 / 12227, WO 94 / 00569, WO 94 / 25585, WO 96 / 14436, WO 97 / 13852, and WO 98 / 24884 and U.S. Pat. No. 5,981, 175. See further Taylor et al. (1992), Chen et al. (1993), Tuaillon et al. (1993), Choi et al. (1993), Lonberg et al. (1994), Taylor et al. (1994), and Tuaillon et al. (1995), Fishwild et al. (1996).

[0985] Kirin has also demonstrated the generation of human antibodies from mice into which large chromosome fragments or entire chromosomes have been introduced by microcell fusion. See European Patent Application Nos. 773 288 and 843 961. Xenerex Biosciences is developing a technology that may generate human antibodies. In this technology, SCID mice are reconstituted with human lymphocytes (e.g., B and / or T cells). The mice are then immunized with an antigen and an immune response to the antigen can be generated. See U.S. Patent Nos. 5,476,996; 5,698,767; and 5,958,765.

[0986] Human anti-mouse antibody (HAMA) responses have led the industry to prepare chimeric or other humanized antibodies. However, it is expected that certain human anti-chimeric antibody (HACA) responses will be observed, especially in long-term or multi-dose use of antibodies. Therefore, it is desirable to provide antibody constructs that include a human binding domain for MSLN and a human binding domain for CD40 in order to eliminate the problems and / or effects of HAMA or HACA responses.

[0987] The terms "(specifically) bind to", "(specifically) recognize", "(specifically) target" and "(specifically) react with" mean that according to the present invention, the binding domain interacts or specifically interacts with a given epitope or a given site on the target molecule (antigen) (here: MSLN and CD40, respectively).

[0988] The term "epitope" refers to a site on an antigen to which a binding domain, such as 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 is sometimes also referred to herein as an "antigenic structure" or "antigenic determinant". Thus, a binding domain is an "antigenic interaction site". The binding / interaction is also understood to be defined as "specific recognition".

[0989] An "epitope" can be formed by contiguous amino acids or non-contiguous amino acids juxtaposed by the tertiary folding of a protein. A "linear epitope" is an epitope that comprises a recognition by a primary sequence of amino acids. A linear epitope typically includes at least 3 or at least 4, and more typically at least 5 or at least 6 or at least 7, such as about 8 to about 10 amino acids in a unique sequence.

[0990] In contrast to linear epitopes, "conformational epitopes" are epitopes in which the primary amino acid sequence that constitutes the epitope is not the only defining component of the epitope that is recognized (e.g., an epitope in which the primary amino acid sequence is not necessarily recognized by a binding domain). Typically, a conformational epitope comprises an increased number of amino acids relative to a linear epitope. With respect to recognition of conformational epitopes, a binding domain recognizes the three-dimensional structure of an antigen, preferably a peptide or protein or fragment thereof (in the context of the present invention, an antigenic structure of a binding domain is contained within the MSLN protein). For example, when a protein molecule folds to form a three-dimensional structure, certain amino acids and / or polypeptide backbones that form a conformational epitope are juxtaposed, enabling antibodies to recognize the epitope. Methods for determining epitope conformation 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.

[0991] The following describes a method for epitope mapping: When a region (contiguous stretch of amino acids) in the human MSLN protein is exchanged or replaced with the corresponding region of its non-human and non-primate MSLN antigen (e.g. mouse MSLN, but others such as chicken, rat, hamster, rabbit, etc. are also possible), a reduction in binding of the binding domain is expected to occur, unless the binding domain is cross-reactive for the non-human, non-primate MSLN used. The reduction is preferably at least 10%, 20%, 30%, 40% or 50% compared to the binding to the corresponding region in the human MSLN protein; more preferably at least 60%, 70% or 80%, and most preferably 90%, 95% or even 100%, thereby setting the binding to the corresponding region in the human MSLN protein to 100%. It is envisaged that the aforementioned human MSLN / non-human MSLN chimera is expressed in CHO cells. The human MSLN / non-human MSLN chimera can also be fused to the transmembrane domain and / or cytoplasmic domain of a different membrane-bound protein (eg, EpCAM), although this technique is not necessary for the methods described in Examples 1 and 2.

[0992] In an alternative or additional method for epitope mapping, several truncated forms of the human MSLN extracellular domain can be generated in order to determine the specific region recognized by the binding domain. In these truncated forms, different extracellular MSLN domains / subdomains or regions are progressively deleted starting from the N-terminus. Truncated MSLN forms can be expressed in CHO cells. It is also envisioned that truncated MSLN forms can be fused with transmembrane domains and / or cytoplasmic domains of different membrane-bound proteins (such as EpCAM). It is also envisioned that truncated MSLN forms can encompass a signal peptide domain at its N-terminus, such as a signal peptide derived from a mouse IgG heavy chain signal peptide. It is further envisioned that truncated MSLN forms can encompass a v5 domain at its N-terminus (after the signal peptide), which allows verification of its correct expression on the cell surface. It is expected that a reduction or loss of binding occurs to those truncated MSLN forms that no longer encompass the MSLN region recognized by the binding domain. The reduction in binding is preferably at least 10%, 20%, 30%, 40%, 50%; more preferably at least 60%, 70%, 80%, and most preferably 90%, 95% or even 100%, thereby setting the binding to the entire human MSLN protein (or its extracellular region or domain) to 100%.

[0993] Another method to determine the contribution of specific residues of the target antigen to recognition by an antibody construct or binding domain is alanine scanning (see, e.g., Morrison KL and Weiss GA. Cur Opin Chem Biol. 2001 Jun;5(3):302-7), in which each residue to be analyzed is replaced with alanine, e.g., via site-directed mutagenesis. Alanine is used because it has a non-bulky, chemically inert methyl functional group, but still mimics the secondary structure references that many other amino acids have. In situations where the size of the mutated residue is desired to be conservative, bulky amino acids (such as valine or leucine) can sometimes be used. Alanine scanning is a mature technique that has been used for a long time.

[0994] The interaction between a binding domain and an epitope or epitope-containing region means that the binding domain exhibits appreciable affinity for the epitope / epitope-containing region on a particular protein or antigen (here: MSLN and CD40, respectively), and generally does not exhibit significant reactivity with proteins or antigens other than MSLN or CD40. "Appreciable affinity" includes binding with an affinity of about 10 -6 Preferably, when the binding affinity is about 10 -12 to 1 0-8 M, 10 -12 Up to 10 -9 M, 10 -12 Up to 10 -10 M, 10-11 Up to 10 -8 M, preferably about 10 -11 Up to 10 -9 M, the binding is considered specific. Whether a binding domain specifically reacts or binds to a target can be easily tested, inter alia, by comparing the reaction of the binding domain with the target protein or antigen with the reaction of the binding domain with proteins or antigens other than MSLN or CD40. Preferably, the binding domains of the invention substantially or not substantially bind to proteins or antigens other than MSLN or CD40 (e.g., one binding domain cannot bind to proteins other than MSLN, and the other binding domain cannot bind to proteins other than CD40).

[0995] The term "substantially / essentially does not bind" or "cannot bind" means that the binding domain of the present invention does not bind to proteins or antigens other than MSLN or CD40, that is, it does not show more than 30%, preferably not more than 20%, more preferably not more than 10%, and particularly preferably not more than 9%, 8%, 7%, 6% or 5% reactivity with proteins or antigens other than MSLN or CD40, thereby setting the binding to MSLN or CD40 to 100%, respectively.

[0996] It is believed that specific binding is affected by the specific motif in the amino acid sequence of the binding domain and the antigen. Therefore, due to the result of their primary, secondary and / or tertiary structure and the secondary modification of the structure, combination is achieved. The specific interaction of the antigen interaction site and its specific antigen can cause the simple combination of the site and the antigen. In addition, the specific interaction of the antigen interaction site and its specific antigen can alternatively or additionally cause the initiation of the signal, for example due to induced antigen conformational changes, antigen oligomerization, etc.

[0997] The Fc region of an antibody interacts with multiple Fc receptors and ligands, conferring a range of important functional properties (called effector functions). For IgG, the Fc region contains the Ig domains CH2 and CH3. An important family of Fc receptors of the IgG isotype is the Fcγ receptors (FcγR). These receptors mediate communication between antibodies and the cell arms of the immune system. In humans, this protein family includes FcγRI (CD64), including isoforms FcγRIa, FcγRIb, and FcγRIc; FcγRII (CD32), including isoforms FcγRlla (including allotypes H131 and R131), FcγRllb (including FcγRllb-1 and FcγRllb-2), and FcγRllc; and FcγRIII (CD16), including isoforms FcγRllla (including allotypes V158 and F158) and FcγRlllb (including allotypes FcγRlilb-NA1 and FcγRlllb-NA2) (Jefferis et al., 2002, Immunol Lett [Immunology Communications] 82:57-65). These receptors usually have an extracellular domain (mediating the binding to Fc), a transmembrane region, and an intracellular domain (which can mediate some signal transduction events in the cell). 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 antigen binding, usually leading to intracellular signal transduction events and important subsequent immune responses, such as the release of inflammatory mediators, B cell activation, endocytosis, phagocytosis and cytotoxic attacks. The ability to mediate cytotoxicity and phagocytic effector functions is a potential mechanism for antibodies to destroy target cells. The cell-mediated reaction in which non-specific cytotoxic cells expressing FcγR recognize the antibodies bound on target cells and subsequently cause target cell lysis is called antibody-dependent cell-mediated cytotoxicity (ADCC). The cell-mediated reaction in which nonspecific cytotoxic cells expressing FcγRs recognize bound antibody on a target cell and subsequently cause phagocytosis of the target cell is called antibody-dependent cell-mediated phagocytosis (ADCP).

[0998] Different IgG subclasses have different affinities for FcγRs, with IgG1 and IgG3 generally binding to the receptors better than IgG2 and IgG4. All FcγRs bind to the same region on the IgG Fc, but with different affinities: the high-affinity binder FcγRI has a Kd of 10-8 M-1 for IgG1, while the low-affinity receptors FcγRII and FcγRIII generally bind at 10-6 and 10-5, respectively.

[0999] By "Fcγ receptor" or "FcγR" as used herein is meant any member of the family of proteins that binds the Fc region of an IgG antibody and is substantially encoded by an FcγR gene. In humans, this family includes, but is not limited to, FcγRI (CD64), including isoforms FcγRIa, FcγRIb, and FcγRIc; FcγRII (CD32), including isoforms FcγRlla (including allotypes H131 and R131), FcγRllb (including FcγRllb-1 and FcγRllb-2), and FcγRllc; and FcγRIII (CD16), including isoforms FcγRllla (including allotypes V158 and F158) and FcγRlllb (including allotypes FcγRlllb-NA1 and FcγRlllb-NA2) (Jefferis et al., 2002, Immunol Lett [Immunology Communications] 82:57-65), as well as any undiscovered human FcγRs or FcγR isoforms or allotypes. FcγR can be from any organism, including but not limited to human, mouse, rat, rabbit and monkey. 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 FcγRs or FcγR isoforms or allotypes.

[1000] Binding to an FcγR can be performed directly by determining the affinity of the antibody for the FcγR. Alternatively, binding can be determined indirectly by measuring the release of cytokines in a cell-based assay.

[1001] In order to promote the association of a specific heavy chain with its cognate light chain, both the heavy chain and the light chain may include complementary amino acid substitutions. As used herein, "complementary amino acid substitutions" refer to the substitution pairing of a positively charged amino acid in one chain with a negatively charged amino acid in another chain. For example, in some embodiments, the heavy chain comprises at least one amino acid substitution to introduce a charged amino acid, and the corresponding light chain comprises at least one amino acid substitution to introduce a charged amino acid, wherein the charged amino acid introduced into the heavy chain has an 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) may be introduced into the first light chain (LC1) and one or more negatively charged residues (e.g., aspartic acid or glutamic acid) may be introduced into the companion heavy chain (HC1) at the LC1 / HC1 binding interface, and one or more negatively charged residues (e.g., aspartic acid or glutamic acid) may be introduced into the second light chain (LC2), and one or more positively charged residues (e.g., lysine, histidine, or arginine) may be introduced into the companion heavy chain (HC2) at the LC2 / HC2 binding interface. When oppositely charged residues (polarity) at the interface attract, electrostatic interactions will guide LC1 to pair with HC1 and LC2 to pair with HC2. Heavy / light chain pairs with identically charged residues (polarity) at the interface (e.g., LC1 / HC2 and LC2 / HC1) will repel, leading to inhibition of unwanted HC / LC pairing.

[1002] In these and other embodiments, the CH1 domain of the heavy chain or the CL domain of the light chain comprises an amino acid sequence different from the wild-type IgG amino acid sequence, so that one or more positively charged amino acids in the wild-type IgG amino acid sequence are 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 comprises an amino acid sequence different from the wild-type IgG amino acid sequence, so that one or more negatively charged amino acids in the wild-type IgG amino acid sequence are replaced by one or more positively charged amino acids. In some embodiments, the CH1 domain of the first and / or second heavy chain in the multispecific antibody construct is selected from F126, P127, L128, A141, L145, K147, D148, H168, F170, P171, V173, Q175, S176, S183, V185 and K213. One or more amino acids at EU positions are replaced by charged amino acids. In certain embodiments, the heavy chain residues substituted with negatively or positively charged amino acids are S183 (EU numbering system). In some embodiments, S183 is replaced by a positively charged amino acid. In alternative embodiments, S183 is replaced by a negatively charged amino acid. For example, in one embodiment, S183 is replaced by a negatively charged amino acid (e.g., S183E) in the first heavy chain, and S183 is replaced by a positively charged amino acid (e.g., S183K) in the second heavy chain.

[1003] 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 multispecific antibody construct at positions selected from F116, F118, S121, D122, E123, Q124, S131, V133, L135, N137, N138, Q160, S162, T164, S174, and S176 (in EU numbering in kappa light chains) are replaced with a charged amino acid. 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 are substituted with a charged amino acid at a position selected from T116, F118, S121, E123, E124, K129, T131, V133, L135, S137, E160, T162, S165, Q167, A174, S176, and Y178 (in EU numbering in the lambda chain). In some embodiments, the residue substituted with a negatively or positively charged amino acid is S176 of the CL domain of a kappa or lambda light chain (EU numbering system). In certain embodiments, S176 of the CL domain is substituted with a positively charged amino acid. In alternative embodiments, S176 of the CL domain is substituted with a negatively charged amino acid. In one embodiment, S176 is substituted with a positively charged amino acid (eg, S176K) in the first light chain, and S176 is substituted with a negatively charged amino acid (eg, S176E) in the second light chain.

[1004] In addition to the complementary amino acid substitutions in the CH1 and CL domains or as an alternative, the variable regions of the light and heavy chains in the multispecific antibody construct may include 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 the multispecific antibody construct comprises an amino acid sequence different from the wild-type IgG amino acid sequence, so that one or more positively charged amino acids in the wild-type IgG amino acid sequence are 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 comprises an amino acid sequence different from the wild-type IgG amino acid sequence, so that one or more negatively charged amino acids in the wild-type IgG amino acid sequence are replaced by one or more positively charged amino acids.

[1005] The V region interface residues (i.e., amino acid residues mediating the assembly of VH and VL regions) in the VH region 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 can be replaced by 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 replaced by a positively charged amino acid (e.g., lysine). In an alternative embodiment, the amino acid at EU position 39 in the VH region of the first and / or second heavy chain is replaced by a negatively charged amino acid (e.g., glutamic acid). In some embodiments, the amino acid at EU position 39 in the VH region of the first heavy chain is replaced by 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 replaced by 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 replaced by 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 replaced by 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 replaced by 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 replaced by a positively charged amino acid (e.g., G44K).

