JP7378567B2 - LAG-3 binding molecules and methods of use thereof - Google Patents

Description

Cross-References to Related Applications This application is based on U.S. patent application Ser. The above-mentioned patent applications are incorporated by reference into this application in their entirety.

Sequence Listing References This application contains one or more sequence listings pursuant to Title 37, Code of Federal Regulations, Section 1.821 et seq. Created May 18, size: 105,774 bytes), the above file is incorporated by reference into this application in its entirety.

The present invention provides selected anti-LAG-3 antibodies: LAG-3 mAb 1, LAG-3 mAb 2, LAG-3 mAb 3, LAG-3 mAb 4, which can bind to both cynomolgus monkey LAG-3 and human LAG-3. , LAG-3 mAb 5 or LAG-3 mAb 6. The present invention particularly relates to humanized or chimeric versions of such antibodies, or LAG-3 binding fragments of such anti-LAG-3 antibodies (particularly immunoconjugates, diabodies, BiTEs, bispecific antibodies, etc.). ). The invention particularly relates to such LAG-3 binding molecules that can further bind to epitopes of molecules involved in the regulation of immune checkpoints present on the surface of immune cells. The invention also relates to methods of using such LAG-3 binding molecules for detection of LAG-3 or stimulation of an immune response. The present invention also provides for the use of LAG-3 binding molecules comprising one or more LAG-3 binding domains of selected anti-LAG-3 antibodies as described above to stimulate such immune responses in a subject. It relates to combination therapy, administered in combination with one or more additional molecules effective to further enhance, stimulate or upregulate the response.

Description of related technology I. Cell-Mediated Immune Response The immune system of humans and other mammals is responsible for providing protection against infection and disease. Such protection is provided by both humoral and cell-mediated immune responses. Humoral immune responses result in the production of antibodies and other biomolecules that can recognize and neutralize foreign targets (antigens). In contrast, cell-mediated immune responses involve the activation of macrophages, natural killer (NK) cells, and antigen-specific cytotoxic T lymphocytes by T cells, and the release of various cytokines in response to antigen recognition ( Non-patent document 1).

The ability of T cells to optimally mediate immune responses against antigens requires two distinct signaling interactions (2, 3). First, the antigen present on the surface of an antigen-presenting cell (APC) is transferred to an antigen-specific naive CD4 ⁺ T cell.
It must be presented to cells. Such presentation delivers a signal through the T-cell receptor (TCR) that directs the T-cell to mount an immune response that is specific for the presented antigen. . A series of costimulatory and inhibitory signals mediated by interactions between APCs and distinct T cell surface molecules then trigger first T cell activation and proliferation and ultimately T cell inhibition. do. Thus, the first signal confers specificity to the immune response, and the second signal serves to determine the nature, strength and duration of said response.

The immune system is tightly regulated by co-stimulatory and co-inhibitory ligands and receptors. These molecules provide a second signal for T cell activation and also provide a balanced network of positive and negative signals to maximize the immune response to infection while limiting immunity to self ( Non-patent documents 4, 5). Inhibitory pathways important for maintaining self-tolerance and modulating the duration and amplitude of immune responses are collectively referred to as immune checkpoints. Of particular importance is the binding between the B7.1 (CD80) and B7.2 (CD86) ligands of antigen-presenting cells and the CD28 and CTLA-4 receptors of CD4 ⁺ T lymphocytes (Non-Patent Document 6, 1, 7). Binding of B7.1 or B7.2 to CD28 stimulates T cell activation, and binding of B7.1 or B7.2 to CTLA-4 inhibits said activation (Non-patent Documents 1, 7, 8). CD28 is constitutively expressed on the surface of T cells (Non-Patent Document 9), whereas CTLA-4 expression is rapidly upregulated following T-cell activation (Non-Patent Document 10). . Because CTLA-4 is a high-affinity receptor (6), binding first initiates T-cell proliferation (through CD28) and then inhibits T-cell proliferation (through early expression of CTLA-4); This dampens the effect of proliferation when it is no longer needed.

Further investigations into the ligands of the CD28 receptor led to the identification and characterization of a set of related B7 molecules (the “B7 Superfamily” [6, 8, 12, 13, 3; Several members of the above families are currently known: B7.1 (CD80), B7.2 (CD86), inducible co-stimulatory factor ligand (ICOS-L), programmed death-1. ligand (PD-L1; B7-H1), programmed death-2 ligand (PD-L2; B7-DC), B7-H3, B7-H4 and B7-H6 (Non-patent Documents 12, 17).

II. Lymphocyte Activation Gene-3 (“LAG-3”)
Lymphocyte activation gene 3 encodes a cell surface receptor protein called "LAG-3" or CD223 (Non-Patent Document 18). LAG-3 is activated CD4 ⁺
and CD8 ⁺ T cells as well as NK cells and is constitutively expressed by plasmacytoid dendritic cells. LAG-3 is not expressed by B cells, monocytes or any other cell type tested (19).

LAG-3 is known to be closely related to the T cell co-receptor CD4 (Non-Patent Documents 18, 20, 21, 22). Like CD4, LAG-3 also binds to MHC class II molecules, but with significantly higher binding affinity (23, 24, 25).

Studies have shown that LAG-3 plays an important role in down-regulating T cell proliferation, function and homeostasis (19, 26, 27, 28, 29, 30).

Studies have suggested that inhibition of LAG-3 function by antibody blockade can reverse LAG-3-mediated immune system inhibition and partially restore effector function (20, 31). LAG-3 has been shown to downregulate T cell proliferation and control the size of the memory T cell pool by inhibiting TCR-induced calcium flux (32, 33).

Dong, C. et al. (2003) “Immune Regulation by Novel Costimulatory Molecules,” Immunolog. Res. 28(1):39‐48 Viglietta, V. et al. (2007) “Modulating Co‐Stimulation,” Neurotherapeutics 4:666‐675 Korman, A.J. et al. (2007) “Checkpoint Blockade in Cancer Immunotherapy,” Adv. Immunol. 90:297‐339 Wang, L. et al. (2011) “VISTA, A Novel Mouse Ig Superfamily Ligand That Negatively Regulates T‐Cell Responses,” J. Exp. Med. 10.1084/jem.20100619:1‐16 Lepenies, B. et al. (2008) “The Role Of Negative Costimulators During Parasitic Infections,” Endocrine, Metabolic & Immune Disorders - Drug Targets 8:279-288 Sharpe, A.H. et al. (2002) “The B7‐CD28 Superfamily,” Nature Rev. Immunol. 2:116‐126 Lindley, P.S. et al. (2009) “The Clinical Utility Of Inhibiting CD28‐Mediated Costimulation,” Immunol. Rev. 229:307‐321 Greenwald, R.J. et al. (2005) “The B7 Family Revisited,” Ann. Rev. Immunol. 23:515‐548 Gross, J., et al. (1992) “Identification And Distribution Of The Costimulatory Receptor CD28 In The Mouse,” J. Immunol. 149:380-388 Linsley, P. et al. (1996) “Intracellular Trafficking Of CTLA4 And Focal Localization Towards Sites Of TCR Engagement,” Immunity 4:535-543 Coyle, A.J. et al. (2001) “The Expanding B7 Superfamily: Increasing Complexity In Costimulatory Signals Regulating T‐Cell Function,” Nature Immunol. 2(3):203‐209 Collins, M. et al. (2005) “The B7 Family Of Immune‐Regulatory Ligands,” Genome Biol. 6:223.1‐223.7 Loke, P. et al. (2004) “Emerging Mechanisms Of Immune Regulation: The Extended B7 Family And Regulatory T‐Cells.” Arthritis Res. Ther. 6:208‐214 Flies, D.B. et al. (2007) “The New B7s: Playing a Pivotal Role in Tumor Immunity,” J. Immunother. 30(3):251‐260 Agarwal, A. et al. (2008) “The Role Of Positive Costimulatory Molecules In Transplantation And Tolerance,” Curr. Opin. Organ Transplant. 13:366‐372 Wang, S. et al. (2004) “Co‐Signaling Molecules Of The B7‐CD28 Family In Positive And Negative Regulation Of T Lymphocyte Responses,” Microbes Infect. 6:759‐766) Flajnik, M.F. et al. (2012) “Evolution Of The B7 Family: Co‐Evolution Of B7H6 And Nkp30, Identification Of A New B7 Family Member, B7H7, And Of B7's Historical Relationship With The MHC,” Immunogenetics 64(8): 571‐90 Triebel, F. et al. (1990) “LAG‐3, A Novel Lymphocyte Activation Gene Closely Related To CD4,” J. Exp. Med. 171(5):1393‐1405 Workman, C.J. et al. (2009) “LAG‐3 Regulates Plasmacytoid Dendritic Cell Homeostasis,” J. Immunol. 182(4):1885‐1891 Grosso, J.F. et al. (2009) “Functionally Distinct LAG‐3 and PD‐1 Subsets on Activated and Chronically Stimulated CD8 T‐Cells,” J. Immunol. 182(11):6659‐6669 Huang, C.T. et al. (2004) “Role Of LAG‐3 In Regulatory T‐Cells,” Immunity 21:503‐513 Berger, R. et al. (2008) “Phase I Safety And Pharmacokinetic Study Of CT-011, A Humanized Antibody Interacting With PD-1, In Patients With Advanced Hematologic Malignancies,” Clin. Cancer Res. 14(10):3044 -3051 Workman, C.J. et al. (2002) “Phenotypic Analysis Of The Murine CD4‐Related Glycoprotein, CD223 (LAG‐3),” Eur. J. Immunol. 32:2255‐2263 Huard, B. et al. (1995) “CD4/Major Histocompatibility Complex Class II Interaction Analyzed With CD4‐ And Lymphocyte Activation Gene‐3 (LAG‐3)‐Ig Fusion Proteins,” Eur. J. Immunol. 25:2718‐ 2721 Huard, B. et al. (1994) “Cellular Expression And Tissue Distribution Of The Human LAG‐3‐ Encoded Protein, An MHC Class II Ligand,” Immunogenetics 39:213‐21 Workman, C.J. et al. (2002) “Cutting Edge: Molecular Analysis Of The Negative Regulatory Function Of Lymphocyte Activation Gene‐3,” J. Immunol. 169:5392‐5395 Workman, C.J. et al. (2003) “The CD4‐Related Molecule, LAG‐3 (CD223), Regulates The Expansion Of Activated T‐Cells,” Eur. J. Immunol. 33:970‐979 Workman, C.J. (2005) “Negative Regulation Of T‐Cell Homeostasis By Lymphocyte Activation Gene‐3 (CD223),” J. Immunol. 174:688‐695 Hannier, S. et al. (1998) “CD3/TCR Complex‐Associated Lymphocyte Activation Gene‐3 Molecules Inhibit CD3/TCR Signaling,” J. Immunol. 161:4058‐4065 Huard, B. et al. (1994) “Lymphocyte‐Activation Gene 3/Major Histocompatibility Complex Class II Interaction Modulates The Antigenic Response Of CD4+ T Lymphocytes,” Eur. J. Immunol. 24:3216‐3221 Grosso, J.F. et al. (2007) “LAG‐3 Regulates CD8+ T‐Cell Accumulation And Effector Function During Self And Tumor Tolerance,” J. Clin. Invest. 117:3383‐3392 Matsuzaki, J. et al. (2010) “Tumor‐Infiltrating NY‐ESO‐1‐Specific CD8+ T‐Cells Are Negatively Regulated By LAG‐3 and PD‐1 In Human Ovarian Cancer,” Proc. Natl. Acad. Sci. (U.S.A.) 107(17):7875-7880 Workman C.J., et al. (2004) “Lymphocyte Activation Gene‐3 (CD223) Regulates The Size Of The Expanding T‐Cell Population Following Antigen Activation in vivo,” J. Immunol. 172:5450‐5455

However, despite all the advances to date as described above, improved compositions that can more powerfully direct the body's immune system to attack cancer cells or pathogen-infected cells, especially at relatively low therapeutic concentrations, have been developed. There continues to be demand for. This is because, although the adaptive immune system can be a powerful protective mechanism against cancer and disease, it can be hampered by immunosuppressive mechanisms in the tumor microenvironment, such as the expression of LAG-3. Furthermore, co-inhibitory molecules expressed by tumor cells, immune cells, and stromal cells in the tumor environment may predominantly attenuate T cell responses against cancer cells. Thus, there continues to be a need for potent LAG-3 binding molecules. In particular, a single agent that exhibits a desired binding kinetic profile, binds to different LAG-3 epitopes, and/or can provide improved therapeutic value for patients with cancer or other diseases and conditions. There continues to be a need for active LAG-3 binding molecules. The present invention is directed to these and other goals.

The present invention provides selected anti-LAG-3 antibodies: LAG-3 mAb 1, LAG-3 mAb 2, LAG-3 mAb 3, LAG-3 mAb 4, which can bind to both cynomolgus monkey LAG-3 and human LAG-3. , LAG-3 mAb 5 or LAG-3 mAb 6. The present invention particularly relates to humanized or chimeric versions of such antibodies, or LAG-3 binding fragments of such anti-LAG-3 antibodies (particularly immunoconjugates, diabodies, BiTEs, bispecific antibodies, etc.). ). The invention particularly relates to such LAG-3 binding molecules that can further bind to epitopes of molecules involved in the regulation of immune checkpoints present on the surface of immune cells. The invention also relates to methods of using such LAG-3 binding molecules for detection of LAG-3 or stimulation of an immune response. The present invention also provides for the use of LAG-3 binding molecules comprising one or more LAG-3 binding domains of selected anti-LAG-3 antibodies as described above to stimulate such immune responses in a subject. It relates to combination therapy, administered in combination with one or more additional molecules effective to further enhance, stimulate or upregulate the response.

In particular, the present invention provides a LAG-3 binding molecule capable of binding both human LAG-3 and cynomolgus monkey LAG-3, said LAG-3 binding molecule comprising three heavy chain CDR domains, namely CDR _H 1 , CDR _H 2 and CDR _H 3, and three light chain CDR domains, namely CDR _L 1, CDR _L 2 and CDR _L 3:
(A) (1) The above CDR _H 1 domain, CDR _H 2 domain and CDR _H 3 domain are:
heavy chain CDRs of LAG-3 mAb 1, having the amino acid sequences: SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO: 10, respectively;
(2) The above CDR _L 1 domain, CDR _L 2 domain, and CDR _L 3 domain are:
light chain CDRs of LAG-3 mAb 1, having the amino acid sequences: SEQ ID NO: 13, SEQ ID NO: 14 and SEQ ID NO: 15, respectively;
or (B) (1) The above CDR _H 1 domain, CDR _H 2 domain and CDR _H 3 domain are
heavy chain CDRs of hLAG-3 mAb 1, having the amino acid sequences: SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO: 10, respectively;
(2) The above CDR _L 1 domain, CDR _L 2 domain, and CDR _L 3 domain are:
light chain CDRs of hLAG-3 mAb 1, having the amino acid sequences: SEQ ID NO: 28, SEQ ID NO: 14 and SEQ ID NO: 15, respectively;
or (C) (1) The above CDR _H 1 domain, CDR _H 2 domain and CDR _H 3 domain are
heavy chain CDRs of LAG-3 mAb 2, having the amino acid sequences: SEQ ID NO: 31, SEQ ID NO: 32, and SEQ ID NO: 33, respectively;
(2) The above CDR _L 1 domain, CDR _L 2 domain, and CDR _L 3 domain are:
light chain CDRs of LAG-3 mAb 2, having the amino acid sequences: SEQ ID NO: 36, SEQ ID NO: 37 and SEQ ID NO: 38, respectively;
or (D) (1) The above CDR _H 1 domain, CDR _H 2 domain and CDR _H 3 domain are
heavy chain CDRs of LAG-3 mAb 3, having the amino acid sequences: SEQ ID NO: 41, SEQ ID NO: 42 and SEQ ID NO: 43, respectively;
(2) The above CDR _L 1 domain, CDR _L 2 domain, and CDR _L 3 domain are:
light chain CDRs of LAG-3 mAb 3, having the amino acid sequences: SEQ ID NO: 46, SEQ ID NO: 47 and SEQ ID NO: 48, respectively;
or (E) (1) the above CDR _H 1 domain, CDR _H 2 domain and CDR _H 3 domain,
heavy chain CDRs of LAG-3 mAb 4, having the amino acid sequences: SEQ ID NO: 51, SEQ ID NO: 52, and SEQ ID NO: 53, respectively;
(2) The above CDR _L 1 domain, CDR _L 2 domain, and CDR _L 3 domain are:
light chain CDRs of LAG-3 mAb 4, having the amino acid sequences: SEQ ID NO: 56, SEQ ID NO: 57 and SEQ ID NO: 58, respectively;
(F) (1) The above CDR _H 1 domain, CDR _H 2 domain and CDR _H 3 domain are
heavy chain CDRs of LAG-3 mAb 5, having the amino acid sequences: SEQ ID NO: 61, SEQ ID NO: 62, and SEQ ID NO: 63, respectively;
(2) The above CDR _L 1 domain, CDR _L 2 domain, and CDR _L 3 domain are:
light chain CDRs of LAG-3 mAb 5, having the amino acid sequences: SEQ ID NO: 66, SEQ ID NO: 67 and SEQ ID NO: 68, respectively;
or (G) (1) The above CDR _H 1 domain, CDR _H 2 domain and CDR _H 3 domain are
heavy chain CDRs of LAG-3 mAb 6 VH1, each having the amino acid sequence: SEQ ID NO: 71, SEQ ID NO: 72, and SEQ ID NO: 73;
(2) The above CDR _L 1 domain, CDR _L 2 domain, and CDR _L 3 domain are:
light chain CDRs of LAG-3 mAb 6, having the amino acid sequences: SEQ ID NO: 76, SEQ ID NO: 77 and SEQ ID NO: 78, respectively;
or (H) (1) The above CDR _H 1 domain, CDR _H 2 domain and CDR _H 3 domain are
heavy chain CDRs of LAG-3 hAb 6, having the amino acid sequences: SEQ ID NO: 71, SEQ ID NO: 72, and SEQ ID NO: 73, respectively;
(2) The above CDR _L 1 domain, CDR _L 2 domain, and CDR _L 3 domain are:
Light chain CDRs of hLAG-3 hAb 6, having amino acid sequences: SEQ ID NO: 87, SEQ ID NO: 77, and SEQ ID NO: 78, respectively.

The invention further relates to embodiments of such LAG-3 binding molecules, wherein the heavy chain variable domain has the amino acid sequence of SEQ ID NO: 6, SEQ ID NO: 29, SEQ ID NO: 39, SEQ ID NO: 49 or SEQ ID NO: 59.

The present invention further relates to embodiments of such LAG-3 binding molecules, wherein the light chain variable domain has the amino acid sequence of SEQ ID NO: 11, SEQ ID NO: 34, SEQ ID NO: 44, SEQ ID NO: 54 or SEQ ID NO: 64.

The invention further relates to embodiments of such LAG-3 binding molecules, wherein the heavy chain variable domain has the amino acid sequence of SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 79 or SEQ ID NO: 80.

The present invention further provides a LAG-3 binding molecule in which the light chain variable domain has the amino acid sequence of SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 83 or SEQ ID NO: 85. Regarding embodiments.

The invention further relates to all such LAG-3 binding molecule embodiments, wherein said molecule is an antibody, in particular said molecule is a chimeric or humanized antibody.

The present invention further relates to embodiments in which said LAG-3 binding molecule is a bispecific binding molecule capable of simultaneously binding human LAG-3 and a second epitope, in particular said second epitope is a Epitopes of molecules involved in the control of immune checkpoints present on the surface (in particular, the second epitope is B7-H3, B7-H4, BTLA, CD40, CD40L, CD47, CD70, CD80, CD86, CD94, CD137, CD137L, CD226, CTLA-4, galectin-9, GITR, GITRL, HHLA2, ICOS, ICOSL, KIR, LAG-3, LIGHT, MHC class I or II, NKG2a, NKG2d, OX40, OX40L, PD1H, an epitope of PD-1, PD-L1, PD-L2, PVR, SIRPa, TCR, TIGIT, TIM-3 or VISTA, and most particularly said second epitope is CD137, OX40, PD-1, TIGIT or an epitope of TIM-3).

The invention further provides that said molecule is a diabody, and in particular said diabody is a covalent complex comprising two or three or four or five or more polypeptide chains. The present invention relates to embodiments of LAG-3 binding molecules such as. The invention further provides such a method, wherein said molecule is a trivalent binding molecule, and said trivalent binding molecule is a covalent complex comprising three, four, five or more polypeptide chains. Embodiments of anti-LAG-3 binding molecules. The present invention further relates to embodiments of such LAG-3 binding molecules, wherein said molecule comprises an Fc region. The present invention further relates to embodiments of such LAG-3 binding molecules, wherein said molecules comprise an albumin binding domain, in particular a deimmunized albumin binding domain.

The present invention further provides that the molecule comprises an Fc (Fraction Crystallizable) region, the Fc region is a mutant Fc region, and the mutant Fc region has an affinity for FcγR. or more particularly, the modifications include:
(1) L234A;
(2) L235A;
(3) L234A and L235A;
(4) M252Y; M252Y and S254T;
(5) M252Y and T256E;
(6) M252Y, S254T and T256E; or (7) K288D and H435K;
This numbering relates to embodiments of all such LAG-3 binding molecules, wherein the numbering is that of the EU index according to Kabat.

The present invention further relates to embodiments in which any of the LAG-3 binding molecules described above is used to stimulate a T cell-mediated immune response. The present invention further relates to embodiments in which any of the LAG-3 binding molecules described above is used in the treatment of diseases or conditions associated with a suppressed immune system, particularly cancer or infectious diseases.

The invention particularly relates to the use as described above in the treatment or diagnosis or prognosis of cancers, including: adrenal cancer; AIDS-related cancers; alveolar soft tissue sarcoma; astrocytic tumors; bladder cancer; bone cancer; brain and spinal cord cancer. Cancer; metastatic brain tumor; breast cancer; carotid bulb tumor; cervical cancer; chondrosarcoma; chordoma; chromogenic renal cell carcinoma; clear cell carcinoma; colon cancer; colorectal cancer; cutaneous benign fibrous histiocytoma; desmoplasia small round cell tumor; ependymoma; Ewing tumor; extraosseous myxoid chondrosarcoma; osseous fibroplasia; fibrous dysplasia; gallbladder or cholangiocarcinoma; gastrointestinal cancer; gestational trophoblastic disease; embryonic Cellular tumor; head and neck cancer; hepatocellular carcinoma; pancreatic islet cell tumor; Kaposi's sarcoma; kidney cancer, leukemia, lipoma/benign liposome tumor, liposarcoma/malignant liposome tumor, liver cancer; lymphoma; lung cancer; medulloblastoma; melanoma ; meningioma; multiple endocrine tumors; multiple myeloma; myelodysplastic syndrome; neuroblastoma; neuroendocrine tumor; ovarian cancer; pancreatic cancer; papillary thyroid cancer; parathyroid tumor; childhood cancer; peripheral nerve sheath Tumor; pheochromocytoma; pituitary tumor; prostate cancer; posterior uveal melanoma; rare hematological disorder; renal metastatic carcinoma; rhabdoid tumor; rhabdomyosarcoma; sarcoma; skin cancer; soft tissue sarcoma; squamous cell characterized by the presence of cancer cells selected from the group consisting of cancer; gastric cancer; synovial sarcoma; testicular cancer; thymic cancer; thymoma; metastatic thyroid cancer; and uterine cancer cells.

The invention particularly relates to the use as described above in the treatment or diagnosis or prognosis of cancers, wherein said cancers include: colorectal cancer; hepatocellular carcinoma; glioma; kidney cancer; breast cancer; multiple myeloma; bladder cancer; blastoma; sarcoma; non-Hodgkin lymphoma; non-small cell lung cancer; ovarian cancer; pancreatic cancer; rectal cancer; acute myeloid leukemia (AML); chronic myeloid leukemia (CML); acute B-lymphoblastic leukemia (B- ALL); chronic lymphocytic leukemia (CLL); hairy cell leukemia (HCL); blastic plasmacytoid dendritic cell neoplasm (BPDCN); mantle cell leukemia (MCL) and small lymphocytic lymphoma (SLL) non-Hodgkin's lymphoma (NHL), including Hodgkin's lymphoma; systemic mastocytosis; or Burkitt's lymphoma.

The present invention further relates to embodiments in which any of the LAG-3 binding molecules described above is detectably labeled and used for the detection of LAG-3.

FIG. 1 is a schematic diagram of a representative covalent diabody with two epitope binding sites consisting of two polypeptide chains, each with an E-coil or K-coil heterodimer-promoting domain. As shown in Figure 3B, cysteine residues may be present in the linker and/or in the heterodimer-promoting domain. VL and VH domains that recognize the same epitope are indicated using the same shading or fill pattern. Figure 2 shows a representative covalent diabody with two epitope binding sites consisting of two polypeptide chains with CH2 and CH3 domains, respectively, such that the linked chains form all or part of the Fc region. FIG. 2 is a schematic diagram of a molecule. VL and VH domains that recognize the same epitope are indicated using the same shading or fill pattern. Figures 3A-3C are schematic diagrams of a representative quadrivalent diabody with four epitope binding sites made up of two pairs of polypeptide chains (ie, four polypeptide chains total). One polypeptide of each pair has CH2 and CH3 domains such that the linked chains form all or part of an Fc region. VL and VH domains that recognize the same epitope are indicated using the same shading or fill pattern. The two pairs of polypeptide chains may be identical. In embodiments where the VL and VH domains recognize different epitopes (as shown in Figures 3A-3C), the resulting molecule has four epitope binding sites and is bispecific, with each epitope it binds On the other hand, it is divalent. In embodiments where the VL and VH domains recognize the same epitope (e.g., the same VL domain CDR and the same VH domain CDR are used for both chains), the resulting molecule has four epitope binding sites. It is monospecific and quadrivalent for a single epitope. Alternatively, the two pairs of polypeptides may be different. In embodiments in which the VL and VH domains of each pair of polypeptides recognize different epitopes (as shown in Figure 3C), the resulting molecule has four epitope binding sites and is tetraspecific, binding monovalent for each epitope. FIG. 3A shows an Fc diabody containing a peptide heterodimer-promoting domain that includes cysteine residues. FIG. 3B shows an Fc region-containing diabody, which contains E-coil and K-coil heterodimer-promoting domains, including cysteine residues and a linker (with an optional cysteine residue). Figure 3C shows an Fc region-containing diabody containing antibody CH1 and CL domains. Figures 4A and 4B are schematic diagrams of a representative covalent diabody molecule with two epitope binding sites consisting of three polypeptide chains. Two of the polypeptide chains have CH2 and CH3 domains such that the linked chains form all or part of an Fc region. The polypeptide chain comprising the VL and VH domains further comprises a heterodimerization promoting domain. VL and VH domains that recognize the same epitope are indicated using the same shading or fill pattern. FIG. 5 is a schematic diagram of a representative covalent diabody with four epitope binding sites consisting of five polypeptide chains. Two of the polypeptide chains have CH2 and CH3 domains such that the linked chains form an Fc region comprising all or part of the Fc region. The polypeptide chain comprising the VL and VH domains further comprises a heterodimerization promoting domain. VL and VH domains that recognize the same epitope are indicated using the same shading or fill pattern. Figures 6A-6F are schematic diagrams of representative Fc region-containing trivalent binding molecules with three epitope binding sites. Figures 6A and 6B show domains of trivalent binding molecules comprising two diabody- and Fab-type binding domains, respectively, with different domain orientations, with the diabody-type binding domain being N-terminal or C-terminal to the Fc region. . The molecules of Figures 6A and 6B comprise four chains. Figures 6C and 6D show, respectively, two diabody-type binding domains N-terminal to the Fc region and a Fab-type binding domain or scFv-type binding domain in which the light and heavy chains are linked via a polypeptide linker spacer. 3 shows domains of a trivalent binding molecule comprising: The trivalent binding molecules of Figures 6E and 6F each have two diabody-type binding domains C-terminal to the Fc region and a linked Fab-type binding domain, or an scFv in which the diabody-type binding domains are present. 2 schematically depicts the domains of a trivalent binding molecule, comprising a type binding domain. The trivalent binding molecules of Figures 6C-6F comprise three chains. VL and VH domains that recognize the same epitope are indicated using the same shading or fill pattern. Figures 7A-7C show the binding properties of LAG-3 mAb 1 and LAG-3 mAb 6 to cynomolgus LAG-3. hLAG-3 mAb 6(1.1) (Figure 7A), hLAG-3 mAb 1(1) demonstrates that hLAG-3 mAb 6(1.1) exhibits relatively good binding to cynomolgus monkey LAG-3. Biacore binding curves (RU; response units) of .4) (Figure 7B) and LAG-3 mAb A (Figure 7C). Figures 8A-8D show that LAG-3 mAb 1 and LAG-3 mAb 6 bind to different epitopes than that bound by the reference antibody LAG 3 mAb A. In the absence of competing antibody (FIG. 8A), in the presence of an excess of LAG-3 mAb 1 (FIG. 8B), in the presence of an excess of LAG-3 mAb 6 (FIG. 8C), or in the presence of LAG-3 mAb A. Binding profiles (RU; response units) of labeled LAG-3 mAb 1, LAG-3 mAb 6 and LAG-3 mAb A at the bottom (FIG. 8D). Figures 9A-9B show that isolated antibodies inhibit the binding of soluble human LAG-3 (shLAG-3) to MHC class II as determined by ELISA assay. Figure 9A shows the inhibition curves of LAG-3 mAb 1, LAG-3 mAb 2, LAG-3 mAb 3, LAG-3 mAb 4 and LAG-3 mAb 5. Figure 9B shows the inhibition curves (RLU; relative luminescence units) of LAG-3 mAb A, humanized hLAG-3 1 (1.4) and hLAG-3 6 (1.1). Figures 10A-10C show that isolated antibodies inhibit the binding of shLAG-3 on the surface of MHC class II expressing Daudi cells. FIG. 10A shows the inhibition curves of LAG-3 mAb 1, LAG-3 mAb 2, LAG-3 mAb 3, LAG-3 mAb 4, LAG-3 mAb 5 and the reference antibody LAG-3 mAb A. FIG. 10B shows the inhibition curves of LAG-3 mAb 1, LAG-3 mAb 6 and LAG-3 mAb A. FIG. 10C shows LAG-3 mAb 1 and humanized hLAG-3 1(1.4), hLAG-3 1(1.2), hLAG-3 1(2.2), hLAG-3 1(1. The inhibition curve of 1) is shown. Each figure represents a separate experiment (MFI: mean fluorescence intensity). Figures 11A-11C show that LAG-3 expression is upregulated by stimulated CD4+ T cells. Flow cytometric analysis of LAG-3 expression on unstimulated CD4+ T cells (FIG. 11A) and CD3/CD28 bead-stimulated CD4+ T cells from two different donors (FIGS. 11B and 11C) at days 11 and 14. . All cells were co-stained with anti-CD4 antibody. Figure 12 (Panels AF) shows that isolated anti-LAG-3 antibodies bind to stimulated but not unstimulated T cells. CD3/CD28-stimulated CD4+ T cells (panels A to C) labeled with LAG-3 mAb 1 (panels A and D), LAG-3 mAb 2 (panels B and E), or LAG-3 mAb 3 (panels C and F). ) and flow cytometry analysis of unstimulated CD4+ T cells (panels D-F). All cells were co-stained with anti-CD4 antibody. Figure 13 (Panels AD) shows that isolated anti-LAG-3 antibodies bind to stimulated but not unstimulated T cells. CD3/CD28 stimulated CD4+ T cells (panels A-B) and unstimulated CD4+ T cells (panels C-D) labeled with LAG-3 mAb 4 (panels A and C) or LAG-3 mAb 5 (panels B and D). ) Flow cytometry analysis. All cells were co-stained with anti-CD4 antibody. Figure 14 (Panels A-D) shows LAG-3 and PD- 1 shows that expression of 1 is upregulated. from a representative donor 48 hours after primary stimulation (Panels AB) labeled with anti-LAG-3/anti-CD3 antibody (Panel A) or anti-PD-1/anti-CD3 antibody (Panel B). Day 5 SEB stimulation of SEB-stimulated PBMCs and labeled with anti-LAG-3/anti-PD-1 antibodies treated with SEB alone (panel C) or with isotype control antibody (panel D) during secondary stimulation. (Panels C-D) Flow cytometry analysis of cells. Figure 15 (Panels AD) shows that LAG-3 and PD-1 expression is upregulated in SEB-stimulated PBMCs from representative donor D:53724. from a representative donor 48 hours after primary stimulation (Panels AB) labeled with anti-LAG-3/anti-CD3 antibody (Panel A) or anti-PD-1/anti-CD3 antibody (Panel B). Day 5 SEB stimulation of SEB-stimulated PBMCs and labeled with anti-LAG-3/anti-PD-1 antibodies treated with SEB alone (panel C) or with isotype control antibody (panel D). ) Flow cytometry analysis of cells. Figures 16A-16B show that LAG-3 mAb 1 can stimulate cytokine production to levels comparable to treatment with anti-PD-1 antibodies. IFNγ (FIG. 16A) and TNFα (FIG. 16B) secretion profiles from SEB-stimulated PBMCs treated with anti-LAG-3 or anti-PD-1 antibodies. Figure 17 shows that LAG-3 mAb 1 can stimulate cytokine production to levels comparable to treatment with anti-PD-1 antibodies, and that treatment with LAG-3 mAb 1 in combination with anti-PD-1 antibodies increases cytokine release. shows that the maximum enhancement of IFNγ secretion profile from SEB-stimulated PBMCs treated with anti-LAG-3 and anti-PD-1 antibodies alone and in combination. Figures 18A-18B show anti-LAG-3 antibodies hLAG-3 mAb 6 (1.1) and LAG-3 against endogenous LAG-3 expressed on SEB-stimulated cynomolgus monkey PBMCs from two donors (Figures 18A and 18B). -3 Shows mAb A binding. Anti-PD-1 antibody PD-1 mAb A was included as a positive control for SEB stimulation.

The present invention provides selected anti-LAG-3 antibodies: LAG-3 mAb 1, LAG-3 mAb 2, LAG-3 mAb 3, LAG-3 mAb 4, which can bind to both cynomolgus monkey LAG-3 and human LAG-3. , LAG-3 mAb 5 or LAG-3 mAb 6. The present invention particularly relates to humanized or chimeric versions of such antibodies, or LAG-3 binding fragments of such anti-LAG-3 antibodies (particularly immunoconjugates, diabodies, BiTEs, bispecific antibodies, etc.). ). The invention particularly relates to such LAG-3 binding molecules that can further bind to epitopes of molecules involved in the regulation of immune checkpoints present on the surface of immune cells. The invention also relates to methods of using such LAG-3 binding molecules for detection of LAG-3 or stimulation of an immune response. The present invention also provides for the use of LAG-3 binding molecules comprising one or more LAG-3 binding domains of selected anti-LAG-3 antibodies as described above to stimulate such immune responses in a subject. It relates to combination therapy, administered in combination with one or more additional molecules effective to further enhance, stimulate or upregulate the response.

I. Antibodies and Binding Domains Thereof Antibodies of the invention are capable of immunospecifically binding to targets such as carbohydrates, polynucleotides, lipids, polypeptides, etc. by means of at least one epitope recognition site located in the variable domain of the immunoglobulin molecule. It is a globulin molecule.

As used herein, the term "antibody" means that a specific domain or portion or form (an "epitope") of a polypeptide or protein or Refers to an immunoglobulin molecule that can immunospecifically bind to non-protein molecules. Epitope-containing molecules may have immunogenic activity whereby they induce an antibody-producing response in an animal; such molecules are called "antigens." Epitope-containing molecules do not necessarily have to be immunogenic.

The binding domains of the invention bind epitopes in an "immunospecific" manner. As used herein, an antibody, diabody, or other epitope binding molecule binds to a region (i.e., an epitope) of another molecule more frequently and more rapidly than to alternative epitopes. A substance is said to bind "immunospecifically" if it reacts or links for a longer period of time and/or with higher affinity. For example, an antibody that specifically binds to one LAG-3 epitope will bind with higher affinity, avidity, and more rapidly than it binds to other LAG-3 epitopes or non-LAG-3 epitopes. and/or an antibody that binds to the LAG-3 epitope for a longer period of time. Reading this definition, it is also understood that, for example, an antibody (or moiety or epitope) that immunospecifically binds to a first target may or may not bind specifically or preferentially to a second target. Ru. Thus, "immunospecific binding" does not necessarily require (although it can include) exclusive binding. Generally, but not necessarily, references to binding mean "specific" binding. Two molecules are said to be able to bind to each other in a "physiospecific" manner if their binding exhibits specificity such that the receptor binds to the respective ligands of these two molecules. Ru. The ability of an antibody to immunospecifically bind to an epitope can be determined, for example, by immunoassay.

Natural antibodies (such as IgG antibodies) consist of two light chains complexed with two heavy chains. Each light chain contains a variable domain (VL) and a constant domain (CL). Each heavy chain contains a variable domain (VH), three constant domains (CH1, CH2 and CH3), and a "Hinge" domain ("H") located between the CH1 and CH2 domains. . Thus, the basic structural unit of naturally occurring immunoglobulins (eg, IgG) is a trimer with a light chain and two heavy chains, usually expressed as a glycoprotein of about 150,000 Da. The amino-terminal ("N-terminal") portion of each chain contains approximately 100-110 variable domains that play a major role in antigen recognition. The carboxy-terminal ("C-terminal") portion of each chain defines a constant region, with light chains having a single constant domain and heavy chains typically having three constant domains and one hinge domain. have
Therefore, the structure of the light chain of an IgG molecule is n-VL-CL-c, and the structure of the IgG heavy chain is n-VH-CH1-H-CH2-CH3-c, where n and c are respectively (represents the N-terminus and C-terminus of the polypeptide). The ability of an antibody to bind to an epitope on an antigen depends on the presence of the antibody's VL and VH domains and its amino acid sequence. The interaction of an antibody light chain with an antibody heavy chain, and in particular with its VL and VH domains, forms one of the two epitope binding sites of a natural antibody. A natural antibody can bind only one epitopic species (ie, it is monospecific), but it can bind multiple copies of that species (ie, exhibits bivalency or multivalency). The variable domain of an IgG molecule consists of multiple complementarity determining regions (CDRs) containing residues that contact epitopes, and non-CDR segments called framework segments (FRs), which generally consist of CDR loops. Maintaining the structure of the CDRs allows for such contacts (although certain framework residues may also contact the antigen). Therefore, the V _L and V H domains have the structure n-FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4-c. Polypeptides that are (or can function as) the first, second and third CDRs of an antibody light chain are herein defined as the CDR _L 1 domain, C
They are called the DR _L 2 domain and the CDR _L 3 domain. Similarly, polypeptides that are (or can function as) the first, second and third CDRs of an antibody heavy chain are herein defined as the CDR _H 1 domain, respectively; They are called CDR _H 2 domain and CDR _H 3 domain. Therefore, the terms CDR _L 1 domain, CDR _L 2 domain, CDR _L 3 domain, CDR _H 1 domain, CDR _H 2 domain, and CDR _H 3 domain, when incorporated into a protein, make the protein light. The protein, whether an antibody with chains and heavy chains or diabodies or single chain binding molecules (e.g. scFv, BiTe, etc.), or another type of protein, can be made to bind to a specific epitope. It is targeted at polypeptides. Thus, as used herein, the term "epitope binding domain" refers to the portion of an epitope binding molecule that is responsible for the ability of such a molecule to immunospecifically bind to an epitope. . The epitope-binding fragment can contain one, two, three, four, five or all six of the CDR domains of the antibody and is capable of immunospecifically binding to such an epitope, while It may exhibit immunospecificity, affinity or selectivity for an epitope different from that of the antibody described above. Preferably, however, the epitope binding fragment contains all six of the CDR domains of the antibody. Epitope-binding fragments of antibodies may be a single polypeptide chain (e.g., scFv) or may comprise two or more polypeptide chains, each having an amino and a carboxyl terminus (e.g., diabodies, Fab fragments, Fab ₂ fragment etc.).

As used herein, the term "antibody" refers to: monoclonal antibodies; multispecific antibodies; human antibodies; humanized antibodies; synthetic antibodies; chimeric antibodies; polyclonal antibodies; camelized antibodies; scFv); single chain antibodies; immunologically active antibody fragments (e.g.: Fab fragments; Fab'fragments;F(ab') ₂ fragments; Fv fragments; fragments containing V _L and/or V _H domains, or A complementarity determining region (CDR) of a VL domain (i.e. CDR _L 1, CDR _L 2 and/or CDR _L 3) or a complementarity determining region (CDR) of a VH domain (i.e. CDR _H ) that specifically binds to a certain antigen etc. 1, CDR _H 2 and/or CDR _H 3); bifunctional; or multifunctional antibodies; disulfide-bonded bispecific Fvs (sdFv); intracellular antibodies; and epitope-binding fragments of any of the foregoing. In particular, the term "antibody" is intended to encompass immunoglobulin molecules and immunologically active fragments of immunoglobulin molecules, ie, molecules that contain an epitope binding site. Immunoglobulin molecules can be of any type (e.g. IgG, IgE, IgM, IgD, IgA and IgY), class (e.g. IgG ₁ , IgG ₂ , IgG ₃ , IgG ₄ , IgA ₁ and IgA ₂ ) or subclass. (
For example, US Published Patent No. 20040185045; US Published Patent No. 20050037000; US Published Patent No. 20050064514; US Published Patent No. 20050215767; US Published Patent No. 20070004909; and US Published Patent No. 20070244303). In recent decades, there has been a renewed interest in the therapeutic potential of antibodies, making them one of the leading classes of biotechnologically derived drugs (Chan, CE et al. (2009) “The Use Of Antibodies In The Treatment Of Infectious Diseases,” Singapore Med. J. 50(7):663-666). More than 200 antibody-based drugs have been approved for use or are in development.

The anti-LAG-3 antibodies of the present invention are humanized versions of the antibodies LAG-3 mAb 1, LAG-3 mAb 2, LAG-3 mAb 3, LAG-3 mAb 4, LAG-3 mAb 5, or LAG-3 mAb 6. , including chimeric or camelized variants.

The term "chimeric antibody" means that the heavy and/or light chain portions are identical to or identical to an antibody or antibody class or subclass derived from a species (e.g., mouse), so long as they exhibit the desired biological activity. Refers to an antibody that is homologous, but the remainder is identical or homogeneous to an antibody of another species (eg, human) or an antibody class or subclass. Chimeric antibodies of interest herein include "primatized" antibodies with variable domain antigen binding sequences derived from non-human primates (e.g., Old World monkeys, apes, etc.) and human constant region sequences. .

As used herein, the term "monoclonal antibody"
``antibody'' refers to one antibody of a population of substantially homogeneous antibodies, i.e. the individual antibodies making up said population are identical except for the possibility of antibodies having naturally occurring mutations, which may be present in trace amounts. It is. Also, as used herein, the term "polyclonal antibody" refers to an antibody obtained from a heterogeneous population of antibodies. The term "monoclonal" refers to a substantially homogeneous population of antibodies. These characteristics of antibodies are not to be construed as requiring production of the antibodies by any particular method (e.g., by hybridomas, phage selection, recombinant expression, transgenic animals, etc.). The term includes whole immunoglobulins as well as fragments such as those mentioned above in the definition of "antibody". Methods for making monoclonal antibodies are known in the art. One method that may be employed is that of Kohler, G. et al. (1975) "Continuous Cultures Of Fused Cells Secreting Antibody Of Predefined Specificity," Nature 256:495-497, or a modification thereof. Typically, monoclonal antibodies are expressed in mice, rats or rabbits. The antibodies are produced by immunizing an animal with an immunogenic amount of cells, cell extracts, or protein preparations containing the desired epitope. Immunogens can be, but are not limited to, primary cells, cultured cell lines, cancer cells, proteins, peptides, nucleic acids, or tissues. Cells that may be used for immunization may be cultured for a period of time (eg, at least 24 hours) prior to their use as immunogen. The cells may be used as immunogens alone or in combination with non-denaturing adjuvants such as Ribi (e.g. Jennings, VM (1995) “Review of Selected Adjuvants Used in Antibody Production,” ILAR J. 37(3): 119-125). Generally, cells must be maintained in an intact and preferably viable condition when used as an immunogen. Intact cells may enable the immunized animal to detect antigens better than ruptured cells. The use of denaturing or strong adjuvants, such as Freund's adjuvant, may rupture the cells and is therefore not recommended. The immunogen may be administered multiple times at periodic intervals, such as once every two weeks or once a week, or may be administered to maintain viability in the animal (e.g., during tissue recombination). . Alternatively, existing monoclonal antibodies and other equivalent antibodies with immunospecificity for the desired pathogenic epitope can be recombinantly sequenced and produced by any means known in the art. In one embodiment, such antibodies are sequenced and the polynucleotide sequences are subsequently cloned into vectors for expression or propagation. Sequences encoding antibodies of interest are maintained in vectors in host cells, which can then be expanded and frozen for future use. Such antibody polynucleotide sequences may be used in monospecific or multispecific (e.g., bispecific, trispecific, and tetraspecific) molecules of the invention, as well as affinity-optimized antibodies, chimeric It may be used for genetic engineering to improve the affinity or other characteristics of antibodies by producing antibodies, humanized antibodies and/or caninized antibodies.

The term "scFv" refers to a single chain variable domain fragment. scFv molecules are created by joining light or heavy chain variable domains using short linking peptides. Bird et al. (1988) ("Single-Chain Antigen-Binding Proteins," Science 242:423-426) reported that approximately 3.5 nm between the carboxy terminus of one variable domain and the amino terminus of the other variable domain. An example of a connecting peptide that fills in is described. Linkers of other sequences have also been designed and used (Bird et al. (1988), "Single-Chain Antigen-Binding Proteins," Science 242:423-426). Linkers can be modified for additional functions, such as attaching drugs or attachments to solid supports. Single chain variants can be produced recombinantly or synthetically. For synthetic production of scFv, automated synthesizers can be used. For recombinant production of scFv, a suitable plasmid containing a polynucleotide encoding the scFv is introduced into a suitable host cell, such as a eukaryotic cell such as a yeast, plant, insect or mammalian cell, or a prokaryotic cell such as E. coli. can. Polynucleotides encoding scFvs of interest can be generated by conventional manipulations such as ligation of polynucleotides. The resulting scFv can be isolated using standard protein purification techniques known in the art.

The invention particularly encompasses humanized variants of the anti-LAG-3 antibodies of the invention, and multispecific binding molecules comprising the same. The term "humanized" antibody refers to a chimeric molecule, generally prepared using recombinant techniques, that combines the epitope binding site of an immunoglobulin from a non-human species with the remaining structure and/or structure of a human immunoglobulin. Refers to a chimeric molecule that has the immunoglobulin structure of the molecule based on its sequence. The epitope binding site may comprise a complete variable domain fused to a constant domain, or only the CDRs grafted onto appropriate framework regions within the variable domain. The epitope binding site may be wild type or modified by one or more amino acid substitutions. This reduces the constant region as an immunogen in the human individual, but leaves the possibility of an immune response against foreign variable regions (LoBuglio, AF et al. (1989) “Mouse/Human Chimeric Monoclonal Antibody In Man: Kinetics And Immune Response,” Proc. Natl. Acad. Sci. (USA) 86:4220-4224). Another approach focuses not only on providing human-derived constant regions, but also on modifying and reshaping the variable regions to be as close to the human form as possible. The variable regions of both heavy and light chains are known to contain three CDRs flanked by four framework regions (FRs) that change in response to the antigen in question and determine binding capacity. It is assumed that the framework regions are relatively conserved in a given species and also provide a scaffold for the CDRs. When preparing a non-human antibody against a particular antigen, the variable region can be "reshaped" or "humanized" by grafting the CDRs from the non-human antibody into the FRs present in the human antibody to be modified. "can. Application of this approach to various antibodies: Sato, K.
et al. (1993) “Reshaping A Human Antibody To Inhibit The Interleukin 6-Dependent Tumor Cell Growth,” Cancer Res 53:851-856. Riechmann, L. et al. (1988) “Reshaping Human Antibodies for Therapy,” Nature 332:323-327; Verhoeyen, M. et al. (1988) “Reshaping Human Antibodies: Grafting An Antilysozyme Activity,” Science 239:1534-1536; Kettleborough, CA et al. (1991) “Humanization Of A Mouse Monoclonal Antibody By CDR‐Grafting: The Importance Of Framework Residues On Loop Conformation,” Protein Engineering 4:773‐3783; Maeda, H. et al. (1991) “
Construction Of Reshaped Human Antibodies With HIV-Neutralizing Activity,” Human Antibodies Hybridoma 2:124-134; Gorman, SD et al. (1991) “Reshaping A Therapeutic CD4 Antibody,” Proc. Natl. Acad. Sci. (USA) 88 :4181-4185; Tempest, PR et al. (1991) “Reshaping A Human Monoclonal Antibody To Inhibit Human Respiratory Syncytial Virus Infection in vivo,” Bio/Technology 9:266-271; Co, M.
S. et al. (1991) “Humanized Antibodies For Antiviral Therapy,” Proc. Natl. Acad. Sci. (USA) 88:2869‐2873; Carter, P. et al. (1992) “Humanization Of An Anti‐p185her2 Antibodies For Human Cancer Therapy,” Proc. Natl. Acad. Sci. (USA) 89:4285-4289; and Co, MS et al. (1992) “Chimeric And Humanized Antibodies
With Specificity For The CD33 Antigen,” J. Immunol. 148:1149-1154. In some embodiments, humanized antibodies conserve all CDR sequences (e.g., six CDR sequences from mouse antibodies). A humanized mouse antibody containing all CDRs). In other embodiments, a humanized antibody contains one or more CDRs (one, 2, 3, 4, 5 or 6), which are “derived from” (i.e., derived from) one or more CDRs from the original antibody (i.e., derived from knowledge of the amino acid sequence of such CDR, etc.) is also referred to as one or more CDRs. Polynucleotide sequences encoding the variable domains of antibodies can be used for genetic engineering to generate such derivatives and to improve the affinity or other characteristics of the antibodies. The general principle in humanizing antibodies involves replacing the non-human remainder of the antibody with human antibody sequences while retaining the base sequence of the epitope-binding portion of the antibody. There are four general steps to humanize monoclonal antibodies. The steps are as follows: (1) determining the nucleotide and predicted amino acid sequences of the light chain and heavy chain variable domains of the starting antibody; (2) designing humanized or caninized antibodies; (3) the actual humanization or caninization method/technique; and (4) transfection and expression of the humanized antibody. See, eg, US Patent No. 4,816,567; US Patent No. 5,807,715; US Patent No. 5,866,692; and US Patent No. 6,331,415.

The epitope binding site of a molecule of the invention may comprise a complete variable domain fused to a constant domain, or only the complementarity determining regions (CDRs) of such a variable domain grafted to appropriate framework regions. The epitope binding site may be wild type or may be modified by one or more amino acid substitutions. This eliminates constant regions that are immunogenic in human individuals, but leaves open the possibility of foreign variable domains (LoBuglio, AF et al. (1989) “Mouse/Human Chimeric Monoclonal Antibody In Man: Kinetics And Immune Response ,” Proc. Natl. Acad. Sci. (USA) 86:4220-4224). Another approach focuses not only on providing human-derived constant regions, but also on modifying the variable domains to transform them as closely as possible to the human form. . The variable domains of both heavy and light chains contain three complementarity determining regions (CDRs) flanked by four framework regions (FRs) that change in response to the antigen in question and determine binding capacity. The framework regions are known to be relatively conserved in a given species and are presumed to provide a scaffold for the CDRs. When preparing non-human antibodies against a particular antigen, the variable domains can be "reshaped" or "humanized" by grafting the CDRs from the non-human antibody into the FRs present in the human antibody to be modified. "can. Application of this approach to various antibodies is described in Sato, K. et al. (1993) “Reshaping A Human
Antibody To Inhibit The Interleukin 6‐Dependent Tumor Cell Growth,” Cancer Res 53:851‐856. Riechmann, L. et al. (1988) “Reshaping Human Antibodies for Therapy,” Nature 332:323‐327; Verhoeyen, M. et al. (1988) “Reshaping Human Antibodies: Grafting An Antilysozyme Activity,” Science 239:1534‐1536; Kettleborough, CA et al. (1991) “Humanization Of A Mouse Monoclonal Antibody By CDR‐Graf
ting: The Importance Of Framework Residues On Loop Conformation,” Protein Engineering 4:773‐3783; Maeda, H. et al. (1991) “Construction Of Reshaped Human Antibodies With HIV‐Neutralizing Activity,” Human Antibodies Hybridoma 2:124‐ 134; Gorman, SD et al. (1991) “Reshaping A Therapeutic CD4 Antibody,” Proc. Natl. Acad. Sci. (USA) 88:4181-4185; Tempest, PR et al. (1991) “Reshaping A
Human Monoclonal Antibody To Inhibit Human Respiratory Syncytial Virus Infection in vivo,” Bio/Technology 9:266-271; Co, MS et al. (1991) “Humanized Antibodies For Antiviral Therapy,” Proc. Natl. Acad. Sci. (USA ) 88:2869-2873; Carter, P. et al. (1992) “Humanization Of An Anti-p185her2 Antibody For Human Cancer Therapy,” Proc. Natl. Acad. Sci. (USA) 89:4285-4289; and Co , MS et al. (1992) “Chimeric And Humanized Antibodies With Specificity For The CD33 Antigen,” J. Immunol. 148:1149-1154. In some embodiments, humanized antibodies contain all CDRs. (e.g., a humanized mouse antibody that contains all six CDRs from a mouse antibody). In other embodiments, a humanized antibody contains one or more modified CDRs that differ in sequence relative to the original antibody. Has multiple CDRs (1, 2, 3, 4, 5 or 6).

A number of "humanized" antibody molecules have been described that include epitope binding sites derived from non-human immunoglobulins, fused to rodent or modified rodent variable domains and human constant domains. These include chimeric antibodies with associated complementarity determining regions (CDRs) (e.g. Winter et al. (1991) “Man-made Antibodies,” Nature 349:293-299; Lobuglio et al. (1989) “Mouse /Human Chimeric Monoclonal Antibody In Man: Kinetics And Immune Response,” Proc. Natl. Acad. Sci. (USA) 86:4220-4224 (1989); Shaw et al. (1987) “Characterization Of A Mouse/Human Chimeric Monoclonal Antibody (17-1A) To A Colon Cancer Tumor-Associated Antigen,” J. Immunol. 138:4534-4538; and Brown et al. (1987) “Tumor-Specific Genetically Engineered Murine/Human Chimeric Monoclonal Antibody,” Cancer Res 47:3577-3583). Other references describe rodent CDRs that are grafted and polymerized into human supportive framework regions (FRs) before fusion with appropriate human antibody constant domains (e.g., Riechmann, L. et al. al. (1988) “Reshaping Human Antibodies for Therapy,” Nature 332:323-327; Verhoeyen, M. et al. (1988) “Reshaping Human Antibodies: Grafting An Antilysozyme Activity,” Science 239:1534-1536; and Jones et al. (1986) “Replacing The Complementarity-Determining Regions In A Human Antibody With Those From
A Mouse,” Nature 321:522-525). Other references describe rodent CDRs supported by recombinantly veneered rodent framework regions; e.g. See No. 519,596. These "humanized" molecules are free from undesirable immunological responses to rodent anti-human antibody molecules, which limits the duration and efficacy of therapeutic application of these parts in human recipients. Designed to minimize Other methods that may be used to humanize antibodies include Daugherty et al. (1991) “Polymerase Chain Reaction Facilitates The Cloning, CDR-Grafting, And Rapid Expression Of A Murine Monoclonal Antibody Directed Against The CD18 Component Of Leukocyte. Integrins,
” Nucl. Acids Res. 19:2471-2476 and U.S. Patent No. 6,180,377; U.S. Patent No. 6,054,297; U.S. Patent No. 5,997,867; No. 692.

II. Fcγ receptor (FcγR)
The CH2 and CH3 domains of the two heavy chains interact to form the Fc region, which is the domain recognized by cellular Fc receptors, including but not limited to Fcγ receptors (FcγRs). As used herein, the term "Fc region" is used to define the C-terminal region of an IgG heavy chain. The amino acid sequence of an exemplary human IgG1 CH2-CH3 domain is (SEQ ID NO: 1):
231 240 250 260 270 280
APELLGGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSHED PEVKFNWYVD
290 300 310 320 330
GVEVHNAKTK PREEQYNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKALPA
340 350 360 370 380
PIEKTISKAK GQPREPQVYT LPPSREEMTK NQVSLTCLVK GFYPSDIAVE
390 400 410 420 430
WESNGQPENN YKTTPPVLDS DGSFFLYSKL TVDKSRWQQG NVFSCSVMHE
440 447
ALHNHYTQKS LSLSPG X
, which is numbered by the EU index according to Kabat, where X is lysine (K) or absent.

The amino acid sequence of an exemplary human IgG2 CH2-CH3 domain is (SEQ ID NO: 2): 231 240 250 260 270 280
APPVA-GPSV FLFPPKPKDT LMISRTPEVT CVVVDVSHED PEVQFNWYVD
290 300 310 320 330
GVEVHNAKTK PREEQFNSTF RVVSVLTVVH QDWLNGKEYK CKVSNKGLPA
340 350 360 370 380
PIEKTISKTK GQPREPQVYT LPPSREEMTK NQVSLTCLVK GFYPSDISVE
390 400 410 420 430
WESNGQPENN YKTTPPMLDS DGSFFLYSKL TVDKSRWQQG NVFSCSVMHE
440 447
ALHNHYTQKS LSLSPG X
, which is numbered by the EU index according to Kabat, where X is lysine (K) or absent.

The amino acid sequence of an exemplary human IgG3 CH2-CH3 domain is (SEQ ID NO: 3): 231 240 250 260 270 280
APELLGGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSHED PEVQFKWYVD
290 300 310 320 330
GVEVHNAKTK PREEQYNSTF RVVSVLTVLH QDWLNGKEYK CKVSNKALPA
340 350 360 370 380
PIEKTISKTK GQPREPQVYT LPPSREEMTK NQVSLTCLVK GFYPSDIAVE
390 400 410 420 430
WESSGQPENN YNTTPMLDS DGSFFLYSKL TVDKSRWQQG NIFSCSVMHE
440 447
ALHNRFTQKS LSLSPG X
, which is numbered by the EU index according to Kabat, where X is lysine (K) or absent.

The amino acid sequence of an exemplary human IgG4 CH2-CH3 domain is (SEQ ID NO: 4): 231 240 250 260 270 280
APEFLGGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSQED PEVQFNWYVD
290 300 310 320 330
GVEVHNAKTK PREEQFNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKGLPS
340 350 360 370 380
SIEKTISKAK GQPREPQVYT LPPSQEEMTK NQVSLTCLVK GFYPSDIAVE
390 400 410 420 430
WESNGQPENN YKTTPPVLDS DGSFFLYSRL TVDKSRWQEG NVFSCSVMHE
440 447
ALHNHYTQKS LSLSLG X
, which is numbered by the EU index according to Kabat, where X is lysine (K) or absent.

Throughout this specification, the numbering of residues in the constant region of IgG heavy chains refers to Kabat et al., Sequences of Proteins of Immunological Interest, ^5th Ed. Public Health Service, NH1, MD (1991) (“Kabat”). , which is expressly incorporated herein by reference). "EU index according to Kabat" refers to the numbering of human IgG1 EU antibodies. Amino acids from the variable domains of the mature heavy and light chains of immunoglobulins are designated by the position of the amino acid within the chain, and CDRs are identified as defined by Kabat (Chothia, C. & Lesk, AM). (1987) “Canonical structures for the hypervariable regions of immunoglobrins,”. _J. Mol. Biol. 196:901-917). Kabat describes numerous amino acid sequences for antibodies, identifies amino acid consensus sequences for each subgroup, and assigns a residue number to each amino acid. The Kabat numbering scheme can be extended to antibodies not included in the Kabat summary by referencing conserved amino acids and aligning the antibody in question with one of the consensus sequences in Kabat. It is. The above methods for assigning residue numbers have become standard in the art and readily identify amino acids at equivalent positions in different antibodies, including chimeric or humanized variants. For example, amino acid position 50 of a human antibody light chain occupies the equivalent position as amino acid position 50 of a mouse antibody light chain.

Polymorphisms include a number of different positions within antibody constant regions (e.g., positions 270, 272, 312, 315, 356, and 358, numbered according to the EU index as described in Kabat). (but not limited to the Fc position), there may therefore be slight differences between the sequences presented here and those of the prior art. Polymorphic forms of human immunoglobulins have been well characterized. Currently, 18 Gm allotypes are known: G1m (1, 2, 3, 17) or G1m (a, x, f, z), G2m (23) or G2m (n), G3m (5, 6, 10, 11, 13, 14, 15, 16, 21, 24, 26, 27, 28) or G3m (b1, c3, b3, b0, b3, b4, s, t, g1, c5, u, v, g5 ) (Lefranc, et al., The human IgG subclasses: molecular
analysis of structure, function and regulation. Pergamon, Oxford, pp. 43-78 (1990); Lefranc, G. et al., 1979, Hum. Genet.: 50, 199-211). In particular, it is contemplated that the antibodies of the invention may incorporate any allotype, isoallotype or haplotype of any immunoglobulin gene and are not limited to the allotypes, isoallotypes or haplotypes of the sequences presented herein. . Furthermore, depending on the expression system, the C-terminal amino acid residue of the CH3 domain (in bold above) can be removed post-translationally. Therefore, the C-terminal residue of the CH3 domain is any amino acid residue of the LAG-3 binding molecule of the invention. Specifically encompassed by the invention are LAG-3 binding molecules lacking the C-terminal residue of the CH3 domain. Also specifically encompassed by the invention are structures that include the C-terminal lysine residue of the CH3 domain.

Activating and inhibitory signals are transduced by ligation of the Fc region to cellular Fc receptors (FcγRs). The ability of such ligation to result in opposite functions is due to structural differences between different receptor isoforms. Two distinct domains within the cytoplasmic signaling domain of the receptor, called the immunoreceptor tyrosine system activation motif (ITAM) and the immunoreceptor tyrosine system inhibition motif (ITIM), are responsible for different responses. Recruitment of different cytoplasmic enzymes to these structures determines the outcome of FcγR-mediated cellular responses. The ITAM-containing FcγR complex contains FcγRI, FcγRIIA, FcγRIIIA, while the ITIM-containing complex contains only FcγRIIB. Human neutrophils express the FcγRIIA gene. Clustering of FcγRIIA by immune complexes or specific antibody cross-linking serves to aggregate ITAM with receptor-associated kinases that promote ITAM phosphorylation. ITAM phosphorylation serves as a docking site for Syk kinase, and its activation results in activation of downstream substrates (eg PI ₃ K). Cell activation leads to the release of pro-inflammatory mediators. The FcγRIIB gene is expressed on B lymphocytes, its extracellular domain is 96% identical to FcγRIIA, and it binds IgG complexes in an indistinguishable manner. The presence of ITIM in the cytoplasmic domain of FcγRIIB defines the above-mentioned inhibitory subclass of FcγRs. Recently, the molecular basis of this inhibition has been established. Upon co-ligation with activated FcγRs, ITIM in FcγRIIB becomes phosphorylated and attracts the SH2 domain of inositol polyphosphate 5′-phosphatase (SHIP), which is released as a result of ITAM-containing FcγR-mediated tyrosine kinase activation. phosphoinositol, thereby preventing the influx of intracellular Ca ⁺⁺ . Therefore, cross-linking of FcγRIIB attenuates the activation response to FcγR ligation and inhibits cellular responsiveness. In this way, B cell activation, B cell proliferation and antibody secretion are disrupted.

III. Bispecific Antibodies, Multispecific Diabodies and DART® Diabodies The functionality of antibodies is based on the ability of antibodies to treat two separate and different antigens (or different epitopes of the same antigen).
by generating multispecific antibody-based molecules that can simultaneously bind to the same epitope and/or antigen and/or having higher valencies (i.e., three or more binding sites) for the same epitope and/or antigen. It can be strengthened by

A wide range of recombinant bispecific antibody formats have been developed to provide molecules with higher potency than natural antibodies (e.g. WO 2008/003116; WO 2009/132876; WO 2008/ WO 2007/146968; WO 2009/018386; WO 2012/009544; WO 2013/070565), most of which contain additional epitope-binding fragments (e.g. scFv, VL , VH, etc.) to or within an antibody core (IgA, IgD, IgE, IgG or IgM) or to fuse multiple epitope binding fragments (e.g. two Fab fragments or scFv). Use a linker peptide. An alternative format uses a linker peptide to fuse an epitope binding fragment (e.g. scFv, VL, VH, etc.) to a dimerization domain, such as a CH2-CH3 domain or an alternative polypeptide (International Publication No. 2005/070966; International Publication No. 2006/107786A; International Publication No. 2006/107617A; International Publication No. 2007/046893). Typically, such approaches involve compromises and trade-offs. For example, WO 2013/174873; WO 2011/133886; and WO 2010/136172 disclose that the use of linkers can cause problems in therapeutic settings; teaches trispecific antibodies in which the CL and CH1 domains are switched from their natural positions and the VL and VH domains are diversified so that they can bind more than one antigen (WO 2008/027236 International Publication No. 2010/108127). The molecules disclosed in these documents therefore trade binding specificity for the ability to bind additional antigenic species. WO 2013/163427 and WO 2013/119903 disclose modifying a CH2 domain to contain a fusion protein adduct comprising a binding domain. This document states that CH2 appears to play only a minimal role in mediating effector functions. International Publication No. 2010/028797; International Publication No. 2010028796; and International Publication No. 2010/028795 are Fc
Recombinant antibodies are disclosed in which regions are replaced with additional VL and VH domains to form trivalent binding molecules. WO 2003/025018; and WO 2003012069 disclose recombinant diabodies in which each chain contains an scFv domain. WO 2013/006544 discloses multivalent fab molecules that are synthesized as a single polypeptide chain and then subjected to proteolysis to obtain a heterodimeric structure. The molecules disclosed in these documents therefore exchange all or some of their ability to mediate effector functions for the ability to bind additional antigenic species. International Publication No. 2014/022540; International Publication No. 2013/003652; International Publication No. 2012/162583; International Publication No. 2012/156430; International Publication No. 2011/086091; International Publication No. 2008/024188; International Publication No. WO 2007/024715; WO 2007/075270; WO 1998/002463; WO 1992/022583; and WO 1991/003493 disclose that additional binding domains or functional groups can be added to antibodies or Attaching antibody moieties (e.g. attaching diabodies to the light chain of an antibody, or adding additional VL and VH domains to the light and heavy chains of an antibody, or attaching heterologous fusion proteins to each other) or the step of chaining multiple Fab domains to each other. The molecules disclosed in these documents therefore replace the naive antibody structure with the ability to bind additional antigenic species.

The art further describes such natural antibodies in that they are capable of binding two or more different epitopic species (i.e., can exhibit bispecificity or multispecificity in addition to bivalency or multivalency). (For example, Holliger et al. (1993) “'Diabodies': Small Bivalent And Bispecific Antibody Fragments,” Proc. Natl. Acad. Sci. (USA) 90: 6444-6448; US Patent No. 2004/0058400 (Hollinger et al.); US Patent No. 2004/0220388; WO 02/02781 (Mertens et al.); Alt et al. (1999) FEBS Lett. 454(1-2):90-94; Lu, D. et al. (2005) “A Fully Human Recombinant IgG-Like Bispecific Antibody To Both The Epidermal Growth Factor Receptor And The Insulin-Like Growth Factor Receptor For Enhanced Antitumor Activity ,” J. Biol. Chem. 280(20):19665-19672; WO 02/02781 (Mertens et al.); Olafsen, T. et al. (2004) “Covalent Disulfide-Linked Anti-CEA Diabody Allows Site-Specific Conjugation And Radiolabeling For Tumor Targeting Applications,” Protein Eng. Des. Sel. 17(1):21-27; Wu, A. et al. (2001) “Multimerization Of A Chimeric Anti-CD20 Single Chain Fv -Fv Fusion Protein Is Mediated Through Variable Domain Exchange,” Protein Engineering 14(2):1025-1033; Asano et al. (2004) “A Diabody For Cancer Immunotherapy And Its Functional Enhancement By Fusion Of Human Fc Domain,” Abstract 3P -683, J. Biochem. 76(8):992; Takemura, S. et al. (2000) “Construction Of A Diabody (Small Recombinant Bispecific Antibody) Using A Refolding System,” Protein Eng. 13(8):583 -588; Baeuerle, PA et al. (2009) “Bispecific T-Cell Engaging Antibodies For
Cancer Therapy,” Cancer Res. 69(12):4941-4944).

Diabody design is based on antibody derivatives known as single chain variable domain fragments (scFv). Such molecules are created by linking light and/or heavy chain variable domains via short linking peptides. Linkers can be modified for additional functions such as attachment of drugs or attachment of rigid supports. Single chain variants can be produced recombinantly or synthetically. For synthetic production of scFv, automated synthesizers can be used. For recombinant production of scFv, a suitable plasmid containing a polynucleotide encoding the scFv can be introduced into a suitable host cell, which is a eukaryotic cell such as a yeast, plant, insect or mammalian cell, or a prokaryotic cell such as E. coli. Polynucleotides encoding scFvs of interest can be generated by conventional manipulations such as ligation of polynucleotides. The resulting scFv can be isolated using standard protein purification techniques known in the art.

The provision of non-monospecific diabodies offers significant advantages over antibodies, including, but not limited to, the ability to co-ligate and co-exist multiple cells expressing different epitopes. provide. Bispecific diabodies therefore have a wide range of uses, including therapy and immunodiagnostics. Bispecificity allows for significant flexibility in the design and processing of diabodies for a variety of applications, allowing for increased avidity of multimeric antigens, cross-linking of multiple different antigens, and the presence of both target antigens. provides directional targeting to specific cell types based on Diabody molecules known in the art have also found particular use in the field of tumor imaging due to their high valency, low dissociation rate and rapid clearance from circulation (for small size diabodies of ~50 kDa or less). (Fitzgerald et al. (1997)
“Improved Tumour Targeting By Disulphide Stabilized Diabodies Expressed In Pichia pastoris,” Protein Eng. 10:1221).