[1006] The V region interface residues in the VL region (i.e., amino acid residues mediating 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 can be replaced with charged amino acids (preferably with those in the VH region of the homologous heavy chain with opposite charges). In some embodiments, the amino acid at EU position 100 in the VL region of the first and / or second light chain is replaced by a positively charged amino acid (e.g., lysine). In an alternative embodiment, the amino acid at EU position 100 in the VL region of the first and / or second light chain is replaced by a negatively charged amino acid (e.g., 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).

[1007] Any constant domain may be modified to contain one or more of the charge pair mutations described above to facilitate proper assembly of multispecific antibody constructs.

[1008] As used herein, the term "Fc region" refers to the C-terminal region of an immunoglobulin heavy chain, which can be produced by papain digestion of a complete antibody. The Fc region of an immunoglobulin generally comprises two constant domains, i.e., a CH2 domain and a CH3 domain, and optionally comprises a CH4 domain. In certain embodiments, the Fc region is an Fc region from an IgG1, IgG2, IgG3, or IgG4 immunoglobulin. In certain embodiments, the Fc region comprises CH2 and CH3 domains from human IgG1 or human IgG2 immunoglobulins. The Fc region can retain effector functions, such as C1q binding, complement dependent cytotoxicity (CDC), Fc receptor binding, antibody dependent cell-mediated cytotoxicity (ADCC), and phagocytosis. In other embodiments, the Fc region can be modified to reduce or eliminate effector functions, as described in more detail herein.

[1009] The heavy chain constant region or Fc region of the multispecific antibody constructs described herein may comprise one or more amino acid substitutions that affect the glycosylation and / or effector function of the antigen binding protein. One of the functions of the immunoglobulin Fc region is to communicate with the immune system when the immunoglobulin binds to its target. This is generally referred to as "effector function". Communication leads to antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP) and / or complement-dependent cytotoxicity (CDC). ADCC and ADCP are mediated by binding to Fc receptors on the surface of immune system cells via the Fc region. CDC is mediated by binding to proteins of the complement system, such as C1q, via Fc. In some embodiments, the multispecific antibody constructs of the present invention comprise one or more amino acid substitutions in the constant region that enhance the effector function (including ADCC activity, CDC activity, ADCP activity and / or clearance rate or half-life) of the antigen binding protein. Exemplary amino acid substitutions (EU numbering) that can enhance effector function include, but are not limited to, E233L, L234I, L234Y, L235S, G236A, S239D, F243L, F243V, P247I, D280H, K290S, K290E, K290N, K290Y, R292P, E294L, Y296W, S298A, S298D, S 298V, 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 foregoing.

[1010] In other embodiments, the multispecific antibody constructs of the present invention include one or more amino acid substitutions that reduce effector function in the constant region. Exemplary 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 of the foregoing.

[1011] Glycosylation can promote the effector function of antibodies, particularly IgG1 antibodies. Therefore, in some embodiments, the multispecific antibody constructs of the present invention contain one or more amino acid substitutions that affect the level or type of glycosylation of the binding protein. The glycosylation of polypeptides is usually N-linked or O-linked. N-linked refers to the connection of the carbohydrate moiety to the side chain of an asparagine residue. The tripeptide sequences asparagine-X-serine and asparagine-X-threonine (wherein X is any amino acid except proline) are recognition sequences for enzymatic connection of the carbohydrate moiety to the asparagine side chain. Therefore, the presence of any of these tripeptide sequences in a polypeptide creates a potential glycosylation site. O-linked glycosylation refers to the connection 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.

[1012] 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 the antigen binding protein can generally be accomplished by altering the amino acid sequence so that it contains one or more of the above tripeptide sequences (for N-linked glycosylation sites). The alteration can also be made by adding one or more serine or threonine residues to the starting sequence or by replacing one or more serine or threonine residues (for O-linked glycosylation sites). For convenience, the amino acid sequence of the antigen binding protein can be altered by changes at the DNA level, particularly by mutating the DNA encoding the target polypeptide at preselected bases, thereby generating codons that will translate into the desired amino acids.

[1013] The present invention also encompasses the generation of multispecific antibody construct molecules with altered carbohydrate structures that cause changes in effector activity, including antigen binding proteins that exhibit improved ADCC activity without or with reduced fucosylation. Various methods of reducing or eliminating fucosylation are known in the art. For example, ADCC effector activity is mediated by binding the antibody molecule to the FcγRIII receptor, which has been shown to depend on the glycosylated carbohydrate structure of the N-linked residue N297 of the CH2 domain. Compared to native, fucosylated antibodies, non-fucosylated antibodies bind to this receptor with increased affinity and more effectively trigger FcγRIII-mediated effector function. For example, recombinant production of non-fucosylated antibodies in CHO cells in which α-1,6-fucosyltransferase has been knocked out results in antibodies with a 100-fold increased ADCC activity (see Yamane-Ohnuki et al., Biotechnol Bioeng. [Biotechnology and Bioengineering] 87 (5): 614-22, 2004). Similar effects can be achieved by reducing the activity of α-1,6-fucosyltransferase or other enzymes in the fucosylation pathway, for example by siRNA or antisense RNA treatment, engineering the cell line to knock out one or more enzymes, or culturing with 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 a low degree of fucosylation (see Shields et al., J Biol Chem. 277(30): 26733-40, 2002; and Shinkawa et al., J Biol Chem. 278(5): 3466-73, 2003). It has also been determined that increasing the amount of bisected carbohydrate, for example by recombinant production of antibodies in cells overexpressing the GnTIII enzyme, can increase ADCC activity (see Umana et al., Nat Biotechnol. 17(2): 176-80, 1999).

[1014] In other embodiments, the glycosylation of the multispecific antibody construct described herein is reduced or eliminated by, for example, removing one or more glycosylation sites from the Fc region of the binding protein. The amino acid substitution that eliminates or changes the glycosylation site of N-connection can reduce or eliminate the glycosylation of the N-connection of the antigen-binding protein. In certain embodiments, the multispecific antibody construct described herein includes a mutation at position N297 (EU numbering), such as N297Q, N297A or N297G. In certain embodiments, the multispecific antibody construct described herein includes mutations at positions L234 and L235 (EU numbering), such as L234A and L235A. In a specific embodiment, the multispecific antibody construct of the present invention includes an Fc region with an N297G mutation from a human IgG1 antibody. In order to improve the stability of the molecule containing the N297 mutation, the Fc region of the molecule can be further engineered. For example, in some embodiments, one or more amino acids in the Fc region are substituted with cysteine ​​to promote the formation of disulfide bonds in the dimer state. Corresponding to the residues of V259, A287, R292, V302, L306, V323 or I332 (EU numbering) in the IgG1 Fc region, it can therefore be substituted with cysteine. In one embodiment, specific residue pairs are substituted with cysteine ​​so that they preferentially form disulfide bonds with each other, thereby limiting or preventing disulfide bond confusion. In certain embodiments, pairs include, but are not limited to, A287C and L306C, V259C and L306C, R292C and V302C, and V323C and I332C. In a specific embodiment, the multispecific antibody constructs described herein include Fc regions with R292C and V302C mutations from human IgG1 antibodies. In such embodiments, the Fc region may also include N297G mutations.

[1015] In one embodiment, the heavy chain comprises an amino acid substitution selected from the group consisting of:

[1016] (i) N297G or N297A;

[1017] (ii) L234A and L235A; and

[1018] (iii) R292C and V302C;

[1019] The amino acid numbering is according to the EU numbering of Kabat.

[1020] In one embodiment, the heavy chain comprises N297G, R292C, and V302C mutations, wherein the amino acid numbering is according to EU numbering as in Kabat.

[1021] It may also be desirable to modify the multispecific antibody constructs of the invention to increase serum half-life, for example by incorporating or adding a salvage receptor binding epitope (e.g., by mutating the appropriate region or by incorporating the epitope into a peptide tag, which is then fused to the antigen binding protein at either end or in the middle, for example by DNA or peptide synthesis; see, e.g., WO96 / 32478) or adding molecules such as PEG or other water-soluble polymers, including polysaccharide polymers. The salvage receptor binding epitope preferably constitutes a region in which any one or more amino acid residues from one or two loops in the Fc region are transferred to an analogous position in the antigen binding protein. In one embodiment, three or more amino acid residues from one or two loops in the Fc region are transferred. In one embodiment, the epitope is taken from the CH2 domain of the Fc region (e.g., an IgG Fc region) and transferred to the CH1, CH3, or VH region of the antigen binding protein, or more than one such region. Alternatively, the epitope is taken from the CH2 domain of the Fc region and transferred to the CL region or VL region, or both, of the antigen binding protein. For a description of Fc variants and their interaction with salvage receptors, see International Publication Nos. WO 97 / 34631 and WO 96 / 32478.

[1022] In certain embodiments of the multispecific antibody constructs of the present invention, the binding domain located at the amino terminus of the Fc region (i.e., the amino terminal binding domain) is a Fab fragment fused to the amino terminus of the Fc region through a peptide linker as described herein or through an immunoglobulin hinge region. "Immunoglobulin hinge region" refers to the amino acid sequence of the CH1 domain and the CH2 domain connecting the immunoglobulin heavy chain. The hinge region of human IgG1 is generally defined as an amino acid sequence from about Glu216 or about Cys226 to about Pro230. The hinge regions of other IgG isotypes can be aligned with the IgG1 sequence by placing the first and last cysteine ​​residues that form the inter-heavy chain disulfide bond in the same position, and are determined for those skilled in the art. In some embodiments, the amino terminal binding domain is connected to the amino terminus of the Fc region through the human IgG1 hinge region. In other embodiments, the amino terminal binding domain is connected to the amino terminus of the Fc region through the human IgG2 hinge region. In one embodiment, the amino terminal binding domain (e.g., Fab fragment) is fused to the Fc region through the carboxyl terminus of the CH1 region of Fab.

[1023] 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 terminal binding domain) is a Fab fragment. In such an embodiment, Fab is fused to the carboxyl terminus of the Fc region (e.g., the carboxyl terminus of the CH3 domain) through a peptide linker via the amino terminus of the VH region of the Fab fragment or is otherwise connected to the carboxyl terminus of the Fc region (e.g., the carboxyl terminus of the CH3 domain). Therefore, in one embodiment, Fab is fused to the Fc region via the amino terminus of the VH region of Fab, so that the resulting fusion protein comprises a CH2 domain, a CH3 domain, a peptide linker, a VH region, and a CH1 region from the N-terminus to the C-terminus.

[1024] The peptide linker connecting the Fc region to the carboxyl terminal Fab can be any peptide linker described herein. In a specific embodiment, the peptide linker connecting the Fc region to the carboxyl terminal Fab fragment is at least 5 amino acids in length. In other embodiments, the peptide linker connecting the Fc region to the carboxyl terminal Fab fragment is at least 8 amino acids in length. A particularly suitable peptide linker for connecting the Fc region to the carboxyl terminal Fab fragment is a glycine-serine linker, such as (Gly x Ser) n , wherein x=3 or 4 and n=2, 3, 4, 5 or 6 (SEQ ID NO: 923). In one embodiment, the peptide linker connecting the Fc region to the carboxy-terminal Fab fragment is a L10(G4S)2 linker (SEQ ID NO: 888). In another embodiment, the peptide linker connecting the Fc region to the carboxy-terminal Fab fragment is a L9 or G3SG4S linker (SEQ ID NO: 924).

[1025] In some embodiments of the antigen-binding proteins of the present invention, wherein 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 can be fused to the amino terminus of the Fc region through a peptide linker or an immunoglobulin hinge region as described herein. In some embodiments, the amino terminal Fab fragment is connected to the amino terminus of the Fc region through a human IgG1 hinge region. In other embodiments, the amino terminal Fab fragment is connected to the amino terminus of the Fc region through a human IgG2 hinge region. In one embodiment, the amino terminal Fab fragment is fused to the Fc region through the carboxyl terminus of the CH1 region of the Fab.

[1026] In some embodiments, the multispecific antibody construct of the present invention comprises a first antibody that specifically binds to a first target, wherein one polypeptide chain (e.g., heavy chain (VH2-CH1)) from a Fab fragment of a second antibody (that specifically binds to a second target) is fused to the carboxyl terminus of the heavy chain of the first antibody. The multispecific antibody construct in this embodiment also comprises a polypeptide chain (e.g., light chain (VL2-CL)) containing the other half of the Fab fragment from the second antibody. This format is referred to herein as an "IgG-Fab" format, and an embodiment of this type of molecule is schematically shown in Figure 1 In. Therefore, in certain embodiments, the present invention includes a bispecific, multivalent antigen-binding protein comprising: (i) a light chain from a first antibody, (ii) a heavy chain from a first antibody, wherein the heavy chain is fused at its carboxyl terminus to a first polypeptide comprising the VH-CH1 domain of a second antibody via a peptide linker to form a modified heavy chain, and (iii) a second polypeptide comprising the VL-CL domain of a second antibody. When dimerized, the multispecific antibody construct is a homohexamer consisting of two polypeptide chains comprising two modified heavy chains, two light chains (from the first antibody) and the other half of the Fab fragment (Fd fragment) from the second antibody. In one embodiment, the first polypeptide (fused to the carboxyl terminus of the heavy chain) comprises the VH and CH1 domains from the second antibody, and the second polypeptide comprises the VL and CL domains from the second antibody.

[1027] Charge pair mutations or complementary amino acid substitutions as described herein may be introduced into the Fab region of the first antibody (Fab 1) or the second antibody (Fab 2) to promote correct heavy chain-light chain pairing. For example, in some embodiments, the amino acid at EU position 38 of the VL domain in Fab1 is replaced by 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 by 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 by a positively charged amino acid (e.g., lysine) and the amino acid at EU position 39 of the VH domain in Fab1 is replaced by a negatively charged amino acid (e.g., glutamic acid). In certain embodiments, the amino acid at EU position 38 of the VL domain in Fab2 is replaced by 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 by a positively charged amino acid (e.g., lysine). In other embodiments, 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).

[1028] In the embodiment where the VH-CH1 region (i.e., Fd fragment) from the second antibody is fused to the heavy chain of the first antibody, the heavy chain from the first antibody comprises an S183E mutation (EU numbering), the light chain from the first antibody comprises an S176K mutation (EU numbering), the light chain from the second antibody comprises an S176E mutation (EU numbering), and the Fd region from the second antibody (fused to the C-terminus of the heavy chain from the first antibody) comprises an S183K mutation (EU numbering). In other embodiments, the heavy chain from the first antibody comprises a G44E mutation (EU) and an S183E mutation (EU numbering), the light chain from the first antibody comprises a G100K mutation (EU) and an S176K mutation (EU numbering), the light chain from the second antibody comprises a G100E mutation (EU) and an S176E mutation (EU numbering), and the Fd region from the second antibody (fused to the C-terminus of the heavy chain from the first antibody) comprises a G44K mutation (EU) and an S183K mutation (EU numbering). The charges in the foregoing examples can be reversed as long as the charges on the corresponding light or heavy chains are also reversed so that the correct heavy / light chain pair has opposite charges.

[1029] "Corresponding" as related to VH2 and the second CH1 domain means that if there is no linker, the amino acid residues of VH2 and the second CH1 domain are counted from the C-terminus of the first heavy chain. If there is a peptide linker, the amino acid residues of VH2 and the second CH1 domain are counted from the C-terminus of the peptide linker. In both cases, the amino acid residues are not counted from the N-terminus of the first heavy chain. Specifically, for VH2 and the second CH1 domain, the count starts at the first amino acid residue of the VH2 domain. The counting of amino acid residues is performed using the EU or AHo convention.