The bispecificity of diabodies allows their use for co-linking different cells, for example cross-linking cytotoxic T cells and tumor cells (Staerz et al. (1985)
“Hybrid Antibodies Can Target Sites For Attack By T Cells,” Nature 314:628-631, and Holliger et al. (1996) “Specific Killing Of Lymphoma Cells By Cytotoxic T-Cells Mediated By A Bispecific Diabody,” Protein Eng. 9 :299-305; Marvin et al. (2005) “Recombinant Approaches To IgG-Like Bispecific Antibodies,” Acta Pharmacol. Sin. 26:649-658). Alternatively or additionally, bispecific diabodies can be used to coligate receptors on the surfaces of different cells or on a single cell. Colligation of different cells and/or receptors is useful for modulating effector function and/or immune cell signaling. Multispecific molecules (e.g., bispecific diabodies) containing an epitope binding site include B7-H3 (CD276), which is expressed on T lymphocytes, natural killer (NK) cells, antigen-presenting cells, or other mononuclear cells. ), B7-H4 (VTCN1), BTLA (CD272), CD3, CD8, CD16, CD27, CD32, CD40, CD40L, CD47, CD64, CD70 (CD27L), CD80 (B7-1), CD86 (B7-2) , CD94 (KLRD1), CD137 (4-1BB), CD137L (4-1BBL), CD226, CTLA-4 (CD152), Galectin-9, GITR, GITRL, HHLA2, ICOS (CD278), ICOSL (CD275), Killer Activating receptor (KIR), LAG-3 (CD223), LIGHT (TNFSF14, CD258), MHC class I or II, NKG2a, NKG2d, OX40 (CD134), OX40L (CD134L), PD1H, PD-1 (CD279) , PD-L1 (B7-H1, CD274), PD-L2 (B7-CD, CD273), PVR (NECL5, CD155), SIRPa, TCR, TIGIT, TIM-3 (HAVCR2), and/or VISTA (PD- 1H) can be directed against any immune cell surface determinant. In particular, epitope binding sites directed to cell surface receptors involved in the regulation of immune checkpoints (or their ligands) respond to a subject's immune response by antagonizing or blocking the inhibitory signaling of immune checkpoint molecules. Useful in the generation of bispecific or multispecific binding molecules that stimulate, upregulate or enhance. Molecules involved in immune checkpoint regulation include, but are not limited to, B7-H3, B7-H4, BTLA, CD40, CD40L, CD47, CD70, CD80, CD86, CD94, CD137, CD137L, CD226, CTLA -4, Galectin-9, GITR, GITRL, HHLA2, ICOS, ICOSL, KIR, LAG-3, LIGHT, MHC class I or II, NKG2a, NKG2d, OX40, OX40L, PD1H, PD-1, PD-L1, PD -L2, PVR, SIRPa, TCR, TIGIT, TIM-3 and/or VISTA.

However, the above-mentioned advantages come at a significant cost. Formation of such non-monospecific diabodies requires successful assembly of two or more separate and different polypeptides (i.e., formation of such non-monospecific diabodies requires that the diabodies are heterodimers of different polypeptide chain species). (needs to be formed by body formation). This fact is in contrast to monospecific diabodies, which are formed by homodimerization of identical polypeptide chains. Because at least two different polypeptides (i.e., two polypeptide species) must be provided to form a non-monospecific diabody, and homodimerization of such polypeptides is not possible with inert molecules. (Takemura, S. et al. (2000) “Construction Of A Diabody (Small Recombinant Bispecific Antibody) Using A Refolding System,” Protein Eng. 13(8):583-588
), the production of such polypeptides must be accomplished in a manner that prevents covalent binding (i.e., prevents homodimerization) between polypeptides of the same species (Takemura, S. et al. (2000) “Construction Of A Diabody (Small Recombinant Bispecific Antibody) Using A Refolding System,” Protein Eng. 13(8):583-588). Accordingly, the art teaches non-covalent linkage of such polypeptides (eg Olafsen et al.
(2004) “Covalent Disulfide-Linked Anti-CEA Diabody Allows Site-Specific Conjugation And Radiolabeling For Tumor Targeting Applications,” Prot. Engr. Des. Sel. 17:21-27; Asano et al. (2004) “A Diabody For Cancer Immunotherapy And Its Functional Enhancement By Fusion Of Human Fc Domain,” Abstract 3P-683, J. Biochem.
76(8):992; Takemura, S. et al. (2000) “Construction Of A Diabody (Small Recombinant Bispecific Antibody) Using A Refolding System,” Protein Eng. 13(8):583-588; Lu, D. et al. (2005) “A Fully Human Recombinant IgG-Like Bispecific Antibody To Both The Epidermal Growth Factor Receptor And The Insulin-Like Growth Factor Receptor For Enhanced Antitumor Activity,” J. Biol. Chem. 280(20):19665- (see 19672).

However, the art recognizes that bispecific diabodies composed of non-covalently linked polypeptides are unstable and easily degrade into non-functional monomers (e.g. Lu, D. et al. (2005) “A Fully Human Recombinant IgG-Like Bispecific Antibody To Both The Epidermal Growth Factor Receptor And The Insulin-Like Growth Factor Receptor For Enhanced Antitumor Activity,” J. Biol. Chem. 280(20 ):19665-19672).

In spite of this challenge, the field has developed stable covalent heterodimeric non -monospecific diabodies called DART® ( Dual Affinity Re -targeting Reagents ) diabodies. successfully developed bodies (e.g., US Published Patent No. 2013-0295121; US Published Patent No. 2010-0174053; and US Published Patent No. 2009-0060910; European Published Patent No. 2714079; European Published Patent No. 2601216; European Publication No. 2376109; European Publication No. 2158221; and International Publication No. 2012/162068; International Publication No. 2012/018687; International Publication No. 2010/080538; International Publication No. 2008/157379; 2006/113665 and Sloan, DD et al. (2015) “Targeting HIV Reservoir in Infected CD4 T Cells by Dual-Affinity Re-targeting Molecules (DARTs) that Bind HIV Envelope and Recruit Cytotoxic T Cells,” PLoS Pathog. 11 (11):e1005233. doi: 10.1371/journal.ppat.1005233; Al Hussaini, M. et al. (2015)
“Targeting CD123 In AML Using A T-Cell Directed Dual-Affinity Re-Targeting (DART(R)) Platform,” Blood 127(1):122-131; Chichili, GR et al. (2015) “A CD3xCD123 Bispecific DART For Redirecting Host T Cells To Myelogenous Leukemia: Preclinical Activity And Safety In Nonhuman Primates,” Sci. Transl. Med. 7(289):289ra82; Moore, PA et al. (2011) “Application Of Dual Affinity Retargeting Molecules To Achieve Optimal Redirected T-Cell Killing Of B-Cell Lymphoma,” Blood 117(
17):4542-4551; Veri, MC et al. (2010) “Therapeutic Control Of B Cell Activation Via Recruitment Of Fcgamma Receptor IIb (CD32B) Inhibitory Function With A Novel Bispecific Antibody Scaffold,” Arthritis Rheum. 62(7): 1933-1943; Johnson, S. et al. (2010) “Effector Cell Recruitment With Novel Fv-Based Dual-Affinity Re-Targeting Protein Leads To Potent Tumor Cytolysis And in vivo B-Cell Depletion,” J. Mol. Biol. 399(3):436-449; Marvin, JS et al. (2005) “Recombinant Approaches To IgG-Like Bispecific Antibodies,” Acta Pharmacol. Sin. 26:649-658; Olafsen, T. et al. (2004) “Covalent Disulfide-Linked Anti-CEA Diabody Allows Site-Specific Conjugation And Radiolabeling For Tumor Targeting Applications,” Prot. Engr. Des. Sel. 17:21-27; Holliger, P. et al. (1993) “'Diabodies' : Small Bivalent And Bispecific Antibody Fragments,” Proc. Natl. Acad. Sci. (USA) 90:6444-644). Such diabodies comprise two or more covalently complexed polypeptides, one or more cysteine residues capable of forming a disulfide bond, thereby covalently linking the two polypeptide chains. and processing steps into each of the employed polypeptide species. For example, the addition of a cysteine residue to the C-terminus of such a structure has been shown to allow disulfide bonds between polypeptide chains, without interfering with the binding properties of the divalent molecule. Stabilize the resulting heterodimer.

The two polypeptides of the simplest bispecific DART® diabody each comprise three domains. The first polypeptide comprises (in the N-terminus to C-terminus direction): (i) a first domain comprising the binding region of the light chain variable domain (VL1) of a first immunoglobulin; (ii) a second domain; a second domain comprising a binding region of an immunoglobulin heavy chain variable domain (VH2); and (iii) a heterodimer of a cysteine residue (or cysteine-containing domain) with said second polypeptide of said diabody. and a heterodimerization promoting domain that serves to promote incorporation and covalently link the first and second polypeptides of the diabody to each other. The second polypeptide comprises (in the N-terminus to C-terminus direction): (i) a first domain comprising the binding region of a second immunoglobulin light chain variable domain (VL2); (ii) a first a second domain comprising a binding region of an immunoglobulin heavy chain variable domain (VH1); and (iii) a cysteine residue (or cysteine-containing domain) and said heterodimerization promoting domain of said first polypeptide chain. and a complementary heterodimer-promoting domain that promotes heterodimerization with the first polypeptide chain. The cysteine residue (or cysteine-containing domain) of the third domain of the second polypeptide chain facilitates covalent binding of the second polypeptide chain to the first polypeptide chain of the diabody. play a role. Such molecules are stable, potent, and capable of binding two or more antigens simultaneously. In one embodiment, the third domain of the first and second polypeptides each contains a cysteine residue that serves to link the polypeptides together by a disulfide bond. Figure 1 provides a schematic representation of such a diabody, which utilizes an E-coil/K-coil heterodimer-promoting domain and a cysteine-containing linker for covalent attachment. As presented in FIGS. 2 and 3A-3C, one or both of the polypeptides may further have a CH2-CH3 domain sequence, thereby providing a link between the two diabody polypeptides. complexation forms an Fc region that can bind to Fc receptors on cells such as B lymphocytes, dendritic cells, natural killer cells, macrophages, neutrophils, eosinophils, basophils, and mast cells. Ru. As will be presented in more detail below, the CH2 and/or CH3 domains of such polypeptide chains need not be identical in sequence; advantageously, the conjugation between these two polypeptide chains Qualified to promote.

Numerous variants of such molecules have been described (e.g., U.S. Published Patent No. 2015/0175697; U.S. Published Patent No. 2014/0255407; U.S. Published Patent No.
4/0099318; US Published Patent No. 2013/0295121; US Published Patent No. 2010/0174053; and US Published Patent No. 2009/0060910; European Published Patent No. 2714079; No. 2376109; European Publication No. 2158221; and International Publication No. 2012/162068; International Publication No. 2012/018687; International Publication No. 2010/080538). These Fc region-containing DART® diabodies may include two pairs of polypeptide chains. The first polypeptide chain comprises (in the N-terminus to C-terminus direction): (i) a first domain comprising the binding region of the light chain variable domain (VL1) of a first immunoglobulin; (ii) a second domain; (iii) a second domain comprising a binding region of an immunoglobulin heavy chain variable domain (VH2); a third domain that serves to promote dimerization and covalently link the first and second polypeptides of the diabody to each other; and (iv) a CH2-CH3 domain. The second polypeptide comprises (in the N-terminus to C-terminus direction): (i) a first domain comprising the binding region of a second immunoglobulin light chain variable domain (VL2); (ii) a first a second domain comprising a binding region of an immunoglobulin heavy chain variable domain (VH1); and (iii) heterodimerization of a cysteine residue (or cysteine-containing domain) with the first polypeptide chain. and a heterodimer-promoting domain. Here, the two first polypeptides are complexed with each other to form an Fc region. Figures 3A-3C present schematic diagrams of three variants of such diabodies that utilize different heterodimerization promoting domains.

Other Fc region-containing DART® diabodies may include three polypeptide chains. The first polypeptide of such a DART® diabody contains three domains: (i) a VL1-containing domain; (ii) a VH2-containing domain; and (iii) a domain containing a CH2-CH3 sequence. do. The second polypeptide of such a DART® diabody is: (i) a VL2-containing domain; (ii) a VH1-containing domain; and (iii) a heterodimer with the first polypeptide chain of the diabody. Contains domains that promote assembly and covalent binding. The third polypeptide of such DART® diabodies comprises the CH2-CH3 sequence. Thus, the first and second polypeptide chains of such a DART® diabody, associated together, have a VL1/VH1 binding site capable of binding an epitope and a second polypeptide chain capable of binding an epitope. Forms a VL2/VH2 binding site. These relatively complex DART® molecules also have cysteine-containing domains that function to form covalent complexes. The first and second polypeptides are thus linked to each other via a disulfide bond involving cysteine residues within their respective third domains. In particular, the first and third polypeptide chains are complexed with each other to form an Fc region stabilized by disulfide bonds. Figures 4A-4B present a schematic diagram of such a diabody containing three polypeptide chains.

Still other Fc region-containing DART® diabodies comprise binding regions derived from up to three different immunoglobulin light and heavy chain variable domains (referred to as VL1/VH1, VL2/VH2 and VL3/VH3). The polypeptide chain may contain five polypeptide chains. For example, the first polypeptide chain of such a diabody may contain: (i) a VH1-containing domain; (ii) a CH1-containing domain; and (iii) a domain containing a CH2-CH3 sequence. The second and fifth polypeptide chains of such a diabody may contain: (i) a VL1-containing domain; and (ii) a CL-containing domain. The third polypeptide chain of such a diabody is: (i) a VH1-containing domain; (ii) a CH1-containing domain; (iii) a domain containing a CH2-CH3 sequence; (iv) a VL2-containing domain; (v) and (vi) a heterodimer-promoting domain, the heterodimer-promoting domain promoting dimerization of the third and fourth chains. The fourth polypeptide of such a diabody is: (i) a VL3-containing domain; (ii) a VH2-containing domain; and (iii) heterodimerizing and covalent with the third polypeptide chain of said diabody. It may contain domains that promote binding. Here, the first and third polypeptides are complexed with each other to form an Fc region. These relatively complex DART® molecules also have cysteine-containing domains that function to form covalent complexes, whereby each polypeptide chain is linked by disulfide bonds involving cysteine residues. Binds to at least one additional polypeptide chain. Preferably, such domains are aligned in an N-terminal to C-terminal direction. Figure 5 presents a schematic diagram of such a diabody containing five polypeptide chains.

For applications where a tetravalent molecule is desired but an Fc is not required, an alternative configuration is known in the art, which consists of two identical chains each having a VH1, VL2, VH2, and VL2 domain. including, but not limited to, quadrivalent tandem antibodies, also called "TandAbs", formed by homodimerization of No. 2010-0099853; US Published Patent No. 2011-020667; US Published Patent No. 2013-0189263; European Published Patent No. 1078004; European Published Patent No. 237186
6; European Publication No. 2361936; and European Publication No. 1293514; International Publication No. 1999/057150; International Publication No. 2003/025018; and International Publication No. 2013/013700).

Recently, trivalent structures incorporating two diabody-type binding domains and one non-diabody-type binding domain as well as an Fc region have been described (e.g. PCT Application No. PCT/US15/33076, entitled "Tri-Specific "Binding Molecules and Methods of Use Thereof", filed May 29, 2015; and PCT Application No. PCT/US15/33081, entitled "Tri
-Specific Binding Molecules That Specifically Bind to Multiple Cancer Antigens and Methods of Use Thereof'', filed May 29, 2015). Such trivalent molecules may be utilized to generate monospecific, bispecific or trispecific molecules. Figures 6A-6F present schematic diagrams of such trivalent molecules containing three or four polypeptide chains.

IV. LAG-3 Binding Molecules of the Invention Preferred LAG-3 binding molecules of the invention include antibodies, diabodies, BiTEs, etc., which contain continuous or discontinuous (e.g. conformational) portions of human LAG-3 ( epitope). The LAG-3 binding molecules of the invention preferably also exhibit the ability to bind LAG-3 molecules of one or more non-human species, especially primate species (and especially primate species such as cynomolgus monkeys). A representative human LAG-3 polypeptide (NCBI sequence NP_002277.4; containing a 22 amino acid residue signal sequence (underlined) and a 503 amino acid residue mature protein) has the amino acid sequence (SEQ ID NO: 5):
MWEAQFLGLL FLQPLWVAPV KP LQPGAEVP VVWAQEGAPA QLPCSPTIPL
QDLSLLRRAG VTWQHQPDSG PPAAAPGHPL APGPHPAAPS SWGPRPRRYT
VLSVGPGGLR SGRLPLQPRV QLDERGRQRG DFSLWLRPAR RADAGEYRAA
VHLRDRALSC RLRLRLGQAS MTASPPGSLR ASDWVILNCS FSRPDRPASV
HWFRNRGQGR VPVRESPHHH LAESFLFLPQ VSPMDSGPWG CILTYRDGFN
VSIMYNLTVL GLEPPTPLTV YAGAGSRVGL PCRLPAGVGT RSFLTAKWTP
PGGGPDLLVT GDNGDFTLRL EDVSQAQAGT YTCHIHLQEQ QLNATVTLAI
ITVTPKSFGS PGSLGKLLCE VTPVSGQERF VWSSLDTPSQ RSFSGPWLEA
QEAQLLSQPW QCQLYQGERL LGAAVYFTEL SSPGAQRSGR APGALPAGHL
LLFLILGVLS LLLLVTGAFG FHLWRRQWRP RRFSALEQGI HPPQAQSKIE
ELEQEPEPEP EPEPEPEPEP EPEQL
has.

In certain embodiments, LAG-3 binding molecules of the invention are characterized by any(s) of the following criteria:
(1) specifically binds to human LAG-3 endogenously expressed on the surface of stimulated human T cells;
(2) specifically binds to human LAG-3 with an equilibrium binding constant (K _D ) of 40 nM or less;
(3) specifically binds to human LAG-3 with an equilibrium binding constant (K _D ) of 0.5 nM or less;
(4) specifically binds to non-human primate LAG-3 (e.g. cynomolgus macaque LAG-3);
(5) specifically binds to non-human primate LAG-3 with an equilibrium binding constant (K _D ) of 50 nM or less;
(6) specifically binds to non-human primate LAG-3 with an equilibrium binding constant (K _D ) of 5 nM or less;
(7) inhibiting (i.e. blocking or interfering with) the binding of LAG-3 to MHC class II;
(8) stimulate immune response;
(9) stimulate antigen-specific T cell responses as a single agent;
(10) synergize with anti-PD-1 antibodies to stimulate antigen-specific T cell responses;
(11) binds to the same epitope of LAG-3 as the anti-LAG-3 antibody LAG-3 mAb 1 or LAG-3 mAb 6; and/or (12) LAG-3 binding (as determined by, e.g., Biacore analysis) does not compete with anti-LAG-3 antibody 257F (BMS986016, Medarex/BMS).

As used herein, the term "antigen specific T-cell response" refers to the response of a T-cell that results from stimulation of the T-cell by an antigen for which the T-cell is specific. Point. Non-limiting examples of T cell responses to antigen-specific stimulation include proliferation and cytokine production (eg, TNF-α, IFN-γ production). The ability of a molecule to stimulate an antigen-specific T cell response can be determined using, for example, the Staphylococcus aureus enterotoxin type B antigen ("SEB") stimulated PBMC assay described herein.

Preferred LAG-3 binding molecules of the present invention include mouse anti-LAG-3 monoclonal antibodies "LAG-3 mAb 1", "LAG-3 mAb 2", "LAG-3 mAb 3", "LAG-3 mAb 4", "LAG-3 mAb 5" and/or "LAG-3 mAb 6" VH and/or VL domain, more preferably, the CDR _H of the VH domain of such anti-LAG-3 monoclonal antibody and/or one, two or all of the CDR _L of the VL domain. Such preferred LAG-3 binding molecules include bispecific (or multispecific) antibodies, chimeric or humanized antibodies, BiTe, diabodies, etc., and binding molecules with variant Fc regions.

The invention inter alia:
(A) (1) Anti-LAG-3 antibody LAG-3 mAb 1 or hLAG-3 mAb 1
Three CDR _H of the VH domain of VL4;
(2) Three CDR _L of the VL domain of anti-LAG-3 antibody LAG-3 mAb 1;
(3) three CDR _H of the VH domain of the anti-LAG-3 antibody LAG-3 mAb 1 and three CDR _L of the VL domain of the anti-LAG-3 antibody LAG-3 mAb 1;
(4) VH domain of anti-LAG-3 antibody LAG-3 mAb 1;
(5) VL domain of anti-LAG-3 antibody LAG-3 mAb 1;
(6) VH and VL domains of anti-LAG-3 antibody LAG-3 mAb 1;
(B) (1) Three CDRs of the VH domain of anti-LAG-3 antibody LAG-3 mAb 2
_H ;
(2) Three CDR _L of the VL domain of anti-LAG-3 antibody LAG-3 mAb 2;
(3) three CDR _H of the VH domain of anti-LAG-3 antibody LAG-3 mAb 2 and three CDR _L of the VL domain of anti-LAG-3 antibody LAG-3 mAb 2;
(4) VH domain of anti-LAG-3 antibody LAG-3 mAb 2;
(5) VL domain of anti-LAG-3 antibody LAG-3 mAb 2;
(6) VH and VL domains of anti-LAG-3 antibody LAG-3 mAb 2;
(C) (1) Three CDR _H of the VH domain of anti-LAG-3 antibody LAG-3 mAb 3;
(2) Three CDR _L of the VL domain of anti-LAG-3 antibody LAG-3 mAb 3;
(3) three CDR _H of the VH domain of anti-LAG-3 antibody LAG-3 mAb 3 and three CDR _L of the VL domain of anti-LAG-3 antibody LAG-3 mAb 3;
(4) VH domain of anti-LAG-3 antibody LAG-3 mAb 3;
(5) VL domain of anti-LAG-3 antibody LAG-3 mAb 3;
(6) VH and VL domains of anti-LAG-3 antibody LAG-3 mAb 3;
(D) (1) Three CDR _H of the VH domain of anti-LAG-3 antibody LAG-3 mAb 4;
(2) Three CDR _L of the VL domain of anti-LAG-3 antibody LAG-3 mAb 4;
(3) three CDR _H of the VH domain of anti-LAG-3 antibody LAG-3 mAb 4, and three CDR _L of the VL domain of anti-LAG-3 antibody LAG-3 mAb 4;
(4) VH domain of anti-LAG-3 antibody LAG-3 mAb 4;
(5) VL domain of anti-LAG-3 antibody LAG-3 mAb 4;
(6) VH and VL domains of anti-LAG-3 antibody LAG-3 mAb 4;
(E) (1) Three CDR _H of the VH domain of anti-LAG-3 antibody LAG-3 mAb 5;
(2) Three CDR _L of the VL domain of anti-LAG-3 antibody LAG-3 mAb 5;
(3) three CDR _H of the VH domain of anti-LAG-3 antibody LAG-3 mAb 5 and three CDR _L of the VL domain of anti-LAG-3 antibody LAG-3 mAb 5;
(4) VH domain of anti-LAG-3 antibody LAG-3 mAb 5;
(5) VL domain of anti-LAG-3 antibody LAG-3 mAb 5;
(6) VH and VL domains of anti-LAG-3 antibody LAG-3 mAb 5;
(F) (1) Three CDR _H of the VH domain of anti-LAG-3 antibody LAG-3 mAb 6;
(2) Three CDR _L of the VL domain of anti-LAG-3 antibody LAG-3 mAb 6;
(3) three CDR _H of the VH domain of anti-LAG-3 antibody LAG-3 mAb 6 and three CDR _L of the VL domain of anti-LAG-3 antibody LAG-3 mAb 6;
(4) VH domain of anti-LAG-3 antibody LAG-3 mAb 6;
(5) VL domain of anti-LAG-3 antibody LAG-3 mAb 6;
(6) VH and VL domains of anti-LAG-3 antibody LAG-3 mAb 6;
(G) (1) Three CDR _L of the VL domain of anti-LAG-3 antibody hLAG-3 mAb 1 VL4;
(2) Three CDR _H of the VH domain of the anti-LAG-3 antibody LAG-3 mAb 1 and three CDRs of the VL domain of the anti-LAG-3 antibody hLAG-3 mAb 1 VL4.
DR _L
or LAG-3 mAb 1, LAG-3 mAb 2, LAG-3 mAb 3, LAG-3 mAb 4, LAG-3 mAb 5, or LAG-3 It relates to a LAG-3 binding molecule that binds to or competes for binding to the epitope to which mAb 6 immunospecifically binds.

A. Anti-LAG-3 antibody LAG-3 mAb 1
1. Mouse anti-human antibody LAG-3 mAb 1
The amino acid sequence of the VH domain of LAG-3 mAb 1 (SEQ ID NO: 6) is shown below (CDR _H residues are underlined).
QIQLVQSGPE LKKPGETVKI SCKASGYTFR NYGMN WVKQA PGKVLKWMG W
INTYTGESTY ADDFEG RFAF SLGTSASTAY LQINILKNED TATYFCAR ES
LYDYYSMDY W GQGTSVTVSS
CDR _H 1 of LAG-3 mAb 1 (SEQ ID NO: 8): NYGMN
CDR _H 2 of LAG-3 mAb 1 (SEQ ID NO: 9): WINTYTGESTYADDFEG
CDR _H 3 of LAG-3 mAb 1 (SEQ ID NO: 10): ESLYDYYSMDY

An exemplary polynucleotide encoding the VH domain of LAG-3 mAb 1 is SEQ ID NO: 7 (nucleotides encoding CDR _H residues are underlined).
:
cagatccagt tggtgcagtc tggacctgag ctgaagaagc ctggagagac
agtcaagatc tcctgcaagg cttctgggta taccttcaga aactatggaa
tgaac tgggt gaagcaggct ccaggaaagg ttttaaagtg gatgggc tgg
ataaacacct acactggaga gtcaacatat gctgatgact tcgaggga cg
gtttgccttc tctttgggaa cctctgccag cactgcctat ttgcagatca
acatcctcaa aaatgaggac acggctacat atttctgtgc aaga gaatcc
ctctatgatt actattctat ggactac tgg ggtcaaggaa cctcagtcac
cgtctcctca
It is.

The amino acid sequence of the VL domain of LAG-3 mAb 1 (SEQ ID NO: 11) is shown below (CDR _L residues are underlined):
DVVVTQTPLT LSVTIGQPAS ISC KSSQSLL HSDGKTYLN W LLQRPGQSPE
RLIY LVSELD S GVPDRFTGS GSGTDFTLKI SRVEAEDLGV YYC WQGTHFP
YT FGGGTKLE IK
CDR _L 1 of LAG-3 mAb 1 (SEQ ID NO: 13): KSSQSLLHSDGKTYLN
CDR _L 2 of LAG-3 mAb 1 (SEQ ID NO: 14): LVSELDS
CDR _L 3 of LAG-3 mAb 1 (SEQ ID NO: 15): WQGTHFPYT

An exemplary polynucleotide encoding the VL domain of LAG-3 mAb 1 is SEQ ID NO: 12 (nucleotides encoding CDR _L residues are underlined):
gatgttgtgg tgacccagac tccactcact ttgtcggtta ccattggaca
accagcctcc atctctcttgc a agtcaagtca gagcctctta catagtgatg
gaaagacata ttt gaattgg ttgttacaga ggccaggcca gtctccagag
cgcctaatct at ctggtgtc tgaactggac tct ggagtcc ctgacaggtt
cactggcagt ggatcaggga cagatttcac actgaaaatc agcagagtgg
aggctgagga tttgggagtt tattattgc t ggcaaggtac acattttccg
tacacg ttcg gaggggggac caagctggaa ataaaa
It is.

2. Humanization of anti-LAG-3 antibody LAG-3 mAb 1 to form "hLAG-3 mAb 1" By humanizing the mouse anti-human LAG-3 antibody LAG-3 mAb 1 described above, human recipient demonstrated the ability to humanize anti-LAG-3 antibodies to reduce antigenicity upon administration to. The humanization results in two humanized VH domains, referred to herein as "hLAG-3 mAb 1 VH-1" and "hLAG-3 mAb 1 VH-2", and "hLAG-3 mAb 1 VL". Four humanized VL domains were obtained, designated "hLAG-3 mAb 1 VL-1", "hLAG-3 mAb 1 VL-2", "hLAG-3 mAb 1 VL-3", and "hLAG-3 mAb 1 VL-4". Ta. Any of the humanized VL domains described above may be paired with the humanized VH domains described above. Therefore, any antibody comprising one of the humanized VL domains paired with the humanized VH domain is commonly referred to as "hLAG-3 mAb 1" and is a combination of humanized VH/VL domains. Certain combinations are designated by reference to specific VH/VL domains; for example, a humanized antibody comprising hLAG-3 mAb 1 VH-1 and hLAG-3 mAb 1 VL-2 is specifically designated as “hLAG-3 mAb 1 VL-2”. 3 mAb 1 (1.2).

The amino acid sequence of the VH domain VH-1 of hLAG-3 mAb 1 (SEQ ID NO: 16) is shown below (CDR _H residues are underlined):
QVQLVQSGAE VKKPGASVKV SCKASGYTFT NYGMN WVRQA PGQGLEWMG W
INTYTGESTY ADDFEG RFVF SMDTSASTAY LQISSLKAED TAVYYCAR ES
LYDYYSMDY W GQGTTVTVSS

An exemplary polynucleotide encoding hLAG-3 mAb 1 VH-1 is SEQ ID NO: 17 (nucleotides encoding CDR _H residues are underlined)
:
caggtgcaac tggttcaatc cggcgccgag gtgaaaaagc ctggcgcctc
cgtgaaagtg tcctgtaagg catctgggta tacgttcaca aattatggta
tgaac tgggt gcgacaggca ccagggcagg gactggaatg gatgggg tgg
atcaatactt atacaggcga gagtacttat gctgacgatt tcgaggc ag
atttgtcttc tccatggaca ccagcgctag taccgcttat ctccagatta
gttctctcaa ggcggaggac acagctgttt attattgtgc ccgc gagagt
ttgtatgact actatagcat ggattac tgg ggacaaggta caaccgtgac
agtgagttcc
It is.

The amino acid sequence of the VH domain VH-2 of hLAG-3 mAb 1 (SEQ ID NO: 18) is shown below (CDR _H residues are underlined):
QVQLVQSGAE VKKPGASVKV SCKASGYTFT NYGMN WVRQA PGQGLEWMG W
INTYTGESTY ADDFEG RFVF SMDTSASTAY LQISSLKAED TAVYFCAR ES
LYDYYSMDY W GQGTTVTVSS

An exemplary polynucleotide encoding hLAG-3 mAb 1 VH-2 is SEQ ID NO: 19 (nucleotides encoding CDR _H residues are underlined)
:
caggtgcaac tggttcaatc cggcgccgag gtgaaaaagc ctggcgcctc
cgtgaaagtg tcctgtaagg catctgggta tacgttcaca aattatggta
tgaac tgggt gcgacaggca ccagggcagg gactggaatg gatgggg tgg
atcaatactt atacaggcga gagtacttat gctgacgatt tcgaggc ag
atttgtcttc tccatggaca ccagcgctag taccgcttat ctccagatta
gttctctcaa ggcggaggac acagctgttt atttctgtgc ccgc gagagt
ttgtatgact actatagcat ggattac tgg ggacaaggta caaccgtgac
agtgagttcc
It is.

The amino acid sequence of the VL domain VL-1 of hLAG-3 mAb 1 (SEQ ID NO: 20) is shown below (CDR _L residues are underlined):
DIVMTQTPLS LSVTPGQPAS ISC KSSQSLL HSDGKTYLN W LLQKPGQSPE
RLIY LVSELD S GVPDRFSGS GSGTDFTLKI SRVEAEDVGV YYC WQGTHFP
YT FGGGTKVE IK

An exemplary polynucleotide encoding hLAG-3 mAb 1 VL-1 is SEQ ID NO: 21 (nucleotides encoding CDR _L residues are underlined)
:
gatatcgtta tgactcagac accactgtca ctgagtgtga ccccaggtca
gcccgctagt atttcctgt a aatcatccca gtccctcctg catagcgatg
gaaagaccta tttgaac tgg cttctgcaga aaccaggcca aagtccagag
agattgatct ac ctcgtttc agaactcgac agt ggagtgc ccgatcgctt
ctcagggtcc ggctctggga ctgattttac tctcaagatc tcaagagtgg
aggccgagga cgtcggggtt tactactgt t ggcagggtac ccacttccct
tataca tttg gcggaggcac aaaagtggag attaaa
It is.

The amino acid sequence of the VL domain VL-2 of hLAG-3 mAb 1 (SEQ ID NO: 22) is shown below (CDR _L residues are underlined):
DIVMTQTPLS LSVTPGQPAS ISC KSSQSLL HSDGKTYLN W LLQRPGQSPE
RLIY LVSELD S GVPDRFSGS GSGTDFTLKI SRVEAEDVGV YYC WQGTHFP
YT FGGGTKVE IK

An exemplary polynucleotide encoding hLAG-3 mAb 1 VL-2 is SEQ ID NO: 23 (nucleotides encoding CDR _L residues are underlined)
:
gatatcgtta tgactcagac accactgtca ctgagtgtga ccccaggtca
gcccgctagt atttcctgt a aatcatccca gtccctcctg catagcgatg
gaaagaccta tttgaac tgg cttctgcaga gaccaggcca aagtccagag
agattgatct ac ctcgtttc agaactcgac agt ggagtgc ccgatcgctt
ctcagggtcc ggctctggga ctgattttac tctcaagatc tcaagagtgg
aggccgagga cgtcggggtt tactactgt t ggcagggtac ccacttccct
tataca tttg gcggaggcac aaaagtggag attaaa
It is.

The amino acid sequence of the VL domain VL-3 of hLAG-3 mAb 1 (SEQ ID NO: 24) is shown below (CDR _L residues are underlined):
DIVMTQTPLS LSVTPGQPAS ISC KSSQSLL HSDGKTYLN W LLQKPGQPPE
RLIY LVSELD S GVPDRFSGS GSGTDFTLKI SRVEAEDVGV YYC WQGTHFP
YT FGGGTKVE IK

An exemplary polynucleotide encoding hLAG-3 mAb 1 VL-3 is SEQ ID NO: 25 (nucleotides encoding CDR _L residues are underlined)
:
gatatcgtta tgactcagac accactgtca ctgagtgtga ccccaggtca
gcccgctagt atttcctgt a aatcatccca gtccctcctg catagcgatg
gaaagaccta tttgaac tgg cttctgcaga aaccaggcca accgccagag
agattgatct ac ctcgtttc agaactcgac agt ggagtgc ccgatcgctt
ctcagggtcc ggctctggga ctgattttac tctcaagatc tcaagagtgg
aggccgagga cgtcggggtt tactactgt t ggcagggtac ccacttccct
tataca tttg gcggaggcac aaaagtggag attaaa

The amino acid sequence of the VL domain VL-4 of hLAG-3 mAb 1 (SEQ ID NO: 26) is shown below (CDR _L residues are underlined):
DIVMTQTPLS LSVTPGQPAS ISC KSSQSLL HSDAKTYLN W LLQKPGQPPE
RLIY LVSELD S GVPDRFSGS GSGTDFTLKI SRVEAEDVGV YYC WQGTHFP
YT FGGGTKVE IK

An exemplary polynucleotide encoding hLAG-3 mAb 1 VL-4 is SEQ ID NO: 27 (nucleotides encoding CDR _L residues are underlined)
:
gatatcgtta tgactcagac accactgtca ctgagtgtga ccccaggtca
gcccgctagt atttcctgt a aatcatccca gtccctcctg catagcgatg
caaagaccta tttgaac tgg cttctgcaga aaccaggcca accgccagag
agattgatct ac ctcgtttc agaactcgac agt ggagtgc ccgatcgctt
ctcagggtcc ggctctggga ctgattttac tctcaagatc tcaagagtgg
aggccgagga cgtcggggtt tactactgt t ggcagggtac ccacttccct
tataca tttg gcggaggcac aaaagtggag attaaa
It is.

CDR _L 1 of the VL domain VL-4 of hLAG-3 mAb 1 contains an amino acid substitution of glycine to alanine and has the amino acid sequence: KSSQSLLHSD A KTYLN (SEQ ID NO: 28) (the substituted alanine is underlined). ing). It is contemplated that similar substitutions could be incorporated into any of the LAG-3 mAb 1 CDR _L 1 domains described above.