[1030] In certain embodiments: a) VH1 comprises a Q39E mutation and the first CH1 domain comprises a S183K mutation, using EU numbering; b) VH2 comprises a Q39K mutation and the second CH1 domain comprises a S183E mutation, using EU numbering; c) VL1 comprises a Q38K mutation and the first CL domain comprises a S176E mutation, using EU numbering; and d) VL2 comprises a Q38E mutation and the second CL domain comprises a S176K mutation, using EU numbering.

[1031] In certain embodiments: a) the first CH1 domain comprises G44E and S183K mutations using EU numbering; b) the second CH1 domain comprises G44K and S183E mutations using EU numbering; c) the first CL domain comprises G100K and S176E mutations using EU numbering; and d) the second CL domain comprises G100E and S176K mutations using EU numbering.

[1032] In certain embodiments: a) VH1 comprises a Q39K mutation and the first CH1 domain comprises a S183E mutation, using EU numbering; b) VH2 comprises a Q39E mutation and the second CH1 domain comprises a S183K mutation, using EU numbering; c) VL1 comprises a Q38E mutation and the first CL domain comprises a S176K mutation, using EU numbering; and d) VL2 comprises a Q38K mutation and the second CL domain comprises a S176E mutation, using EU numbering.

[1033] In certain embodiments: a) the first CH1 domain comprises G44K and S183E mutations using EU numbering; b) the second CH1 domain comprises G44E and S183K mutations using EU numbering; c) the first CL domain comprises G100E and S176K mutations using EU numbering; and d) the second CL domain comprises G100K and S176E mutations using EU numbering.

[1034] In certain embodiments, the first heavy chain is fused to VH2 via a peptide linker. In certain embodiments, the peptide linker comprises 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 sequences can also be written as: GGGSGGGS (SEQ ID NO:916), GGGGSGGGGS (SEQ ID NO:888), GGGSGGGSGGGS (SEQ ID NO:917), GGGGSGGGGSGGGGS (SEQ ID NO:889), GGGSGGGSGGGSGGGS (SEQ ID NO:918), GGGGSGGGGSGGGGSGGGGS (SEQ ID NO:918) NO:890), GGGSGGGSGGGSGGGSGGGS (SEQ ID NO:919), GGGGSGGGGSGGGGSGGGGSGGGGS (SEQ ID NO:920), GGGSGGGSGGGSGGGSGGGSGGGS (SEQ ID NO:921), and GGGGSGGGGSGGGGSGGGGSGGGGSGGGGS (SEQ ID NO:922).

[1035] Additionally or alternatively, the correct heavy-light chain pairing can be promoted 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 comprise the VL domain and the CH1 domain from the second antibody, and the second polypeptide may comprise the VH domain and the CL domain from the second antibody. In other embodiments, the first polypeptide (fused to the carboxyl terminus of the heavy chain) may comprise the VH domain and the CL domain from the second antibody, and the second polypeptide may comprise the VL domain and the CH1 domain from the second antibody.

[1036] In another aspect, the present invention relates to a multispecific antibody construct comprising:

[1037] 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 wherein the hinge-CH2-CH3 polypeptide is linked to a second heavy chain variable region (VH2), wherein the VH2 is linked to a second CH1 domain; wherein

[1038] i) the VH1 or first CH1 domain comprises at least one amino acid substitution to introduce a positively charged amino acid at a residue selected from the group consisting of positions 39, 44, and 183, using EU numbering; and

[1039] ii) the VH2 or second CH1 domain comprises at least one amino acid substitution to introduce 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

[1040] 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 wherein the VL1 or the first CL domain comprises at least one amino acid substitution to introduce a negatively charged amino acid at a residue selected from the group consisting of positions 38, 100, and 176, using EU numbering; and

[1041] 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 wherein the VL2 or the second CL domain comprises at least one amino acid substitution to introduce a positively charged amino acid at a residue selected from the group consisting of positions 38, 100, and 176, using EU numbering;

[1042] wherein VH1 and VL1 interact to bind a first antigen and wherein VH2 and VL2 interact to bind a second antigen;

[1043] in:

[1044] The first antigen is human CD40 (SEQ ID NO: 1) and the second antigen is human mesothelin ("MSLN"; SEQ ID NO: 2); or

[1045] The first antigen is human MSLN (SEQ ID NO: 2) and the second antigen is human CD40 (SEQ ID NO: 1).

[1046] In one aspect, the present invention relates to a multispecific antibody construct comprising:

[1047] 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 wherein the hinge-CH2-CH3 polypeptide is linked to a second heavy chain variable region (VH2), wherein the VH2 is linked to a second CH1 domain; wherein

[1048] i) the VH1 or first CH1 domain comprises at least one amino acid substitution to introduce a negatively charged amino acid at a residue selected from the group consisting of positions 39, 44, and 183, using EU numbering; and

[1049] ii) the VH2 or second CH1 domain comprises at least one amino acid substitution to introduce 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

[1050] 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 wherein the VL1 or the first CL domain comprises at least one amino acid substitution to introduce a positively charged amino acid at a residue selected from the group consisting of positions 38, 100, and 176, using EU numbering; and

[1051] 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 wherein the VL2 or the second CL domain comprises at least one amino acid substitution to introduce a negatively charged amino acid at a residue selected from the group consisting of positions 38, 100, and 176, using EU numbering;

[1052] wherein VH1 and VL1 interact to bind a first antigen and wherein VH2 and VL2 interact to bind a second antigen;

[1053] in:

[1054] The first antigen is human CD40 (SEQ ID NO: 1) and the second antigen is human mesothelin ("MSLN"; SEQ ID NO: 2); or

[1055] The first antigen is human MSLN (SEQ ID NO: 2) and the second antigen is human CD40 (SEQ ID NO: 1).

[1056] In one embodiment, the hinge-CH2-CH3 polypeptide is linked to VH2 via a peptide linker.

[1057] In one embodiment, the peptide linker comprises 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).

[1058] In one embodiment,

[1059] a) the VH1 or first CH1 domain comprises a mutation selected from the group consisting of: Q39K, G44K, and S183K, using EU numbering;

[1060] b) the VH2 or second CH1 domain comprises a mutation selected from the group consisting of: Q39E, G44E, and S183E, using EU numbering;

[1061] c) the VL1 or first CL domain comprises a mutation selected from the group consisting of: Q38E, G100E, and S176E, using EU numbering; and

[1062] d) VL2 or the second CL domain comprises a mutation selected from the group consisting of: Q38K, G100K, and S176K, using EU numbering.

[1063] In one embodiment,

[1064] a) the first CH1 domain contains an S183K mutation, using EU numbering;

[1065] b) the second CH1 domain contains the S183E mutation, using EU numbering;

[1066] c) the first CL domain comprises an S176E mutation, using EU numbering; and

[1067] d) The second CL domain contains the S176K mutation, using EU numbering.

[1068] In one embodiment,

[1069] a) VH1 comprises a Q39K mutation and the first CH1 domain comprises a S183K mutation, using EU numbering;

[1070] b) VH2 comprises a Q39E mutation and the second CH1 domain comprises an S183E mutation, using EU numbering;

[1071] c) VL1 comprises a Q38E mutation and the first CL domain comprises an S176E mutation, using EU numbering; and

[1072] d) VL2 comprises a Q38K mutation and the second CL domain comprises a S176K mutation, using EU numbering.

[1073] In one embodiment,

[1074] a) the first CH1 domain contains G44K and S183K mutations, using EU numbering;

[1075] b) the second CH1 domain contains G44E and S183E mutations, using EU numbering;

[1076] c) the first CL domain comprises G100E and S176E mutations, using EU numbering; and

[1077] d) The second CL domain contains G100K and S176K mutations, using EU numbering.

[1078] In one embodiment,

[1079] a) the VH1 or first CH1 domain comprises a mutation selected from the group consisting of: Q39E, G44E, and S183E, using EU numbering;

[1080] b) the VH2 or second CH1 domain comprises a mutation selected from the group consisting of: Q39K, G44K, and S183K, using EU numbering;

[1081] c) the VL1 or first CL domain comprises a mutation selected from the group consisting of: Q38K, G100K, and S176K, using EU numbering; and

[1082] d) VL2 or the second CL domain comprises a mutation selected from the group consisting of: Q38E, G100E, and S176E, using EU numbering.

[1083] In one embodiment,

[1084] a) the first CH1 domain contains the S183E mutation, using EU numbering;

[1085] b) the second CH1 domain contains an S183K mutation, using EU numbering;

[1086] c) the first CL domain comprises an S176K mutation, using EU numbering; and

[1087] d) The second CL domain contains the S176E mutation, using EU numbering.

[1088] In one embodiment,

[1089] a) VH1 comprises a Q39E mutation and the first CH1 domain comprises a S183E mutation, using EU numbering;

[1090] b) VH2 comprises a Q39K mutation and the second CH1 domain comprises a S183K mutation, using EU numbering;

[1091] c) VL1 comprises a Q38K mutation and the first CL domain comprises a S176K mutation, using EU numbering; and

[1092] d) VL2 comprises a Q38E mutation and the second CL domain comprises a S176E mutation, using EU numbering.

[1093] In one embodiment,

[1094] a) the first CH1 domain contains G44E and S183E mutations, using EU numbering;

[1095] b) the second CH1 domain contains G44K and S183K mutations, using EU numbering;

[1096] c) the first CL domain comprises G100K and S176K mutations, using EU numbering; and

[1097] d) The second CL domain contains G100E and S176E mutations, using EU numbering.

[1098] In one embodiment, the hinge-CH2-CH3 polypeptide comprises an amino acid substitution selected from the group consisting of:

[1099] (i) N297G or N297A;

[1100] (ii) L234A and L235A; and

[1101] (iii) R292C and V302C;

[1102] The amino acid numbering is according to the EU numbering of Kabat.

[1103] In one embodiment, the hinge-CH2-CH3 polypeptide comprises N297G, R292C, and V302C mutations, wherein the amino acid numbering is according to EU numbering as in Kabat.

[1104] 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

[1105] VL1 comprises CDRL1, CDRL2, and CDRL3 selected from the group consisting of:

[1106] SEQ ID NOs: 58, 59, and 60, respectively;

[1107] SEQ ID NOs: 64, 65, and 66, respectively;

[1108] SEQ ID NOs: 70, 71, and 72, respectively;

[1109] SEQ ID NOs: 76, 77, and 78, respectively;

[1110] SEQ ID NOs: 82, 83, and 84, respectively;

[1111] SEQ ID NOs: 88, 89, and 90, respectively;

[1112] SEQ ID NOs: 94, 95, and 96, respectively;

[1113] SEQ ID NOs: 100, 101, and 102, respectively;

[1114] SEQ ID NOs: 106, 107, and 108, respectively;

[1115] SEQ ID NOs: 112, 113, and 114, respectively;

[1116] SEQ ID NOs: 118, 119, and 120, respectively;

[1117] SEQ ID NOs: 124, 125, and 126, respectively; and

[1118] SEQ ID NOs: 130, 131, and 132, respectively;

[1119] VH1 comprises CDRH1, CDRH2, and CDRH3 selected from the group consisting of:

[1120] SEQ ID NOs: 136, 137, and 138, respectively;

[1121] SEQ ID NOs: 142, 143, and 144, respectively;

[1122] SEQ ID NOs: 148, 149, and 150, respectively;

[1123] SEQ ID NOs: 154, 155, and 156, respectively;

[1124] SEQ ID NOs: 160, 161, and 162, respectively;

[1125] SEQ ID NOs: 166, 167, and 168, respectively;

[1126] SEQ ID NOs: 172, 173, and 174, respectively;

[1127] SEQ ID NOs: 178, 179, and 180, respectively;

[1128] SEQ ID NOs: 184, 185, and 186, respectively;

[1129] SEQ ID NOs: 190, 191, and 192, respectively;

[1130] SEQ ID NOs: 196, 197, and 198, respectively;

[1131] SEQ ID NOs: 202, 203, and 204, respectively; and

[1132] SEQ ID NOs: 208, 209, and 210, respectively;

[1133] VL2 comprises CDRL1, CDRL2, and CDRL3 selected from the group consisting of:

[1134] SEQ ID NOs: 230, 231, and 232, respectively;

[1135] SEQ ID NOs: 236, 237, and 238, respectively;

[1136] SEQ ID NOs: 242, 243, and 244, respectively; and

[1137] SEQ ID NOs: 248, 249, and 250, respectively;

[1138] and VH2 comprises CDRH1, CDRH2, and CDRH3 selected from the group consisting of:

[1139] SEQ ID NOs: 254, 255, and 256, respectively;

[1140] SEQ ID NOs: 260, 261, and 262, respectively;

[1141] SEQ ID NOs: 266, 267, and 268, respectively; and

[1142] They are SEQ ID NO: 272, 273, and 274 respectively.

[1143] In one embodiment,

[1144] 1) VL1 and VH1 are selected from the group consisting of:

[1145] a) a VL1 comprising CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 58, 59, and 60, respectively; and a VH1 comprising CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 136, 137, and 138, respectively;

[1146] b) a VL1 comprising CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 64, 65, and 66, respectively; and a VH1 comprising CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 142, 143, and 144, respectively;

[1147] c) a VL1 comprising CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 70, 71, and 72, respectively; and a VH1 comprising CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 148, 149, and 150, respectively;

[1148] d) a VL1 comprising CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 76, 77, and 6780, respectively; and a VH1 comprising CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 154, 155, and 156, respectively;

[1149] e) a VL1 comprising CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 82, 83, and 84, respectively; and a VH1 comprising CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 160, 161, and 162, respectively;

[1150] f) a VL1 comprising CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 88, 89, and 90, respectively; and a VH1 comprising CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 166, 167, and 168, respectively;

[1151] g) a VL1 comprising CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 94, 95, and 96, respectively; and a VH1 comprising CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 172, 173, and 174, respectively;

[1152] h) a VL1 comprising CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 100, 101, and 102, respectively; and a VH1 comprising CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 178, 179, and 180, respectively;

[1153] i) a VL1 comprising CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 106, 107, and 108, respectively; and a VH1 comprising CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 184, 185, and 186, respectively;

[1154] j) a VL1 comprising CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 112, 113, and 114, respectively; and a VH1 comprising CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 190, 191, and 192, respectively;

[1155] k) a VL1 comprising CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 118, 119, and 120, respectively; and a VH1 comprising CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 196, 197, and 198, respectively;

[1156] l) a VL1 comprising CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 124, 125, and 126, respectively; and a VH1 comprising CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 202, 203, and 204, respectively; and

[1157] m) a VL1 comprising CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 130, 131, and 132, respectively; and a VH1 comprising CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 208, 209, and 210, respectively;

[1158] and

[1159] 2) VL2 and VH2 are selected from the group consisting of:

[1160] a) a VL2 comprising CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 230, 231, and 232, respectively; and a VH2 comprising CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 254, 255, and 256, respectively;

[1161] b) a VL2 comprising CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 236, 237, and 238, respectively; and a VH2 comprising CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 260, 261, and 262, respectively;

[1162] c) a VL2 comprising CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 242, 243, and 244, respectively; and a VH2 comprising CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 266, 267, and 268, respectively; and

[1163] d) a VL2 comprising CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 248, 249, and 250, respectively; and a VH2 comprising CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 272, 273, and 274, respectively.

[1164] In one embodiment:

[1165] VL1 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 5, 9, 13, 17, 21, 25, 29, 33, 37, 41, 45, 49, and 53;

[1166] VH1 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 6, 10, 14, 18, 22, 26, 30, 34, 38, 42, 46, 50, and 54;

[1167] VL2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 213, 217, 221, and 225;

[1168] VH2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 214, 218, 222, and 226.

[1169] In one embodiment:

[1170] 1) VL1 and VH1 are selected from the group consisting of:

[1171] a) VL1 comprising SEQ ID NO: 5 and VH1 comprising SEQ ID NO: 6;

[1172] b) VL1 comprising SEQ ID NO:9 and VH1 comprising SEQ ID NO:10;

[1173] c) a VL1 comprising SEQ ID NO: 13 and a VH1 comprising SEQ ID NO: 14;

[1174] d) VL1 comprising SEQ ID NO: 17 and VH1 comprising SEQ ID NO: 18;

[1175] e) a VL1 comprising SEQ ID NO: 21 and a VH1 comprising SEQ ID NO: 22;

[1176] f) VL1 comprising SEQ ID NO:25 and VH1 comprising SEQ ID NO:26;

[1177] g) VL1 comprising SEQ ID NO:29 and VH1 comprising SEQ ID NO:30;

[1178] h) a VL1 comprising SEQ ID NO: 33 and a VH1 comprising SEQ ID NO: 34;

[1179] i) VL1 comprising SEQ ID NO: 37 and VH1 comprising SEQ ID NO: 38;

[1180] j) VL1 comprising SEQ ID NO:41 and VH1 comprising SEQ ID NO:42;

[1181] k) a VL1 comprising SEQ ID NO:45 and a VH1 comprising SEQ ID NO:46;

[1182] 1) VL1 comprising SEQ ID NO: 49 and VH1 comprising SEQ ID NO: 50; and

[1183] m) a VL1 comprising SEQ ID NO:53 and a VH1 comprising SEQ ID NO:54;

[1184] and

[1185] 2) VL2 and VH2 are selected from the group consisting of:

[1186] a) VL2 comprising SEQ ID NO: 213 and VH2 comprising SEQ ID NO: 214;

[1187] b) VL2 comprising SEQ ID NO: 217 and VH2 comprising SEQ ID NO: 218;

[1188] c) VL2 comprising SEQ ID NO: 221 and VH2 comprising SEQ ID NO: 222; and

[1189] d) VL2 comprising SEQ ID NO:225 and VH2 comprising SEQ ID NO:226.

[1190] In one embodiment, the first antigen is human MSLN (SEQ ID NO: 2) and the second antigen is human CD40 (SEQ ID NO: 1); wherein:

[1191] VL1 comprises CDRL1, CDRL2, and CDRL3 selected from the group consisting of:

[1192] SEQ ID NOs: 230, 231, and 232, respectively;

[1193] SEQ ID NOs: 236, 237, and 238, respectively;

[1194] SEQ ID NOs: 242, 243, and 244, respectively; and

[1195] SEQ ID NOs: 248, 249, and 250, respectively;

[1196] VH1 comprises CDRH1, CDRH2, and CDRH3 selected from the group consisting of:

[1197] SEQ ID NOs: 254, 255, and 256, respectively;

[1198] SEQ ID NOs: 260, 261, and 262, respectively;

[1199] SEQ ID NOs: 266, 267, and 268, respectively; and

[1200] SEQ ID NOs: 272, 273, and 274, respectively;

[1201] VL2 comprises CDRL1, CDRL2, and CDRL3 selected from the group consisting of:

[1202] SEQ ID NOs: 58, 59, and 60, respectively;

[1203] SEQ ID NOs: 64, 65, and 66, respectively;

[1204] SEQ ID NOs: 70, 71, and 72, respectively;

[1205] SEQ ID NOs: 76, 77, and 78, respectively;

[1206] SEQ ID NOs: 82, 83, and 84, respectively;

[1207] SEQ ID NOs: 88, 89, and 90, respectively;

[1208] SEQ ID NOs: 94, 95, and 96, respectively;

[1209] SEQ ID NOs: 100, 101, and 102, respectively;

[1210] SEQ ID NOs: 106, 107, and 108, respectively;

[1211] SEQ ID NOs: 112, 113, and 114, respectively;

[1212] SEQ ID NOs: 118, 119, and 120, respectively;

[1213] SEQ ID NOs: 124, 125, and 126, respectively; and

[1214] SEQ ID NOs: 130, 131, and 132, respectively;

[1215] and

[1216] VH2 comprises CDRH1, CDRH2, and CDRH3 selected from the group consisting of:

[1217] SEQ ID NOs: 136, 137, and 138, respectively;

[1218] SEQ ID NOs: 142, 143, and 144, respectively;

[1219] SEQ ID NOs: 148, 149, and 150, respectively;

[1220] SEQ ID NOs: 154, 155, and 156, respectively;

[1221] SEQ ID NOs: 160, 161, and 162, respectively;

[1222] SEQ ID NOs: 166, 167, and 168, respectively;

[1223] SEQ ID NOs: 172, 173, and 174, respectively;

[1224] SEQ ID NOs: 178, 179, and 180, respectively;

[1225] SEQ ID NOs: 184, 185, and 186, respectively;

[1226] SEQ ID NOs: 190, 191, and 192, respectively;

[1227] SEQ ID NOs: 196, 197, and 198, respectively;

[1228] SEQ ID NOs: 202, 203, and 204, respectively; and

[1229] They are SEQ ID NOs: 208, 209, and 210, respectively.

[1230] In one embodiment,

[1231] 1) VL1 and VH1 are selected from the group consisting of:

[1232] a) a VL1 comprising CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 230, 231, and 232, respectively; and a VH1 comprising CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 254, 255, and 256, respectively;

[1233] b) a VL1 comprising CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 236, 237, and 238, respectively; and a VH1 comprising CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 260, 261, and 262, respectively;

[1234] c) a VL1 comprising CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 242, 243, and 244, respectively; and a VH1 comprising CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 266, 267, and 268, respectively; and

[1235] d) a VL1 comprising CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 248, 249, and 250, respectively; and a VH1 comprising CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 272, 273, and 274, respectively;

[1236] and

[1237] 2) VL2 and VH2 are selected from the group consisting of:

[1238] a) a VL2 comprising CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 58, 59, and 60, respectively; and a VH2 comprising CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 136, 137, and 138, respectively;

[1239] b) a VL2 comprising CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 64, 65, and 66, respectively; and a VH2 comprising CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 142, 143, and 144, respectively;

[1240] c) a VL2 comprising CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 70, 71, and 72, respectively; and a VH2 comprising CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 148, 149, and 150, respectively;

[1241] d) a VL2 comprising CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 76, 77, and 6780, respectively; and a VH2 comprising CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 154, 155, and 156, respectively;

[1242] e) a VL2 comprising CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 82, 83, and 84, respectively; and a VH2 comprising CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 160, 161, and 162, respectively;

[1243] f) a VL2 comprising CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 88, 89, and 90, respectively; and a VH2 comprising CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 166, 167, and 168, respectively;

[1244] g) a VL2 comprising CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 94, 95, and 96, respectively; and a VH2 comprising CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 172, 173, and 174, respectively;

[1245] h) a VL2 comprising CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 100, 101, and 102, respectively; and a VH2 comprising CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 178, 179, and 180, respectively;

[1246] i) a VL2 comprising CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 106, 107, and 108, respectively; and a VH2 comprising CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 184, 185, and 186, respectively;

[1247] j) a VL2 comprising CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 112, 113, and 114, respectively; and a VH2 comprising CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 190, 191, and 192, respectively;

[1248] k) a VL2 comprising CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 118, 119, and 120, respectively; and a VH2 comprising CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 196, 197, and 198, respectively;

[1249] 1) a VL2 comprising CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 124, 125, and 126, respectively; and a VH2 comprising CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 202, 203, and 204, respectively; and

[1250] m) a VL2 comprising CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 130, 131, and 132, respectively; and a VH2 comprising CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 208, 209, and 210, respectively.

[1251] In one embodiment,

[1252] VL1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 213, 217, 221, and 225;

[1253] VH1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 214, 218, 222, and 226;

[1254] VL2 comprises an amino acid sequence selected from the group consisting of: SEQ ID NO: 5, 9, 13, 17, 21, 25, 29, 33, 37, 41, 45, 49, and 53; and

[1255] VH2 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 6, 10, 14, 18, 22, 26, 30, 34, 38, 42, 46, 50, and 54.

[1256] In one embodiment,

[1257] 1) VL1 and VH1 are selected from the group consisting of:

[1258] a) VL1 comprising SEQ ID NO: 213 and VH1 comprising SEQ ID NO: 214;

[1259] b) VL1 comprising SEQ ID NO: 217 and VH1 comprising SEQ ID NO: 218;

[1260] c) a VL1 comprising SEQ ID NO: 221 and a VH1 comprising SEQ ID NO: 222; and

[1261] d) VL1 comprising SEQ ID NO: 225 and VH1 comprising SEQ ID NO: 226;

[1262] and

[1263] 2) VL2 and VH2 are selected from the group consisting of:

[1264] a) VL2 comprising SEQ ID NO: 5 and VH2 comprising SEQ ID NO: 6;

[1265] b) VL2 comprising SEQ ID NO: 9 and VH2 comprising SEQ ID NO: 10;

[1266] c) VL2 comprising SEQ ID NO: 13 and VH2 comprising SEQ ID NO: 14;

[1267] d) VL2 comprising SEQ ID NO: 17 and VH2 comprising SEQ ID NO: 18;

[1268] e) VL2 comprising SEQ ID NO: 21 and VH2 comprising SEQ ID NO: 22;

[1269] f) VL2 comprising SEQ ID NO:25 and VH2 comprising SEQ ID NO:26;

[1270] g) VL2 comprising SEQ ID NO:29 and VH2 comprising SEQ ID NO:30;

[1271] h) VL2 comprising SEQ ID NO:33 and VH2 comprising SEQ ID NO:34;

[1272] i) VL2 comprising SEQ ID NO: 37 and VH2 comprising SEQ ID NO: 38;

[1273] j) VL2 comprising SEQ ID NO:41 and VH2 comprising SEQ ID NO:42;

[1274] k) VL2 comprising SEQ ID NO:45 and VH2 comprising SEQ ID NO:46;

[1275] 1) VL2 comprising SEQ ID NO: 49 and VH2 comprising SEQ ID NO: 50; and

[1276] m) VL2 comprising SEQ ID NO:53 and VH2 comprising SEQ ID NO:54.

[1277] The present invention includes one or more isolated nucleic acids encoding the multispecific antibody constructs and components thereof described herein. The nucleic acid molecules of the present invention include DNA and RNA in single-stranded and double-stranded forms, and corresponding complementary sequences. DNA includes, for example, cDNA, genomic DNA, chemically synthesized DNA, DNA amplified by PCR, and combinations thereof. The nucleic acid molecules of the present invention include full-length genes or cDNA molecules, and combinations of fragments thereof. In one embodiment, the nucleic acid of the present invention is derived from a human source, but the present invention also includes nucleic acids derived from non-human species.

[1278] "Isolated nucleic acid" is used interchangeably with "isolated polynucleotide" herein, and in the case of isolating nucleic acid from a naturally occurring source, refers to a nucleic acid that has been separated from the adjacent gene sequence present in the genome of the organism from which the nucleic acid is isolated. In the case of nucleic acid synthesized by template enzymatic method or chemical synthesis, such as PCR product, cDNA molecule or oligonucleotide, it should be understood that the nucleic acid obtained by such method is an isolated nucleic acid. An isolated nucleic acid molecule refers to a nucleic acid molecule in the form of an independent fragment or as a component of a larger nucleic acid construct. In one embodiment, the nucleic acid is substantially free of contaminating endogenous substances. The nucleic acid molecule is derived from a substantially pure form and can utilize standard biochemical methods (such as Sambrook et al., Molecular Cloning: A Laboratory Manual [Molecular Cloning: Laboratory Manual], 2nd Edition, Cold Spring Harbor Laboratory [Cold Spring Harbor Laboratory], Cold Spring Harbor [Cold Spring Harbor], NY [New York] (1989) to identify, operate and recover the amount or concentration of its component nucleotide sequence separated at least once DNA or RNA. Such sequences are provided and / or constructed in the form of an open reading frame that does not contain internal non-translated sequences or introns that are typically present in eukaryotic genes. Non-translated DNA sequences may be present at 5' or 3' of the open reading frame, where these sequences do not interfere with the manipulation or expression of the coding region. Unless otherwise specified, the left-hand end of any single-stranded polynucleotide sequence discussed herein is the 5' end; the left-hand direction of a double-stranded polynucleotide sequence is referred to as 5'. The 5' to 3' direction of addition of a nascent RNA transcript is referred to as the transcription direction; the sequence region from the 5' end to the 5' end of the RNA transcript on a DNA strand having the same sequence as the sequence of the RNA transcript is referred to as the "upstream sequence"; the sequence region from the 3' end to the 3' end of the RNA transcript on a DNA strand having the same sequence as the sequence of the RNA transcript is referred to as the "downstream sequence".

[1279] In one aspect, the invention relates to a polynucleotide encoding the light chain of an antibody construct of the invention.

[1280] In one aspect, the invention relates to a polynucleotide encoding a heavy chain fusion protein of an antibody construct of the invention.

[1281] In one aspect, the invention relates to a vector comprising a polynucleotide encoding a light chain of an antibody construct, a polynucleotide encoding a heavy chain of an antibody construct, or both.

[1282] In one aspect, the invention relates to a host cell transformed or transfected with a vector or a polynucleotide encoding a light chain of an antibody construct and a polynucleotide encoding a heavy chain of an antibody construct.

[1283] In one aspect, the present invention relates to a method for producing an antibody construct of the present invention, the method comprising culturing a host cell comprising a polynucleotide encoding a light chain and further comprising a polynucleotide encoding a heavy chain fusion protein under conditions allowing expression of the antibody construct, and recovering the produced antibody construct from the culture.

[1284] As outlined herein, site-specific mutagenesis can be performed on nucleotides in the DNA encoding the polypeptide using cassette or PCR mutagenesis, or other techniques well known in the art, to produce DNA encoding variants, and then recombinant DNA is expressed in cell culture to prepare variants of the antigen-binding proteins described herein. However, determined techniques can also be used to prepare antigen-binding proteins comprising variant CDRs having up to about 100-150 residues by in vitro synthesis. These variants typically exhibit qualitative biological activity identical to naturally occurring analogs, such as binding to antigens. Such variants include, for example, deletions and / or insertions and / or substitutions of residues within the amino acid sequence of the antigen-binding protein. Any combination of deletions, insertions and substitutions is performed to obtain the final construct, provided that the final construct has the desired characteristics. Amino acid changes may also change the post-translational processing of the antigen-binding protein, such as changing the number or position of glycosylation sites. In certain embodiments, the preparation of antigen-binding protein variants is intended to modify amino acid residues that are directly involved in epitope binding. In other embodiments, for the purposes discussed herein, it is necessary to modify residues that are not directly involved in epitope binding or residues that are not involved in epitope binding in any way. Mutagenesis in any CDR region and / or framework region is contemplated. One of ordinary skill in the art can employ covariance analysis to design useful modifications in the amino acid sequence of an antigen binding protein. See, e.g., 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 Publication No. 2008 / 0318207A1; Glaser et al., U.S. Patent Publication No. 2009 / 0048122A1; Urech et al., WO 2008 / 110348A1; Borras et al., WO 2009 / 000099 A2. Such modifications, as determined by analysis of covariance, may improve the potency, pharmacokinetics, pharmacodynamics, and / or manufacturability characteristics of the antigen binding protein.

[1285] Nucleotide sequences of the present invention. It will be appreciated by those skilled in the art that, due to the degeneracy of the genetic code, it is possible to obtain a very large number of nucleic acids that all encode the CDRs herein (and the heavy and light chains or other components of the antigen-binding proteins described herein). Therefore, by simply modifying one or more codon sequences to identify a specific amino acid sequence in a manner that does not change the amino acid sequence of the encoded protein, those skilled in the art can make a variety of different nucleic acids.