B. Anti-LAG-3 antibody LAG-3 mAb 2
The amino acid sequence of the VH domain of LAG-3 mAb 2 (SEQ ID NO: 29) is shown below (CDR _H residues are underlined):
EVQLQQSGPE LVKPGASVKI SCKTSGYTFT DYNIH WLRQS HGESLEWIG Y
IYPYSGDIGY NQKFKN RATL TVDNSSSTAY MDLRSLTSED SAVFYCAR WH
RNYFGPWFAY WGQGTPVTVS A
CDR _H 1 of LAG-3 mAb 2 (SEQ ID NO: 31): DYNIH
VH CDR _H 2 of LAG-3 mAb 2 (SEQ ID NO: 32): YIYPYSGDIGYNQKFKN
VH CDR _H 3 of LAG-3 mAb 2 (SEQ ID NO: 33): WHRNYFGPWFAY

An exemplary polynucleotide encoding the VH domain of LAG-3 mAb 2 is SEQ ID NO: 30 (nucleotides encoding CDR _H residues are underlined):
gaggtccagc ttcagcagtc aggacctgag ctggtgaaac ctggggcctc
agtgaagatt tcctgcaaga cttctggata cacatttact gactacaaca
tacac tggtt gaggcagagc catggagaga gccttgagtg gattgga tat
atttatcctt acagtggtga tattggatac aaccagaagt tcaagaac ag
ggccacattg actgtagaca attcctccag cacagcctac atggatctcc
gcagcctgac atctgaagac tctgcagtct tttactgtgc aaga tggcac
aggaactact ttggcccctg gtttgcttac tggggccaag ggactccggt
cactgtctct gca
It is.

The amino acid sequence of the VL domain of LAG-3 mAb 2 (SEQ ID NO: 34) is shown below (CDR _L residues are underlined):
DIVLTQSPAS LAVSLGQRAT ISC KASQSVD YDGESYMN WY QQKPGQPPKL
LIY VVSNLES GIPARFSGSG SGTDFTLNIH PVEEEDAATY YC QQSSEDPL
T FGAGTKLEL K
CDR _L 1 of LAG-3 mAb 2 (SEQ ID NO: 36): KASQSVDYDGESYMN
CDR _L 2 of LAG-3 mAb 2 (SEQ ID NO: 37): VVSNLES
CDR _L 3 of LAG-3 mAb 2 (SEQ ID NO: 38): QQSSEDPLT

An exemplary polynucleotide encoding the VL domain of LAG-3 mAb 2 is SEQ ID NO: 35 (nucleotides encoding CDR _L residues are underlined):
gacattgtgc tgacccaatc tccagcttct ttggctgtgt ctctagggca
gagggccacc atctctcctgc a aggccagcca aagtgttgat tatgatggtg
aaagttatat gaac tggtac caacagaaac caggacagcc acccaaactc
ctcatttat g ttgtatccaa tctagaatct gggatcccag ccaggtttag
tggcagtggg tctgggacag acttcaccct caacatccat cctgtggagg
aggaggatgc tgcaacctat tac tgtcagc aaagtagtga ggatccgctc
acgttcggtg ctgggaccaa gctggagctg aaa
It is.

C. Anti-LAG-3 antibody LAG-3 mAb 3
The amino acid sequence of the VH domain of LAG-3 mAb 3 (SEQ ID NO: 39) is shown below (CDR _H residues are underlined):
EVRLQQSGPE LVKPGASVKI SCKASGYTFT DYNIH WVRQS HGQSLEWIG Y
IYPYNGDTGY NQKFKT KATL TVDNSSNTAY MELRSLASED SAVYYCTR WS
RNYFGPWFAY WGQGTLVTVS A
CDR _H 1 of LAG-3 mAb 3 (SEQ ID NO: 41): DYNIH
CDR _H2 of LAG-3 mAb 3 (SEQ ID NO: 42): YIYPYNGDTGYNQKFKT
CDR _H 3 of LAG-3 mAb 3 (SEQ ID NO: 43): WSRNYFGPWFAY

An exemplary polynucleotide encoding the VH domain of LAG-3 mAb 3 is SEQ ID NO: 40 (nucleotides encoding CDR _H residues are underlined):
gaggtccggc ttcagcagtc aggacctgag ctggtgaaac ctggggcctc
agtgaagata tcctgcaagg cttctggata cacattcact gactacaaca
ttcac tgggt gaggcagagc catggacaga gccttgagtg gattgga tat
atttatcctt ataatggtga tactggctac aaccagaagt tcaagacc aa
ggccacattg actgtagaca attcctccaa cacagcctac atggaactcc
gcagcctggc atctgaagac tctgcagtct attactgtac aaga tggagc
aggaactact ttggcccctg gtttgcttac tggggccaag ggactctggt
cactgtctct gca
It is.

The amino acid sequence of the VL domain of LAG-3 mAb 3 (SEQ ID NO: 44) is shown below (CDR _L residues are underlined):
DIVLTQSPTS LAVSLGQRAT ISC KASQSVD YDGDSYMN WY QQKPGQPPKL
LIY AASNLES GIPARFSGSG SGTDFTLNIH PVEEEDAATY YC QQSSEDPL
T FGAGTKLEL K
CDR _L1 of LAG-3 mAb 3 (SEQ ID NO: 46): KASQSVDYDGDSYMN
CDR _L2 of LAG-3 mAb 3 (SEQ ID NO: 47): AASNLES
CDR _L3 of LAG-3 mAb 3 (SEQ ID NO: 48): QQSSEDPLT

An exemplary polynucleotide encoding the VL domain of LAG-3 mAb 3 is SEQ ID NO: 45 (nucleotides encoding CDR _L residues are underlined):
gacattgtgc tgacccaatc tccaacttct ttggctgtgt ctctagggca
gagggccacc atctctcctgc a aggccagcca aagtgttgat tatgatggtg
atagttatat gaac tggtat caacagaaac caggacagcc acccaaactc
ctcatctat g ctgcatccaa tctagaatct gggatcccag ccaggtttag
tggcagtggg tctgggacag acttcaccct caacatccat cctgtggagg
aggaggatgc tgcaacctat tactgt cagc aaagtagtga ggatccgctc
acg ttcggtg ctgggaccaa gctggagctg aaa
It is.

D. Anti-LAG-3 antibody LAG-3 mAb 4
The amino acid sequence of the VH domain of LAG-3 mAb 4 (SEQ ID NO: 49) is shown below (CDR _H residues are underlined):
EVQLHQSGPE LVKPGASVKI SCKTSGYTFT DYNIH WVKQS HGKSLEWIG Y
IYPYNGDAGY NQNFKT KATL TVDNSSSTAY MELRSLTSED SAVYYCAR WN
MNYFGPWFAY WGQGTLVTVS A
CDR _H 1 of LAG-3 mAb 4 (SEQ ID NO: 51): DYNIH
CDR _H2 of LAG-3 mAb 4 (SEQ ID NO: 52): YIYPYNGDAGYNQNFKT
CDR _H 3 of LAG-3 mAb 4 (SEQ ID NO: 53): WNMNYFGPWFAY

An exemplary polynucleotide encoding the VH domain of LAG-3 mAb 4 is SEQ ID NO: 50 (nucleotides encoding CDR _H residues are underlined):
gaggtccagc ttcaccagtc aggacctgag ctggtgaaac ctggggcctc
agtgaagata tcctgcaaga cttctggata cactttcact gactacaaca
tacact gggt gaagcagagc catggaaaga gccttgagtg gattgga tat
atttatcctt acaatggtga tgctggctac aaccagaact tcaagacc aa
ggccacattg actgtagaca attcctccag cacagcctac atggagctcc
gcagcctgac atctgaggac tctgcagtct attactgtgc aaga tggaac
atgaactact ttggcccctg gtttgcttac tggggccaag ggactctggt
cactgtctct gcg
It is.

The amino acid sequence of the VL domain of LAG-3 mAb 4 (SEQ ID NO: 54) is shown below (CDR _L residues are underlined):
DIVLTQSPAS LAVSLGQRAT ISC KASQSVD YDGVTYIN WY QQKPGQPPKL
LIF AASNLES GIPARFSGSG SGTDFTLNIH PVEEEDAATY YC QQSNEDPL
T FGAGTKLEL K
CDR _L1 of LAG-3 mAb 4 (SEQ ID NO: 56): KASQSVDYDGVTYIN
CDR _L2 of LAG-3 mAb 4 (SEQ ID NO: 57): AASNLES
CDR _L3 of LAG-3 mAb 4 (SEQ ID NO: 58): QQSNEDPLT

An exemplary polynucleotide encoding the VL domain of LAG-3 mAb 4 is SEQ ID NO: 55 (nucleotides encoding CDR _L residues are underlined):
gacattgtgc tgacccaatc tccagcttct ttggctgtgt ctctagggca
gagggccacc atctctcctgc a aggccagcca aagtgttgat tatgatggtg
ttacttatat caac tggtac caacagaaac caggacagcc acccaaactc
ctcatcttt g ctgcatccaa tctagaatct gggatcccag ccaggtttag
tggcagtggg tctgggacag acttcaccct caacatccat cctgtggagg
aggaggatgc tgcaacctat tactgt cagc aaagtaatga ggatccgctc
acg ttcggtg ctgggaccaa gctggagctg aaa
It is.

E. Anti-LAG-3 antibody LAG-3 mAb 5
The amino acid sequence of the VH domain of LAG-3 mAb 5 (SEQ ID NO: 59) is shown below (CDR _H residues are underlined):
EVQLQQSGPE LVKPGASVKI SCKASGYTFT DYNIH WVKQS PGKSLEWIG Y
IYPYSGDFGY NQKFKS KATL TVDNSSSTAY MDLRSLTSED SAVFYCAR WH
RNYFGPWFAY WGQGTLVTVS A
CDR _H 1 of LAG-3 mAb 5 (SEQ ID NO: 61): DYNIH
CDR _H 2 of LAG-3 mAb 5 (SEQ ID NO: 62): YIYPYSGDFGYNQKFKS
CDR _H3 of LAG-3 mAb 5 (SEQ ID NO: 63): WHRNYFGPWFAY

An exemplary polynucleotide encoding the VH domain of LAG-3 mAb 5 is SEQ ID NO: 60 (nucleotides encoding CDR _H residues are underlined):
gaggtccagc ttcagcagtc aggacctgag ctggtgaaac ctggggcctc
agtgaagatt tcctgcaaag cttctggata cacatttact gactacaaca
tacac tgggt gaagcagagc cctggaaaga gccttgaatg gattgga tat
atttatcctt acagtggtga ttttggatac aaccagaagt tcaagagc aa
ggccacattg actgtagaca attcctccag cacagcctac atggatctcc
gcagcctgac atctgaggac tctgcagtct tttactgtgc aaga tggcac
aggaactact ttggcccctg gtttgcttac tggggccaag ggactctggt
cactgtctct gca
It is.

The amino acid sequence of the VL domain of LAG-3 mAb 5 (SEQ ID NO: 64) is shown below (CDR _L residues are underlined):
DIVLTQSPAS LAVSLGQRAT ISC KASQSVD YDGESYMN WY QQKPGQPPKL
LIY VVSNLES GIPARFSGSG SGTDFTLNIH PVEEEDAATY YC QQSSEDPL
T FGAGTKLEL K
CDR _L 1 of LAG-3 mAb 5 (SEQ ID NO: 66): KASQSVDYDGESYMN
CDR _L 2 of LAG-3 mAb 5 (SEQ ID NO: 67): VVSNLES
CDR _L 3 of LAG-3 mAb 5 (SEQ ID NO: 68): QQSSEDPLT

An exemplary polynucleotide encoding the VL domain of LAG-3 mAb 5 is SEQ ID NO: 65 (nucleotides encoding CDR _L residues are underlined):
gacattgtgc tgacccaatc tccagcttct ttggctgtgt ctctagggca
gagggccacc atctctcctgc a aggccagcca aagtgttgat tatgatggtg
aaagttatat gaac tggtac caacagaaac caggacagcc acccaaactc
ctcatttat g ttgtttccaa tctagaatct gggatcccag ccaggtttag
tggcagtggg tctgggacag acttcaccct caacatccat cctgtggagg
aggaggatgc tgcaacctat tactgt cagc aaagtagtga ggatccgctc
acg ttcggtg ctgggaccaa gctggagctg aaa
It is.

F. Anti-LAG-3 antibody LAG-3 mAb 6
1. Mouse anti-human antibody LAG-3 mAb 6
The amino acid sequence of the VH domain of LAG-3 mAb 6 (SEQ ID NO: 69) is shown below (CDR _H residues are underlined):
EVLLQQSGPE LVKPGASVKI PCKASGYTFT DYNMD WVKQS HGESLEWIG D
INPDNGVTIY NQKFEG KATL TVDKSSSTAY MELRSLTSED TAVYYCAR EA
DYFYFDY WGQ GTTLTVSS
LAG-3 mAb 6 CDR _H 1 (SEQ ID NO: 71): DYNMD
CDR _H 2 of LAG-3 mAb 6 (SEQ ID NO: 72): DINPDNGVTIYNQKFEG
CDR _H 3 of LAG-3 mAb 6 (SEQ ID NO: 72): EADYFYFDY

An exemplary polynucleotide encoding the VH domain of LAG-3 mAb 6 is SEQ ID NO: 70 (nucleotides encoding CDR _H residues are underlined):
gaggtcctgc tgcaacagtc tggacctgag ctggtgaagc ctggggcttc
agtgaagata ccctgcaagg cttctggata cacattcact gactacaaca
tggac tgggt gaagcagagc catggagaga gccttgagtg gattgga gat
attaatcctg acaatggtgt tactatctac aaccagaagt ttgaggc aa
ggccacactg actgtagaca agtcctccag tacagcctac atggagctcc
gcagcctgac atctgaggac actgcagtct attactgtgc aaga gaggcg
gattacttct actttgacta c tggggccaa ggcaccactc tcacagtctc
ctca
It is.

The amino acid sequence of the VL domain of LAG-3 mAb 6 (SEQ ID NO: 74) is shown below (CDR _L residues are underlined):
DIVMTQSHRF MSTSVGDRVS ITC KASQDVS SVVA WYQQKP GQSPKLLIF S
ASYRYT GVPD RFTGSGSGTD FTFTISSVQA ADLAVYYC QQ HYSTPWT FGG
GTKLEIK
CDR _L 1 of LAG-3 mAb 6 (SEQ ID NO: 76): KASQDVSSVVA
CDR _L 2 of LAG-3 mAb 6 (SEQ ID NO: 77): SASYRYT
CDR _L 3 of LAG-3 mAb 6 (SEQ ID NO: 78): QQHYSTPWT

An exemplary polynucleotide encoding the VL domain of LAG-3 mAb 6 is SEQ ID NO: 75 (nucleotides encoding CDR _L residues are underlined):
gacattgtga tgacccagtc tcacagattc atgtccacat cagttggaga
cagggtcagc atcacctgc a aggccagtca ggatgtgagt tctgttgtag
cc tggtatca acagaaacca ggacaatctc ctaaattact gattttt tcg
gcatcctacc ggtacact gg agtccctgat cgcttcactg gcagtggatc
tgggacggat ttcactttca ccatcagcag tgtgcaggct gcagacctgg
cagtttatta ctgt cagcaa cattatagta ctccgtggac g ttcggtgga
ggcaccaagc tggaaatcaa a
It is.

2. Humanization of the anti-LAG-3 antibody LAG-3 mAb 6 to form "hLAG-3 mAb 6" Humanization of the mouse anti-LAG-3 antibody LAG-3 mAb 6 described above and administration to human recipients demonstrated the ability to humanize anti-LAG-3 antibodies to reduce their antigenicity. Due to humanization, "hLAG-3 mAb 6 VH-1" and "hLAG-3
Two humanized VH domains, referred to as "mAb 6 VH-2" and two humanized VL domains, herein referred to as "hLAG-3 mAb 6 VL-1" and "hLAG-3 mAb 6 VL-2" were obtained. It was done. Any of the humanized VL domains can be paired with one of the humanized VH domains. Therefore, any antibody comprising one of the humanized VL domains paired with the humanized VH domain is generally referred to as "hLAG-3 mAb 6" and is a specific combination of humanized VH/VL domains. are named with reference to a specific VH/VL domain. For example, a humanized antibody comprising hLAG-3 mAb 6 VH-1 and hLAG-3 mAb 6 VL-2 is specifically referred to as "hLAG-3 mAb 6(1.2)."

The amino acid sequence of the VH domain VH-1 of hLAG-3 mAb 6 (SEQ ID NO: 79) is shown below (CDR _H residues are underlined):
QVQLVQSGAE VKKPGASVKV SCKASGYTFT DYNMD WVRQA PGQGLEWMG D INPDNGVTIY NQKFEG RVTM TTDTSTSTAY MELRSLRSDD TAVYYCAR EA DYFYFDY WGQ GTTLTVSS

An exemplary polynucleotide encoding hLAG-3 mAb 6 VH-1 is SEQ ID NO: 80 (nucleotides encoding CDR _H residues are underlined)
:
caggtccagc tggtgcagtc tggcgccgaa gtgaagaaac ctggcgcaag
cgtgaaggtg tcctgcaagg ccagcggcta caccttcacc gactacaaca
tggac tgggt ccgacaggcc ccaggacagg gcctggaatg gatgggc gac
atcaacccccg acaacggcgt gaccatctac aaccagaaat tcgagggc ag
agtgaccatg accaccgaca ccagcaccag caccgcctac atggaactgc
ggtccctgcg gagcgacgac accgccgtgt actactgcgc caga gaggcc
gactacttct acttcgacta c tggggccag ggcaccaccc tgaccgtgtc
ctcc
It is.

The amino acid sequence of the VH domain VH-2 of hLAG-3 mAb 6 (SEQ ID NO: 81) is shown below (CDR _H residues are underlined):
EVQLVESGGG LVKPGGSLRL SCAASGFTFS DYNMD WVRQA PGKGLEWVS D
INPDNGVTIY NQKFEG RFTI SRDNAKNSLY LQMNSLRAED TAVYYCAR EA
DYFYFDY WGQ GTTLTVSS

An exemplary polynucleotide encoding hLAG-3 mAb 6 VH-2 is SEQ ID NO: 82 (nucleotides encoding CDR _H residues are underlined)
:
gaggtccagc tggtggaatc tggcggcgga ctggtcaagc ctggcggcag
cctgagactg agctgcgctg ccagcggctt caccttcagc gactacaaca
tggac tgggt ccgacaggcc cctggcaagg gcctggaatg ggtgtcc gac
atcaacccccg acaacggcgt gaccatctac aaccagaagt tcgagggc cg
gttcaccatc agccgggaca acgccaagaa cagcctgtac ctgcagatga
acagcctgcg ggccgaggac accgccgtgt actactgcgc caga gaggcc
gactacttct acttcgacta c tggggccag ggcaccaccc tgaccgtgtc
ctcc
It is.

The amino acid sequence of the VL domain VL-1 of hLAG-3 mAb 6 (SEQ ID NO: 83) is shown below (CDR _L residues are underlined):
DIQMTQSPSS LSASVGDRVT ITC RASQDVS SVVA WYQQKP GKAPKLLIY S
ASYRYT GVPS RFSGSGSGTD FTLTISSLQP EDFATYYC QQ HYSTPWT FGG
GTKLEIK

An exemplary polynucleotide encoding hLAG-3 mAb 6 VL-1 is SEQ ID NO: 84 (nucleotides encoding CDR _L residues are underlined)
:
gacatccaga tgacccagag ccccagcagc ctgagcgcca gcgtgggcga
cagagtgacc atcacctgt c gggccagcca ggatgtgtcc agcgtggtgg
cc tggtatca gcagaagccc ggcaaggccc ccaagctgct gatctac agc
gccagctacc ggtacaca gg cgtgcccagc agattcagcg gcagcggctc
cggcaccgac ttcaccctga ccatcagcag cctgcagccc gaggacttcg
ccacctacta ctgc cagcag cactacagca ccccctggac c ttcggcgga
ggcaccaagc tggaaatcaa g
It is.

The amino acid sequence of the VL domain VL-2 of hLAG-3 mAb 6 (SEQ ID NO: 85) is shown below (CDR _L residues are underlined):
DIVMTQSPSS LSASVGDRVT ITC RASQDVS SVVA WYQQKP GKAPKLLIY S
ASYRYT GVPD RFSGSGSGTD FTFTISSLQP EDIAVYYC QQ HYSTPWT FGG
GTKLEIK

An exemplary polynucleotide encoding hLAG-3 mAb 6 VL-2 is SEQ ID NO: 86 (nucleotides encoding CDR _L residues are underlined)
:
gacatcgtga tgaccgag ccccagcagc ctgagcgcca gcgtgggcga
cagagtgacc atcacctgt c gggccagcca ggatgtgtcc agcgtggtgg
cc tggtatca gcagaagccc ggcaaggccc ccaagctgct gatctac agc
gccagctacc ggtacaca gg cgtgcccgat agattcagcg gcagcggctc
cggcaccgac ttcaccttca ccatcagcag cctgcagccc gaggacatcg
ccgtttacta ctgc cagcag cactacagca ccccctggac c ttcggcgga
ggcaccaagc tggaaatcaa g
It is.

CDR _L 1 of the VL domains VL-1 and VL-2 of hLAG-3 mAb 2 contains an amino acid substitution of lysine to arginine and has the amino acid sequence: R ASQDVSSVVA (SEQ ID NO: 87) (the substituted arginine is underlined). ). It is contemplated that similar substitutions could be incorporated into any of the LAG-3 mAb 6 CDR _L 1 domains described above.

Some changes to the amino acid sequences of the VH and/or VL domains presented herein are possible. For example, subcloning can be facilitated by substituting the C-terminal amino acid residues of the VH and/or VL domains described herein.

V. Anti-LAG-3 antibody LAG-3 mAb 1, LAG-3 mAb 2, LAG-3
mAb 3, LAG-3 mAb 4, LAG-3 mAb 5 and/or LAG-3
mAb 6 and their derivatives with engineered Fc regions In conventional immune function, the interaction of antibody-antigen complexes with cells of the immune system is characterized by a number of factors, including antibody-dependent cytotoxicity, mast cell degranulation, and phagocytosis. It produces a wide range of responses ranging from effector functions to immunomodulatory signals such as those controlling lymphocyte proliferation and antibody secretion. All of these interactions are initiated by the binding of the Fc region of the antibody or immune complex to specialized cell surface receptors on hematopoietic cells. The diversity of cellular responses triggered by antibodies and immune complexes is afforded by the structural heterogeneity of the three Fc receptors: FcγRI (CD64), FcγRII (CD32) and FcγRIII (CD16). FcγRI (CD64), FcγRIIA (CD32A) and FcγRIII (CD16) are activating (ie, immune system enhancing) receptors; FcγRIIB (CD32B) is an inhibitory (ie, immune system attenuating) receptor. Furthermore, interaction with neonatal Fc receptors (FcRn) mediates recycling of IgG molecules from endosomes to the cell surface and release into the blood. Exemplary IgG1 (SEQ ID NO: 1), IgG2 (SEQ ID NO: 2), IgG3 (SEQ ID NO: 3) and IgG4 (SEQ ID NO: 4) amino acid sequences have been previously presented.

Modifications of the Fc region usually lead to phenotypic changes, such as changes in serum half-life, changes in stability, changes in sensitivity to cellular enzymes, or changes in effector function. It may be desirable to modify antibodies or other binding molecules of the invention with respect to effector function to enhance the effectiveness of such molecules, for example in the treatment of cancer. In certain cases, reduction or elimination of effector function is desirable, for example, for antibodies whose mechanism of action involves blocking or antagonizing rather than killing cells bearing the target antigen. Increased effector function targets undesirable cells such as tumors and foreign cells that express low levels of FcγR, such as tumor-specific B cells that have low levels of FcγRIIB (e.g. non-Hodgkin's lymphoma, CLL and Burkitt's lymphoma). generally desirable in cases. In the embodiments described above, molecules of the invention with conferred or altered effector function activity are useful for the treatment and/or prevention of diseases, disorders or infections in which enhancement of the efficiency of effector function activity is desired.

In certain embodiments, a LAG-3 binding molecule of the invention comprises an Fc region that has one or more modifications (e.g., substitutions, deletions, or insertions) to the sequence of amino acids of a wild-type Fc region (e.g., SEQ ID NO: 1). , said modification reduces the affinity and binding activity of said Fc region, and thus of the molecules of the invention, for one or more FcγR receptors. In another embodiment, a molecule of the invention comprises an Fc region with one or more modifications to the amino acids of a wild-type Fc region, wherein said modification is one of said Fc regions and thus of a molecule of the invention. or increase affinity and binding activity for multiple FcγR receptors. In other embodiments, the molecule comprises a mutant Fc region, wherein the mutant has antibody-dependent cell-mediated cytotoxicity ( ADCC) activity and/or increase binding to FcγRIIA. In an alternative embodiment, the molecule comprises a mutant Fc region, wherein the mutant has no Fc region or a higher ADCC activity (or other effector function) than a molecule comprising a wild-type Fc region. conferring or mediating a decrease in and/or an increase in binding to FcγRIIB. In some embodiments, the invention encompasses a LAG-3 binding molecule comprising a mutant Fc region, wherein the mutant Fc region has a greater ability to bind to any FcγR than an equivalent molecule comprising a wild-type Fc region. Shows no detectable binding. In other embodiments, the invention encompasses LAG-3 binding molecules comprising a mutant Fc region, wherein the mutant Fc region is a single FcγR, preferably one of FcγRIIA, FcγRIIB, or FcγRIIIA. It only binds to. Such increased affinity and/or avidity preferably occurs in cells expressing low levels of FcγR when no avidity of the parent molecule (without a modified Fc region) is detectable in the cell. or a density of 30,000 to 20,000 molecules/cell, a density of 20,000 to 10,000 molecules/cell, a density of 10,000 to 5,000 molecules/cell, a density of 5,000 to 1,000 molecules/cell, a density of 1,000 to 200 molecules/cell, or a density of 200 molecules/cell. Less than a cell (but at least 1
The extent of detectable FcγR binding or FcγR-associated activity is assessed in vitro in cells expressing non-FcγR receptor target antigens at a density of 0, 50, 100, or 150 molecules/cell).

LAG-3 binding molecules of the invention may comprise variant Fc regions with altered affinity for activating and/or inhibiting Fcγ receptors. In one embodiment, the LAG-3 binding molecule is a mutant form that has increased affinity for FcγRIIB and decreased affinity for FcγRIIIA and/or FcγRIIA relative to an equivalent molecule with a wild-type Fc region. It includes an Fc region. In another embodiment, a LAG-3 binding molecule of the invention has decreased affinity for FcγRIIB and increased affinity for FcγRIIIA and/or FcγRIIA relative to an equivalent molecule with a wild-type Fc region. , comprising a mutant Fc region. In yet another embodiment, a LAG-3 binding molecule of the invention has reduced affinity for FcγRIIB and also has reduced affinity for FcγRIIIA and/or FcγRIIA relative to a comparable molecule with a wild-type Fc region. It has a mutant Fc region. In yet another embodiment, a LAG-3 binding molecule of the invention has an unchanged affinity for FcγRIIB and an altered affinity for FcγRIIIA and/or FcγRIIA relative to an equivalent molecule with a wild-type Fc region. It has a mutant Fc region with reduced (or increased) sex.

In certain embodiments, a LAG-3 binding molecule of the invention comprises a variant Fc region with altered affinity for FcγRIIIA and/or FcγRIIA such that the immunoglobulin has enhanced effector function. Non-limiting examples of effector cell functions include antibody-dependent cell-mediated cytotoxicity, antibody-dependent cell phagocytosis, phagocytosis, opsonization, opsonophagocytosis, cell binding, rosette formation, C1q binding, and complementarity determination. Cell-mediated cytotoxicity.

In certain preferred embodiments, the change in affinity or effector function is at least 2-fold, preferably at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8 times, at least 9 times, at least 10 times, at least 50 times, or at least 100 times. In other embodiments of the invention, the variant Fc region is at least 65%, preferably at least 70%, at least 75%, at least 80%, at least 85%, at least 90% relative to the molecule comprising the wild type Fc region. , immunospecifically binds to one or more FcRs with at least 95%, at least 100%, at least 125%, at least 150%, at least 175%, at least 200%, at least 225%, or at least 250% higher affinity. do. Such measurements can be in vivo or in vitro assays, and in preferred embodiments are in vitro assays such as ELISA or surface plasmon resonance assays.

In various embodiments, the LAG-3 binding molecules of the invention include a variant Fc region, wherein the variant stimulates at least one activity of an FcγR receptor or stimulates at least one activity of an FcγR receptor. antagonize. In a preferred embodiment, the molecule inhibits one or more activities of FcγRIIB, such as B cell receptor-mediated signaling, B cell activation, B cell proliferation, antibody production, B cell intracellular calcium influx, cell cycle progression, FcγRIIB-mediated inhibition of FcεRI signaling, phosphorylation of FcγRIIB, SHIP recruitment, SHIP phosphorylation and coupling with Shc, or activation of one or more downstream molecules in the FcγRIIB signaling pathway (e.g. MAP kinase , JNK, p38, or Akt). In another embodiment, a LAG-3 binding molecule of the invention comprises a variant that stimulates one or more activities of FcεRI, such as mast cell activation, calcium mobilization, degranulation, cytokine production, or serotonin release. .

In certain embodiments, the molecule comprises two or more IgG isotypes (e.g., IgG1,
IgG2, IgG3 and IgG4). As used herein, an Fc region is described as being of a particular IgG isotype if its amino acid sequence has the highest homology with that particular IgG isotype compared to other IgG isotypes. Ru. The various IgG isotypes mentioned above are described, for example, in Flesch, and Neppert (1999) J. Clin. Lab. Anal. 14: 141-156; Chappel et al. (1993) J. Biol. Chem. 33:25124-25131; Chappel et al. al. (1991) Proc. Natl. Acad. Sci. (USA) 88:9036-9040; or its hinge as described in Bruggemann et al. (1987) J. Exp. Med 166: 1351-1361. and/or differences in the amino acid sequence of the Fc region exhibit different physical and functional properties, including serum half-life, complement fixation, FcγR binding affinity, and effector function activity (eg, ADCC, CDC, etc.). This type of variant Fc region can be used alone or in combination with amino acid modifications to affect Fc-mediated effector function and/or binding activity. In combination, the amino acid modification and the IgG hinge/Fc region can exhibit similar functionality (e.g., increased affinity for FcγRIIA) and may act additively, or more preferably synergistically, to The effector functionality of the molecules of the invention can be modified compared to molecules of the invention that contain a type Fc region. In other embodiments, the amino acid modification and the IgG Fc region can exhibit opposite functionality (e.g., increased and decreased affinity for FcγRIIA, respectively) and can also be used in combinations of the same isotype, without an Fc region or with a wild-type It can act to selectively modulate or reduce the specific functionality of molecules of the invention compared to molecules of the invention that contain an Fc region.

In a preferred specific embodiment, the LAG-3 binding molecule of the invention comprises a mutant Fc region, said mutant Fc region comprising at least one amino acid modification relative to the wild-type Fc region, thereby making said mutant Substitutions in which the Fc region makes direct contact with FcγRs based on crystallographic and structural analyzes of Fc-FcR interactions such as those disclosed in Sondermann et al. (2000) Nature 406:267-73 In the absence of this molecule, the affinity of the molecule for FcR changes. Examples of positions within the Fc region that make direct contact with FcγRs are amino acid residues 234-239 (hinge region), amino acid residues 265-269 (B/C loop), amino acid residues 297-299 (C'/E loop). ) and amino acid residues 327-332 (F/G loop). In some embodiments, the molecules of the invention include modifications of at least one residue that does not make direct contact with the FcγR, e.g., is not within the Fc-FcγR binding site, based on structural and crystallographic analysis. Contains a type Fc region.

Variant Fc regions are known in the art and affect the effector functions exhibited by molecules of the invention comprising an Fc region (or a portion thereof), as functionally assayed, for example, in NK-dependent or macrophage-dependent assays. Any known variant Fc region may be used in the present invention to impart or modify Fc regions. For example, Fc region variants identified as altered effector functions have been described in WO 04/063351; WO 06/088494; Any suitable variant disclosed in WO 07/021841; WO 07/106707; and WO 2008/140603 may be used in the molecules of the invention.

In certain embodiments, a LAG-3 binding molecule of the invention comprises a variant Fc region having one or more amino acid modifications in one or more regions, wherein the one or more modifications Altering the ratio of the affinity of an Fc region for an activating FcγR (such as FcγRIIA or FcγRIIIA) to an inhibitory FcγR (such as FcγRIIB) (relative to a wild-type Fc region):

Particularly preferred are LAG-3 binding molecules of the invention having a mutant Fc region in which the affinity ratio of the mutant Fc region (relative to the wild type Fc region) is greater than 1. Such molecules may be useful in therapeutic or prophylactic treatment of diseases, disorders, or infections, such as cancer or infectious diseases, in which enhancement of the efficiency of FcγR-mediated effector cell functions (e.g., ADCC) is desired, or amelioration of symptoms thereof. It has a specific use in providing. In contrast, mutant Fc regions with affinity ratios less than 1 mediate reduced efficiency of effector cell function. Table 1 lists exemplary single, double, triple, quadruple and quintuple mutations according to whether their affinity ratios are greater than or less than one.

In certain specific embodiments, in the variant Fc region, any amino acid modification at any of positions 235, 240, 241, 243, 244, 247, 262, 263, 269, 298, 328, or 330 (e.g. substitution), and preferably one or more of the following residues: A240, I240, L241, L243, H244, N298, I328 or V330. In different specific embodiments, in the variant Fc region, 268, 269, 270, 272, 276, 278, 283, 285, 286, 289, 292, 293, 301, 303, 305, 307, 309, 331, Any amino acid modification (e.g. substitution) at any of positions 333, 334, 335, 337, 338, 340, 360, 373, 376, 416, 419, 430, 434, 435, 437, 438 or 439, preferably The following residues: H280, Q280, Y280, G290, S290, T290, Y290, N294, K295, P296, D298, N298, P298, V298, I300 or L300.

In certain preferred embodiments, 255, 256, 258, 267, 268, 269, 270, 272, 276, 278, 280, 283, 285, 286, 289 , 290, 292, 293, 294, 295, 296, 298, 300, 301, 303, 305, 307, 309, 312, 320, 322, 326, 329, 330, 332, 331, 333, 334, 335, 337 , 338, 339, 340, 359, 360, 373, 376, 416, 419, 430, 434, 435, 437, 438 or 439. Preferably, the variant Fc region has any one of the following residues: A256, N268, Q272, D286, Q286, S286, A290, S290, A298, M301, A312, E320, M320, Q320, R320, E322, A326, D326, E326, N326, S326, K330, T339, A333, A334, E334, H334, L334, M334, Q334, V334, K335, Q335, A359, A360 or A430.

In different embodiments, in a variant Fc region that binds (via its Fc region) to an FcγR with reduced affinity, 252, 254, 265, 268, 269, 270, 278, 289, 292, 293, 294, 295, 296, 298, 300, 301, 303, 322, 324, 327, 329, 333, 335, 338, 340, 373, 376, 382, 388, 389, 414, 416, 419, 434, 435, 437, Any amino acid modification (eg, substitution) at either position 438 or 439.

In different embodiments, 280, 283, 285, 286, 290, 294, 295, 298, 300, 301, 305 in a variant Fc region that binds (via its Fc region) to an FcγR with enhanced affinity. , 307, 309, 312, 315, 331, 333, 334, 337, 340, 360, 378, 398, or 430. In different embodiments, any one of the following residues in the variant Fc region that binds FcγRIIA with enhanced affinity: A255, A256, A258, A267, A268, N268, A272, Q272, A276. , A280, A283, A286, A286, D286, D286, Q286, S286, S290, S290, S301, M320, Q320, Q320, Q320, R320, E322, D326, E326, S326, A 331, Q335, A337 or A430.