[1286] The present invention also includes vectors comprising one or more nucleic acids encoding one or more components (e.g., variable regions, light chains, heavy chains, modified heavy chains, and Fd fragments) of the multispecific antibody constructs of the present invention. The term "vector" refers to any molecule or entity (e.g., nucleic acid, plasmid, phage, or virus) used to transfer protein coding information to a host cell. Examples of vectors include, but are not limited to, plasmids, viral vectors, non-episomal mammalian vectors, and expression vectors, such as recombinant expression vectors. As used herein, the term "expression vector" or "expression construct" refers to a recombinant DNA molecule containing a desired coding sequence and suitable nucleic acid control sequences necessary for expressing an operably linked coding sequence in a specific host cell. The expression vector may include, but is not limited to, sequences that affect or control transcription, translation, and, when introns are present, RNA splicing of the coding region operably linked thereto. The nucleic acid sequences necessary for expression in prokaryotes include promoters, optionally operator sequences, ribosome binding sites, and possible other sequences. Eukaryotic cells are known to utilize promoters, enhancers, and terminators as well as polyadenylation signals. The secretion signal peptide sequence can also be optionally encoded by an expression vector, operably connected to the target coding sequence, so that the expressed polypeptide can be secreted by the recombinant host cell, if necessary, so as to more easily separate the target polypeptide from the cell. For example, in some embodiments, the signal peptide sequence can be attached / fused to the amino terminus of any polypeptide sequence of the present invention. In certain embodiments, the signal peptide with the amino acid sequence of MDMRVPAQLLGLLLLWLRGARC (SEQ ID NO: 894) is fused to the amino terminus of any polypeptide sequence of the present invention. In other embodiments, the signal peptide with the amino acid sequence of MAWALLLLTLLTQGTGSWA (SEQ ID NO: 895) is fused to the amino terminus of any polypeptide sequence of the present invention. In yet other embodiments, the signal peptide with the amino acid sequence of MTCSPLLLTLLIHCTGSWA (SEQ ID NO: 896) is fused to the amino terminus of any polypeptide sequence 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 may be fused to any polypeptide chain of the invention, for example to facilitate or optimize expression in a particular host cell.

[1287] Typically, the expression vector used in the host cell to produce the bispecific antigen protein of the present invention will contain sequences for plasmid maintenance and for cloning and expressing the exogenous nucleotide sequences encoding the components of the multispecific antibody construct. In certain embodiments, this sequence, collectively referred to as "flanking sequence", typically will include one or more of 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 a nucleic acid encoding a polypeptide to be expressed, and a selectable marker component. These sequences are discussed separately below.

[1288] 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; the oligonucleotide tag sequence encodes polyhistidine (e.g., hexahistidine (SEQ ID NO: 925)), FLAG, HA (hemagglutinin influenza virus), myc, or another "tag" molecule to which an existing commercially available antibody is directed. The tag is typically fused to the antibody when the polypeptide is expressed, and can be used as a means of affinity purification or detection of the polypeptide from the host cell. Affinity purification can be achieved, for example, by column chromatography, using antibodies to the tag as an affinity matrix. Optionally, the tag can then be removed from the purified polypeptide by various means, such as cleavage using certain peptidases.

[1289] The flanking sequences may be homologous (i.e., from the same species and / or strain as the host cell), heterologous (i.e., from a species different from the host cell species or strain), hybrid (i.e., a combination of flanking sequences from more than one source), synthetic, or natural. Thus, the source of the flanking sequences may be any prokaryotic or eukaryotic organism, any vertebrate or invertebrate organism, or any plant, as long as the flanking sequences function in and can be activated by the host cell machinery.

[1290] The flanking sequences that can be used in the vectors of the present invention can be obtained by any of several methods well known in the art. Typically, the flanking sequences that can be used herein will be identified in advance by positioning and / or by restriction endonuclease digestion and therefore can be separated from appropriate tissue sources using appropriate restriction endonucleases. In some cases, the full nucleotide sequence of the flanking sequence can be known. Here, the flanking sequence can be synthesized using conventional nucleic acid synthesis or cloning methods.

[1291] Whether all or only a portion of the flanking sequence is known, it can be obtained using the polymerase chain reaction (PCR) and / or by screening a genomic library with suitable probes, such as oligonucleotides and / or flanking sequence fragments from the same or another species. If the flanking sequence is unknown, a DNA fragment containing the flanking sequence can be isolated from a larger DNA fragment that may contain, for example, a coding sequence or even another gene or genes. Appropriate DNA fragments can be generated by restriction endonuclease digestion, followed by agarose gel purification, Separation is accomplished by column chromatography (Chatsworth, CA) or other methods known to the skilled artisan. The selection of suitable enzymes to accomplish this purpose will be readily apparent to one of ordinary skill in the art.

[1292] The replication origin is typically a part of a commercially available prokaryotic expression vector, and the starting point helps to amplify the vector in a host cell. If the selected vector does not contain a replication origin, it can be chemically synthesized based on a known sequence and connected to the vector. For example, the replication origin from plasmid pBR322 (New England Biolabs, Beverly, MA) in Beverly, Massachusetts) is suitable for most gram-negative bacteria, and various viral starting points (such as SV40, polyoma virus, adenovirus, vesicular stomatitis virus (VSV), or papillomavirus, such as HPV or BPV) can be used for cloning vectors in mammalian cells. In general, mammalian expression vectors do not need a replication origin component (for example, usually only using the SV40 starting point, because it also contains a viral early promoter).

[1293] The transcription termination sequence is typically located at the 3' end of the polypeptide coding region and is used to terminate transcription. Usually, the transcription termination sequence in prokaryotes is a GC-rich fragment followed by a poly-T sequence. Although the sequence is easy to clone from a library or even commercially available as a part of a vector, it can also be easily synthesized using a nucleic acid synthesis method.

[1294] Selectable marker genes encode proteins required for survival and growth of host cells grown in selective media. Typical selectable marker genes encode proteins that (a) confer resistance to antibiotics or other toxins (e.g., ampicillin, tetracycline, or kanamycin for prokaryotic host cells); (b) supplement nutritional deficiencies of cells; or (c) provide important nutrients that are not available from complex media or defined media. Specific selectable markers are kanamycin resistance genes, ampicillin resistance genes, and tetracycline resistance genes. Advantageously, the neomycin resistance gene can also be used to select in both prokaryotic and eukaryotic host cells.

[1295] Other selectable genes can be used to amplify genes to be expressed. Amplification is the process of replicating genes required for the production of proteins necessary for growth or cell survival in tandem within the chromosomes of successive generations of recombinant cells. Examples of selectable markers suitable for mammalian cells include dihydrofolate reductase (DHFR) and promoterless thymidine kinase genes. Mammalian cell transformants are placed under selection pressure, wherein only transformants are uniquely suitable for survival due to the presence of selectable genes in the vector. Selection pressure is by culturing transformed cells under conditions of continuously increasing the concentration of the selector in the culture medium, thereby applying DNA amplification of selectable genes and encoding another gene, such as one or more components of the multispecific antibody construct described herein. Therefore, a larger amount of polypeptides are synthesized by the amplified DNA.

[1296] A ribosome binding site is generally necessary for the initiation of mRNA translation 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 linked to an internal ribosome binding site (IRES), thereby allowing translation of two open reading frames from a single RNA transcript.

[1297] In some cases, such as when glycosylation is needed in a eukaryotic host cell expression system, various pre-sequences or pro-sequences can be operated to improve glycosylation or yield. For example, the peptidase cleavage site of a specific signal peptide can be changed, or a pro-sequence can be added, which can also affect glycosylation. The final protein product can have one or more other amino acids that are easy to express at position-1 (relative to the first amino acid of the mature protein), and these amino acids may not be completely removed. For example, the final protein product may have one or two amino acid residues attached to the amino terminal found in the peptidase cleavage site. Alternatively, when the enzyme is cut in such regions in the mature polypeptide, some enzyme cleavage sites may be used to produce a slightly truncated form of the desired polypeptide.

[1298] The expression and cloning vectors of the present invention typically contain a promoter that is recognized by the host organism and is operably connected to the molecule of the encoded polypeptide. As used herein, the term "operably connected" refers to that two or more nucleic acid sequences are connected in a manner that produces a nucleic acid molecule that can guide the transcription of a given gene and / or the synthesis of a desired protein molecule. For example, a control sequence that is "operably connected" to a protein coding sequence in a vector is connected to the coding sequence so that the expression of the protein coding sequence is achieved under conditions that are compatible with the transcriptional activity of the control sequence. More specifically, if a promoter and / or enhancer sequence (including any combination of cis-acting transcriptional control components) stimulates or regulates the transcription of a coding sequence in a suitable host cell or other expression system, it is operably connected to the coding sequence.

[1299] A promoter is a non-transcribed sequence located upstream (i.e., 5') of the start codon of a structural gene that controls transcription of the structural gene (generally within about 100 to 1000 bp). Promoters are generally grouped into one of two categories: inducible promoters and constitutive promoters. An inducible promoter initiates transcription of the DNA under its control at an increased level in response to a certain change in culture conditions (such as the presence or absence of nutrients, or temperature changes). On the other hand, a constitutive promoter consistently transcribes the gene to which it is operably linked, i.e., has little or no control over gene expression. Many promoters recognized by a variety of potential host cells are well known. The source DNA promoter is removed by restriction enzyme digestion, and the desired promoter sequence is inserted into a vector, and a suitable promoter is operably linked to DNA encoding, for example, a heavy chain, a light chain, a modified heavy chain, or other components of a multispecific antibody construct of the present invention.

[1300] Suitable promoters for use with yeast hosts are also well known in the art. Yeast enhancers are preferably used with yeast promoters. Suitable promoters for mammalian host cells are well known and include, but are not limited to, those obtained from viral genomes such as polyoma virus, transmissible epithelioma virus, adenovirus (such as adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, retrovirus, hepatitis B virus, and simian virus 40 (SV40). Other suitable mammalian promoters include heterologous mammalian promoters, such as heat shock promoters and actin promoters.

[1301] Other promoters of interest include, but are not limited to, 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 Rous sarcoma virus promoter (Rous sarcoma virus promoter). virus) (Yamamoto et al., 1980, Cell 22:787-797); the herpes thymidine kinase promoter (Wagner et al., 1981, Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences of the United States] 78:1444-1445); the promoter and regulatory sequences from the metallothionein gene (Prinster et al., 1982, Nature 296:39-42); and prokaryotic promoters, such as the β-lactamase promoter (Villa-Kamaroff et al., 1978, Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences of the United States] 75:3727-3731); or the tac promoter (DeBoer et al., 1983, Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences of the United States] 80:21-25).Also of interest are the following animal transcriptional control regions, which exhibit tissue specificity and have been used in transgenic animals: the elastase I gene control region, which is 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 control region, which is active in pancreatic β cells (Hanahan, 1985, Nature, 315:115-122); the immunoglobulin gene control region, which is active in lymphoid cells (Grosschedl et al., 1984, Cell, 38:647-651); and the mitochondrial growth factor receptor (MTR) gene, which is active in pancreatic β cells. 658; Adames et al., 1985, Nature 318:533-538; Alexander et al., 1987, Mol. Cell. Biol. 7:1436-1444); a mouse mammary tumor virus control region active in testicular, mammary, lymphoid, and mast cells (Leder et al., 1986, Cell 45:485-495); an albumin gene control region active in liver (Pinkert et al., 1987, Genes and Devel. [Genes and Development] 1: 268-276); the α-fetoprotein gene control region active in the liver (Krumlauf et al., 1985, Mol. Cell. Biol. [Molecular Cell Biology] 5: 1639-1648; Hammer et al., 1987, Science [Science] 253: 53-58); the α1-antitrypsin gene control region active in the liver (Kelsey et al., 1987, Gene ... Devel. [Genes and Development] 1: 161-171); the beta-globin gene control region active in bone marrow cells (Mogram et al., 1985, Nature [Nature] 315: 338-340; Kollias et al., 1986, Cell [Cell] 46: 89-94); the myelin basic protein gene control region active in oligodendrocytes in the brain (Readhead et al., 1987, Cell [Cell] 48: 703-712); the myosin light chain-2 gene control region active in skeletal muscle (Sani, 1985, Nature [Nature] 314: 283-286); and the gonadotropin-releasing hormone gene control region active in the hypothalamus (Mason et al., 1986, Science [Science] 234: 1372-1378).

[1302] Enhancer sequences can be inserted into vectors to increase the transcription of DNA encoding components (e.g., light chain, heavy chain, modified heavy chain, Fd fragment) of multispecific antibody constructs in higher eukaryotic cells. Enhancers are cis-acting elements of DNA, usually about 10-300bp in length, acting on promoters to increase transcription. Enhancers are relatively independent in direction and position, and have been found in 5' and 3' positions of transcription units. Several enhancer sequences (e.g., globulin, elastase, albumin, α-fetoprotein and insulin) known to be available from mammalian genes. However, enhancers from viruses are typically used. SV40 enhancers, cytomegalovirus early promoter enhancers, polyoma enhancers and adenovirus enhancers known in the art are exemplary reinforcing elements for activating eukaryotic promoters. Although enhancers can be positioned at 5' or 3' of the coding sequence in the vector, they are typically located at the site of the promoter 5'. Sequences encoding appropriate natural or heterologous signal sequences (leader sequences or signal peptides) can be incorporated into expression vectors to promote extracellular secretion of antibodies. The choice of signal peptide or leader sequence depends on the type of host cell in which the antibody is to be produced, and heterologous signal sequences can replace natural signal sequences. Examples of signal peptides are described above. Other signal peptides that work in mammalian host cells include the signal sequence of interleukin-7 (IL-7) described in U.S. Patent No. 4,965,195; Cosman et al., 1984, the signal sequence of the interleukin-2 receptor described in Nature 312:768; the interleukin-4 receptor signal peptide described in European Patent No. 0367566; the type I interleukin-1 receptor signal peptide described in U.S. Patent No. 4,968,607; the type II interleukin-1 receptor signal peptide described in EP Patent No. 0460846.

[1303] The provided expression vectors can be constructed from a starting vector (e.g., a commercially available vector). These vectors may or may not contain all desired flanking sequences. In the absence of one or more flanking sequences described herein in the vector, they can be independently obtained and connected to the vector. Methods for obtaining each flanking sequence are well known to those of ordinary skill in the art. The expression vector can be introduced into a host cell, thereby producing a protein encoded by a nucleic acid as described herein, including a fusion protein.

[1304] In certain embodiments, the nucleic acid encoding the different components of the multispecific antibody construct of the present invention can be inserted into the same expression vector. For example, the nucleic acid encoding the anti-first target antigen light chain can be cloned into the same vector as the nucleic acid encoding the anti-first target antigen heavy chain. In such embodiments, the two nucleic acids can be separated by an internal ribosome entry site (IRES) and under the control of a single promoter, so that the light chain and the heavy chain are expressed by the same mRNA transcript. Alternatively, the two nucleic acids can be under the control of two independent promoters, so that the light chain and the heavy chain are expressed by two independent mRNA transcripts. In certain embodiments, the nucleic acid encoding the anti-first target antigen light chain and the anti-first target antigen heavy chain can be cloned into an expression vector, and the nucleic acid encoding the anti-second target antigen light chain and the anti-second target antigen heavy chain can be cloned into a second expression vector.

[1305] Similarly, for IgG-Fab multi-specific antibody constructs, the nucleic acid encoding each of the three components can be cloned into the same expression vector. In certain embodiments, the nucleic acid encoding the light chain of IgG-Fab molecules and the nucleic acid encoding the second polypeptide (comprising the other half of the C-terminal Fab domain) are cloned into an expression vector, and the nucleic acid encoding the modified heavy chain (comprising the fusion protein of half of the heavy chain and Fab domain) is cloned into the second expression vector. In certain embodiments, all components of multi-specific antibody constructs as herein described are expressed from identical host cell populations. For example, even if one or more components are cloned into a separate expression vector, host cells are also co-transfected with two expression vectors so that a cell produces all components of multi-specific antibody constructs.