Preferred variants are 228, 230, 231, 232, 233, 234, 235, 239, 240, 241, 243, 244, 245, 247, 262, 263, 264, 265, 266, 271, 273, 275, 281 , 284, 291, 296, 297, 298, 299, 302, 304, 305, 313, 323, 325, 326, 328, 330 or 332.

Particularly preferred variants include one or more modifications selected from groups A to AI:

Further particularly preferred variants include one or more modifications selected from groups 1 to 105:

In one embodiment, a multi-LAG-3 binding molecule of the invention comprises a variant Fc region having at least one modification in the Fc region. In certain embodiments, said variant Fc domain comprises at least one substitution selected from the group consisting of L235V, F243L, R292P, Y300L, V305I, and P396L, wherein said numbering is the EU index according to Kabat. numbering.

In certain specific embodiments, the variant Fc region comprises:
(A) at least one substitution selected from the group consisting of F243L, R292P, Y300L, V305I, and P396L;
(B) (1) F243L and P396L;
(2) F243L and R292P; and (3) R292P and V305I;
at least two permutations selected from the group consisting of;
(C) (1) F243L, R292P and Y300L;
(2) F243L, R292P and V305I;
(3) F243L, R292P and P396L; and (4) R292P, V305I and P396L;
at least three permutations selected from the group consisting of;
(D) (1) F243L, R292P, Y300L and P396L; and (2) F243L, R292P, V305I and P396L;
or (E) (1) F243L, R292P, Y300L, V305I and P396L; and (2) L235V, F243L, R292P, Y300L and P396L.
At least five permutations selected from the group consisting of.

In another specific embodiment, the variant Fc region comprises the following substitutions:
(A) F243L, R292P, and Y300L;
(B) L235V, F243L, R292P, Y300L, and P396L; or (C) F243L, R292P, Y300L, V305I, and P396L.

In one embodiment, the LAG-3 binding molecules of the invention (compared to the binding exhibited by the wild-type IgG1 Fc region (SEQ ID NO: 1)) include FcγRIA (CD64), FcγRIIA (CD32A), FcγRIIB (CD32B), FcγRIIIA ( CD16a) or FcγRIIIB (CD16b) with reduced binding (or substantially no binding). In one embodiment, a LAG-3 binding molecule of the invention is a mutant that has reduced (or substantially no binding) to an FcγR (e.g., FcγRIIIA) and has reduced (or no) ADCC effector function. type Fc region. In certain embodiments, the variant Fc region comprises at least one substitution selected from the group consisting of L234A, L235A, D265A, N297Q, and N297G. In certain specific embodiments, the variant Fc region comprises the following substitutions: L234A; L235A; L234A and L235A; D265A; N297Q or N297G.

In a different embodiment, a LAG-3 binding molecule of the invention has reduced binding (or substantially no binding) to FcγRIIIA (CD16a) (relative to the binding exhibited by the wild-type IgG1 Fc region (SEQ ID NO: 1)); and/or an Fc region with reduced effector function. In certain specific embodiments, a LAG-3 binding molecule of the invention comprises an IgG2 Fc region (SEQ ID NO: 2) or an IgG4 Fc region (SEQ ID NO: 4). When utilizing the IgG4 Fc region, the present invention provides an IgG4 hinge region S228P substitution (e.g., SEQ ID NO: 117: ESKYGPPCP P CP (Lu et al., (2008) “The Effect Of It also includes the introduction of stabilizing mutations such as A Point Mutation On The Stability Of Igg4 As Monitored By Analytical Ultracentrifugation,” J. Pharmaceutical Sciences 97:960-969). Other stabilizing mutations known in the art may be introduced into the IgG4 Fc region (Peters, P et al., (2012) “Engineering an Improved IgG4 Molecule with Reduced Disulfide Bond Heterogeneity and Increased Fab Domain Thermal Stability,” J. Biol. Chem., 287:24525-24533; WO 2008/145142).

In other embodiments, the invention provides: Jefferis, BJ et al. (2002) Interaction Sites On Human IgG-Fc For FcgammaR: Current Models," Immunol. Lett. 82:57-65; Presta, LG
et al. (2002) "Engineering Therapeutic Antibodies For Improved Function" Biochem. Soc. Trans. 30:487-90; Idusogie, EE et al. (2001) "Engineered Antibodies With Increased Activity To Recruit Complement," J. Immunol 166:2571-75; Shields, RL et al. (2001) "High Resolution Mapping Of The Binding Site On Human IgGl For Fc Gamma RI, Fc Gamma RII, Fc Gamma RIII, And FcRn And Design Of IgGl Variants With Improved Binding To The Fc gamma R " J. Biol. Chem. 276:6591-6604; Idusogie, EE et al. (2000) "Mapping Of The Clq Binding Site On Rituxan, A Chimeric Antibody With A Human IgG Fc," J. Immunol . 164:4178-84; Reddy, MP et al. (2000) "Elimination Of Fc Receptor-Dependent Effector Functions Of A Modified IgG4 Monoclonal Antibody To Human CD4" J. Immunol. 164: 1925-1933; Xu, D. et al. (2000) "In Vitro Characterization of Five Humanized OKT3 Effector Function Variant Antibodies" Cell. Immunol. 200: 16-26; Armour, KL et al. (1999) "Recombinant human IgG Molecules Lacking Fcgamma Receptor I Binding And Monocyte Triggering Activities " Eur. J. Immunol. 29:2613-24; Jefferis, R. et al. (1996) "Modulation Of
Fc(Gamma)R And Human Complement Activation By IgG3-Core Oligosaccharide Interactions " Immunol. Lett. 54: 101-04; Lund, J. et al. (1996) "Multiple Interactions
Of IgG With Its Core Oligosaccharide Can Modulate Recognition By Complement And
Human Fc Gamma Receptor I And Influence The Synthesis Of Its Oligosaccharide Chains " J. Immunol. 157:4963-4969; Hutchins et al. (1995) "Improved Biodistribution, Tumor Targeting, And Reduced Immunogenicity In Mice With A Gamma 4 Variant Of Campath -IH," Proc. Natl. Acad. Sci. (USA) 92: 11980-84; Jefferis, R. et al.
(1995) "Recognition Sites On Human IgG For Fc Gamma Receptors: The Role Of Glycosylation," Immunol. Lett. 44: 111-17; Lund, J. et al. (1995) "Oligosaccharide-Protein Interactions In IgG Can Modulate Recognition By Fc Gamma Receptors " FASEB J. 9: 115-19; Alegre, M . et al. (1994) "A Non-Activating "Humanized" Anti-CD3
Monoclonal Antibody Retains Immunosuppressive Properties In Vivo," Transplantation 57: 1537-1543; Lund et al. (1992) "Multiple Binding Sites On The CH2 Domain Of IgG For Mouse Fc Gamma RII," Mol. Immunol. 29:53-59; Lund et al. (1991) "Human Fc Gamma RI And Fc Gamma RII Interact With Distinct But Overlapping Sites On Human IgG," J. Immunol. 147:2657-2662; Duncan, AR et al. (1988) "Localization Of The Binding Site For The Human High-Affinity Fc Receptor On IgG," Nature 332:563-564; U.S. Patent No. 5,624,821; U.S. Patent No. 5,885,573; U.S. Patent No. 6,194,551 ; U.S. Pat. No. 7,276,586; and U.S. Pat. No. 7,317,091; and any of the known including the use of Fc variants of.

In some embodiments, molecules of the invention may further comprise one or more glycosylation sites, whereby one or more carbohydrate moieties are covalently attached to the molecule. Preferably, molecules of the invention having one or more glycosylation sites and/or one or more modifications in the Fc region exhibit enhanced antibody-mediated effector functions, e.g. enhanced imparting or having a specific ADCC activity. In some embodiments, the present invention further provides compounds that are known to interact directly or indirectly with carbohydrate moieties of the Fc region. , 262, 264, 265, 296, 299, or 301. Amino acids that interact directly or indirectly with carbohydrate moieties of the Fc region are known in the art and are described, for example, in Jefferis et al. (1995) Immunology Letters, 44: 111-7, which is incorporated by reference in its entirety. (incorporated into this application).

In another embodiment, the invention preferably relates to the functionality of the molecule, e.g. targeting molecules or Fc
Includes molecules that have been modified by introducing one or more glycosylation sites into one or more sites of the molecule without altering the binding activity to γR. Glycosylation sites may be introduced into the variable and/or constant regions of the molecules of the invention. As used herein, a "glycosylation site" refers to an antibody to which an oligosaccharide (i.e., a carbohydrate containing two or more monosaccharides linked together) is specifically covalently attached. including any specific amino acid sequence in. Oligosaccharide side chains are typically linked to the antibody backbone via N-linkages or O-linkages. N-linked glycosylation refers to the attachment of oligosaccharide moieties to the side chains of asparagine residues. O-linked glycosylation refers to the attachment of oligosaccharide moieties to hydroxy amino acids such as serine, threonine, etc. Molecules of the invention may comprise one or more glycosylation sites, including N-linked and O-linked glycosylation sites. Any glycosylation site for N-linked or O-linked glycosylation known in the art may be used in accordance with the present invention. An exemplary N-linked glycosylation site that can be used according to the methods of the invention is the amino acid sequence: Asn-X-Thr/Ser, where X can be any amino acid and Thr/Ser can be threonine or serine. show. Such sites or sites may be introduced into the molecules of the invention using methods known in the art to which the invention pertains (e.g., IN VITRO MUTAGENESIS, RECOMBINANT DNA: A SHORT COURSE, JD Watson , et al. WH Freeman and Company, New York, 1983, chapter 8, pp. 106-116 (incorporated in its entirety into this application). Exemplary methods for introducing glycosylation sites into molecules of the invention may include: modifying or mutating the amino acid sequence of the molecule to obtain the desired Asn-X-Thr/Ser sequence. .

In some embodiments, the invention encompasses methods of modifying the carbohydrate content of the molecules of the invention by adding or deleting glycosylation sites. Methods of altering the carbohydrate content of antibodies (and molecules that include antibody domains, eg, Fc regions) are known in the art and are encompassed by the present invention. For example, US Patent No. 6,218,149; European Patent No. 0359096B1; US Patent Application No. 2002/0028486; WO 03/035835; No. 149; US Pat. No. 6,472,511, incorporated herein by reference in its entirety. In other embodiments, the invention encompasses methods of altering the carbohydrate content of the molecules of the invention by deleting one or more endogenous carbohydrate moieties of the molecule. In certain specific embodiments, the invention involves shifting the glycosylation site of the Fc region of the antibody by modifying the position adjacent to position 297. In certain specific embodiments, the invention includes glycosylating position 296 rather than position 297 by modifying position 296.

Effector functions may also be achieved by introducing one or more cysteine residues in the Fc region, allowing for interchain disulfide bonds in this region, with the result that absorption capacity may be improved and/or complement-mediated Can be modified by producing homodimeric antibodies that can increase cell killing and ADCC (Caron, PC et al. (1992) "Engineered Humanized Dimeric Forms Of IgG Are More Effective Antibodies J. Exp. Med. 176 : 1191-1195; Shopes, B. (1992) "A Genetically Engineered Human IgG Mutant With Enhanced Cytolytic Activity," J. Immunol. 148(9):2918-2922). Homodimer with enhanced antitumor activity. Antibodies can also be prepared using heterobifunctional cross-linkers as described in Wolff, EA et al. (1993) "Monoclonal Antibody Homodimers: Enhanced Antitumor Activity In Nude Mice," Cancer Research 53:2560-2565. Alternatively, antibodies can be engineered that have dual Fc domains, thereby enhancing complement lysis and ADCC capabilities (Stevenson, GT et al. (1989) "A Chimeric Antibody With Dual Fc Regions (bisFabFc) Prepared". By Manipulations At The IgG Hinge "Anti-Cancer Drug Design 3:219-230)
.

The serum half-life of molecules of the invention comprising an Fc region can be increased by increasing the binding affinity of the Fc region for FcRn. As used herein, the term "half-life" refers to the pharmacokinetic property of a molecule that is a measure of the average survival time of the molecule after administration. Fifty percent (50%) of a known amount of a molecule is eliminated from a subject's body (e.g., a human patient or other mammal) or a particular body cavity thereof, as measured in the body (i.e., circulating half-life) or other tissue. In general, an increase in half-life leads to an increase in the mean residence time (MRT) of the administered molecule in the circulation.

In some embodiments, the LAG-3 binding molecules of the invention contain at least one amino acid modification relative to the wild-type Fc region and exhibit an increased half-life (relative to the wild-type Fc region). type Fc region.

In some embodiments, the LAG-3 binding molecules of the invention are numbered by the EU index according to Kabat: 238, 250, 252, 254, 256, 257, 256, 265, 272, 286, 288, 303, selected from the group consisting of 305, 307, 308, 309, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, 413, 424, 428, 433, 434, 435 and 436 A variant Fc region is provided that includes a half-life extending amino acid substitution at one or more positions. A number of specific mutations are known in the art that can increase the half-life of Fc region-containing molecules, including M252Y, S254T, T256E, and combinations thereof. For example: U.S. Patent No. 6,277,375; U.S. Patent No. 7,083,784; U.S. Patent No. 7,217,797; U.S. Patent No. 8,088,376; U.S. Published Patent No. 2002/0147311 ; US Publication No. 2007/0148164; and WO 98/23289; WO 2009/058492; and WO 2010/033279, which are incorporated by reference into this application in their entirety. See mutations described in . Fc region-containing molecules with enhanced half-life include Fc region residues 250, 252, 254, 256, 257, 288, 307, 308, 309, 311, 378, 428, 433, 434, 435 and 436. Also included are substitutions in two or more of , especially those selected from T250Q, M252Y, S254T, T256E, K288D, T307Q, V308P, A378V, M428L, N434A, H435K and Y436I.

In certain specific embodiments, the variant Fc region comprises the following substitutions:
(A) 252Y, 254T and 256E;
(B) M252Y and S254T;
(C) M252Y and T256E;
(D) 250Q and 428L;
(E) T307Q and N434A;
(F) A378V and N434A;
(G) N434A and Y436I;
(H) V308P and N434A; or (I) K288D and H435K.

The invention further provides:
Also encompassed are variant Fc regions that include (A) one or more mutations that alter effector function and/or FcγR; and (B) one or more mutations that increase serum half-life.

VI. Bispecific LAG-3 Binding Molecules of the Invention One embodiment of the invention relates to a bispecific binding molecule capable of binding to a "first epitope" and a "second epitope", wherein said first The epitope is an epitope of human LAG-3, and the second epitope is the same or a different epitope of LAG-3, or is present on the surface of an immune cell (such as a T lymphocyte) for immune check. It is an epitope of another molecule involved in the regulation of point. In certain embodiments, the second epitope is preferably not an epitope of LAG-3. In one embodiment, the second epitope is B7-H3, B7-H4, BTLA, CD3, CD8, CD16, CD27, CD32, CD40, CD40L, CD47, CD64, CD70, CD80, CD86, CD94, CD137, CD137L, CD226, CTLA-4, Galectin-9, GITR, GITRL, HHLA2, ICOS, ICOSL, KIR, LAG-3, LIGHT, MHC class I or II, NKG2a, NKG2d, OX40, OX40L, PD1H, PD-1, It is an epitope of PD-L1, PD-L2, PVR, SIRPa, TCR, TIGIT, TIM-3 or VISTA. In certain specific embodiments, the second epitope is CD137, PD-1, OX40, TIGIT or TIM-3. In certain embodiments, such bispecific molecules comprise three or more epitope binding sites. Such bispecific molecules may, for example, bind two or more different epitopes of LAG-3 and at least one epitope of a molecule that is not LAG-3.

The present invention provides LAG-3 and the second epitope (e.g. B7-H3, B7-H4, BTLA, CD40, CD80, CD86, CD137, CTLA-4, ICOS, KIR, MHC class I or II, OX40, PD -1, PD-L1, TCR, TIM-3, etc.). In some embodiments, the bispecific antibody capable of simultaneously binding PD-1 and the second epitope is WO 1998/002463, WO 2005/070966, WO 2006/107786 International Publication No. 2007/024715, International Publication No. 2007/075270, International Publication No. 2006/107617, International Publication No. 2007/046893, International Publication No. 2007/146968, International Publication No. 2008/003103, International Publication No. 2008/003116, International Publication No. 2008/027236, International Publication No. 2008/024188, International Publication No. 2009/132876, International Publication No. 2009/018386, International Publication No. 2010/028797, International Publication No. International Publication No. 2010028796, International Publication No. 2010/028795, International Publication No. 2010/108127, International Publication No. 2010/136172, International Publication No. 2011/086091, International Publication No. 2011/133886, International Publication No. 2012/009544 , WO 2013/003652, WO 2013/070565, WO 2012/162583, WO 2012/156430, WO 2013/174873, and WO 2014/022540 produced using any of the methods described, each of which is incorporated by reference into this application in its entirety.

1. Bispecific Diabodies Without Fc Regions One embodiment of the invention comprises a first polypeptide chain and a second polypeptide chain, most preferably said first polypeptide chain and a second polypeptide chain. Regarding bispecific monovalent diabodies, consisting of peptide chains, the sequence is such that said polypeptide chains are covalently linked to each other to form a first epitope ("Epitope 1") and a second epitope ("Epitope 1"). 2') (these epitopes are not identical to each other) to form covalent diabodies that can bind simultaneously. Such a bispecific diabody therefore has a "VL1"/"VH1" domain (VL _epitope /VH _epitope ) capable of binding to the first epitope.
_VL2 /VH2 domain (VL _{epitope 2} /VH _{epitope 2} ) capable of binding to the second epitope. Notation “VL1” and “V
"H1" refers to the variable light chain domain and the variable heavy chain domain, respectively, that bind to the "first" epitope of such a bispecific diabody. Similarly, the notations "VL2" and "VH2" refer to the variable light and variable heavy chain domains, respectively, that bind to the "second" epitope of such bispecific diabodies. In one embodiment, epitope 1 of such a diabody molecule is an epitope of LAG-3 and epitope 2 of such a diabody molecule is not an epitope of LAG-3 (e.g. B7-H3, B7- H4, BTLA, CD40, CD80, CD86, CD137, CTLA-4, ICOS, KIR, MHC class I or II, OX40, PD-1, PD-L1, TCR, TIM-3, etc.).

The VL domain of the first polypeptide chain of such a LAG-2-binding diabody interacts with the VH domain of the second polypeptide chain to bind to the first antigen (i.e., LAG-3 or the second polypeptide chain). forming a first functional epitope binding site specific for the antigen (containing two epitopes). Similarly, the VL domain of said second polypeptide chain interacts with the VH domain of said first polypeptide chain to generate a second antigen (i.e., an antigen containing said second epitope or LAG-3). forming a second functional epitope binding site specific for. Therefore, the selection of the VL and VH domains of the first and second polypeptide chains is such that the two polypeptide chains of the diabody together target both the LAG-3 epitope and the second epitope. The VL and VH domains are aligned such that they have VL and VH domains capable of binding (ie, they together comprise the VL _LAG-3 /VH _LAG-3 and VL _{function 2} /VH _{function 2} domains). A specific pair of binding domains (i.e. _VL /VH
_{The epitope of the antigen having epitope 1} or the epitope of _{the antigen} having _{epitope 2} is referred to as the first epitope:second epitope of the diabody, regardless of the epitope of the antigen having epitope 1 or the epitope of the antigen having epitope 2. , such notation relates only to the presence and orientation of the domains of the polypeptide chain of the binding molecules of the invention.

The first polypeptide chain of certain embodiments of such bispecific monovalent diabodies is in the N-terminus to C-terminus direction: N-terminus; the VL domain of a monoclonal antibody capable of binding to the first epitope; , or the VL domain of a monoclonal antibody capable of binding to the second epitope (i.e., VL _LAG-3 or VL _epitope ); the first intervening spacer peptide (
Linker 1); (if the first polypeptide chain contains VL _LAG-3 ) the VH domain of a monoclonal antibody capable of binding to the second epitope; or (if the first polypeptide chain contains VL _LAG-3); a VH domain of a monoclonal antibody capable of binding to the first epitope; a second intervening spacer peptide (linker 2) optionally comprising a cysteine residue; a heterodimer-promoting domain; and a C-terminus. (Figure 1).

The second polypeptide chain of this embodiment of the bispecific monovalent diabody is in the N-terminus to C-terminus direction: the N-terminus; the VL domain of a monoclonal antibody capable of binding LAG-3; a VL domain of a monoclonal antibody capable of binding to an epitope of a monoclonal antibody (i.e., VL _LAG-3 or VL _{epitope 2} ; and said VL domain is selected not to be included in said first polypeptide chain of said diabody); an intervening spacer peptide; (Linker 1); (if the second polypeptide chain contains VL _LAG-3 ) the VH domain of a monoclonal antibody capable of binding to the second epitope; or (if the second polypeptide chain contains VL _LAG-3); a VH domain of a monoclonal antibody capable of binding LAG- ₃ ; a second intervening spacer peptide (linker 2) optionally containing a cysteine residue; a heterodimer-promoting domain; Figure 1).

Most preferably, the length of the intervening linker peptide (e.g. Linker 1) separating said VL and VH domains is such that the length substantially or completely inhibits the binding of said VL and VH domains of said polypeptide chain to each other. Selected to prevent Thus, the VL and VH domains of the first polypeptide chain are substantially or not capable of binding to each other. Similarly, the VL and VH domains of the second polypeptide chain are substantially or not capable of binding to each other. A preferred intervening spacer peptide (linker 1) has the sequence (SEQ ID NO: 88): GGGSGGGG.

The length and composition of the second intervening linker peptide (Linker 2) is selected based on the choice of heterodimerization promoting domain. Typically, the second intervening linker peptide (Linker 2) contains 3 to 20 amino acid residues. Particularly when the heterodimer-promoting domain does not contain a cysteine residue, a cysteine-containing second intervening linker peptide (linker 2) is utilized. The cysteine-containing second intervening spacer peptide (linker 2) contains one, two, three or more cysteine residues. A preferred cysteine-containing spacer peptide (linker 2) has the sequence SEQ ID NO: 89: GGCGGG. Alternatively, linker 2 does not contain cysteine (e.g., GGG, GGGS (SEQ ID NO: 90), LGGGSG (SEQ ID NO: 91), GGGGSGGSGGG (SEQ ID NO: 92), ASTKG (SEQ ID NO: 93), LEPKSS (SEQ ID NO: 94), APSSS (SEQ ID NO: 95), etc.), a cysteine-containing heterodimer-promoting domain described below is used. Optionally, both a cysteine-containing linker 2 and a cysteine-containing heterodimer-promoting domain are used.

The heterodimerization promoting domain comprises GVEPKSC (SEQ ID NO: 96) or VEPKSC (SEQ ID NO: 97) or AEPKSC (SEQ ID NO: 98) on one polypeptide chain and GFNRGEC (SEQ ID NO: 99) on the other polypeptide chain. or FNRGEC (SEQ ID NO: 100) (US Patent No. 2007/
No. 0004909).

More preferably, however, the heterodimerization-promoting domain of such a diabody comprises one, two, three or four of the opposite poles, comprising at least 6, at least 7 or at least 8 amino acid residues. formed from two tandem repeating coiled coil domains, whereby the heterodimer-promoting domain has a net charge (Apostolovic, B. et al. (2008) “pH-Sensitivity of the E3/K3 Heterodimeric Coiled Coil,” Biomacromolecules 9 :3173-3180; Arndt, KM et al. (2001) “Helix‐stabilized Fv (hsFv) Antibody Fragments: Substituting the Constant Domains of a Fab Fragment for a Heterodimeric Coiled‐coil Domain,” J. Molec. Biol. 312: 221-228; Arndt, KM et al. (2002) “Comparison of In Vivo Selection and Rational Design of Heterodimeric Coiled Coils,”
Structure 10:1235-1248; Boucher, C. et al. (2010) “Protein Detection By Western Blot Via Coiled-Coil Interactions,” Analytical Biochemistry 399:138-140; Cachia, PJ et al. (2004) “Synthetic Peptide Vaccine Development: Measurement Of
Polyclonal Antibody Affinity And Cross‐Reactivity Using A New Peptide Capture And Release System For Surface Plasmon Resonance Spectroscopy,” J. Mol. Recognit. 17:540‐557; De Crescenzo, GD et al. (2003) “Real‐Time Monitoring of the Interactions of Two‐Stranded de novo Designed Coiled‐Coils: Effect of Chain Length on the Kinetic and Thermodynamic Constants of Binding,” Biochemistry 42:1754‐1763; Fernandez‐Rodriquez, J. et al. (2012) “Induced Heterodimerization And Purification Of Two Target Proteins By A Synthetic Coiled‐Coil Tag,” Protein
Science 21:511‐519; Ghosh, TS et al. (2009) “End‐To‐End And End‐To‐Middle Interhelical Interactions: New Classes Of Interacting Helix Pairs In Protein Structures,” Acta Crystallographica D65:1032‐1041; Grigoryan, G. et al. (2008)
“Structural Specificity In Coiled‐Coil Interactions,” Curr. Opin. Struc. Biol. 18:477‐483; Litowski, JR et al. (2002) “Designing Heterodimeric Two‐Stranded α‐Helical Coiled‐Coils: The Effects Of Hydrophobicity And α‐Helical Propensity On Protein Folding, Stability, And Specificity,” J. Biol. Chem. 277:37272‐37279; Steinkruger, JD et al. (2012) “The d′‐‐d‐‐d′ Vertical Triad
is Less Discriminating Than the a′‐‐a‐‐a′ Vertical Triad in the Antiparallel Coiled‐coil Dimer Motif,” J. Amer. Chem. Soc. 134(5): 2626-2633; Straussman, R. et al. (2007) “Kinking the Coiled Coil - Negatively Charged Residues at the Coiled‐coil Interface,” J. Molec. Biol. 366:1232‐1242; Tripet, B. et al. (2002) “Kinetic Analysis of the Interactions between Troponin C and the C-terminal Troponin I Regulatory Region and Validation of a New Peptide Delivery/Capture System used for Surface Plasmon Resonance,” J. Molec. Biol. 323:345-362; Woolfson, DN (2005) “The Design Of Coiled ‐Coil Structures And Assemblies,” Adv.
Prot. Chem. 70:79‐112; Zeng, Y. et al. (2008) “A Ligand‐Pseudoreceptor System Based On de novo Designed Peptides For The Generation Of Adenoviral Vectors With Altered Tropism,” J. Gene Med. 10: 355-367).

Such repeating coil domains may be complete repeats or may have substitutions. For example, the coil domain of the heterodimer-promoting domain of the first polypeptide chain may include a sequence of eight amino acid residues selected to impart a negative charge to the heterodimer-promoting domain. , the coil domain of the heterodimer-promoting domain of the second polypeptide chain may comprise a sequence of eight amino acid residues selected to impart a positive charge to the heterodimer-promoting domain ( or vice versa). When both polypeptide chains are provided with such heterodimerization-promoting domains, if a coil of opposite charge is used for the other polypeptide chain, either It is not important whether a coil is provided. Positively charged amino acids may be lysine, arginine, histidine, etc., and/or negatively charged amino acids may be glutamic acid, aspartic acid, etc. The positively charged amino acid is preferably lysine and/or the negatively charged amino acid is preferably glutamic acid. Although only a single heterodimer-promoting domain can be employed (as such a domain inhibits homodimerization and thereby promotes heterodimerization), the first and Preferably, both second polypeptide chains contain heterodimerization promoting domains.

In a preferred embodiment, one of the heterodimer-promoting domains comprises four tandem "E-coil" helical domains (SEQ ID NO: 101: E VAAL E K- E VAAL E K- E VAAL E K- E VAAL E K), whose glutamate residues form a negative charge at pH 7, while the other of the heterodimer-promoting domains comprises four tandem "K-coil" helical domains (SEQ ID NO: 102: K VAAL K E- K VAAL K E- K VAAL K E- K VAAL K E), whose lysine residues form a positive charge at pH 7. The presence of such charged domains promotes linkage between the first polypeptide and the second polypeptide, thus promoting heterodimer formation. Particularly preferred is that one of the four tandem "E-coil" helical domains of SEQ ID NO: 101 has a cysteine residue: E VAA CE K- E VAAL E K- E VAAL E K- E VAAL E K (sequence 103), the heterodimer-promoting domain. Similarly, it is particularly preferred that one of said four tandem "K-coil" helical domains of SEQ ID NO: 102 comprises cysteine residues: K VAA CK E- K VAAL K E- K VAAL K E- K VAAL K A heterodimer-promoting domain that has been modified to contain E (SEQ ID NO: 104).

As disclosed in WO 2012/018687, in order to improve the in vivo pharmacokinetic properties of the diabody, the diabody can be coated with a polypeptide of a serum binding protein at one or more of the termini of the diabody. may be modified to contain moieties. Most preferably, the polypeptide portion of such serum binding protein will be placed at the C-terminus of the diabody. Albumin is the most abundant protein in plasma and has a half-life of 19 days in humans. Albumin has multiple small molecule binding sites that allow it to bind non-covalently to other proteins and extend its serum half-life. The albumin binding domain 3 (ABD3) of protein G of streptococcal strain G148 consists of 46 amino acid residues that form a stable triple helical bundle and has broad albumin binding specificity (Johansson, MU et al. (2002) “Structure, Specificity, And Mode Of Interaction For Bacterial Albumin-Binding Modules,” J. Biol. Chem. 277(10):8114-8120). Accordingly, a particularly preferred polypeptide portion of a serum binding protein for improving the in vivo pharmacokinetic properties of diabodies is the albumin binding domain (ABD) from streptococcal protein G, and more preferably the protein of streptococcal strain G148. Albumin binding domain 3 (ABD3) of G (SEQ ID NO: 105): LAEAKVLANR ELDKYGVSDY YKNLIDNAKS AEGVKALIDE ILAALP.

As disclosed in WO 2012/162068 (incorporated into this application by reference), "deimmunized" variants of SEQ ID NO: 105 attenuate or eliminate MHC class II binding. have the ability to Based on the combined mutational results, the following combinations of substitutions appear to be preferred substitutions to form such deimmunized ABDs: 66D/70S+71A; 66S/70S+71A; 66S/70S+79A; 64A/65A/71A; 64A/65A/71A+66S; 64A/65A/71A+66D; 64A/65A/71A+66E; 64A/65A/79A+66S; 64A/65A/79A+66D; 64A/65A/79A+66E. Mutant ABD with modifications L64A, I65A and D79A, or modifications N66S, T70S and D79A. Amino acid sequence:
LAEAKVLANR ELDKYGVSDY YKNLI D ₆₆ NAK S ₇₀ A ₇₁ EGVKALIDE ILAALP (SEQ ID NO: 106)
or amino acid sequence:
LAEAKVLANR ELDKYGVSDY YKN A ₆₄ A ₆₅ NNAKT VEGVKALI A ₇₉ E ILAALP (SEQ ID NO: 107)
or amino acid sequence:
Particularly preferred is a variant deimmunized ABD having LAEAKVLANR ELDKYGVSDY YKNLI S ₆₆ NAK S ₇₀ VEGVKALI A ₇₉ E ILAALP (SEQ ID NO: 108). This is because such deimmunized ABDs exhibit approximately wild-type binding while providing attenuated MHC class II binding. Accordingly, said first polypeptide chain of such a diabody with an ABD is preferably positioned C-terminal to the E-coil (or K-coil) domain of such polypeptide chain, thereby Contains a third linker (linker 3) intervening between the E-coil (or K-coil) domain and the ABD, which is preferably a deimmunized ABD. A preferred sequence for such linker 3 is SEQ ID NO: 90: GGGS.

2. Bispecific Diabodies Containing an Fc Region One embodiment of the invention provides a bispecific diabody containing an Fc region containing LAG-3 and a second epitope (e.g., B7-H3, B7-H4, BTLA, CD40, CD80, CD86, CD137, CTLA-4). , ICOS, KIR, MHC class I or II, OX40, PD-1, PD-L1, TCR, TIM-3, etc.). Addition of an IgG CH2-CH3 domain to one or both of the diabody polypeptide chains, such that conjugation of the diabody chains forms an Fc region, reduces the biological half-life of the diabody. period increases and/or valence changes. Incorporation of IgG CH2-CH3 domains into both of the above diabody polypeptides allows for the formation of a two-chain bispecific Fc region-containing diabody (Figure 2).

Alternatively, incorporation of an IgG CH2-CH3 domain into one of the diabody polypeptides allows for the formation of more complex four-chain bispecific Fc region-containing diabodies (Figures 3A-3C). Although FIG. 3C shows a representative four-chain diabody with a constant light chain (CL) domain and a constant heavy chain CH1 domain, fragments of such domains and other polypeptides may be employed instead ( For example, FIGS. 3A and 3B, US Published Patent No. 2013-0295121; US Published Patent No. 2010-0174053 and US Published Patent No. 2009-0060910; European Published Patent No. 2714079; European Published Patent No. 26012
16; EP 2376109; EP 2158221; and WO 2012/162068; WO 2012/018687; WO 2010/080538). Therefore, for example, instead of the CH1 domain, the amino acid sequence GVEPKSC (SEQ ID NO: 96) derived from the hinge domain of human IgG, VEPKSC (SEQ ID NO: 9
7) or AEPKSC (SEQ ID NO: 98), and instead of the CL domain, the C-terminal 6 amino acids of human kappa light chain, GFNRGEC (SEQ ID NO: 99) or FNRGEC (SEQ ID NO: 100), may be employed. You may do so. A representative peptide containing a four-chain diabody is shown in Figure 3A. Alternatively, or in addition, tandem coil domains of opposite charge, such as "E-coil" helical domains (SEQ ID NO: 101: E VAAL E K- E VAAL E K- E VAAL E K- E VAAL E K or SEQ ID NO: 103: E VAA CE K- E VAAL E K- E VAAL E K- E VAAL E K); and “K-coil” domain (SEQ ID NO: 102: K VAAL K E- K VAAL K E- K VAAL K E- K VAAL K E Alternatively, a peptide comprising SEQ ID NO: 104: K VAA CK E- K VAAL K E- K VAAL K E- K VAAL K E) may be employed. A representative coil domain containing a four-chain diabody is shown in Figure 3B.

Fc region-containing diabody molecules of the invention generally include an intervening linker peptide (linker). Typically, this additional linker contains 3-20 amino acid residues. Additional or alternative linkers that can be employed in Fc region-containing diabody molecules of the invention include: GGGS (SEQ ID NO: 90), LGGGSG (SEQ ID NO: 91), GGGGSGGSGGG (SEQ ID NO: 92), ASTKG (SEQ ID NO: 93), DKTHTCPPCP (SEQ ID NO: 109), LEPKSS (SEQ ID NO: 94), APSSS
(SEQ ID NO: 95), APSSSPME (SEQ ID NO: 110), LEPKSADKTHTCPPC (SEQ ID NO: 11)
1), GGC, and GGG. SEQ ID NO: 94 may be used in place of GGG or GGC to facilitate cloning. Furthermore, an alternative linker by immediately following the amino acid GGG or SEQ ID NO: 94 with SEQ ID NO: 109: GGGDKTHTCPPCP (SEQ ID NO: 112);
and LEPKSSDKTHTCPPCP (SEQ ID NO: 113). Fc region-containing diabody molecules of the invention may incorporate an IgG hinge region in addition to or in place of a linker. Exemplary hinge regions include: EPKSCDKTHTCPPCP from IgG1 (SEQ ID NO: 114
); ERKCCVECPPCP from IgG2 (SEQ ID NO: 115); ESKYGPPCPSCP from IgG4 (SEQ ID NO: 116); and ESKYGPPCPPCP from IgG4 hinge variant (SEQ ID NO: 117) containing stabilizing substitutions to reduce strand exchange. .