[1306] After constructing a vector and inserting one or more nucleic acid molecules encoding each component of the multispecific antibody construct described herein into one or more appropriate sites of one or more vectors, one or more complete vectors can be inserted into appropriate host cells for amplification and / or polypeptide expression. Therefore, the present invention includes isolated host cells, which contain one or more expression vectors encoding the multispecific antibody construct components. As used herein, the term "host cell" refers to a cell that is transformed with nucleic acid or can be transformed with nucleic acid and thus expresses the gene of interest. The term includes the offspring of the parental cell, whether or not the offspring is the same as the original parental cell in morphology or genetic composition, as long as the gene of interest is present. A host cell comprising the isolated nucleic acid of the present invention (operably connected to at least one expression control sequence (e.g., promoter or enhancer) in one embodiment) is a "recombinant host cell".

[1307] The expression vector of the antigen-binding protein can be transformed into the selected host cell by well-known methods, including transfection, infection, calcium phosphate coprecipitation, electroporation, microinjection, liposome transfection, DEAE-dextran mediated transfection or other known techniques. The method will vary in part with the type of host cell used. These methods and other suitable methods are well known to the technician and are described in, for example, Sambrook et al., 2001, supra.

[1308] The host cells, when cultured under appropriate conditions, synthesize antigen binding proteins, which can then be collected from the culture medium (if the host cells secrete it into the culture medium) or directly from the host cells that produced it (if it is not secreted). The selection of an appropriate host cell will depend on various factors, such as the desired expression level, polypeptide modifications (e.g., glycosylation or phosphorylation) that are desired or necessary for activity, and ease of folding into a biologically active molecule.

[1309] Exemplary host cells include prokaryotic cells, yeast, or higher eukaryotic cells. Prokaryotic host cells include eubacteria, such as Gram-negative or Gram-positive organisms, for example, Enterobacteriaceae such as Escherichia, e.g., E. coli, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, e.g., Salmonella typhimurium, Serratia, e.g., Serratia marcescans, and Shigella, and Bacillus, e.g., B. subtilis and B. licheniformis, Pseudomonas, and Streptomyces. Eukaryotic microorganisms such as filamentous fungi or yeast are suitable cloning or expression hosts for recombinant polypeptides. Saccharomyces cerevisiae or common baker's yeast is the most commonly used of the lower eukaryotic host microorganisms. However, a variety of other genera, species and strains are in common use and can be used herein, such as Pichia, e.g., P. pastoris, Schizosaccharomyces pombe; Kluyveromyces, Yarrowia; Candida; Trichoderma reesia; Neurosporacrassa; Schwanniomyces, e.g., Schwanniomyces occidentalis; and filamentous fungi, such as, e.g., Neurospora, Penicillium, Tolypocladium, and Aspergillus hosts, such as A. nidulans and A. niger.

[1310] Host cells for expressing glycosylated antigen binding proteins can be derived from multicellular organisms. Examples of invertebrate cells include plants and insect cells. Many baculovirus strains and variants have been identified, as well as corresponding permissive insect host cells from the following hosts, such as Spodoptera frugiperda (caterpillars), Aedes aegypti (mosquitoes), Aedes albopictus (mosquitoes), Drosophila melanogaster (fruit flies) and Bombyx mori. Multiple virus strains for transfecting such cells are publicly available, for example, the L-1 variant of Autographa californica NPV and the Bm-5 strain of Bombyx mori NPV.

[1311] Vertebrate host cells are also suitable hosts, and recombinant production of antigen-binding proteins by such cells has become routine. Mammalian cell lines that can be used as hosts for expression are well known in the art and include, but are not limited to, immortalized cell lines available from the American Germplasm Collection Center (ATCC), including, but not limited to, Chinese hamster ovary (CHO) cells, including CHOK1 cells (ATCC CCL61), DXB-11, DG-44, and Chinese hamster ovary cells / -DHFR (CHO, Urlaub et al., Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences of the United States] 77:4216, 1980); monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line (293 cells or 293 cells subcloned for growth in suspension culture (Graham et al., J. Gen Virol. [Journal of General Virology] 36:59, 1977); baby hamster kidney cells (BHK, ATCC CCL10); mouse Sertoli cells (TM4, Mather, Biol. Reprod. [Reproduction Biology] 23:243-251, 1980); monkey kidney cells (CV1, ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); Buffalo rat liver cells (BRL 3A, ATCC CRL1442); human lung cells (W138, ATCC CCL 75); human hepatoma cells (Hep G2, HB 8065); mouse mammary tumor (MMT060562, ATCC CCL51); TRI cells (Mather et al., Annals NY Acad. Sci. [Annals of the New York Academy of Sciences] 383:44-68, 1982); MRC 5 cells or FS4 cells; mammalian myeloma cells, and many other cell lines. In certain embodiments, cell lines can be selected by determining which cell lines have high expression levels and constitutively produce the multispecific antibody constructs of the present invention. In another embodiment, cell lines that do not produce autoantibodies but are capable of making and secreting heterologous antibodies can be selected from the B cell lineage. CHO cells are host cells for expressing the multispecific antibody constructs of the present invention.

[1312] Host cells are transformed or transfected with the above nucleic acids or vectors to produce multispecific antibody constructs and cultured in conventional nutrient media appropriately modified for inducing promoters, selecting transformants, or amplifying genes encoding desired sequences. In addition, novel vectors and transfected cell lines having multiple copies of transcription units separated by selectable markers are particularly suitable for expressing antigen binding proteins. Therefore, the present invention also provides a method for preparing the multispecific antibody constructs described herein, the method comprising culturing a host cell containing one or more expression vectors in a culture medium under conditions that allow expression of the multispecific antibody construct encoded by one or more expression vectors described herein; and recovering the multispecific antibody construct from the culture medium.

[1313] Host cells for producing antigen-binding proteins of the invention can be cultured in a variety of culture media. Commercially available culture media such as Ham's F10 (Sigma), Minimal Essential Medium (MEM, Sigma), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle's Medium (DMEM, Sigma) are suitable for culturing host cells. In addition, any of the media described in Ham et al., Meth. Enz. [Methods in Enzymology] 58:44, 1979; Barnes et al., Anal. Biochem. [Analytical Biochemistry] 102:255, 1980; U.S. Pat. Nos. 4,767,704; 4,657,866; 4,927,762; 4,560,655; or 5,122,469; WO 90103430; WO 87 / 00195; or U.S. Pat. No. 30,985 can be used as a culture medium for the host cells. If necessary, any of these media can be supplemented with hormones and / or other growth factors (e.g., insulin, transferrin, or epidermal growth factor), salts (e.g., sodium chloride, calcium, magnesium, and phosphate), buffers (e.g., HEPES), nucleotides (e.g., adenosine and thymidine), antibiotics (e.g., gentamycin TM ) drugs), trace elements (defined as inorganic compounds generally present at final concentrations in the micromolar range), and glucose or an equivalent energy source. Any other necessary supplements may also be included at appropriate concentrations known to those skilled in the art. The culture conditions, such as temperature, pH, and the like, are those previously used with the host cell selected for expression and will be apparent to the skilled artisan.

[1314] After culturing host cells, multispecific antibody constructs can be produced in cells, in the periplasmic space, or directly secreted into the culture medium. If antigen-binding proteins are produced in cells, then as a first step, the granular fragments of host cells or dissolved fragments are removed, for example, by centrifugation or ultrafiltration. Bispecific antigen-binding proteins can be purified using, for example, hydroxyapatite chromatography, cation or anion exchange chromatography, or affinity chromatography, using one or more target antigens or protein A or protein G as affinity ligands. Protein A can be used to purify proteins including polypeptides based on human γ1, γ2 or γ4 heavy chains (Lindmark et al., J. Immunol. Meth. [Journal of Immunology Methods] 62: 1-13, 1983). Protein G is recommended for all mouse isotypes and for human γ3 (Guss et al., EMBO J. [Journal of the European Molecular Biology Association] 5: 15671575, 1986). The matrix to which the affinity ligand is attached is usually agarose, but other matrices can also be used. Mechanically stable matrices such as controlled pore glass or poly(styrenedivinyl)benzene can increase flow rates and reduce processing times compared to agarose. In the case of proteins containing CH3 domains, Bakerbond ABX TM Resins (JT Baker, Phillipsburg, NJ) can be used for purification. Other protein purification techniques such as ethanol precipitation, reverse phase HPLC, chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation can also be used depending on the specific multispecific antibody construct to be recovered.

[1315] The multispecific antibody constructs of the present invention are used to detect one or more target antigens (in a biological sample) and identify cells or tissues expressing one or more target antigens. The multispecific antibody constructs described herein can be used for diagnostic purposes to detect, diagnose or monitor diseases and / or disorders associated with one or more target antigens. Also provided are methods for detecting the presence of one or more target antigens in a sample using classical immunohistological methods known to those skilled in the art (e.g., Tijssen, 1993, Practice and Theory of Enzyme Immunoassays, Vol. 15 (R. H. Burdon and P. H. van Knippenberg, eds., 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.

[1316] One embodiment provides a multispecific antibody construct of the present invention or a multispecific antibody construct produced according to the method of the present invention for use in preventing, treating or ameliorating a tumor or cancer disease or a metastatic cancer disease.

[1317] According to a preferred embodiment of the present invention, the tumor or cancer disease is a solid tumor disease.

[1318] The preparations described herein can be used as pharmaceutical compositions for treating, improving and / or preventing pathological medical conditions described herein in patients in need. The term "treatment" refers to both therapeutic treatment and prophylactic or preventive measures. Treatment includes applying or applying the preparation to the body, separated tissue or cells from a patient with a disease / disorder, a symptom of a disease / disorder or a tendency to a disease / disorder, for the purpose of treating, curing, alleviating, alleviating, changing, remedying, alleviating, improving or affecting a disease, a symptom of a disease or a tendency to a disease.

[1319] As used herein, the term "alleviation" refers to any improvement in the disease state of a patient suffering from a tumor or cancer or metastatic cancer as described herein by administering a multispecific antibody construct according to the present invention to a subject in need thereof. Such improvement may also be considered to slow down or stop the progression of a patient's tumor or cancer or metastatic cancer. As used herein, the term "prevention" means avoiding the onset or recurrence of a patient suffering from a tumor or cancer or metastatic cancer as described herein by administering a multispecific antibody construct according to the present invention to a subject in need thereof.

[1320] 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 conditions or diseases, including those pathological conditions that predispose mammals to the disease in question. A "vegetation" is an abnormal growth of tissue, usually but not always forming a mass. When a mass is also formed, it is usually referred to as a "tumor". A vegetation or tumor may be benign, potentially malignant (precancerous) or malignant. Malignant tumors are usually referred to as cancer. They usually invade and destroy surrounding tissues and can form metastases, i.e., they spread to other parts of the body, tissues or organs. Therefore, the term "metastatic cancer" includes metastases of other tissues or organs outside the original tumor. Lymphomas and leukemias are lymphoid tumors. For the purposes of the present invention, they are also encompassed in the terms "tumor" or "cancer".

[1321] In a preferred embodiment of the present invention, the tumor or cancer disease is a solid tumor disease and the metastatic cancer disease may be derived from any of the aforementioned.

[1322] Preferred tumors or cancer diseases related to the present invention are selected from the group consisting of: breast cancer, carcinoid, 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, the tumor or cancer disease (preferably a solid tumor disease) can be selected from the group consisting of: ovarian cancer, pancreatic cancer, mesothelioma, lung cancer, gastric cancer and triple negative breast cancer. Metastatic cancer diseases can be derived from any of the aforementioned.

[1323] The present invention also provides a method for treating or ameliorating a tumor or cancer disease or a metastatic cancer disease, which comprises the step of administering the multispecific antibody construct of the present invention or the multispecific antibody construct produced according to the method of the present invention to a subject in need thereof.

[1324] The terms "subject in need thereof" or "in need of treatment" include those subjects already suffering from the disorder, as well as those in which the disorder is to be prevented. Subjects in need thereof or "patients" include humans and other mammalian subjects receiving prophylactic or therapeutic treatment.

[1325] The multispecific antibody constructs of the invention are typically designed for specific routes and methods of administration, specific dosages and frequencies of administration, specific treatments of specific diseases, with ranges of bioavailability and persistence, etc. The materials of the composition are preferably formulated at concentrations acceptable to the site of administration.

[1326] Thus, formulations and compositions can be designed according to the present invention for delivery by any suitable route of administration. In the context of the present invention, routes of administration include, but are not limited to

[1327] Topical routes (e.g., epidermal, inhalation, nasal, ocular, auricular / aural, vaginal, mucous membranes);

[1328] Enteral routes (eg, oral, gastrointestinal, sublingual, sublabial, buccal, rectal); and

[1329] Parenteral routes (e.g., intravenous, intraarterial, intraosseous, intramuscular, intracerebral, intraventricular, epidural, intrathecal, subcutaneous, intraperitoneal, extraamniotic, intraarticular, intracardiac, intradermal, intralesional, intrauterine, intravesical, intravitreal, transdermal, intranasal, transmucosal, intrasynovial, intraluminal).

[1330] The pharmaceutical compositions and multispecific antibody constructs of the present invention are particularly suitable for parenteral administration, such as subcutaneous or intravenous delivery, for example by injection such as bolus injection or by infusion such as continuous infusion. The pharmaceutical compositions can be administered using a medical device. Examples of medical devices for administering pharmaceutical compositions are described in U.S. Pat. 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.

[1331] In particular, the present invention provides for the uninterrupted administration of suitable compositions. As a non-limiting example, uninterrupted or substantially uninterrupted, i.e., continuous administration can be achieved by a small pump system worn by the patient for metering the therapeutic agent flowing into the patient's body. The pharmaceutical composition comprising the multispecific antibody construct of the present invention can be administered by using the pump system. Such pump systems are generally known in the art and generally rely on the regular replacement of cartridges containing the therapeutic agent to be infused. When the cartridge is replaced in such a pump system, the uninterrupted flow of the therapeutic agent into the patient's body may be temporarily interrupted. In this case, the administration phase before the cartridge is replaced and the administration phase after the cartridge is replaced will still be considered to be within the meaning of the pharmaceutical means and methods of the present invention, which together constitute a "uninterrupted administration" of such a therapeutic agent.

[1332] The multi-specific antibody construct of the present invention can be used intravenously or subcutaneously continuously or uninterruptedly by a fluid delivery device or a small pump system, and the fluid delivery device or the small pump system include a fluid drive mechanism for driving the fluid to leave the reservoir and a driving mechanism for driving the driving mechanism. The pump system for subcutaneous administration may include a needle or cannula for penetrating the patient's skin and delivering the suitable composition to the patient's body. The pump system can be directly fixed or attached to the patient's skin independently of a vein, an artery or a blood vessel, thereby allowing direct contact between the pump system and the patient's skin. The pump system can be attached to the patient's skin for 24 hours to several days. The pump system can be small in size, and the reservoir is suitable for small volumes. As a non-limiting example, the reservoir volume of the pharmaceutical composition suitable for use can be between 0.1 and 50ml.

[1333] Continuous application can also be carried out transdermal application by the patch worn on the skin, and replaced once at intervals. Those skilled in the art understand the patch system for drug delivery suitable for this purpose. It should be noted that transdermal application is particularly suitable for uninterrupted application, because the replacement of the patch exhausted for the first time can be advantageously placed on the skin surface of the patch exhausted for the first time, for example, a new second patch is placed simultaneously and before removing the patch exhausted for the first time, immediately complete. There will be no problem of flow interruption or battery failure.