As presented in Figures 3A-3C, the diabodies of the invention may include four different chains. The first and third polypeptide chains of such diabodies contain three domains: (i) a VL1-containing domain; (ii) a VH2-containing domain; (iii) a heterodimer-promoting domain; and (iv) a CH2 - Contains a domain containing the CH3 sequence. The second and fourth polypeptide chains contain: (i) a VL2-containing domain; (ii) a VH1-containing domain; and (iii) a heterodimer-promoting domain, wherein the heterodimer-promoting domain / Promote dimerization of the third polypeptide chain and the second/fourth polypeptide chain. The VL and/or VH domains of the third and fourth polypeptide chains and the VL and/or VH domains of the first and second polypeptide chains may be the same or different, thereby Monospecific, bispecific or tetravalent binding is possible. The notations "VL3" and "VH3" refer to the variable light and variable heavy chain domains, respectively, that bind to the "third" epitope ("epitope 3") of such diabodies. Similarly, the notations "VL4" and "VH4" refer to the variable light and variable heavy chain domains, respectively, that bind to the "fourth" epitope ("epitope 4") of such diabodies. The general structure of the polypeptide chains of representative four-chain Fc region-containing diabodies of the invention is presented in Table 2.

In certain specific embodiments, the diabodies of the invention are bispecific, tetravalent (i.e., having four epitope binding sites), Fc-containing diabodies consisting of a total of four polypeptide chains (Figures 3A-3C ). The bispecific, tetravalent, Fc-containing diabodies of the invention have two epitope binding sites immunospecific for LAG-3 (which can bind to the same epitope of LAG-3 or different epitopes of LAG-3). ), and a second epitope (e.g. B7-H3, B7-H4, BTLA, CD40, CD80, CD86, CD137, CTLA-4, ICOS, KIR, MHC class I or II, OX40, PD-1, PD-L1 , TCR, TIM-3, etc.).

In a further embodiment, the bispecific Fc region-containing diabody may include three polypeptide chains. The first polypeptide of such a diabody contains three domains: (i) a VL1-containing domain; (ii) a VH2-containing domain; and (iii) a domain containing a CH2-CH3 sequence. The second polypeptide of such a diabody is: (i) a VL2-containing domain; (ii) a VH1-containing domain; and (iii) heterodimerizing and covalent with the first polypeptide chain of said diabody. Contains domains that promote binding. The third polypeptide of such diabodies contains the CH2-CH3 sequence. The first and second polypeptide chains of such a diabody are thus linked together to provide a VL1/VH1 binding site capable of binding to the first epitope and a VL2/VH1 binding site capable of binding to the second epitope. Forms a VH2 binding site. The first and second polypeptides are linked to each other by a disulfide bond involving cysteine residues in their respective third domains. In particular, the first and third polypeptide chains are complexed with each other to form an Fc region stabilized by disulfide bonds. Such diabodies have enhanced strength. Figures 4A and 4B show the structure of such a diabody. Such Fc region-containing bispecific diabodies may have either of two orientations (Table 3).

In certain specific embodiments, the diabodies of the invention are bispecific, bivalent (i.e., have two epitope binding sites), Fc-containing diabodies consisting of a total of three polypeptide chains (FIGS. 4A-4B). ). The bispecific, bivalent Fc-containing diabodies of the invention have one epitope binding site immunospecific for LAG-3, a second epitope binding site (e.g., B7-H3, B7-H4, BTLA, CD40, CD80, CD86, CD137, CTLA-4, ICOS, KIR, LAG-3 MHC class I or II, OX40, PD-L1, TCR, TIM-3, etc.).

In a further embodiment, the bispecific Fc region-containing diabody may include a total of five polypeptide chains. In certain embodiments, two of the five polypeptide chains have the same amino acid sequence. The first polypeptide chain of such a diabody contains: (i) a VH1-containing domain; (ii) a CH1-containing domain; and (iii) a domain containing a CH2-CH3 sequence. The first polypeptide chain may be an antibody heavy chain containing a VH1 and heavy chain constant region. The second and fifth polypeptide chains of such diabodies contain: (i) a VL1-containing domain; and (ii) a CL-containing domain. Said second and/or fifth polypeptide chain of such a diabody may be a light chain of an antibody containing a VL1 complementary to the VH1 of said first/third polypeptide chain. . The first, second and/or fifth polypeptide chains can be isolated from naturally occurring antibodies. Alternatively, they can be constructed recombinantly. The third polypeptide chain of such a diabody is: (i) a VH1-containing domain; (ii) a CH1-containing domain; (iii) a domain containing a CH2-CH3 sequence; (iv) a VL2-containing domain; (v) and (vi) a heterodimer-promoting domain, the heterodimer-promoting domain promoting dimerization of the third chain and the fourth chain. The fourth polypeptide of such a diabody is: (i) a VL3-containing domain; (ii) a VH2-containing domain; and (iii) heterodimerizing and covalent with the third polypeptide chain of said diabody. Contains domains that promote binding.

Therefore, the first and second polypeptide chains and the third and fifth polypeptide chains of such a diabody are linked to each other to form two VL1/VH1 bonds capable of binding to the first epitope. form a part. The third and fourth polypeptide chains of such a diabody are linked to each other to provide a VL2/VH2 binding site capable of binding a second epitope and a VL3/VH3 binding site capable of binding a third epitope. Form. The first and third polypeptides are bonded to each other through disulfide bonds involving cysteine residues in their respective constant regions. In particular, the first and third polypeptide chains are complexed with each other to form an Fc region. Such diabodies have enhanced strength. FIG. 5 shows the structure of such a diabody. The VL1/VH1, VL2/VH2 and VL3/VH3 domains may be the same or different, allowing binding to be monospecific, bispecific or trispecific. It will be understood. However, as provided herein, these domains preferably connect LAG-3 to a second epitope (or to a second and third epitope (e.g. B7-H3, B7-H4, BTLA, CD40, CD80, CD86, CD137, CTLA-4, ICOS, KIR, LAG-3 MHC class I or II, OX40, PD-L1, TCR, TIM-3, etc.)). The second and third epitopes may be different epitopes of the same antigen molecule or may be epitopes of different antigen molecules. These aspects of the invention are discussed in detail below.

The VL and VH domains of the polypeptide chain are thus selected to form a VL/VH binding site specific for the desired epitope. The VL/VH binding sites formed by linking the polypeptide chains may be the same or different, thereby providing monospecific, bispecific, trispecific or quadruple specificity. Certain four-valent bonds are possible. In particular, the VL and VH domains provide that the bispecific diabody has two binding sites for the first epitope and two binding sites for the second epitope, or three binding sites for the first epitope and two binding sites for the second epitope. Selected to have one binding site for the epitope, or (as shown in Figure 5) two binding sites for the first epitope, one binding site for the second epitope and one binding site for the third epitope. You may do so. Table 4 shows the general structure of the polypeptide chain of a typical 5-chain Fc region-containing diabody of the present invention.

In certain specific embodiments, the diabodies of the invention are bispecific, consisting of a total of five polypeptide chains, having two binding sites for a first epitope and two binding sites for a second epitope; It is a tetravalent (ie, has four epitope binding sites), Fc-containing diabody. In one embodiment, the bispecific, tetravalent, Fc-containing diabodies of the invention contain two epitope binding sites that are immunospecific for LAG-3 (these may be the same epitope of LAG-3 or a second epitope (e.g. B7-H3, B7-H4, BTLA, CD40, CD80, CD86, CD137, CTLA-4, ICOS, KIR, LAG-3 MHC class I or II) , OX40, PD-L1, TCR, TIM-3, etc.). In another embodiment, the bispecific, tetravalent, Fc-containing diabodies of the invention contain three epitope binding sites that are immunospecific for LAG-3 (these may be the same epitope of LAG-3 or a second epitope (e.g. B7-H3, B7-H4, BTLA, CD40, CD80, CD86, CD137, CTLA-4, ICOS, KIR, LAG-3 MHC class I or II, OX40, PD-L1, TCR, TIM-3, etc.). In another embodiment, the bispecific, tetravalent, Fc-containing diabodies of the invention have one epitope binding site immunospecific for LAG-3, and a second epitope binding site (e.g., B7-H3, B7- Three epitope binding specific for H4, BTLA, CD40, CD80, CD86, CD137, CTLA-4, ICOS, KIR, LAG-3 MHC class I or II, OX40, PD-L1, TCR, TIM-3, etc.) Includes parts.

3. Bispecific Trivalent Binding Molecules Comprising an Fc Region A further embodiment of the invention provides a bispecific trivalent binding molecule comprising an Fc region and capable of simultaneously binding a first epitope, a second epitope and a third epitope. , a trivalent binding molecule, wherein at least one of said epitopes is not identical to another of said epitopes. Such bispecific diabodies therefore have a "VL1"/"VH1" domain capable of binding a first epitope, a "VL2"/"VH2" domain capable of binding a second epitope, and a third epitope. Contains "VL3"/"VH3" domains that can bind. In one embodiment, one or two of the epitopes are of LAG-3 and another of the epitopes is not an epitope of LAG-3 (e.g. B7 -H3, B7-H4, BTLA, CD40, CD80, CD86, CD137, CTLA-4, ICOS, KIR, LAG-3, MHC class I or II, OX40, PD-1, PD-L1, TCR, TIM-3 etc.). Such bispecific trivalent binding molecules comprise three epitope binding sites, two of which are diabody-type binding domains providing binding site A and binding site B; 6A-6F and PCT Application No. PCT/US15/33081 and PCT Application No. PCT/US15/33076).

Typically, a trivalent binding molecule of the invention comprises four different polypeptide chains (see Figures 6A-6B); or by “dividing” these polypeptides to form additional polypeptide chains, or by linking fewer or additional polypeptide chains by disulfide bonds. A larger number of polypeptide chains can be included. Figures 6B-6F illustrate this aspect of the invention by schematically showing a molecule with three polypeptide chains. As presented in FIGS. 6A-6F, the trivalent binding molecules of the invention are characterized in that the diabody-type binding domain is either N-terminal (FIGS. 6A, 6C and 6D) or C-terminal (FIGS. 6B, 6E and 6D) to the Fc region. 6F).

In certain embodiments, the first polypeptide chain of such trivalent binding molecules of the invention comprises: (i) a VL1-containing domain; (ii) a VH2-containing domain; (iii) a heterodimer-promoting domain; and (iv) Contains a domain containing a CH2-CH3 sequence. The VL1 and VL2 domains are located N-terminally or C-terminally to the CH2-CH3 containing domain, as presented in Table 5 (FIGS. 6A and 6B). The second polypeptide chain of such embodiments contains: (i) a VL2-containing domain; (ii) a VH1-containing domain; and (iii) a heterodimer-promoting domain. The third polypeptide chain of such embodiments contains: (i) a VH3-containing domain; (ii) a CH1-containing domain; and (iii) a domain containing a CH2-CH3 sequence. The third polypeptide chain may be an antibody heavy chain containing a VH3 and a heavy chain constant region. The fourth polypeptide of such embodiments contains: (i) a VL3-containing domain; and (ii) a CL-containing domain. The fourth polypeptide chain may be an antibody light chain containing a VL3 that is complementary to the VH3 of the third polypeptide chain. The third or fourth polypeptide chain can be isolated from naturally occurring antibodies. Alternatively, they may be constructed recombinantly, synthetically, or by other means.

The variable light chain domains of said first and second polypeptide chains are separated from the variable heavy chain domains of such polypeptide chains by an intervening spacer linker, said intervening spacer linker separating these VL1/VH2 (or These VL2/VH1) domains have a length that is too short to allow them to be linked together to form an epitope binding site that can bind to a first or second epitope. A preferred intervening spacer peptide (linker 1) for this purpose has the sequence (SEQ ID NO: 14): GGGSGGGG. Other domains of the trivalent binding molecule may be separated by one or more intervening spacer peptides, optionally containing cysteine residues. Exemplary linkers useful in generating trivalent binding molecules are provided herein and also in PCT Application No. PCT/US15/33081; and PCT Application No. PCT/US15/33076. There is. The first and second polypeptide chains of such a trivalent binding molecule are thus linked together to form a VL1/VH1 binding site capable of binding a first epitope and a VL2/VH2 binding site capable of binding a second epitope. Form a binding site. The third and fourth polypeptide chains of such a trivalent binding molecule are linked together to form a VL3/VH3 binding site capable of binding a third epitope.

As mentioned above, trivalent binding molecules of the invention may include three polypeptides. A trivalent binding molecule comprising three polypeptide chains can be obtained by linking the N-terminal domain of a fourth polypeptide to the VH3-containing domain of a third polypeptide. Alternatively, a third polypeptide of the trivalent binding molecule of the invention contains three domains: (i) a VL3-containing domain; (ii) a VH3-containing domain; and (iii) a domain containing a CH2-CH3 sequence. A chain is utilized in which VL3 and VH3 are linked by an intervening spacer peptide of sufficient length (at least 9 or more amino acid residues) to allow these domains to link to form an epitope binding site. separated from each other.

The VL1/VH1, VL2/VH2 and VL3/VH3 domains of such diabody molecules may be the same or different, thereby making them monospecific, bispecific or trispecific. It will be appreciated that combinations are possible. However, as provided herein, these domains are preferably selected to bind LAG-3 and a second epitope (or a second and a third epitope) (preferably said epitope is , B7-H3, B7-H4, BTLA, CD40, CD80, CD86, CD137, CTLA-4, ICOS, KIR, LAG-3 MHC class I or II, OX40, PD-L1, TCR, TIM-3, etc. epitopes ).

In particular, the VL and VH domains are such that the trivalent binding molecule has two binding sites for the first epitope and one binding site for the second epitope, or one binding site for the first epitope and two binding sites for the second epitope. or one binding site for the first epitope, one binding site for the second epitope and one binding site for the third epitope. The general structure of the polypeptide chain of a representative trivalent binding molecule of the invention is presented in FIGS. 6A-6F and Table 5.

One embodiment of the invention provides two epitope binding sites for LAG-3 and molecules other than LAG-3 (e.g. B7-H3, B7-H4, BTLA, CD40, CD80, CD86, CD137, CTLA-4, ICOS). , KIR, LAG-3, MHC class I or II, OX40, PD-L1, TCR, TIM-3, etc.). Concerning molecules. The two epitope binding sites for LAG-3 may bind to the same epitope or different epitopes. Another embodiment of the invention provides one epitope binding site for LAG-3 and molecules other than LAG-3 (e.g. B7-H3, B7-H4, BTLA, CD40, CD80, CD86, CD137, CTLA-4, bispecific trivalent with two epitope binding sites for a second antigen present on ICOS, KIR, LAG-3, MHC class I or II, OX40, PD-L1, TCR, TIM-3, etc.) Concerning binding molecules. The two epitope binding sites for said second antigen may bind to the same epitope of said antigen or to different epitopes (eg the same or different epitopes of LAG-3). As mentioned above, such bispecific trivalent binding molecules may contain three or four polypeptide chains.

VII. Constant Domains and Fc Regions Provided herein are antibody constant domains useful in generating LAG-3 binding molecules (eg, antibodies, diabodies, trivalent binding molecules, etc.) of the invention.

A preferred CL domain is a human IgG CLK domain. An exemplary human CLK domain amino acid sequence is (SEQ ID NO: 118):
RTVAAPSVFI FPPSDEQLKS GTASVVCLLN NFYPREAKVQ WKVDNALQSG
NSQESVTEQD SKDSTYSLSS TLTLSKADYE KHKVYACEVT HQGLSSPVTK
SFNRGEC
It is.

Alternatively, an exemplary CL domain is a human IgG CLλ domain. The amino acid sequence of an exemplary human CLλ domain is (SEQ ID NO: 119):
QPKAAPSVTL FPPSSEELQA NKATLVCLIS DFYPGAVTVA WKADSSPVKA
GVETTPSKQS NNKYAASSYL SLTPEQWKSH RSYSCQVTHE GSTVEKTVAP
TECS
It is.

As provided herein, the LAG-3 binding molecules of the invention may comprise an Fc region. The Fc region of such molecules of the invention may be of any isotype (eg, IgG1, IgG2, IgG3 or IgG4). LAG-3 binding molecules of the invention may further comprise a CH1 domain and/or a hinge region. When present, said CH1 domain and/or hinge region may be of any isotype (e.g. IgG1, IgG2, IgG3 or IgG4), preferably of the desired Fc region. It is of the same isotype as

An exemplary CH1 domain is a human IgG1 CH domain. The amino acid sequence of an exemplary human IgG1 CH1 domain is (SEQ ID NO: 120):
ASTKGPSVFP LAPSSKSTSG GTAALGCLVK DYFPEPVTVS WNSGALTSGV
HTFPAVLQSS GLYSLSSVVT VPSSSLGTQT YICNVNHKPS NTKVDKRV
It is.

An exemplary CH1 domain is a human IgG2 CH domain. The amino acid sequence of an exemplary human IgG2 CH1 domain is (SEQ ID NO: 121):
ASTKGPSVFP LAPCSRSTSE STAALGCLVK DYFPEPVTVS WNSGALTSGV
HTFPAVLQSS GLYSLSSVVT VPSSNFGTQT YTCNVDHKPS NTKVDKTV
It is.

An exemplary CH1 domain is a human IgG4 CH1 domain. The amino acid sequence of an exemplary human IgG4 CH1 domain is (SEQ ID NO: 122):
ASTKGPSVFP LAPCSRSTSE STAALGCLVK DYFPEPVTVS WNSGALTSGV
HTFPAVLQSS GLYSLSSVVT VPSSSLGTKT YTCNVDHKPS NTKVDKRV
It is.

One exemplary hinge region is the human IgG1 hinge region. An exemplary human IgG1 hinge region amino acid sequence is (SEQ ID NO: 114): EPKSCDKTHTCPPCP.

Another exemplary hinge region is the human IgG2 hinge region. An exemplary human IgG2 hinge region amino acid sequence is (SEQ ID NO: 115): ERKCCVECPPCP.

Another exemplary hinge region is the human IgG4 hinge region. An exemplary human IgG4 hinge region amino acid sequence is (SEQ ID NO: 116): ESKYGPPCPSCP. As described herein, the IgG4 hinge region may include stabilizing mutations such as a S228P substitution. An exemplary stabilized IgG4 hinge region amino acid sequence is (SEQ ID NO: 117): ESKYGPPCP P CP.

The Fc region of Fc region-containing molecules (e.g. antibodies, diabodies and trivalent molecules) of the invention can be a complete Fc region (e.g. a complete IgG Fc region) or only a fragment of an Fc region. . Optionally, the Fc region of the Fc region-containing molecules of the invention does not include a C-terminal lysine amino acid residue. In particular, the Fc region of the Fc region-containing molecules of the invention may be an engineered variant Fc region. The Fc region of the bispecific Fc region-containing molecules of the invention may have the ability to bind to one or more Fc receptors (e.g., one or more FcγRs), but more preferably, the above-described mutant whether the type Fc region has altered binding to FcγRIA (CD64), FcγRIIA (CD32A), FcγRIIB (CD32B), FcγRIIIA (CD16a), or FcγRIIIB (CD16b) (relative to the binding exhibited by the wild-type Fc region); or have a reduced or no ability to bind to one or more inhibitory receptors. Thus, the Fc region of a bispecific Fc region-containing molecule of the invention may include some or all of the CH2 domain and/or some or all of the CH3 domain of a complete Fc region, or (e.g., a complete may include variant CH2 and/or variant CH3 sequences (which may include one or more insertions and/or one or more deletions to the CH2 or CH3 domain of the Fc region). Such Fc regions may comprise non-Fc polypeptide portions, or may comprise portions of a complete Fc region that do not occur naturally, or may comprise non-naturally occurring orientations of the CH2 and/or CH3 domains ( For example, two CH2 domains or two CH3 regions, or a CH3 domain and a CH2 domain linked together in the direction from the N-terminus to the C-terminus, etc.).

Fc domain modifications that manifest as altered effector function are known in the art, including modifications that increase binding to activating receptors (e.g., FcγRIIA (CD16A)) and modifications that decrease binding to inhibitory receptors. (For example, FcγRIIB (CD32B)) ” Cancer Res. 57(18):8882-8890). Exemplary variants of the human IgG1 Fc region with decreased binding to CD32B and/or increased binding to CD16A contain the F243L, R292P, Y300L, V305I or P296L substitutions. These amino acid substitutions may occur in any combination or subcombination within the human IgG1 Fc region. In one embodiment, the human IgG1 Fc region variant contains the F243L, R292P and Y300L substitutions. In another embodiment, the human IgG1 Fc region variant contains the F243L, R292P, Y300L, V305I and P296L substitutions.

In particular, regarding the CH2-CH3 domains of the polypeptide chains of the Fc region-containing molecules of the invention (relative to the binding exhibited by the wild-type IgG1 Fc region (SEQ ID NO: 1)), FcγRIA (CD64), FcγRIIA (CD32A), FcγRIIB ( Preferably, the binding to CD32B), FcγRIIIA (CD16a), or FcγRIIIB (CD16b) is reduced (or substantially not bound to these). Variant Fc regions and forms of mutations that can mediate such altered binding are described above. In certain specific embodiments, an Fc region-containing molecule of the invention comprises an IgG Fc region with reduced ADCC effector function. In certain preferred embodiments, the CH2-CH3 domains of the first and/or third polypeptide chains of such molecules have any one of the following substitutions: L234A, L235A, N297Q, and N297G, 2 Contains one or three. In another embodiment, the human IgG Fc region variants include N297Q substitution, N297G substitution, L234A and L235
Contains the A substitution or the D265A substitution because these mutations abolish FcR binding. Alternatively, CH2 of an Fc region that has inherently low binding (or almost no binding) to FcγRIIIA (CD16a) (compared to the binding exhibited by the wild-type IgG1 Fc region (SEQ ID NO: 1)) and/or has a naturally low effector function. - Utilizes CH3 domain. In certain specific embodiments, an Fc region-containing molecule of the invention comprises an IgG2 Fc region (SEQ ID NO: 2) or an IgG4 Fc region (SEQ ID NO: 4). When utilizing an IgG4 Fc region, the invention also encompasses the introduction of stabilizing mutations, such as the hinge region S228P substitution described above (see, eg, SEQ ID NO: 117). Since the N297G, N297Q, L234A, L235A and D265A substitutions abolish effector function, these substitutions are preferably not employed in situations where effector function is desired.

Preferred IgG1 sequences for the CH2 and CH3 domains of LAG-3 binding molecules of the invention include the L234A/L235A substitution (SEQ ID NO: 123):
APE AA GGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSHED PEVKFNWYVD
GVEVHNAKTK PREEQYNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKALPA
PIEKTISKAK GQPREPQVYT LPPSREEMTK NQVSLTCLVK GFYPSDIAVE
WESNGQPENN YKTTPPVLDS DGSFFLYSKL TVDKSRWQQG NVFSCSVMHE
ALHNHYTQKS LSLSPG X
where X is lysine (K) or absent.

In particular, it is preferred for the Fc region of the polypeptide chain of the Fc region-containing molecules of the invention to have an increased serum half-life (relative to the half-life exhibited by the corresponding wild-type Fc region). Variant Fc regions and mutant forms with increased serum half-life are described above. In certain preferred embodiments, the CH2-CH3 domains of the first and/or third polypeptide chains of such Fc region-containing molecules have any one of the following substitutions: M252Y, S254T and T256E, 2 Contains one or three. The invention further provides:
The present invention comprises a variant Fc region comprising (A) one or more mutations that alter effector function and/or FcγR; and (B) one or more mutations that increase serum half-life. Fc region-containing molecules.

Preferred IgG1 sequences for the CH2 and CH3 domains of Fc region-containing molecules of the invention are substituted L234A/L235A/M252Y/S254T/T256E (SEQ ID NO: 124):
APE AA GGPSV FLFPPKPKDT L Y I T R E PEVT CVVVDVSHED PEVKFNWYVD
GVEVHNAKTK PREEQYNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKALPA
PIEKTISKAK GQPREPQVYT LPPSREEMTK NQVSLTCLVK GFYPSDIAVE
WESNGQPENN YKTTPPVLDS DGSFFLYSKL TVDKSRWQQG NVFSCSVMHE
ALHNHYTQKS LSLSPG X
where X is lysine (K) or absent.

Preferred IgG4 sequences for the CH2 and CH3 domains of Fc region-containing molecules of the invention include M252Y/S254T/T256E substitutions (SEQ ID NO: 125):
APEFLGGPSV FLFPPKPKDT L Y I T R E PEVT CVVVDVSQED PEVQFNWYVD
GVEVHNAKTK PREEQFNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKGLPS
SIEKTISKAK GQPREPQVYT LPPSQEEMTK NQVSLTCLVK GFYPSDIAVE
WESNGQPENN YKTTPPVLDS DGSFFLYSRL TVDKSRWQEG NVFSCSVMHE
ALHNHYTQKS LSLSLG X
where X is lysine (K) or absent.

For diabodies and trivalent binding molecules where the first and third polypeptide chains are not identical, between the CH2-CH3 domains of the two first polypeptide chains or the CH2-CH3 of the two third polypeptide chains. It is desirable to reduce or prevent the occurrence of homodimerization between CH3 domains. The CH2 and/or CH3 domains of such polypeptide chains need not be identical in sequence, but are advantageously modified to facilitate complex formation between the two polypeptide chains. For example, domains that are similarly mutated due to steric hindrance by introducing amino acid substitutions (preferably by amino acids containing bulky side chain groups forming "knob", e.g. tryptophan) in the CH2 or CH3 domains. The altered domain can be paired with a complementary or adaptively mutated domain (eg, glycine substitution), or "hole." Such a series of mutations can be made to any pair of polypeptides containing CH2-CH3 domains that form the Fc region. Methods of protein processing to reduce homodimerization and promote heterodimerization are known in the art, particularly for processing immunoglobulin-like molecules, and are encompassed herein (e.g., Ridgway et al. et al. (1996) “'Knobs‐Into‐Holes' Engineering Of Antibody CH3 Domains For Heavy Chain Heterodimerization,” Protein Engr. 9:617‐621; Atwell et al. (1997) “Stable Heterodimers From Remodeling The Domain Interface Of A Homodimer Using A Phage Display Library,” J. Mol. Biol. 270: 26-35; and Xie et al. (2005) “A New Format Of Bispecific Antibody: Highly Efficient Heterodimerization, Expression And Tumor Cell Lysis,” J. See Immunol. Methods 296:95-101, each of which is incorporated herein by reference in its entirety. Preferably, the "knob" is engineered into the CH2-CH3 domain of the first polypeptide chain and the "hole" is engineered into the CH2-CH3 domain of the third polypeptide chain of the diabody comprising these polypeptide chains. be done. The "knob" will therefore serve to prevent homodimerization of the first polypeptide chain via its CH2 and/or CH3 domains. The third polypeptide chain preferably contains a "hole" substituent so that it heterodimerizes with the first polypeptide chain and homodimerizes with itself. This strategy may be utilized for diabodies and trivalent binding molecules containing three, four or five chains as described above, where the "knob" is the CH2-CH3 domain of the first polypeptide chain. The "hole" is processed into the CH2-CH3 domain of the third polypeptide chain.

A preferred knob is generated by modifying the IgG Fc region to contain the modifying group T366W. Preferred holes are created by modifying the IgG Fc region to contain the modifying groups T366S, L368A and Y407V. To aid in purifying hole-carrying third polypeptide chain homodimers from bispecific heterodimeric Fc region-containing molecules, the CH2 and CH3 domains of the third polypeptide chain are preferably The protein A binding site of is mutated by an amino acid substitution at position 435 (H435R). In this way, the hole-carrying third polypeptide chain homodimer does not bind to protein A, whereas the bispecific monovalent Fc diabody binds to the protein A binding site of the first polypeptide chain. remains capable of binding to protein A via In an alternative embodiment, the hole-carrying third polypeptide chain may incorporate amino acid substitutions at positions 434 and 435 (N434A/N435K).

Preferred IgG1 amino acid sequences for the CH2 and CH3 domains of the first polypeptide chain of Fc region-containing molecules of the invention include the "knob-carrying" sequence (SEQ ID NO: 126):
APE AA GGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSHED PEVKFNWYVD
GVEVHNAKTK PREEQYNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKALPA
PIEKTISKAK GQPREPQVYT LPPSREEMTK NQVSL W C L VK GFYPSDIAVE
WESNGQPENN YKTTPPVLDS DGSFFLYSKL TVDKSRWQQG NVFSCSVMHE
ALHN H YTQKS LSLSPG
where X is lysine (K) or absent.

A second polypeptide chain of an Fc region-containing molecule of the invention having two polypeptide chains (or a third polypeptide chain of an Fc region-containing molecule having three, four or five polypeptide chains) A preferred IgG1 amino acid sequence for the CH2 and CH3 domains is the "hole-carrying" sequence (SEQ ID NO: 127):
APE AA GGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSHED PEVKFNWYVD
GVEVHNAKTK PREEQYNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKALPA
PIEKTISKAK GQPREPQVYT LPPSREEMTK NQVSL S C A VK GFYPSDIAVE
WESNGQPENN YKTTPPVLDS DGSFFL V SKL TVDKSRWQQG NVFSCSVMHE
ALHN R YTQKS LSLSPG
where X is lysine (K) or absent.

As mentioned above, the CH2-CH3 domains of SEQ ID NO: 126 and SEQ ID NO: 127 contain a substitution at position 234 by alanine and a substitution at position 235 by alanine, thus reducing the binding exhibited by the wild-type Fc region (SEQ ID NO: 1). form an Fc region with reduced binding (or substantially no binding) to FcγRIA (CD64), FcγRIIA (CD32A), FcγRIIB (CD32B), FcγRIIIA (CD16a), or FcγRIIIB (CD16b). The invention also encompasses such CH2-CH3 domains further comprising one or more half-life extending amino acid substitutions. In particular, the present invention encompasses such hole-carrying and knob-carrying CH2-CH3 domains further comprising M252Y/S254T/T256E.

Preferably, the first polypeptide chain has a "knob-carrying" CH2-CH3 sequence, such as that of SEQ ID NO: 126. However, it will be appreciated that a "hole-carrying" CH2-CH3 domain (e.g. SEQ ID NO: 127) can be employed in the first polypeptide chain, in which case a "knob-carrying" CH2-CH3 domain (e.g. SEQ ID NO: 126) is the second polypeptide chain of an Fc region-containing molecule of the invention having two polypeptide chains (or the third polypeptide chain of an Fc region-containing molecule having three, four or five polypeptide chains) Adopted inside.

As mentioned above, the invention encompasses Fc region-containing molecules (e.g. antibodies and Fc region-containing diabodies) having wild-type CH2 and CH3 domains, or having CH2 and CH3 domains containing combinations of the above-mentioned substitutions. . An exemplary amino acid sequence of an IgG1 CH2-CH3 domain including such variants is (SEQ ID NO: 128):
APEX ₁ X ₂ GGPSV FLFPPKPKDT LX ₃ IX ₄ RX ₅ PEVT CVVVDVSHED
PEVKFNWYVD GVEVHNAKTK PREEQYNSTY RVVSVLTVLH
QDWLNGKEYK CKVSNKALPA PIEKTISKAK GQPREPQVYT
LPPSREEMTK NQVSLX ₆ CX ₇ VK GFYPSDIAVE WESNGQPENN
YKTTPPVLDS DGSFFLX ₈ SKL TVDKSRWQQG NVFSCSVMHE
ALHX ₉ X ₁₀ YTQKS LSLSPGX ₁₁
and here:
(a) Both X ₁ and X ₂ are L (wild type), or both are A (lower FcγR binding);
(b) X ₃ , X ₄ and X ₅ are respectively M, S and T (wild type) or Y, T and E (half-life extended);
(c) X ₆ , X ₇ and X ₈ are respectively T, L and Y (wild type) or W, L and Y (knob) or S, A and V (hole);
(d) X ₉ and X ₁₀ are respectively N and H (wild type), or N and R (no protein A binding), or A and K (no protein A binding);
(e) X ₁₁ is K or absent.

In other embodiments, the invention relates to WO 2007/110205; WO 2011/143545; WO 2012/058768; WO 2013/06867, all of which are incorporated herein by reference in their entirety. CH2 and / or a LAG-3 binding molecule comprising a CH3 domain.

VIII. Reference antibody A. Reference Anti-LAG-3 Antibodies To evaluate and characterize the novel anti-LAG-3 binding molecules of the present invention, the following reference antibodies were employed: 25F7 (BMS-986016, Medarex/BMS, herein referred to as “LAG-3 -3 mAb A).

1.25F7 (“LAG-3 mAb A”)
The amino acid sequence (SEQ ID NO: 129) of the VH domain of 25F7 ("LAG-3 mAb A") is shown below (CDR _H residues are underlined):
QVQLQQWGAG LLKPSETLSL TCAVYGGSFS DYYWN WIRQP PGKGLEWIG E
INHNGNTNSN PSLKS RVTLS LDTSKNQFSL KLRSVTAADT AVYYCAF GYS
DYEYNWFDP W GQGTLVTVSS

The amino acid sequence (SEQ ID NO: 130) of the VL domain of 25F7 ("LAG-3 mAb A") is shown below (CDR _L residues are underlined):
EIVLTQSPAT LSLSPGERAT LSC RASQSIS SYLA WYQQKP GQAPRLLIY D
ASNRAT GIPA RFSGSGSGTD FTLTISSLEP EDFAVYYC QQ RSNWPLT FGQ
GTNLEIK

B. Reference Anti-PD-1 Antibodies Reference antibodies may be used to evaluate and characterize the novel anti-LAG-3 binding molecules of the present invention in combination with anti-PD-1 antibodies. Antibodies immunospecific for PD-1 are known (e.g. US Pat. No. 8,008,449; US Pat. No. 8,552,154; WO 2012/135408; WO 2012/145549). and WO 2013/014668): Nivolumab (also known as 5C4, BMS-936558, ONO-4538, MDX-1106, referred to herein as "PD-1 mAb 1"; Bristol-Myers Squibb is an OPDIVO Pembrolizumab (previously known as Lambrolizumab, also known as MK-3475, SCH-900475, commercially available as KEYTRUDA® by Merck), herein referred to as “PD-1 mAb 2” ; EH12.2H7 (Dana Farber), referred to herein as "PD-1 mAb 3"; and pidilizumab (also known as CT-011 (CureTech)), referred to herein as "PD-1 mAb 4".

1. Nivolumab (“PD-1 mAb 1”)
The amino acid sequence of the heavy chain variable domain of PD-1 mAb 1 is the amino acid sequence (SEQ ID NO: 131) (CDR _H residues are underlined):
QVQLVESGGG VVQPGRSLRL DCKASGITFS NSGMH WVRQA PGKGLEWVA V
IWYDGSKRYY ADSVKG RFTI SRDNSKNTLF LQMNSLRAED TAVYYCAT ND
DY WGQGTLVT VSS
has.

The amino acid sequence of the light chain variable domain of PD-1 mAb 1 is the amino acid sequence (SEQ ID NO: 132) (CDR _L residues are underlined):
EIVLTQSPAT LSLSPGERAT LSC RASQSVS SYLA WYQQKP GQAPRLLIY D
ASNRAT GIPA RFSGSGSGTD FTLTISSLEP EDFAVYYC QQ SSNWPRT FGQ
GTKVEIK
has.

2. Pembrolizumab (“PD-1 mAb 2”)
The amino acid sequence of the heavy chain variable domain of PD-1 mAb 2 is the amino acid sequence (SEQ ID NO: 133) (CDR _H residues are underlined):
QVQLVQSGVE VKKPGASVKV SCKASGYTFT NYYMY WVRQA PGQGLEWMG G
INPSNGGTNF NEKFKN RVTL TTDSSTTTAY MELKSLQFDD TAVYYCAR RD
YRFDMGFDY W GQGTTVTVSS
has.