[1334] If the pharmaceutical composition has been lyophilized, the lyophilized material is first reconstituted in a suitable liquid prior to administration. The lyophilized material can be reconstituted in, for example, bacteriostatic water for injection (BWFI), normal saline, phosphate buffered saline (PBS), or the same formulation in which the protein was prior to freeze drying.

[1335] The compositions of the invention can be administered to a subject at an appropriate dose, which can be determined, for example, by a dose escalation study administering increasing doses of a multispecific antibody construct of the invention that exhibits cross-species specificity as described herein for non-chimpanzee primates, such as macaques. As described above, the multispecific antibody constructs of the invention that exhibit cross-species specificity as described herein can be advantageously used in the same form in preclinical trials in non-chimpanzee primates and as a drug for use in humans. The dosage regimen will be determined by the attending physician and clinical factors. As is well known in the medical arts, the dosage for any one patient depends on many factors, including the patient's size, body surface area, age, the specific compound to be administered, sex, time and route of administration, general health, and other drugs administered concurrently.

[1336] The term "effective dose" or "effective dosage" is defined as an amount sufficient to achieve or at least partially achieve the desired effect. The term "therapeutically effective dose" is defined as an amount sufficient to cure or at least partially prevent the disease and its complications in a patient already suffering from the disease. The amount or dosage effective for this purpose will depend on the condition to be treated (indication), the multispecific antibody construct delivered, the treatment context and goal, the severity of the disease, previous therapy, the patient's clinical history and response to the therapeutic agent, the route of administration, the patient's size (weight, body surface or organ size) and / or condition (age and general health) and the general state of the patient's autoimmune system. The appropriate dosage can be adjusted according to the judgment of the attending physician so that it can be administered to the patient once or over a series of administrations, and the goal is to obtain the best therapeutic effect.

[1337] A therapeutically effective amount of the multispecific antibody constructs of the invention preferably results in a reduction in the severity of disease symptoms, an increase in the frequency or duration of disease symptom-free periods, or prevention of impairment or disability due to disease suffering. For the treatment of tumors expressing MSLN, a therapeutically effective amount of the multispecific antibody constructs of the invention, e.g., anti-MSLN / anti-CD40 multispecific antibody constructs, preferably will inhibit cell growth 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% relative to untreated patients. The ability of a compound to inhibit tumor growth can be evaluated in an animal model predictive of efficacy in human tumors.

[1338] The pharmaceutical composition can be administered as the sole therapeutic agent, or in combination with other therapies such as anti-cancer therapies (e.g., other protein and non-protein drugs) as needed. These drugs can be administered simultaneously with the composition comprising the multispecific antibody construct of the invention as defined herein, or at time-limited intervals and doses before or after administration of the multispecific antibody construct.

[1339] As used herein, the term "effective and non-toxic dose" refers to a tolerable dose of a multispecific antibody construct of the invention that is high enough to cause pathological cell depletion, tumor elimination, tumor shrinkage, or disease stabilization without or substantially without major toxic effects. Such an effective and non-toxic dose can be determined, for example, by a dose escalation study as described in the art, and the effective and non-toxic dose should be below the dose that induces severe adverse events (dose-limiting toxicity, DLT).

[1340] As used herein, the term "toxicity" refers to the toxic effects of a drug manifested in adverse events or serious adverse events. These side effects may refer to the general lack of tolerance of the drug after administration and / or the lack of local tolerance. Toxicity may also include teratogenic or carcinogenic effects caused by the drug.

[1341] As used herein, the term "safety", "in vivo safety" or "tolerance" defines the administration of a drug without causing serious adverse events (local tolerance) immediately after administration and without causing serious adverse events during a longer medication period. "Safety", "in vivo safety" or "tolerance" can be regularly assessed, for example, during treatment and during follow-up. Measurements include clinical assessments (e.g., organ performance) and laboratory abnormality screening. Clinical assessments can be performed, and deviations from normal findings can be recorded / coded according to NCI-CTC and / or MedDRA standards. Organ performance can include standards such as allergy / immunology, blood / bone marrow, arrhythmias, coagulation, etc., as described, for example, in the Common Terminology Standards for Adverse Events v3.0 (CTCAE). Laboratory parameters that can be tested include, for example, hematology, clinical chemistry, coagulation profiles, and urinalysis, as well as examinations of other body fluids (e.g., serum, plasma, lymph or spinal fluid, fluid, etc.). Safety can thus be assessed, for example, by physical examination, imaging techniques, i.e. ultrasound, X-ray, CT scan, magnetic resonance imaging (MRI), other measures using technical equipment (i.e. electrocardiogram), vital signs, by measuring laboratory parameters and recording adverse events. For example, non-chimpanzee primates can be examined for adverse events in the uses and methods according to the invention by histopathological and / or histochemical methods. The above terms are also referred to in, for example, Preclinical safety evaluation of biotechnology-derived pharmaceuticals S6; ICH Harmonised Tripartite Guideline; ICH Steering Committee meeting on July 16, 1997.

[1342] In further embodiments, the present invention provides a kit comprising a multispecific antibody construct of the present invention, a multispecific antibody construct produced according to a method of the present invention, a polynucleotide of the present invention, a vector of the present invention and / or a host cell of the present invention.

[1343] In the context of the present invention, the term "kit" means two or more components (one of which corresponds to the multispecific antibody construct, pharmaceutical composition, vector or host cell of the present invention) packaged together in a container, receptacle or otherwise. Thus, a kit can be described as a group of products and / or utensils sufficient to achieve a specific purpose, which can be sold as a single unit.

[1344] The kit may comprise one or more vessels (e.g., vials, ampoules, containers, syringes, bottles, bags) of any suitable shape, size and material (preferably waterproof, e.g., plastic or glass) containing a multispecific antibody construct or pharmaceutical composition of the invention at a suitable administration dose (see above). The kit may further comprise instructions for use (e.g., in the form of a single leaflet or installation manual), a device for administering the multispecific antibody construct of the invention (e.g., a syringe, pump, infusion set, etc.), a device for reconstituting the multispecific antibody construct of the invention and / or a device for diluting the multispecific antibody construct of the invention.

[1345] The present invention also provides a kit for a single-dose administration unit. The kit of the present invention may also contain a first vessel comprising a dried / lyophilized multispecific antibody construct and a second vessel comprising an aqueous formulation. In certain embodiments of the present invention, a kit containing a single-chamber and multi-chamber prefilled syringe (e.g., a liquid syringe and a lyophilized syringe) is provided.

[1346] Examples

[1347] Generation and characterization of anti-mesothelin antibodies

[1348] Anti-mesothelin antibody production.

[1349] As previously described (US20170029502A1) or by Transgenic mice were immunized to produce fully human antibodies against human mesothelin (U.S. Pat. Nos. 6,114,598; 6,162,963; 6,833,268; 7,049,426; 7,064,244, which are incorporated herein by reference in their entireties; Green et al., 1994, Nature Genetics 7:13-21; Mendez et al., 1997, Nature Genetics 15:146-156; Green and Jakobovits, 1998, J. Ex. Med, 188:483-495; Kellerman and Green, Current Opinion in Biotechnology 13, 593-597, 2002). Animals of the strain were used for these immunizations. Animals were immunized with alternating soluble human mesothelin-His and cynomolgus mesothelin-His. Animals with the highest antigen-specific serum native titers for human mesothelin and cynomolgus mesothelin were used to generate hybridomas (Kohler and Milstein, 1975). Pooled lymphocytes from the spleen and / or draining lymph nodes (from each harvest) were dissociated from lymphoid tissue by grinding in a suitable culture medium (e.g., Dulbecco's Modified Eagle Medium (DMEM); Invitrogen, Carlsbad, CA). B cells were selected and / or expanded using standard methods and fused with appropriate fusion partners using techniques known in the art. Hybridoma supernatants that bind to human mesothelin and cynomolgus mesothelin were then selected for further characterization.

[1350] Sequencing of anti-mesothelin antibodies.

[1351] for Antibodies were derived, RNA (total RNA or mRNA) was purified from wells containing hybridoma cells producing anti-mesothelin antibodies using Qiagen RNeasy mini or Invitrogen mRNAcatcher plus kits. The purified RNA was used to amplify antibody heavy and light chain variable region (V) genes using cDNA synthesis via reverse transcription followed by polymerase chain reaction (RT-PCR). Fully human antibody gamma heavy chains were obtained using the Qiagen One-Step Reverse Transcriptase PCR Kit (Qiagen). Fully human kappa light chains were obtained using the Qiagen One-Step Reverse Transcriptase PCR Kit (Qiagen). Amino acid sequences were deduced from corresponding nucleic acid sequences by bioinformatics methods. The derived amino acid sequences were then analyzed to determine the germline sequence origin of the antibody and to identify deviations from the germline sequence. The amino acid sequences corresponding to the complementary determining regions (CDRs) of the sequenced antibodies were aligned, and these alignments were used to group pure lines by similarity.

[1352] Antibody production.

[1353] Selected and sequenced anti-mesothelin antibody heavy and light chains were subcloned into mammalian expression vectors and individual antibodies were produced and purified. All antibodies were re-converted with the human IgG1 SEFL2 (REF) heavy chain sequence.

[1354] Binding of antibodies to human and cynomolgus monkey mesothelin.

[1355] Binding of anti-mesothelin antibodies to human mesothelin was demonstrated by flow cytometry on CHO cells engineered to express human mesothelin (huMSLN-CHO). CHO cells transfected with human MSLN were incubated with various concentrations of purified anti-mesothelin antibodies, washed, and labeled with a fluorescently conjugated secondary antibody specific for human IgG. The cells were then analyzed by flow cytometry. The percentage of fluorescent positive cells was plotted against antibody concentration ( Figure 1 ) and EC50 was determined using Prism GraphPad software (Table 1). The binding affinity of anti-human mesothelin antibodies to soluble forms of recombinant human and cynomolgus monkey (cyno) mesothelin was also measured by Octet assay (Table 1), quantifying the association rate (Kon), dissociation rate (Kdis) and equilibrium binding constant (KD). These data indicate that all anti-MSLN antibodies bind to both human and cynomolgus monkey mesothelin.

[1356] Table 1. Binding of anti-MSLN antibodies to human and cynomolgus monkey mesothelin (MSLN)

[1357]

[1358]

[1359] Generation and characterization of anti-CD40 agonist antibodies

[1360] Anti-CD40 antibody production.

[1361] Through Transgenic mice were immunized to produce fully human antibodies against human CD40 (U.S. Pat. Nos. 6,114,598; 6,162,963; 6,833,268; 7,049,426; 7,064,244, which are incorporated herein by reference in their entireties; Green et al., 1994, Nature Genetics 7:13-21; Mendez et al., 1997, Nature Genetics 15:146-156; Green and Jakobovits, 1998, J. Ex. Med, 188:483-495; Kellerman and Green, Current Opinion in Biotechnology 13, 593-597, 2002). Animals of different strains are used for these immunizations. A variety of immunogens and immunization routes are used to generate anti-human CD40 immune responses. For soluble recombinant protein immunization, mice are immunized with alternating soluble human CD40-Fc and cynomolgus macaque CD40-Fc. For cell-based immunization, CHO-S cells are transiently transfected with wild-type human CD40 or cynomolgus macaque CD40 as a source of immunogen. Animals are immunized with these transiently transfected CHO cells. Animals with the highest antigen-specific serum natural titers for human CD40 and cynomolgus macaque CD40 are used to produce hybridomas (Kohler and Milstein, 1975). Pooled lymphocytes (from each harvest) from the spleen and / or draining lymph nodes are dissociated from lymphoid tissue by grinding in a suitable culture medium (e.g., Dulbecco's Modified Eagle Medium (DMEM); Invitrogen, Carlsbad, CA)). B cells are selected and / or expanded using standard methods and fused with appropriate fusion partners using techniques known in the art. Hybridoma supernatants are identified that bind to human and cynomolgus CD40.

[1362] Antibody production.

[1363] Hybridoma supernatants showing binding to human and cynomolgus CD40 were selected for generation of purified anti-CD40 antibodies using techniques known in the art (Table 2).

[1364] Table 2. Anti-CD40 Antibodies

[1365]

[1366]

[1367] Anti-CD40 antibody that binds to human and cynomolgus monkey CD40.

[1368] The binding affinity of anti-human CD40 antibodies to human and cynomolgus (cyno) CD40 was measured by Octet assay (Table 3), quantifying the association rate (Kon), dissociation rate (Kdis) and equilibrium binding constant (KD). The cross-reactivity of anti-CD40 antibodies to related TNF receptor superfamily (TNFRSF) members was evaluated by expressing human TNFR1, TNFR2, TNFR10 or TNFR14 on HEK293 cells (by transient transfection of expression vectors encoding these genes). The binding of the displayed anti-human CD40 antibodies to these transfected cells was determined by flow cytometry. The calculated geometric mean of the fluorescence intensity relative to the control cells indicated the minimal binding of anti-CD40 to other TNFRSF members (Table 4). The cross-reactivity of anti-CD40 antibodies to cynomolgus and mouse CD40 was calculated by expressing cynomolgus or mouse CD40 on HEK293 cells (by transient transfection of expression vectors encoding these genes). Binding of the displayed anti-human CD40 antibodies to cells overexpressing cynomolgus monkey and mouse CD40 was determined by flow cytometry. The calculated geometric mean of fluorescence intensity relative to control cells indicated robust binding of anti-CD40 to cynomolgus monkey (cyno) CD40, but not to mouse CD40 (Table 5). Overall, these data indicate strong and equivalent binding of anti-CD40 antibodies to human and cynomolgus CD40, but minimal binding to related TNFRSF members or mouse CD40.

[1369] Table 3. Binding affinity of anti-CD40 antibodies to human and cynomolgus monkey CD40

[1370]

[1371]

[1372] Table 4. Presence of anti-CD40 antibodies binding to relevant TNF receptor superfamily members (fold relative to control values)

[1373] Antibody ID TNFR1 TNFR2 TNFR10 TNFR14 29H10 0.98 0.88 0.89 0.84 4G7 0.97 0.90 0.87 0.89 33H6 1.00 0.81 0.77 0.80 35F11 1.00 0.85 0.73 0.74 30A12 1.07 0.89 0.91 0.96 36F3 0.99 0.92 0.86 0.88 39C2 1.00 0.90 0.86 0.89 33H9 1.03 0.93 0.89 0.88 37A6 0.99 0.87 0.88 0.87

[1374] Table 5. Species cross-reactivity of anti-CD40 antibodies to cynomolgus monkey and mouse CD40 (fold relative to control value)

[1375] Antibody ID Cynomolgus monkey Mouse 29H10 71 0.8 4G7 57 0.9 33H6 74 0.9 35F11 76 0.8 30A12 71 1.3 36F3 82 0.9 39C2 87 0.9 33H9 73 0.9 37A6 92 0.9

[1376] Cross-link-dependent activation of CD40 by anti-CD40 antibodies.

[1377] Stimulation of human B cells by the CD40 receptor results in activation and proliferation. Purified primary human B cells were seeded into 384-well assay plates and incubated with IL-4 / IL-21 (as co-mitogens), different concentrations of anti-CD40 antibodies, and protein G in the presence of ( Figure 2 ) or does not exist ( Figure 2 The cells were incubated for 5 days and cell proliferation was examined using CellTiter-Glo. Since all anti-CD40 antibodies examined were IgG4, an unrelated IgG4 isotype antibody was included as a negative control. EC50 and maximum activity percentage were calculated (Table 6). All anti-CD40 antibodies induced robust proliferation of B cells in the presence of protein G but had minimal activity in the absence of protein G, indicating that anti-CD40 antibodies that bind only to CD40 are insufficient to stimulate CD40 and that additional cross-linked antibodies are required.