The amino acid sequence of the light chain variable domain of PD-1 mAb 2 is the amino acid sequence (SEQ ID NO: 134) (CDR _L residues are underlined):
EIVLTQSPAT LSLSPGERAT LSC RASKGVS TSGYSYLH WY QQKPGQAPRL
LIY LASYLES GVPARFSGSG SGTDFTLTIS SLEPEDFAVY YC QHSRDLPL
T FGGGTKVEI K
has.

3. EH12.2H7 (“PD-1 mAb 3”)
The amino acid sequence of the heavy chain variable domain of PD-1 mAb 3 is the amino acid sequence (SEQ ID NO: 135) (CDR _H residues are underlined):
QVQLQQSGAE LAKPGASVQM SCKASGYSFT SSWIH WVKQR PGQGLEWIG Y
IYPSTGFTEY NQKFKD KATL TADKSSSTAY MQLSSLTSED SAVYYCA RWR
DSSGYHAMDY WGQGTSVTVSS
has.

The amino acid sequence of the light chain variable domain of PD-1 mAb 3 is the amino acid sequence (SEQ ID NO: 136) (CDR _L residues are underlined):
DIVLTQSPAS LTVSLGQRAT ISC RASQSVS TSGYSYMH WY QQKPGQPPKL
LIK FGSNLES GIPARFSGSG SGTDFTLNIH PVEEEDTATY YC QHSWEIPY
T FGGGTKLEI K
has.

4. Pigilizumab (“PD-1 mAb 4”)
The amino acid sequence of the heavy chain variable domain of PD-1 mAb 4 is the amino acid sequence (SEQ ID NO: 137) (CDR _H residues are underlined):
QVQLVQSGSE LKKPGASVKI SCKASGYTFT NYGMN WVRQA PGQGLQWMG W
INTDSGESTY AEEFKG RFVF SLDTSVNTAY LQITSLTAED TGMYFCVR VG
YDALDY WGQG TLVTVSS
has.

The amino acid sequence of the light chain variable domain of PD-1 mAb 4 is the amino acid sequence (SEQ ID NO: 138) (CDR _L residues are underlined):
EIVLTQSPSS LSASVGDRVT ITC SARSSVS YMH WFQQKPG KAPKLWIY RT
SNLAS GVPSR FSGSGSGTSY CLTINSLQPE DFATYYC QQR SSFPLT FGGG
TKLEIK
has.

IX. Methods of Production Anti-LAG-3 polypeptides, and other LAG-3 agonists, antagonists, and modulators, can be produced by methods known in the art, such as synthetically or recombinantly, such as LAG-3 mAb 1, LAG-3 mAb 2, Can be produced from polynucleotides and/or sequences of LAG-3 mAb 3, LAG-3 mAb 4, LAG-3 mAb 5 or LAG-3 mAb 6 antibodies. One method of producing such peptide agonists, antagonists, and modulators involves chemical synthesis of the polypeptide, followed by treatment under appropriate oxidative conditions to obtain the native conformation, i.e., the correct disulfide bond. accompanied by. This can be accomplished using methodologies known to those skilled in the art (e.g. Kelley, RF et al. (1990) In: Genetic Engineering Principles and Methods, Setlow, JK Ed., Plenum Press, NY, vol. 12, pp 1 -19; See Stewart, JM et al. (1984) Solid Phase Peptide Synthesis, Pierce Chemical Co., Rockford, IL; U.S. Patent No. 4,105,603; U.S. Patent No. 3,972,859; No. 3,842,067; and see also U.S. Pat. No. 3,862,925).

Polypeptides of the invention can be conveniently prepared using solid phase peptide synthesis (Merrifield, B. (1986) “Solid Phase Synthesis,” Science 232(4748):341-347; Houghten, RA (1985) “General Method For The Rapid Solid‐Phase Synthesis Of Large Numbers Of
Peptides: Specificity Of Antigen‐Antibody Interaction At The Level Of Individual Amino Acids,” Proc. Natl. Acad. Sci. (USA) 82(15): 5131‐5135; Ganesan, A. (2006) “Solid‐Phase Synthesis In The Twenty‐First Century,” Mini Rev. Med.
Chem. 6(1):3-10).

In yet another alternative, LAG-3 mAb 1, LAG-3 mAb 2, LAG-3
A LAG-3 mAb having one or more of the CDRs of mAb 3, LAG-3 mAb 4, LAG-3 mAb 5 or LAG-3 mAb 6, or for binding to human LAG-3 or a soluble form thereof. 1. A fully human antibody that competes with LAG-3 mAb 2, LAG-3 mAb 3, LAG-3 mAb 4, LAG-3 mAb 5, or LAG-3 mAb 6 to express a specific human immunoglobulin protein. It can be obtained using a commercially available processed mouse. For humanization or production of human antibodies, genetically modified animals engineered to produce more desirable (eg, fully human antibodies) or more robust immune responses may also be used. Examples of such technologies are XENOMOUSE™ (Abgenix, Inc., Fremont, CA) and HUMAb-Mouse® and TC MOUSE™ (both Medarex, Inc., Princeton, NJ). .

In one alternative, antibodies may be recombinantly produced and expressed using methods known in the art. Antibodies can be produced by first isolating the produced antibody from a host animal, obtaining the gene sequence, and using the gene sequence to recombinantly express the antibody in host cells (eg, CHO cells). Another method that can be employed is to express the antibody sequences in plants (eg tobacco) or transgenic milk. Suitable methods for recombinantly expressing antibodies in plants or milk have been disclosed (eg Peeters et al.
(2001) "Production Of Antibodies And Antibody Fragments In Plants," Vaccine 19:2756; Lonberg, N. et al. (1995) "Human Antibodies From Transgenic Mice," Int. Rev. Immunol 13:65-93; and Pollock et a/. (1999) "Transgenic Milk As A Method For The Production Of Recombinant Antibodies," J. Immunol Methods 231 : 147-157). Suitable methods for making antibody derivatives, eg, chimeric, humanized, single chain, etc., are known in the art. In another alternative, antibodies can be produced recombinantly by phage display technology (e.g., U.S. Pat. No. 5,565,332; U.S. Pat. No. 5,580,717; U.S. Pat. No. 5,733,743; No. 6,265,150; and Winter, G. et al. (1994) "Making Antibodies By Phage Display Technology," Annu. Rev. Immunol. 12.433-455).

Antibodies or proteins of interest may be subjected to sequencing by Edman degradation, which is known to those skilled in the art. Peptide information generated from mass spectrometry or Edman degradation can be used to design probes or primers used to clone proteins of interest.

An alternative method of cloning the protein of interest is to clone the CDRs of LAG-3 mAb 1, LAG-3 mAb 2, LAG-3 mAb 3, LAG-3 mAb 4, LAG-3 mAb 5 or LAG-3 mAb 6. or for binding to human LAG-3, LAG-3 mAb 1, LAG-3 mAb 2, LAG-3 mAb 3, LAG-3 mAb 4, LAG-3 mAb 5 or LAG-3 3 by "panning" with purified LAG-3, or a portion thereof, for cells expressing the antibody or protein of interest that competes with mAb 6. The "panning" procedure involves obtaining a cDNA library from tissues or cells that express LAG-3, overexpressing said cDNA in a second cell type, and producing LAG-3 mAb 1, LAG-3 mAb 2, LAG-3 Screening transfected cells of a second cell type for specific binding to LAG-3 in the presence or absence of mAb 3, LAG-3 mAb 4, LAG-3 mAb 5 or LAG-3 mAb 6. It may be implemented by A detailed description of the methods used in the cloning of mammalian gene coding for cell surface proteins by "panning" can be found in the prior art (e.g. Aruffo, A. et al. (1987) "Molecular Cloning Of A CD28 cDNA By A High-Efficiency COS Cell Expression System " Proc. Natl. Acad. Sci. (U.S.A.) 84:8573-8577 and Stephan, J. et al. (1999) "Selective Cloning Of Cell Surface Proteins Involved In Organ Development : Epithelial Glycoprotein Is Involved In Normal Epithelial Differentiation" Endocrinol. 140:5841-5854).

Vectors containing the polynucleotide of interest can be: electroporation; transfection using calcium chloride, rubidium chloride, calcium phosphate, DEAE-dextran or other substances; biolistic bombardment; lipofection; can be introduced into the host cell by any of a number of suitable means, including (if the agent is an infectious agent). The choice of vector or polynucleotide introduction often depends on the characteristics of the host cell.

Any host cell capable of overexpressing heterologous DNA can be used to isolate genes encoding antibodies, polypeptides, or proteins of interest. Non-limiting examples of suitable mammalian host cells include, but are not limited to, COS, HeLa and CHO cells. Preferably, the host cell is 5-fold higher, more preferably 10-fold higher, more preferably 20-fold higher, more preferably Express cDNA at 50 times higher, more preferably 100 times higher levels. Screening of host cells for specific binding to LAG-3 is preferably performed by immunoassay or FACS. Cells that overexpress antibodies or proteins of interest can be identified.

The invention includes polypeptides comprising the amino acid sequences of antibodies of the invention. Polypeptides of the invention can be produced by procedures known in the art. The polypeptides can be produced by proteolysis or other degradation of antibodies, by recombinant methods as described above (ie, single or fusion polypeptides), or by chemical synthesis. Polypeptides of the antibodies described above, particularly relatively short polypeptides of up to about 50 amino acids, are conveniently produced by chemical synthesis. Chemical synthesis methods are known in the art and are commercially available. For example, anti-LAG-3 polypeptides may be produced by automated polypeptide synthesizers employing solid phase methods.

The present invention provides LAG-3 mAb 1, LAG-3 mAb 2, LAG-3 mAb 3, LAG-3 mAb 4, LAG-3 mAb 5 or LAG-3 mAb 6 antibodies, and Variants of any binding polypeptide fragments, including functionally equivalent antibodies and fusion polypeptides that do not significantly affect the properties of the molecule, as well as variants with enhanced or decreased activity. good. Modifications of polypeptides are routinely practiced in the art and need not be described in detail herein. Examples of modified polypeptides include polypeptides with conservative substitutions of amino acid residues, one or more deletions or additions that do not significantly alter functional activity to the detriment, or the use of chemical analogs. can be mentioned. Amino acid residues that can be conservatively substituted for each other include: glycine/alanine; serine/threonine; valine/isoleucine/leucine; asparagine/glutamine; aspartic acid/glutamic acid; lysine/arginine; and phenylalanine/tyrosine. but not limited to. These polypeptides also include glycosylated and non-glycosylated polypeptides, as well as polypeptides with other post-conversion modifications, such as glycosylation with different sugars, acetylation and phosphorylation. Preferably, amino acid substitutions are conservative, ie, the substituted amino acid has similar chemical properties as the original amino acid. Such conservative substitutions are known in the art, examples of which are described above. Amino acid modifications may range from changing or modifying one or more amino acids to completely redesigning a region such as a variable domain. Variable domain changes alter binding affinity and/or specificity. Other methods of modification include the use of linking techniques known in the art, including, but not limited to, enzymatic means, oxidative substitution, and chelation. Modifications can be used, for example, to add labels for immunoassays, eg to add radioactive moieties for radioimmunoassays, and the like. Modified polypeptides can be made using procedures established in the art and screened using standard assays known in the art.

The invention also provides one or more of the polypeptides of the invention, or LAG-3 mAb 1, LAG-3 mAb 2, LAG-3 mAb 3, LAG-3 mAb 4, LAG-3 mAb 5 or LAG-3 Also included are fusion proteins comprising the mAb 6 antibody. In one embodiment, a fusion polypeptide is provided comprising a light chain, a heavy chain, or both a light chain and a heavy chain. In another embodiment, the fusion polypeptide contains a heterologous immunoglobulin constant region. In another embodiment, the fusion polypeptide contains the light and heavy chain variable domains of an antibody produced from a publicly deposited hybridoma. For the purposes of the present invention, an antibody fusion protein specifically binds to LAG-3 and another amino acid sequence to which said antibody fusion protein is not attached within the native molecule, e.g. a heterologous sequence from another region or a homologous sequence. contains one or more polypeptide domains.

X. Uses of LAG-3 binding molecules of the invention The present invention provides LAG-3 binding molecules of the invention (e.g., anti-LAG-3 antibodies, anti-LAG-3 bispecific diabodies, etc.), Encompasses compositions, including pharmaceutical compositions, comprising a polypeptide, a polynucleotide comprising a sequence encoding such a molecule or polypeptide, and other agents described herein.

As previously discussed, LAG-3 plays an important role in down-regulating T cell proliferation, function and homeostasis. LAG-3 binding molecules of the invention have the ability to reverse LAG-3-mediated immune system inhibition by inhibiting LAG-3 function. Therefore, LAG-3 mAb 1, LAG-3 mAb 2, LAG-3 mAb 3, LAG-3 mAb 4, LAG-3 mAb 5 and LAG-3 mAb 6, their humanized derivatives, and their LAG-3 by blocking LAG-3-mediated immune system inhibition using molecules that contain binding fragments (e.g., bispecific diabodies, etc.) or molecules that compete for binding with such antibodies. can promote the activation of

The bispecific LAG-3 binding molecules of the present invention that bind LAG-3 and another molecule (e.g. PD-1) present on the cell surface involved in the regulation of immune checkpoints include LAG-3 and the above-mentioned Enhances the immune system by blocking immune system inhibition mediated by immune checkpoint molecules. Accordingly, the LAG-3 binding molecules of the invention are useful for enhancing an immune response (eg, a T cell-mediated immune response) in a subject. In particular, the LAG-3 binding molecules of the present invention can be used to treat any disease associated with the immune system being suppressed in an undesirable manner, including cancers and diseases associated with the presence of pathogens (e.g. bacterial, fungal, viral or protozoal infections). A disease or condition can be treated.

Cancers that can be treated with the LAG-3 binding molecules of the present invention include: adrenal gland tumors; AIDS-related cancers; alveolar soft tissue sarcomas; astrocytic tumors; bladder cancer; bone cancer; brain and spinal cord cancer; metastases. breast cancer; carotid bulb tumor; cervical cancer; chondrosarcoma; chordoma; chromophobe renal cell carcinoma; clear cell carcinoma; colorectal cancer; benign fibrous histiocytoma of the skin; desmoplastic Small round cell tumor; ependymomas; Ewing tumor; extraosseous myxoid chondrosarcoma; osseous fibroplasia; fibrous bone dysplasia; gallbladder or cholangiocarcinoma; gastrointestinal cancer; gestational trophoblastic disease; germ cell tumor ; head and neck cancer; hepatocellular carcinoma; pancreatic islet cell tumor; Kaposi's sarcoma; renal cancer; leukemia; lipoma/benign lipoma; liposarcoma/malignant lipoma; liver cancer; lymphoma; lung cancer; medulloblastoma; melanoma; meninges multiple endocrine tumors; multiple myeloma; myelodysplastic syndrome; neuroblastoma; neuroendocrine tumor; ovarian cancer; pancreatic cancer; papillary thyroid cancer; parathyroid tumor; childhood cancer; peripheral nerve sheath tumor; pituitary tumor; prostate cancer; posterior uveal melanoma; rare hematological disorder; renal metastatic cancer; rhabdoid tumor; rhabdomyosarcoma; sarcoma; skin cancer; soft tissue sarcoma; squamous cell carcinoma; gastric cancer; Mention may be made of cancers characterized by the presence of cancer cells selected from the group consisting of membranous sarcoma; testicular cancer; thymic cancer; thymoma; thyroid metastatic cancer; and uterine cancer. The trispecific binding molecules of the invention can be applied to colorectal cancer, hepatocellular carcinoma, glioma, kidney cancer, breast cancer, multiple myeloma, bladder cancer, neuroblastoma; sarcoma, non-Hodgkin's lymphoma, non-small cell lung cancer. , can be used to treat ovarian cancer, pancreatic cancer and rectal cancer.

In particular, the LAG-3 binding molecules of the invention are suitable for colorectal cancer, hepatocellular carcinoma, glioma, kidney cancer, breast cancer, multiple myeloma, bladder cancer, neuroblastoma. ; Can be used to treat sarcoma, non-Hodgkin's lymphoma, non-small cell lung cancer, ovarian cancer, pancreatic cancer and rectal cancer.

Pathogen-related diseases that can be treated with LAG-3 binding molecules of the invention include chronic viral, bacterial, fungal and parasitic infections. Chronic infections that can be treated with LAG-3 binding molecules of the invention include: Epstein-Barr virus; hepatitis A virus (HAV); hepatitis B virus (HBV); hepatitis C virus (HCV); herpesviruses (e.g. HSV). -1, HSV-2, HHV-6, CMV); Human immunodeficiency virus (HIV); Vesicular stomatitis virus (VSV); Bacillus; Citrobacter; Cholera; Diphtheria; Enterobacter; Neisseria gonorrhoeae; Helicobacter pylori; Klebsiella; Legionella; Meningococcus; Mycobacteria; Pseudomonas; Streptococcus pneumoniae; Rickettsia; Salmonella; Serratia; Staphylococcus; Streptococcus; Tetanus; Aspergillus (Aspergillus fumigatus, Aspergillus niger, etc.); Blastomyces dermatitidis albicans, Candida krusei, Candida glabrata, Candida tropicalis, etc.); Cryptococcus neoformans; Genus Mucorae (Mucor, Yumice mold, Arachnoscabi); Sporothrix schenkii; Paracoccidioides brasiliensis; Coccidioides imchis; Histoplasma capsulatum; Leptospirosis; Borrelia burgdorferi; Helminth parasites (hookworms, tapeworms, trematodes, flatworms (e.g. Schistosoma)); - Donovan Leishmania.

LAG-3 binding molecules of the invention can be combined with immunogenic agents such as tumor vaccines. Such vaccines may contain purified tumor antigens (including recombinant proteins, peptides and carbohydrate molecules), autologous or allogeneic tumor cells. A number of tumor vaccine strategies have been described (e.g. Palena, C., et al., (2006) “Cancer vaccines: preclinical studies and novel strategies,” Adv. Cancer Res. 95, 115-145; Mellman, I. , et al. (2011) “Cancer immunotherapy comes of age,” Nature 480, 480-489; Zhang, X.
M. et al. (2008) “The Anti-Tumor Immune Response Induced By A Combination of MAGE-3/MAGE-n-Derived Peptides,” Oncol. Rep. 20, 245-252; Disis, ML et al. (2002 ) “Generation of T-cell Immunity to the HER-2/neu Protein After Active Immunization with HER-2/neu Peptide-Based Vaccines,” J. Clin. Oncol. 20:2624-2632; Vermeij, R. et al. (2012) “Potentiation of a p53-SLP Vaccine By Cyclophosphamide
In Ovarian Cancer: A Single-Arm Phase II Study.” Int. J. Cancer 131:E670-E680
reference). LAG-3 binding molecules of the invention can be combined with chemotherapy regimes. In these instances, it may be possible to reduce the dose of chemotherapy reagents administered (Mokyr. MB et al. (1998) “Realization Of The Therapeutic Potential Of CTLA‐4 Blockade
In Low‐Dose Chemotherapy‐Treated Tumor‐Bearing Mice,” Cancer Research 58: 5301‐5304).

The LAG-3 binding molecules of the invention can be combined with other immunostimulatory molecules, such as antibodies that activate host immune responses to increase the level of T cell activation. In particular, anti-PD-1 antibodies, anti-PD-L1 antibodies and/or anti-CTLA-4 antibodies have been demonstrated to activate the immune system (e.g. del Rio, ML. et al. (2005) “Antibody -Mediated Signaling Through PD-1 Costimulates T Cells And Enhances CD28-Dependent Proliferation,” Eur. J. Immunol 35:3545-3560; Barber, DL et al. (2006) “Restoring Function In Exhausted CD8 T Cells During Chronic Viral Infection ,” Nature 439, 682-687; Iwai, Y. et al. (2002) “Involvement of PD-L1 On Tumor Cells In The Escape
From Host Immune System And Tumor Immunotherapy by PD-L1 blockade,” Proc. Natl
Acad. Sci. USA 99, 12293-12297; see Leach, DR, et al., (1996) “Enhancement Of Antitumor Immunity By CTLA-4 Blockade,” Science 271, 1734-1736). Further immunostimulatory molecules that can be combined with the LAG-3 binding molecules of the invention include: antibodies against molecules on the surface of dendritic cells (DCs) that activate dendritic cell (DC) function and antigen presentation; T cell helpers; anti-CD40 antibodies that can replace the activity; and activating antibodies against T cell co-stimulatory molecules such as PD-L1, CTLA-4, OX-40 4-1BB and ICOS (e.g. Ito et al. (2000) “Effective Priming Of Cytotoxic T Lymphocyte Precursors By Subcutaneous Administration Of Peptide Antigens In Liposomes Accompanied By Anti-CD40 And Anti-CTLA-4 Antibodies,” Immunobiology 201:527-40; U.S. Patent No. 5,811,
No. 097; Weinberg et al. (2000) “Engagement of the OX-40 Receptor In Vivo Enhances Antitumor Immunity,” Immunol 164:2160-2169; Melero et al. (1997) “Monoclonal Antibodies Against The 4-1BB T-Cell Activation Molecule Eradicate Established Tumors,” Nature Medicine 3: 682-685; Hutloff et al. (1999) “ICOS is An Inducible T-Cell Co-Stimulator Structurally And Functionally Related to CD28,” Nature 397: 263-266; and Moran , AE et al. (2013) “The TNFRs OX40, 4-1BB, and CD40 As Targets For Cancer Immunotherapy,” Curr Opin Immunol. 2013 Apr; 25(2): 10.10
16/j.coi.2013.01.004) and/or from various costimulatory protein receptors (e.g. CD28, 4-1BB, ICOS, OX40, etc.) that play a role in stimulating T cells upon antigen binding. Stimulated chimeric antigen receptors (CARs) comprising an antigen-binding domain directed to a disease antigen fused to one or more intracellular signaling domains (e.g., Tettamanti, S.
et al. (2013) “Targeting Of Acute Myeloid Leukaemia By Cytokine-Induced Killer
Cells Redirected With A Novel CD123-Specific Chimeric Antigen Receptor,” Br. J. Haematol. 161:389-401; Gill, S. et al. (2014) “Efficacy Against Human Acute Myeloid Leukemia And Myeloablation Of Normal Hematopoiesis In A Mouse Model Usage
Chimeric Antigen Receptor-Modified T Cells,” Blood 123(15): 2343-2354; Mardiros, A. et al. (2013) “T Cells Expressing CD123-Specific Chimeric Antigen Receptors Exhibit Specific Cytolytic Effector Functions And Antitumor Effects Against Human Acute Myeloid Leukemia,” Blood 122:3138-3148; Pizzitola, I. et al. (2014)
(See “Chimeric Antigen Receptors Against CD33/CD123 Antigens Efficiently Target Primary Acute Myeloid Leukemia Cells in vivo,” Leukemia 28(8):1596-1605).

LAG-3 binding molecules of the invention can be combined with inhibitory chimeric antigen receptors (iCARs) to direct targeted immunotherapeutic responses. iCARs include: disease fused to one or more intracellular signaling domains from various inhibitory protein receptors (e.g., CTLA-4, PD-1, etc.) that play a role in limiting T cell responses upon antigen binding; Antigen-binding domains targeting antigens (e.g. Fedorov V.D. (2013) “PD-1- and CTLA-4-Based Inhibitory Chimeric Antigen Receptors (iCARs) Divert Off-Target Immunotherapy Responses,” Sci Tranl Med. 5(215): 215ra172).

In particular, the anti-LAG-3 antibody of the present invention is used in combination with an anti-CD137 antibody, an anti-OX40 antibody, an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-TIGIT antibody, an anti-TIM-3 antibody and/or a cancer vaccine.

In addition to therapeutic uses, the LAG-3 binding molecules of the invention can be removably labeled and used to detect LAG-3 in a sample or image LAG-3 on cells.

XI. Pharmaceutical Compositions The compositions of the present invention include bulk pharmaceutical compositions (e.g., impure or non-sterile compositions) that can be used in the manufacture of pharmaceutical compositions, as well as pharmaceutical compositions that can be used in the preparation of unit dosage forms (i.e., compositions suitable for administration to patients). These compositions contain a prophylactically or therapeutically effective amount of a LAG-3 binding molecule of the invention, or a combination of such an agent and a pharmaceutically acceptable carrier. Preferably, compositions of the invention include a prophylactically or therapeutically effective amount of one or more of the LAG-3 binding molecules of the invention and a pharmaceutically acceptable carrier. The invention particularly provides that the LAG-3 binding molecule is: LAG-3 mAb 1, LAG-3 mAb 2, LAG-3 mAb 3, LAG-3 mAb 4, LAG-3 mAb 5 or LAG-3
mAb 6 antibody; humanized LAG-3 mAb 1; LAG-3 mAb 2, LAG-3 mAb 3, LAG-3 mAb 4, LAG-3 mAb 5 or LAG-3 mAb 6 antibody; A pharmaceutical composition that is a LAG-3 binding fragment, or wherein the LAG-3 binding molecule is a bispecific LAG-3 diabody (e.g., a LAG-3 x PD-1 bispecific diabody) Contain things. Specifically included are: LAG-3 mAb 1 comprising 3 CDR _L and 3 CDR _H ; or LAG-3 mAb 2 comprising 3 CDR _L and 3 CDR _H ; or LAG-3 mAb 3 or contains 3 CDR _L and 3 CDR _{H of} LAG-3 mAb 4; or contains 3 CDR _L and 3 _{CDR H} of LAG-3 mAb 5. or a molecule containing the three CDR _L and three CDR _H of LAG-3 mAb 6.

The present invention also encompasses the pharmaceutical composition described above, further comprising a second therapeutic antibody specific for a particular cancer antigen (e.g., a tumor-specific monoclonal antibody), and a pharmaceutically acceptable carrier. .

In certain specific embodiments, the term "pharmaceutically acceptable" refers to a compound that has been approved by a federal regulatory agency or state government for use in animals, and more particularly in humans, or has been approved by the United States Pharmacopoeia or means listed in another generally accepted pharmacopoeia. The term "carrier" refers to a diluent, adjuvant (eg, Freund's adjuvant (complete and incomplete)), excipient, or vehicle used in the administration of a therapeutic agent. These pharmaceutical carriers can be sterile liquids, such as water and oils, including petroleum, animal, vegetable, or synthetic oils, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dry skim milk, glycerol, propylene. , glycol, water, ethanol, etc. If desired, the composition can also contain small amounts of wetting or emulsifying agents, or pH buffering agents. These compositions can take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained release formulations, and the like.

Generally, the ingredients of the compositions of the invention are mixed separately or together in unit dosage form, e.g., as a lyophilized powder or water-free concentrate in an airtight container such as an ampoule or sachet indicating the amount of active agent. Supplied as is. If the composition is administered by infusion, the composition can be dispensed using an infusion bottle containing sterile pharmaceutical grade water or saline. If the composition is to be administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients can be mixed prior to administration.

Compositions of the invention can be formulated as neutral or salt forms. Pharmaceutically acceptable salts include: those formed with anions derived from hydrochloric acid, phosphoric acid, acetic acid, oxalic acid, tartaric acid, etc., and sodium, potassium, ammonium, calcium, ferric hydroxide, Examples include, but are not limited to, those formed with cations derived from isopropylamine, triethylamine, 2-methylaminoethanol, histidine, procaine, and the like.

The invention also provides LAG-3 binding molecules of the invention (and more preferably LAG-3 mAb 1, LAG-3 mAb 2, LAG-3 mAb 3, LAG-3 mAb 4, LAG-3 mAb 5 or LAG-3 -3 mAb 6 antibody; humanized LAG-3 mAb 1, LAG-3 mAb 2, LAG-3 mAb 3, LAG-3 mAb 4, LAG-3 mAb 5 or LAG-3 mAb 6 antibody; any such a LAG-3 binding fragment of an antibody; or one in which the LAG-3 binding molecule is a bispecific LAG-3 diabody (e.g. a LAG-3 x PD-1 bispecific diabody); Alternatively, a pharmaceutical pack or kit comprising multiple containers is also provided. Particularly included are the three CDR _L of LAG-3 mAb 1, alone or with a pharmaceutically acceptable carrier:
and 3 CDR _H ; or 3 CDR _L and 3 CDR _H of LAG-3 mAb 2; or 3 CDR _L and 3 CDR _H of LAG-3 mAb 3; or LAG-3 Contains 3 CDR _L and 3 CDR _H of mAb 4; or 3 CDR _L and 3 CDR _H of LAG-3 mAb 5; or 3 CDR _L and 3 CDR _H of LAG-3 mAb 6. It is a molecule containing. Additionally, one or more other prophylactic or therapeutic agents useful in treating the disease can also be included in the pharmaceutical pack or kit. The invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions of the invention. Optionally, such container or containers may be associated with a notification in a form prescribed by a government agency regulating the manufacture, use or sale of pharmaceutical or biological products, and as described above. The notification reflects the agency's approval of manufacture, use, or sale for human administration.

The present invention provides kits that can be used in the methods described above. The kit can include any of the LAG-3 binding molecules of the invention. The kit may further include in the one or more containers one or more other prophylactic and/or therapeutic agents useful for the treatment of cancer; One or more cytotoxic antibodies that bind to one or more cancer antigens of interest can be included. In certain embodiments, the other prophylactic or therapeutic agent is a chemotherapeutic agent. In other embodiments, the prophylactic or therapeutic agent is a biological or hormonal therapy.

XII. Methods of Administration Compositions of the invention can be administered to a subject to treat a disease, disorder or infection by administering to a subject an effective amount of a fusion protein or conjugate molecule of the invention or a pharmaceutical composition comprising a fusion protein or conjugate molecule of the invention. can be provided for the treatment, prevention, and amelioration of one or more symptoms associated with the disease. In preferred embodiments, the composition is substantially purified (ie, substantially free of substances that limit the effectiveness of the composition or produce undesirable side effects). In certain specific embodiments, the subject is an animal, preferably a non-primate (e.g., a bovine, an equid, a feline, a canine, a rodent, etc.) or a primate (e.g., a monkey such as a cynomolgus monkey, a human, etc.). It is a mammal. In certain preferred embodiments, the subject is a human.

For example, encapsulation into liposomes, particulate microcapsules, recombinant cells capable of expressing antibodies or fusion proteins; receptor-mediated endocytosis (e.g. Wu et al. (1987) “Receptor-Mediated In Vitro Gene Transformation By A Soluble DNA A variety of delivery systems are known, such as the construction of nucleic acids as part of retroviruses or other vectors (see "Carrier System," J. Biol. Chem. 262:4429-4432); Can be used for administration.

Methods of administering the molecules of the invention include: parenteral administration (e.g. intradermal, intramuscular, intraperitoneal, intravenous and subcutaneous); epidural; and mucosal (e.g. intranasal and oral routes). but not limited to. In certain specific embodiments, the LAG-3 binding molecules of the invention are administered intramuscularly, intravenously or subcutaneously. The compositions may be administered by any convenient route, such as by infusion or bolus injection, by absorption through epithelial or mucocutaneous layers (such as oral mucosa, rectal and intestinal mucosa, etc.), and may be administered by any other biologically active route. May be administered with an agent. Administration can be systemic or local. Additionally, pulmonary administration can also be employed, eg, by use of an inhaler or nebulizer and formulation with aerosolizing agents. For example, US Patent No. 6,019,968; US Patent No. 5,985,320; US Patent No. 5,985,309; US Patent No. 5,934,272; US Patent No. 5,874,064. ; U.S. Patent No. 5,855,913; U.S. Patent No. 5,290,540; U.S. Patent No. 4,880,078; International Publication No. 92/19244; International Publication No. 97/32572; International Publication No. See WO 97/44013; WO 98/31346; WO 99/66903, each of which is incorporated by reference into this application.

The present invention also provides for packaging the LAG-3 binding molecules of the present invention in airtight containers such as ampoules or sachets indicating the amount of the molecule. In one embodiment, the molecule is supplied as a sterile lyophilized powder or anhydrous concentrate in an airtight container and can be reconstituted, eg, with water or saline, to a concentration suitable for administration to a subject. Preferably, the LAG-3 binding molecules of the invention are supplied as a sterile lyophilized powder in an airtight container.

The lyophilized LAG-3 binding molecule of the invention should be stored at 2°C to 8°C in its original container, and the molecule should be stored within 12 hours, preferably within 6 hours, after reconstitution. It should be administered within 5 hours, within 3 hours or within 1 hour. In an alternative embodiment, such molecules are supplied in liquid form in an airtight container that indicates the amount and concentration of the molecule, fusion protein, or conjugate molecule. Preferably, such LAG-3 binding molecules, if provided in liquid form, are supplied in an airtight container.

Amounts of the compositions of the invention effective for treating, preventing and ameliorating one or more symptoms associated with a disorder can be determined by standard clinical techniques. The precise dose to be employed in a regimen will also depend on the route of administration and the severity of the condition, and should be decided according to the judgment of the practitioner and each patient's circumstances. Effective doses can be extrapolated from dose-response curves obtained from in vitro or animal model test systems.

As used herein, an "effective amount" of a pharmaceutical composition means, in one embodiment, to: reduce symptoms caused by a disease; symptoms of an infection (e.g., viral load, fever, pain, sepsis, etc.); ) or attenuation of symptoms caused by a disease that attenuates cancer symptoms (e.g. proliferation of cancer cells, presence of tumor, tumor metastasis, etc.); thereby increasing the QOL of people suffering from the disease; to treat the disease; Benefits including, but not limited to, reducing the amount of other medications required; enhancing the effect of another medication, such as by targeting and/or internalization; slowing disease progression; and/or prolonging the survival of an individual. or in an amount sufficient to obtain the desired result.

An effective amount can be administered in one or more doses. For the purposes of the present invention, an effective amount of a drug, compound or pharmaceutical composition is intended to reduce, directly or indirectly, the propagation of the presence of (or its effects on) a virus and to reduce the development of a viral disease and/or or in an amount sufficient to cause a delay. In some embodiments, an effective amount of an agent, compound or pharmaceutical composition may or may not be achieved in combination with another agent, compound or pharmaceutical composition. Accordingly, an "effective amount" can be considered in the context of administering one or more chemotherapeutic agents, and a single agent may be used in combination with one or more other agents to achieve a desired result. An effective amount can be considered to be given if it achieves the desired or desired result. Although individual requirements vary, determination of optimal ranges of effective amounts of each component is within the ability of those skilled in the art.

For LAG-3 binding molecules (eg, antibodies, diabodies, etc.) encompassed by the present invention, the dosage administered to a patient is preferably determined based on the weight (kg) of the recipient subject. For LAG binding molecules encompassed by the invention, the dosage administered to a patient will typically be at least about 0.01 μg/kg body weight, at least about 0.05 μg/kg body weight, at least about 0.1 μg/kg body weight. , at least about 0.2 μg/kg body weight, at least about 0.5 μg/kg body weight, at least about 1 μg/kg body weight, at least about 2 μg/kg body weight, at least about 3 μg/kg body weight, at least about 5 μg/kg body weight, at least about 10 μg /kg body weight, at least about 20 μg/kg body weight, at least about 30 μg/kg body weight, at least about 50 μg/kg body weight, at least about 100 μg/kg body weight, at least about 250 μg/kg body weight, at least about 750 μg/kg body weight, at least about 1. 5 mg/kg body weight, at least about 3 mg/kg body weight, at least about 5 mg/kg body weight, or at least about 10 mg/kg, at least about 30 mg/kg, at least about 50 mg/kg, at least about 75 mg/kg, at least about 100 mg/kg, At least about 125 mg/kg, at least about 150 mg/kg body weight or more. The calculated dose is administered based on the patient's weight at baseline. Significant (≧10%) change in body weight from baseline or established stable body weight shall prompt recalculation of dose.