[1378] Table 6. Activity of anti-CD40 antibodies with or without cross-linking in human B cell function assays

[1379]

[1380] Effect of anti-CD40 antibodies on the binding of human CD40 ligand to human CD40.

[1381] To evaluate the ability of anti-CD40 antibodies to block the interaction of CD40 ligand with CD40, 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 transfection with an expression vector encoding this gene). The percent 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 fluorescence intensity. All anti-CD40 antibodies showed minimal effects on the ability of CD40L to bind to the CD40 receptor (Table 7).

[1382] Table 7. Anti-CD40 antibodies do not block CD40-CD40L interaction

[1383]

[1384] Effects of anti-human CD40 antibodies on CD40L-induced B cell activation.

[1385] Some antibodies specific for TNFRSF members have been shown to enhance activation of the receptor by its normal ligand, possibly by aggregating 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 the presence or absence of 1 ug / ml of the indicated CD40 antibodies. The cells were incubated for 5 days and cell proliferation was examined using CellTiter-Glo. No anti-CD40 antibodies induced a significant change in the EC50 of CD40 ligand to stimulate proliferation of human B cells (Table 8).

[1386] Table 8. Anti-CD40 antibodies do not enhance the activity of CD40L

[1387]

[1388]

[1389] Sequencing of anti-CD40 antibodies.

[1390] RNA (total RNA or mRNA) was purified from wells containing hybridoma cells producing anti-mesothelin antibodies using Qiagen RNeasy mini or Invitrogen mRNAcatcher plus kits. The purified RNA was used to amplify antibody heavy and light chain variable region (V) genes using cDNA synthesis via reverse transcription followed by polymerase chain reaction (RT-PCR). Fully human antibody γ heavy chains were obtained using the Qiagen One-Step Reverse Transcriptase PCR Kit (Qiagen). Fully human κ light chains were obtained using the Qiagen One-Step Reverse Transcriptase PCR Kit (Qiagen). Amino acid sequences were deduced from corresponding nucleic acid sequences by bioinformatics methods. The derived amino acid sequences were then analyzed to determine the germline sequence origin of the antibody and to identify deviations from the germline sequence. The amino acid sequences corresponding to the complementary determining regions (CDRs) of the sequenced antibodies were aligned, and these alignments were used to group pure lines by similarity.

[1391] Optimization of anti-CD40 antibody sequences.

[1392] Engineering of selected anti-CD40 antibodies was performed by standard recombinant DNA techniques to remove potential sequence liabilities in the variable regions of the antibody heavy and light chains. These antibodies were produced by overexpression in cell lines and purified using techniques known in the art. The purified antibodies were then evaluated in a human B cell proliferation assay with and without protein G cross-linking, as described above. EC50 and percent maximum activity were calculated (Table 9), which showed that the engineered antibody variants maintained cross-linking-dependent agonist activity similar to their parent antibody sequences.

[1393] Table 9. Functional activities of engineered variants of anti-CD40 antibodies

[1394]

[1395]

[1396] Generation and characterization of a CD40 agonist bivalent bispecific antibody targeting mesothelin

[1397] Bispecific antibody generation.

[1398] To generate a mesothelin-dependent CD40 agonist, an IgG-Fab format ( Figure 3 A) or IgG-scFv format ( Figure 3 Figure B) shows a bivalent bispecific antibody that binds to human mesothelin and human CD40.

[1399] Generation and evaluation of a mesothelin x CD40 bispecific agonist antibody in IgG-Fab format.

[1400] Panels of the mesothelin x CD40 bispecific antibodies were generated in IgG-Fab format (Table 10) and evaluated for binding to human and cynomolgus CD40 and soluble forms of human and cynomolgus mesothelin using the Octet assay (Table 11). The association rate (K) was calculated. on ), dissociation rate (K dis ), and the equilibrium binding constant (K D). Most bispecific antibodies showed high affinity binding to both human and cynomolgus CD40 and mesothelin. Next, these bispecific antibodies were evaluated for their ability to induce mesothelin-dependent activation of human B cells. Human mesothelin-expressing CHO cells were seeded in 96-well plates. The next day, different concentrations of mesothelin x CD40 IgG-Fab antibodies were added to the wells with purified human B cells, and the plates were incubated for another 48 hours. The upregulation of CD86 (a marker of CD40-mediated B cell activation) was quantified on B cells by flow cytometry. The EC50 value for CD86 upregulation was calculated, and this value shows that most mesothelin x CD40 IgG-Fab bispecific antibodies are capable of B cell activation in the presence of human mesothelin-expressing CHO cells (Table 12).

[1401] Table 10. Anti-CD40xMSLN IgG-Fab molecules

[1402]

[1403]

[1404]

[1405]

[1406] Table 12. Functional activity of anti-CD40xMSLN IgG-Fab molecules in human B cells in the presence of CHO cells expressing human mesothelin

[1407]

[1408]

[1409] Generation and evaluation of a mesothelin x CD40 bispecific agonist antibody in IgG-scFv format.

[1410] Small panels of mesothelin x CD40 bispecific antibodies were produced in IgG-scFv format (Table 13) and evaluated for binding to human and cynomolgus CD40 and soluble forms of human and cynomolgus mesothelin using the Octet assay (Table 14). The association rate (Kon), dissociation rate (Kdis), and equilibrium binding constant (KD) were calculated. Most bispecific antibodies showed high affinity binding to both human and cynomolgus CD40 and mesothelin. Next, these bispecific antibodies were evaluated for their ability to induce mesothelin-dependent activation of human B cells. Human mesothelin-expressing CHO cells were seeded in 96-well plates. The next day, different concentrations of mesothelin x CD40 IgG-scFv antibodies were added to the wells with purified human B cells, and the plates were incubated for an additional 48 hours. The upregulation of CD86 (a marker for CD40-mediated B cell activation) was quantified on B cells by flow cytometry. EC50 values ​​for CD86 upregulation were calculated and showed that most of the mesothelin x CD40 IgG-scFv bispecific antibodies were capable of B cell activation in the presence of human mesothelin-expressing CHO cells (Table 15).

[1411] Table 13. Anti-CD40xMSLN IgG-scFv molecules

[1412]

[1413]

[1414]

[1415]

[1416]

[1417] Table 15. Functional activity of anti-CD40xMSLN IgG-scFv molecules in human B cells in the presence of CHO cells expressing human mesothelin

[1418] Antibody ID EC50(nM) 6028-2 0.012240302 6030-2 0.010141789 6032-2 0.009349861 6043-2 0.007758607 6044-2 0.005001671 6033-2 0.006030707 6034-2 0.007262489 6035-2 0.016708182 6056-2 0.004577206 6039-2 0.003317003 6040-2 0.002665906 6041-2 0.004479453 6042-2 0.003988474 6045-2 0.004663271 6046-2 0.004723116 6047-2 0.005131571 6048-2 0.004342491 6049-2 0.005853663 6050-2 0.003388309 6051-2 0.003753076 6052-2 0.003155443 6055-2 0.004340411 6054-2 0.003331971 6053-2 0.004033184 6023-2 0.002805537

[1419] Sequence optimization of a mesothelin x CD40 bispecific agonist antibody in IgG-scFv format

[1420] The engineering of the mesothelin x CD40 bispecific IgG-scFv antibodies was selected by standard recombinant DNA technology to remove potential sequence liabilities in the variable regions of the heavy and light chains of the antibodies. These antibodies were produced by overexpression in a cell line and purified using techniques known in the art. Next, these bispecific antibodies were evaluated for their ability to induce mesothelin-dependent activation of human B cells. Human mesothelin-expressing CHO cells were seeded in 96-well plates. The next day, different concentrations of mesothelin x CD40 IgG-scFv sequence variant antibodies were added to the wells with purified human B cells, and the plates were incubated for another 48 hours. The upregulation of CD86 (a marker of CD40-mediated B cell activation) was quantified on B cells by flow cytometry. The EC50 value for CD86 upregulation was calculated, and this value shows that most mesothelin x CD40 IgG-scFv sequence variant bispecific antibodies maintain the ability to induce B cell activation in the presence of human mesothelin-expressing CHO cells (Table 16).

[1421] Table 16. Functional activity of sequence optimized anti-CD40xMSLN IgG-scFv molecules in human B cells in the presence of CHO cells expressing human mesothelin

[1422]

[1423]

[1424] Effects of different levels of mesothelin on the activity of selected mesothelin x CD40 bispecific antibodies in a human B cell function assay

[1425] MC38 cells were engineered to express different levels of human mesothelin ranging from low (MC38.MSLN1) to high (MC38.MSLN5). Given that MC38 cells are derived from mice, they do not express human mesothelin ( Figure 4). Parental MC38 cells and MC38 cells expressing different levels of human mesothelin were seeded into 96-well plates and cultured overnight. The next day, mesothelin x CD40 bispecific antibodies were added to the cells at different concentrations. The isolated human B cells were then added to the wells and the plates were incubated for another 48 hours. The upregulation of CD86 in B cells was assessed by flow cytometry and used as a measure of B cell activation. EC50 and maximum activity (Emax) values ​​were calculated, and it was shown that the mesothelin x CD40 bispecific antibody could induce B cell activation in all levels of MSLN expression (Table 17). As measured by the geometric mean fluorescence intensity of CD86 upregulation in B cells, cell lines expressing low levels of mesothelin induce lower maximum activity of mesothelin x CD40 bispecific antibodies. In the presence of parental MC38 cells, the mesothelin x CD40 bispecific antibody was inactive, which is consistent with the mesothelin-dependent activity of these molecules for CD40 agonist activity.

[1426]

[1427]

[1428] Effects of varying levels of mesothelin on the activity of select mesothelinxCD40 bispecific antibodies in a monocyte-derived dendritic cell functional assay.

[1429] MC38 cells engineered to express different levels of human mesothelin ( Figure 4 ) or parental MC38 cells were seeded in 96-well plates and cultured overnight. The next day, the mesothelin x CD40 bispecific antibody was added to the cells at different 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 another 48 hours. The supernatant from each well was collected and the concentration of IL12p40 secreted from dendritic cells was quantified using an ELISA kit. EC50 and maximum activity (Emax) values ​​were calculated, and it was shown that the mesothelin x CD40 bispecific antibody could induce dendritic cell activation in high and low levels of MSLN expression (Table 17). Cell lines expressing low levels of mesothelin induce lower maximum activity of the mesothelin x CD40 bispecific antibody. In the presence of parental MC38 cells, the mesothelin x CD40 bispecific antibody was inactive, which is consistent with the mesothelin-dependent activity of these molecules for CD40 agonist activity.

[1430] Mesothelin x CD40 bispecific antibodies were selected for activity in a cynomolgus B cell function assay using cynomolgus CHO cells expressing mesothelin.

[1431] CHO cells engineered to express cynomolgus mesothelin were seeded in 96-well plates and cultured overnight. The next day, select mesothelin x CD40 bispecific antibodies were added to the cells at different concentrations. Separated cynomolgus B cells were then added to the wells and the plates were incubated for another 48 hours. The upregulation of CD23 in cynomolgus B cells was assessed by flow cytometry and used as a measure of CD40-induced B cell activation. EC50 was calculated, and this value showed that the mesothelin x CD40 bispecific antibody could induce activation of cynomolgus B cells in the presence of cynomolgus mesothelin (Table 18).

[1432] Table 18. Activity of anti-CD40xMSLN bispecific molecules in cynomolgus B cells stimulated with CHO cells expressing cynomolgus mesothelin

[1433] Antibody ID EC50(nM) 6041 0.0012 8765 0.0013 8766 0.0016 8767 0.0014 8945 0.0018 8947 0.0014

[1434] Pharmacokinetics and stability of a mesothelin x CD40 bispecific antibody in mice.

[1435] To characterize the stability of the Mesothelin x CD40 bispecific antibodies in vivo, selected Mesothelin x CD40 bispecific molecules were injected into mice (1 mg / kg dose). Animals were bled at different time points after injection and serum was separated. Immunoassays were used to quantify the concentration of serum Mesothelin x CD40 bispecific antibodies, either captured with human CD40 and detected with anti-human IgG, or captured with human mesothelin and detected with anti-human IgG. The combination of these two assays provides the ability to detect molecular degradation or instability. CD40 or Mesothelin capture assays produced comparable results, indicating that the molecules remained intact over the course of 14 days in vivo ( Figure 5 ). All molecules except one had similar pharmacokinetic ...

Claims

1. An antigen-binding protein that specifically binds to and activates human CD40 whose amino acid sequence is shown in SEQ ID NO: 1, wherein the antigen-binding protein is an antibody or a functional fragment thereof, and the antibody or the functional fragment thereof comprises a light chain variable region VL and a heavy chain variable region VH, wherein: The VL and VH are selected from the group consisting of: a) a VL comprising CDRL1, CDRL2, and CDRL3 selected from the group consisting of amino acids 91 to 101 of SEQ ID NOs: 100, 101, and 370, respectively; and a VH comprising CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 178, 179, and 180, respectively; and b) a VL comprising CDRL1, CDRL2, and CDRL3 selected from the group consisting of SEQ ID NOs: 100, 101, and 102, respectively; and a VH comprising CDRH1, CDRH2, and CDRH3 selected from the group consisting of SEQ ID NOs: 178, 179, and 180, respectively.

2. The antigen binding protein of claim 1, wherein The VL comprises an amino acid sequence selected from the group consisting of amino acids 1 to 112 of SEQ ID NO: 370 or SEQ ID NO: 33; and The VH comprises the amino acid sequence of SEQ ID NO:

34.

3. The antigen-binding protein of claim 1, wherein the VH comprises a human framework region FR sequence, and the VL comprises a human framework region FR sequence.

4. The antigen binding protein of claim 1, wherein the antigen binding protein comprises a human constant domain.

5. The antigen binding protein of claim 4, wherein the antigen binding protein comprises: (a) human light chain constant domain CL; and / or (b) Human heavy chain constant domain CH1.

6. The antigen binding protein of claim 5, wherein the CL is kappa CL or lambda CL; and / or wherein the CH1 is IgG CH1.

7. The antigen binding protein of claim 1, wherein the antigen binding protein comprises a human heavy chain Fc domain. The antigen binding protein of claim 7 , wherein the Fc is IgG1 Fc.

9. The antigen binding protein of claim 1, comprising a VH and a VL, wherein the VH comprises SEQ ID NO: 34 and the VL comprises amino acids 1 to 112 of SEQ ID NO:

370.

10. The antigen binding protein of any one of claims 1-9, wherein the heavy chain comprises an amino acid substitution selected from the group consisting of: (i) N297G or N297A; (ii) L234A and L235A; and (iii) R292C and V302C; The amino acid numbering is according to the EU numbering of Kabat.

11. The antigen binding protein of claim 10, wherein the heavy chain comprises N297G, R292C and V302C mutations, wherein the amino acid numbering is according to EU numbering as in Kabat.

12. The antigen binding protein of any one of claims 1-9 and 11, wherein the antigen binding protein further comprises a light chain CL polypeptide linked to the VL, wherein the CL polypeptide is selected from the group consisting of SEQ ID NO: 883 and SEQ ID NO:

884.

13. The antigen binding protein of any one of claims 1-9 and 11, wherein the antigen binding protein further comprises a heavy chain CH1-hinge-CH2-CH3 polypeptide, wherein the CH1-hinge-CH2-CH3 polypeptide is selected from the group consisting of SEQ ID NO: 885 and SEQ ID NO:

886.

14. The antigen binding protein of any one of claims 1-9 and 11, wherein the antigen binding protein further comprises a second antigen binding portion that specifically binds to a second antigen, wherein the second antigen binding portion is an antibody or a functional fragment thereof and wherein the second antigen is human mesothelin MLSN. The antigen binding protein of claim 6 , wherein the CH1 is IgG1 CH1.

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