In some embodiments, for LAG-3 binding bispecific molecules (e.g., diabodies and trivalent binding molecules) encompassed by the invention, the dosage administered to a patient typically is at least about 0.3 ng/kg/day to about 0.9 ng/kg/day, at least about 1 ng/kg/day to about 3 ng/kg/day, at least about 3 ng/kg/day to about 9 ng/kg/day, at least about 10 ng /kg/day to about 30 ng/kg/day, at least about 30 ng/kg/day to about 90 ng/kg/day, at least about 100 ng/kg/day to about 300 ng/kg/day, at least about 200 ng/kg/day to about 600 ng/kg/day, at least about 300 ng/kg/day to about 900 ng/kg/day, at least about 400 ng/kg/day to about 800 ng/kg/day, at least about 500 ng/kg/day to about 1000 ng/kg/day. at least about 600 ng/kg/day to about 1000 ng/kg/day, at least about 700 ng/kg/day to about 1000 ng/kg/day, at least about 800 ng/kg/day to about 1000 ng/kg/day, at least about 900 ng/kg/day /kg/day to about 1000 ng/kg/day, or at least about 1,000 ng/kg/day. The calculated dose is administered based on the patient's weight at baseline. Significant (≧10%) change in body weight from baseline or established stable body weight shall prompt recalculation of dose.

In another embodiment, the patient is administered a therapeutic regimen comprising one or more doses of a prophylactically or therapeutically effective amount of a LAG-3 binding molecule of the invention as described above, the therapeutic regimen comprising: Administered over 2, 3, 4, 5, 6 or 7 days. In certain embodiments, the therapeutic regimen comprises a prophylactically or therapeutically effective amount of a LAG-3 binding molecule of the invention (and in particular LAG-3 mAb 1, LAG-3 mAb 2, LAG-3 mAb 3, LAG-3 3 mAb 4, LAG-3 mAb 5 or LAG-3 mAb 6 antibody; humanized LAG-3 mAb 1, LAG-3 mAb 2, LAG-3 mAb 3, LAG-3 mAb 4, LAG-3 mAb 5 or LAG -3 mAb 6 antibody; a LAG-3 binding fragment of any such antibody; or a LAG-3 binding molecule is a bispecific LAG-3 diabody (e.g. a LAG-3 x PD-1 bispecific Fc diabody); administration) intermittently (e.g., on days 1, 2, 3, and 4 of a given week, and on days 5 and 6 of the same week). and 7 days without administering a prophylactically or therapeutically effective dose of a trispecific binding molecule encompassed by the invention). Specifically included include 3 CDR _L and 3 CDR _H of LAG-3 mAb 1; or 3 CDR _L and 3 CDR _H of LAG-3 mAb 2; or LAG-3 mAb 3 contains 3 CDR _L and 3 CDR _H of LAG-3 mAb 4; or 3 CDR L and 3 CDR _H of LAG-3 mAb 4
DR _H ; or 3 CDR _L and 3 CDR _H of LAG-3 mAb 5;
or administration (on days 5, 6 and 7 of the same week) of a molecule comprising 3 CDR _L and 3 CDR _H of LAG-3 mAb 6. There are typically one, two, three, four, five or more courses of treatment. Each course may be the same regimen or a different regimen.

In another embodiment, the dose administered is the first quarter, first half of one or more regimens until a daily prophylactically or therapeutically effective amount of said LAG-3 binding molecule is reached. or titrated over the first two-thirds or three-quarters (eg, over the first, second, or third regimen of a four-course treatment). Table 6 provides five examples of the different dosing regimens described above for a typical course of treatment with diabodies.

The dose and frequency of administration of LAG-3 binding molecules of the invention can be reduced or biased by enhancing uptake and tissue penetration of the molecule, for example through modifications such as lipidation.

The dosage of LAG-3 binding molecules of the invention administered to a patient may be calculated for use as a single agent therapy. Alternatively, the molecule is used in combination with other therapeutic compositions and the dosage administered to the patient is lower than if the molecule were used as a single agent therapy.

The pharmaceutical compositions of the present invention may be administered locally to the area in need of treatment, which can be accomplished, for example, but not limited to, by local instillation, by injection, or by using an implant. The implant may be of porous, non-porous or gelatinous material and includes a membrane or fibers, such as a silastic membrane. Preferably, when administering the molecules of the invention, care must be taken to use materials to which the molecules are not absorbed.

Compositions of the invention can be delivered in vesicles, particularly liposomes (Langer (1990) “New Methods Of Drug Delivery,” Science 249:1527-1533); Treat et al., in Liposomes in the Therapy of Infectious Disease. and Cancer, Lopez-Berestein and Fidler (eds.), Liss, New York, pp. 353-365 (1989); see Lopez-Berestein, ibid., pp. 317-327).

Compositions of the invention can be delivered in controlled or sustained release systems. Any technique known to those skilled in the art can be used to produce sustained release formulations containing one or more LAG-3 binding molecules of the invention. For example: US Pat. No. 4,526,938; WO 91/05548; WO 96/20698; Ning et al. (1996) “Intratumoral Radioimmunotheraphy Of A Human Colon Cancer Xenograft Using A Sustained-Release Gel ,” Radiotherapy &Oncology39:179-189;Song et al. (1995) “Antibody Mediated Lung Targeting Of Long-Circulating Emulsions,” PDA Journal of Pharmaceutical Science & Technology
50:372-397;Cleek et al. (1997) “Biodegradable Polymeric Carriers For A bFGF Antibody For Cardiovascular Application,” Pro. Int'l. Symp. Control. Rel. Bioact.
Mater. 24:853-854; and Lam et al. (1997) “Microencapsulation Of Recombinant Humanized Monoclonal Antibody For Local Delivery,” Proc. Int'l. Symp. Control Rel. Bioact. Mater. 24:759-760. (Each of which is incorporated herein by reference in its entirety). In one embodiment, a pump may be used in a controlled release system (Langer,
supra; Sefton, (1987) “Implantable Pumps,” CRC Crit. Rev. Biomed. Eng. 14:201-240; Buchwald et al. (1980) “Long-Term, Continuous Intravenous Heparin Administration By An Implantable Infusion Pump In Ambulatory Patients With Recurrent Venous Thrombosis,” Surgery 88:507-516; and Saudek et al. (1989) “A Preliminary Trial Of The Programmable Implantable Medication System For Insulin Delivery,” N. Engl. J. Med. 321:574-579 reference). In another embodiment, polymeric materials can be used to achieve controlled release of molecules (e.g., Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Pres., Boca Raton, Florida (1974); Controlled Drug Bioavailability, Drug Product Design and Performance, Smolen and Ball (eds.), Wiley, New York (1984);Levy et al. (1985) “Inhibition Of Calcification Of Bioprosthetic Heart Valves By Local Controlled-Release Diphosphonate,” Science228:190 -192;During et al.(1989)“Controlled Release Of Dopamine From A Polymeric Brain Implant: In Vivo Characterization,” Ann. Neurol. 25:351-356;Howard et al.(1989)“Intracerebral Drug Delivery In Rats With Lesion-Induced Memory Deficits,” J. Neurosurg. 7(1):105-112); U.S. Patent No. 5,679,377; U.S. Patent No. 5,916,597; U.S. Patent No. 5,912,015 ; US Pat. No. 5,989,463; US Pat. No. 5,128,326; WO 99/15154; and WO 99/20253). Examples of polymers used in sustained release formulations include poly(2-hydroxyethyl methacrylate), poly(methyl methacrylate), poly(acrylic acid), poly(ethylene-co-vinyl acetate), poly(methacrylic acid). , polyglycolide (PLG), polyanhydrides, poly(N-vinylpyrrolidone), poly(vinyl alcohol), polyacrylamide, poly(ethylene glycol), polylactides (PLA), poly(lactide-co-glycolide) (PLGA) and polyorthoesters. Controlled release systems can be placed close to the therapeutic target (e.g. the lungs) and thus require only a fraction of the systemic dose (e.g. Goodson, in Medical Applications of Controlled Release, supra,
vol.2, pp.115-138 (1984)). Polymeric compositions useful as controlled release implants can be used according to Dunn et al. (see US Pat. No. 5,945,155). This particular method is based on the therapeutic effect of in situ controlled release of bioactive materials from polymeric systems. Implantation can generally be performed at any location within a patient's body in need of therapeutic treatment. Non-polymeric sustained release systems can be used, in which case a non-polymeric implant within the subject's body is used as the drug delivery system. After implantation in the body, the organic solvents of the implant dissipate, disperse, or leach from the composition into the surrounding tissue fluids, and the non-polymeric materials gradually aggregate or precipitate to form a solid microporous matrix. (See US Pat. No. 5,888,533).

Controlled release systems are discussed in a report by Langer (1990, “New Methods Of Drug Delivery,” Science 249:1527-1533). Any technique known to those skilled in the art can be used to produce sustained release formulations containing one or more therapeutic agents of the invention. For example: U.S. Patent No. 4,526,938; WO 91/05548 and WO 96/20698; Ning et al. (1996) “Intratumoral Radioimmunotheraphy Of A Human Colon Cancer Xenograft Using A Sustained-Release Gel,” Radiotherapy &Oncology39:179-189;Song et al.(1995)“Antibody Mediated Lung Targeting Of Long-Circulating Emulsions,” PDA Journal of Pharmaceutical Science &Technology50:372-397;Cleek et al.(1997)“Biodegradable Polymeric Carriers For A bFGF Antibody For Cardiovascular Application,” Pro. Int'l. Symp. Control. Rel. Bioact. Mater. 24:853-854; and Lam et al. (1997) “Microencapsulation Of Recombinant Humanized Monoclonal Antibody For Local
See Delivery,” Proc. Int'l. Symp. Control Rel. Bioact. Mater. 24:759-760, each of which is incorporated by reference in its entirety.

When the composition of the invention is a nucleic acid encoding a LAG-3 binding molecule of the invention, the nucleic acid can be used in a suitable manner to promote expression of the LAG-3 binding molecule it encodes. constructed as part of a nucleic acid expression vector, e.g., by the use of retroviral vectors (see U.S. Pat. No. 4,980,286), or by direct injection, or by the use of microparticle bombardment (e.g., gene gun; Biolistic, Dupont). or by coating with lipids or cell surface receptors or gene transfer agents, or in conjunction with homeobox-like peptides known to enter the cell nucleus (e.g., Joliot et al. (1991) “Antennapedia Homeobox Peptide Regulates Neural Morphogenesis,” Proc. Natl. Acad. Sci. (USA) 88:1864-1868)
The nucleic acid can be administered in vivo by administering the nucleic acid as described above. Alternatively, the nucleic acid can be introduced intracellularly and integrated into host cell DNA for expression by homologous recombination.

Treatment of a subject with a therapeutically or prophylactically effective amount of a LAG-3 binding molecule of the invention can include a single treatment, or preferably can include a series of multiple treatments. In certain preferred examples, the subject uses a diabody as described above for about 1 to 10 weeks, preferably 2 to 8 weeks, more preferably about 3 to 7 weeks, even more preferably about 4, 5, or 6 weeks. Treatments are given once a week for several weeks. Pharmaceutical compositions of the invention can be administered once a day, twice a day or three times a day. Alternatively, the pharmaceutical composition may be administered once a week, twice a week, once every two weeks, once a month, once every six weeks, once every two months, twice a year or once a year. Can be administered once a year. It will also be appreciated that the effective dosage setting of the molecules used in therapy may be increased or decreased over the course of a particular treatment.

Having thus far outlined the invention, it will be more readily understood by reference to the following examples. These examples are provided by way of illustration and are not intended to limit the invention unless expressly stated to the contrary. It will be apparent to those skilled in the art that many modifications, both to materials and methods, may be practiced without departing from the scope of this disclosure.

Example 1 Characterization of anti-LAG-3 monoclonal antibodies LAG-3 mAb 1, LAG-3 mAb 2, LAG-3 mAb 3, LAG-3 mAb 4, LAG-3 mAb 5 and LAG-3 mAb 6 Six mice Monoclonal antibodies were isolated that were capable of immunospecifically binding to both human and cynomolgus monkey LAG-3, and were labeled "LAG-3 mAb 1", "LAG-3 mAb 2", "LAG-3 mAb 3", "LAG -3 mAb 4,” “LAG-3 mAb 5,” and “LAG-3 mAb 6.” The CDRs of these antibodies are known to be different and are described above. When LAG-3 mAb 1 is humanized, it is referred to herein as "hLAG-3 mAb 1 VH-1" and "hLAG-3 mAb 1".
1 VH-2", as well as herein "hLAG-3 mAb 1 VL-1", "hLAG-3 mAb 1 VL-2", "hLAG-3 mAb 1 VL-3". ” and four humanized VL domains called “hLAG-3 mAb 1 VL-4” were obtained. Humanization of LAG-3 mAb 6 also results in two humanized VH domains, herein referred to as "hLAG-3 mAb 6 VH-1" and "hLAG-3 mAb 6 VH-2", and Two humanized VL domains were obtained, designated "hLAG-3 mAb 6 VL-1" and "hLAG-3 mAb 6 VL-2". As discussed above, the humanized heavy and light chain variable domains of a given antibody may be used in any combination, and the particular combination of humanized variable domains may be The humanized antibodies designated by reference and including, for example, hLAG-3 mAb 1 VH-1 and hLAG-3 mAb 1 VL-2 are specifically referred to as "hLAG-3 mAb 1 (1.2)."

Full-length humanized mAbs were constructed as follows: the C-terminus of the humanized VL domain was fused to the N-terminus of the human light chain kappa region to generate light chains, and each light chain was ) Pairing with a heavy chain comprising a humanized VH domain of the same antibody fused to the N-terminus of a human IgG1 constant region containing the substitution or a human IgG4 constant region containing the S228P substitution.

An exemplary human IgG1 constant region amino acid sequence containing L234A/L235A (AA) substitutions is (SEQ ID NO: 139):
ASTKGPSVFP LAPSSKSTSG GTAALGCLVK DYFPEPVTVS WNSGALTSGV
HTFPAVLQSS GLYSLSSVVT VPSSSLGTQT YICNVNHKPS NTKVDKRVEP
KSCDKTHTCP PCPAPE AA GG PSVFLFPPKP KDTLMISRTP EVTCVVVDVS
HEDPEVKFNW YVDGVEVHNA KTKPREEQYN STYRVVSVLT VLHQDWLNGK
EYKCKVSNKA LPAPIEKTIS KAKGQPREPQ VYTLPSREE MTKNQVSLTC
LVKGFYPSDI AVEWESNGQP ENNYKTTPPV LDSDGSFFLY SKLTVDKSRW
QQGNVFSCSV MHEALHNHYT QKSLSLSPG X
where X is lysine (K) or absent.

In SEQ ID NO: 139, amino acid residues 1-98 correspond to the IgG1 CH1 domain (SEQ ID NO: 120), amino acid residues 99-113 correspond to the IgG1 hinge region (SEQ ID NO: 114), and amino acid residues 114-329 correspond to the IgG1 CH1 domain (SEQ ID NO: 120). , corresponds to the IgG1 CH2-CH3 domain (SEQ ID NO: 123) containing the L234A/L235A substitutions (underlined).

An exemplary human IgG4 constant region amino acid sequence containing the S228P substitution is (SEQ ID NO: 140):
ASTKGPSVFP LAPCSRSTSE STAALGCLVK DYFPEPVTVS WNSGALTSGV
HTFPAVLQSS GLYSLSSVVT VPSSSLGTKT YTCNVDHKPS NTKVDKRVES
KYGPPCP P CP APEFLGGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSQED
PEVQFNWYVD GVEVHNAKTK PREEQFNSTY RVVSVLTVLH QDWLNGKEYK
CKVSNKGLPS SIEKTISKAK GQPREPQVYT LPPSQEEMTK NQVSLTCLVK
GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLYSRL TVDKSRWQEG
NVFSCSVMHE ALHNHYTQKS LSLSLG
where X is lysine (K) or absent.

In SEQ ID NO: 140, amino acid residues 1-98 correspond to the IgG4 CH1 domain (SEQ ID NO: 122) and amino acid residues 99-110 correspond to the stabilized IgG4 hinge region (SEQ ID NO: 117) containing the S228P substitution (underlined). Correspondingly, amino acid residues 111-326 correspond to the IgG4 CH2-CH3 domain (SEQ ID NO: 4).

Antibodies LAG-3 mAb 1, LAG-3 mAb 2, LAG-3 mAb 3, LAG-3 mAb 4, LAG-3 mAb 5, LAG-3 mAb 6, multiple humanized LAG-3 mAb A, and reference antibodies The binding kinetics of LAG-3 mAb A was investigated using Biacore analysis. Anti-LAG-3 antibodies were captured and incubated with His-tagged soluble human LAG-3 (shLAG-3-His), and binding kinetics were analyzed by Biacore analysis. The calculated ka, kd and KD are presented in Table 7.

In further studies, we investigated the binding kinetics of the humanized antibodies hLAG-3 mAb 1 (1.4) and hLAG-3 mAb 6 (1.1), as well as the reference antibody LAG-3 mAb A, to both human and cynomolgus monkey LAG-3. was investigated using Biacore analysis. In these studies, soluble LAG-3 fusion proteins (the extracellular domain of human or cynomolgus monkey LAG-3 fused to mouse IgG2a) were captured on Fab2 goat anti-mouse Fc surfaces and incubated with anti-LAG-3 antibodies. The kinetics of binding was then determined by Biacore analysis. The binding curves of LAG-3 mAb A, hLAG-3 mAb 1 (1.4) and hLAG-3 mAb 6 (1.1) binding to cynomolgus monkey LAG-3 are shown in Figures 7A to 7C, respectively, and the calculated ka, kd and KD are presented in Table 8. In another study, the binding kinetics of a bispecific Fc region-containing diabody containing hLAG-3 mAb 6 (1.1) to both human and cynomolgus LAG-3 was investigated using Biacore analysis. In this study, hLAG-3 mAb 6(1.1)-containing diabodies were captured on a Fab ₂ goat anti-human Fc surface and soluble LAG-3 fusion proteins (human or cynomolgus macaque LAG-3 fused to a His tag) were captured on Fab 2 goat anti-human Fc surfaces. ectodomain) and binding kinetics determined by Biacore analysis. The calculated ka, kd and KD are presented in Table 8.

The results demonstrate that LAG-3 mAb 1 and LAG-3 mAb 6 exhibit better binding kinetics than the reference antibody LAG-3 mAb A. Furthermore, humanized LAG-3 mAb 6 exhibits relatively good cross-reactive binding kinetics with cynomolgus LAG-3.

The epitope specificity of LAG-3 mAb 1, LAG-3 mAb 6 and the reference antibody LAG-3 mAb A was tested using Biacore analysis. To determine whether the antibodies bind to different LAG-epitopes, shLAG-3-His was captured by a mouse PentaHis antibody immobilized on a CM5 sensor chip following the manufacturer's recommended procedure. Briefly, carboxyl groups on the sensor chip surface were activated by injection of a solution containing 0.2 M N-ethyl-N-(3diethylamino-propyl)carbodiimide and 0.05 M N-hydroxy-succinimide. did. Anti-PentaHis antibody was injected across the activated CM5 surface in 10 mM sodium acetate, pH 5.0, at a flow rate of 5 μL/min, followed by 1 M ethanol to inactivate any remaining amine-reactive groups. Amine was injected. Binding experiments were performed in HBS-EP buffer containing 10mM HEPES, pH 7.4, 150mM NaCl, 3mM EDTA and 0.005% P20 detergent. After pre-injection of each antibody (LAG-3 mAb 1, LAG-3 mAb 6 and LAG-2 mAb A) over the captured hLAG-3 His for 180 seconds at a flow rate of 5 μL/min and a concentration of 1 μM. , buffer was flushed, and competing antibodies were injected under the same conditions. Competing antibodies for the same epitope were identified by comparing their binding responses to that to hLAG-3 His preinjected with buffer. Regeneration of the immobilized anti-PentaHis surface was performed by pulse injection of 10 mM glycine, pH 1.5. A reference curve was obtained by injection of analyte across the treated surface without immobilized protein. Binding curves were generated by BIAevaluation software v4.1 from real-time sensogram data.

The results of this experiment are shown in Figures 8A-8D. The results of this experiment showed that biotinylated antibody LAG3 mAb A was able to bind to shLAG-3-His even in the presence of excess amounts of non-biotinylated antibodies LAG-3 mAb 1 and LAG-3 mAb 6. ing. In contrast, LAG-3 mAb 1 blocked the binding of LAG-3 mAb 6. The above results therefore indicate that LAG-3 mAb 1 and LAG-3 mAb 6 tend to bind to the same or overlapping epitopes of LAG-3 and compete with each other for binding to LAG-3. Both LAG-3 mAb 1 and LAG-3 mAb 6 were found to bind to epitopes distinct from those bound by LAG-3 mAb A.

To further characterize the anti-LAG3 antibody, the ability of the anti-LAG3 antibody to block binding between LAG-3 and MHC class II was evaluated in two different assays. In one assay, the ability of the antibody to block the binding of soluble human LAG3-Fc fusion protein to MHC class II immobilized on a surface was tested. For this assay, LAG-3 mAb 1, LAG-3 mAb 2, LAG-3 mAb 3, LAG-3 mAb 4, and LAG-3 mAb 5 (0-67 nM, 2-fold serial dilution) were each incubated with soluble human It was mixed with LAG-3-Fc fusion protein (5 μg/mL) and incubated separately with immobilized MHC class II (1 μg/mL). The amount of LAG-3 bound to immobilized MHC class II was assessed using a goat anti-human Fcγ-HRP secondary antibody. In additional experiments, LAG-3 mAb A and the humanized antibodies hLAG-3 mAb 1 (1.4) and hLAG-3 mAb 6 (1.1) (0.0096-7.0 nM, 3-fold serial dilution) was mixed with soluble human LAG-3-His fusion protein (0.2 μg/ml) and assayed for binding to immobilized MHC class II as described above. The results of these experiments are shown in FIGS. 9A and 9B.

In another assay, the ability of anti-LAG-3 antibodies of the invention to block the binding of soluble human LAG3-Fc fusion protein (shLAG-3-Fc) to native MHC class II on the cell surface was tested. For this assay, LAG-3 mAb 1, LAG-3 mAb 2, LAG-3 mAb 3, LAG-3 mAb 4, LAG-3 mAb 5 and the reference antibody LAG-3 mAb A (0.1-26. 7 ng/ml, 2-fold serial dilution) were mixed with biotinylated soluble human LAG-3-Fc fusion protein (0.5 μg/ml) and incubated separately with MHC II-positive Daudi cells. The amount of LAG-3 bound to the surface of the Daudi cells was determined by FACS analysis using a PE-conjugated streptavidin secondary antibody. In further separate experiments, LAG-3 mAb 1, LAG-3 mAb 6 and LAG-3 mAb A; or LAG-3 mAb 1 and the humanized antibodies hLAG-3 mAb 1 (1.4), hLAG-3 mAb 1 (1.2), hLAG-3 mAb 1 (2.2) and hLAG-3 mAb 1 (1.1) were assayed as described above. The results of these experiments are shown in FIGS. 10A-10C, respectively.

The results of these inhibition assays (FIGS. 9A-9B and 10A-10C) show that all anti-LAG-3 antibodies tested blocked the binding of shLAG-3-Fc fusion proteins to MHC class II.

Example 2: Flow Cytometry Methodology In the experiments described below, experiments to determine the expression levels of checkpoint inhibitors: PD-1 and LAG-3 on cells were carried out using appropriately fluorescently labeled Commercially available antibodies (phycoerythrin-cyanine 7 (PE-Cy7) conjugated anti-CD4 [clone SK3] or fluorescein isothiocyanate (FITC) conjugated anti-CD4 [clone RPA-T4], phycoerythrin (PE) conjugated anti-LAG-3 [ Clone 3DS223H], phycoerythrin (PE) conjugated anti-PD-1 [clone EH12.2H7] or allophycocyanin (APC) conjugate (eBiosciences or BioLegend)), and appropriate isotype controls were used. All antibodies were used at the manufacturer's recommended concentrations. Cell staining was performed in FACS buffer (10% FCS in PBS) for 30 minutes on ice in the dark for addition of primary antibodies. After washing twice, cells were stained with appropriate secondary reagents for 30 minutes on ice in the dark or analyzed immediately on a flow cytometer. To exclude dead cells, all samples were treated with a viability dye: 7-aminoactinomycin D (7-AAD) (BD Biosciences or BioLegend) or 4',6-diamidino-2-phenylindole, dihydrochloride ( DAPI) (Life Technologies) was used for co-staining. All samples were analyzed on a FACS Calibur or Fortessa flow cytometer (BD Biosciences) and analyzed using FlowJo software (TreeStar, Ashland, OR).

Example 3: Expression of LAG-3 and antibody binding to stimulated T cells Expression of LAG-3 and isolated LAG-3 antibodies specific for LAG-3 on the surface of CD3/CD28 stimulated T cells The ability to bind to was tested. T cells were obtained from peripheral blood mononuclear cells (PBMC). Briefly, PBMCs were purified from whole blood obtained with informed consent from healthy donors using Ficoll-Paque Plus (GE Healthcare) density gradient centrifugation according to the manufacturer's instructions, followed by T cells were purified using the Dynabeads® non-contact human T cell kit (Life Technologies) according to instructions. For stimulation, isolated T cells were incubated in the presence of recombinant human IL-2 [30U/ml] (Peprotech) and Dynabeads® human T cell activator beads (Life Technologies) according to the manufacturer's instructions. The cells were cultured for 10 to 14 days. Expression of LAG-3 on freshly isolated unstimulated CD4+ T cells and stimulated CD4+ T cells (obtained from culture on day 11 or 14) was determined using FITC-conjugated anti-CD4 and PE-conjugated anti-LAG-3. and tested by flow cytometry as described above.

The results of these studies are shown in Figures 11A-11C. No expression of LAG-3 was observed in unstimulated CD4+ T cells (FIG. 11A). However, in CD3/CD8 stimulated CD4+ T cells from two different donors (D:58468 and D:43530), LAG-3 expression was dramatically increased (FIGS. 11B and 11C).

LAG-3 mAb 1 (Figure 12, Panels A and D), LAG-3 mAb 2 (Figure 12, Panels B and E), LAG-3 mAb 3 (Figure 12, Panels C and F), LAG-3 mAb 4 (FIG. 13, panels A and C) and LAG-3 mAb 5 (FIG. 13, panels B and D) were investigated for their ability to specifically bind to stimulated CD4+ T cells. Stimulated T cells obtained from culture on day 14 (prepared as described above from donor D: 58468) and fresh unstimulated cells (prepared as described above from donor D: 43530) were isolated by The isolated anti-LAG-3 antibody and the following appropriately fluorescently labeled secondary reagents (PC-conjugated anti-mouse IgG (H+L) (Jackson ImmunoResearch Labs)) and FITC-conjugated anti-CD4 were subjected to flow cytometry. did. As shown in FIG. 12, Panels AF and FIG. 13, Panels AD, each anti-LAG-3 antibody tested bound only to stimulated CD4+ T cells and not to unstimulated CD4+ T cells.

The results of these studies demonstrate that LAG-3 is upregulated on stimulated T cells and that the anti-LAG-3 antibodies of the invention specifically bind only to stimulated T cells.

Example 4: Functional Activity of Anti-LAG Antibodies Staphylococcus aureus enterotoxin type B (SEB) is a microbial superantigen that can activate a high proportion of T cells (5-30%). SEB binds MHC II outside the peptide-binding groove and is therefore MHC II-dependent, but neither restricted nor TCR-mediated. SEB stimulation of T cells results in oligoclonal T cell proliferation and cytokine production (although donor variability can be observed). Expression of anti-LAG-3 and anti-PD-1 antibodies alone and in combination on SEB-stimulated PBMCs was tested.

PBMCs purified as described above were cultured alone or with 0.1 ng/ml SEB (Sigma-Aldrich) in RPMI medium + 10% heat-inactivated FBS + 1% penicillin/streptomycin in T-25 bulk flasks. (primary stimulation) and cultured for 2 to 3 days. At the end of the first round of SEB stimulation, PBMCs were washed twice with PBS and treated with medium alone, medium with 0.1 ng/ml SEB and no antibody (secondary stimulation), or SEB and control IgG. The cells were seeded in a 96-well tissue culture plate at a concentration of 1-5×10 ⁵ cells/well in medium containing antibodies and cultured for an additional 2-3 days. Forty-eight hours after primary bulk SEB stimulation, PD-1 and LAG were determined by flow cytometry using PE-conjugated anti-LAG-3 and FITC-conjugated anti-CD3; Cells were tested for -3 expression. On day 5 of subculture in 96-well plates with SEB stimulation, wells treated without antibody or with control antibody were treated with PE-conjugated anti-LAG-3 and APC-conjugated anti-PD-1. and tested for PD-1 and LAG-3 expression using flow cytometry.

Flow cytometry results from two representative donors (D:34765 and D:53724) are shown in Figure 14, panels A-D (D:34765) and Figure 15, panels A-D (D:53724). Shown below. These results demonstrate that LAG-3 and PD-1 are upregulated 48 hours after SEB stimulation, with further enhancement seen on day 5 of culture with SEB stimulation. In these studies, donor 1 had more LAG-3/PD-1 double positive cells after SEB stimulation. Addition of control antibodies after SEB stimulation did not change LAG-3 or PD-1 expression (compare Figure 14, panels C and D with Figure 15, panels C and D).

Upregulation of immune checkpoint proteins LAG-3 and PD-1 after SEB stimulation of PBMC limits cytokine release upon restimulation. LAG-3 mAb 1, LAG-3 mAb 3, LAG-3 mAb 4, LAG-3 mAb 6, a PD-1 monoclonal antibody designated "PD-1 mAb 5", as well as the reference antibody PD-1 mAb 1 (235A/ 235A Fc variant (AA)), PD-1 mAb 2, LAG-3 mAb A, and commercially available anti-LAG3 antibody 17B4 (#LS-C18692, LS Bio, including the name "LAG-3 mAb B"). The ability to enhance cytokine release by point inhibition was tested.

PBMC were seeded with no antibody, with isotype control antibody, or with anti-LAG-3 and anti-PD-1 antibodies alone or in combination during secondary stimulation. Stimulated with SEB as described above. At the end of the secondary stimulation, supernatants were collected and cytokine secretion was measured using the Human DuoSet ELISA kit for IFNγ, TNFα, IL-10 and IL-4 (R&D Systems) according to the manufacturer's instructions. Figures 16A-16B are shown with no antibody or with the following antibodies: isotype control, PD-1 mAb 1, PD-1 mAb 2, LAG-3 mAb A, LAG-3 mAb B, LAG-3 mAb 1, IFNγ (FIG. 16A) and SEB-stimulated PBMCs from a representative donor (D:38166) treated with one of LAG-3 mAb 3, LAG-3 mAb 4, or LAG-3 mAb 6. TNFα (FIG. 16B) secretion profile is shown. For this study, antibodies were used at 0.009, 0.039, 0.156, 0.625, 2.5, 10 and 40 μg/ml. FIG. 17: Without antibody; with isotype control antibody; with PD-1 mAb 2 and/or LAG-3 mAb A; with PD-1 mAb 5 and/or LAG-3 mAb 1. or PD-1 mAb 5 and/or LAG-3 mAb 3 from SEB-stimulated PBMCs from another representative donor (D:58108). For this study, antibodies were used at 10 μg/ml.

The results of these studies showed that anti-PD-1 antibodies significantly inhibited immune system function, as evidenced by increased IFNγ (FIGS. 16A and 17) and TNFα (FIG. 16B) production from SEB-stimulated PBMCs upon restimulation. It has been demonstrated that it has dramatically increased the Surprisingly, LAG-3 mAb 1 was also observed to increase cytokine production across multiple donors to levels comparable to anti-PD-1 antibodies, whereas the reference anti-LAG-3 mAb, LAG -3 mAb A and LAG-3 mAb B, as well as some of the isolated antibodies (LAG-3 mAb 3, LAG-4 and LAG-6), only slightly enhanced cytokine release. Furthermore, the combination of anti-LAG-3 antibody and anti-PD-1 resulted in further enhancement of cytokine release from SEB-stimulated PBMCs upon restimulation (Figure 17). LAG-3 mAb 1 enhanced cytokine release the most when combined with anti-PD-1 antibody compared to LAG-3 mAb 3 and the reference antibody LAG-3 mAb A.

Example 5: Binding to endogenous cynomolgus monkey LAG-3 Humanized antibody hLAG-3 mAb 6 (1.1) and reference antibody LAG-3 mAb A bind to endogenous LAG-3 expressed on cynomolgus monkey PBMCs Performance was investigated by FACS. For this study, PBMC were isolated from donor cynomolgus monkey whole blood and cultured alone or with SEB stimulation (500 ng/mL) as generally described above. After 66 hours of SEB stimulation, cells (unstimulated and stimulated) were stained with hLAG-3 mAb 6 (1.1), LAG-3 mAb A or PD-1 mAb 1 antibodies (10-fold serial dilutions). The above antibody was detected using a goat anti-human Fc-APC labeled secondary antibody. Binding is plotted in Figures 18A-18B for two cynomolgus monkey donors.

SEB stimulation was confirmed by enhanced PD-1 expression as detected by anti-PD-1 antibody PD-1 mAb 1. The results of these studies demonstrate that hLAG-3 mAb 6 (1.1) binds endogenous LAG-3 expressed on the surface of stimulated cynomolgus monkey PBMC.

All publications and patent applications mentioned in this specification are mentioned herein, even if each individual publication or patent application is specifically and independently indicated to be incorporated by reference in its entirety. The same is incorporated herein by reference. Although this invention has been described with respect to specific embodiments thereof, further modifications are possible and this application discloses that such modifications and variations are within the known method or practice of the art to which this invention pertains, and that It is to be understood that the present invention is intended to cover any variations, uses, or modifications of the present invention generally in accordance with its principles, including departures from this disclosure, as applicable to the essential features described above.

The description of the numerical heading <223> in the sequence listing is as follows.
SEQ ID NO: 1: IgG1 Fc region; Xaa217 is lysine (K) or absent SEQ ID NO: 2: IgG2 Fc region; Xaa216 is lysine (K) or absent SEQ ID NO: 3: IgG3 Fc region; or absent SEQ ID NO: 4: IgG4 Fc region; Xaa217 is lysine (K) or absent SEQ ID NO: 5: LAG-3
SEQ ID NO: 6: VH of LAG-3 mAb 1
SEQ ID NO: 7: Polynucleotide encoding the VH of LAG-3 mAb 1 SEQ ID NO: 8: VH CDR1 of LAG-3 mAb 1
SEQ ID NO: 9: VH CDR2 of LAG-3 mAb 1
SEQ ID NO: 10: VH CDR3 of LAG-3 mAb 1
SEQ ID NO: 11: VL of LAG-3 mAb 1
SEQ ID NO: 12: Polynucleotide encoding the VL of LAG-3 mAb 1 SEQ ID NO: 13: VL CDR1 of LAG-3 mAb 1
SEQ ID NO: 14: VL CDR2 of LAG-3 mAb 1
SEQ ID NO: 15: VL CDR3 of LAG-3 mAb 1
SEQ ID NO: 16: VH1 of hLAG-3 mAb 1
SEQ ID NO: 17: Polynucleotide encoding VH1 of hLAG-3 mAb 1 SEQ ID NO: 18: VH2 of hLAG-3 mAb 1
SEQ ID NO: 19: Polynucleotide encoding VH2 of hLAG-3 mAb 1 SEQ ID NO: 20: VL1 of hLAG-3 mAb 1
SEQ ID NO: 21: Polynucleotide encoding VL1 of hLAG-3 mAb 1 SEQ ID NO: 22: VL2 of hLAG-3 mAb 1
SEQ ID NO: 23: Polynucleotide encoding VL2 of hLAG-3 mAb 1 SEQ ID NO: 24: VL3 of hLAG-3 mAb 1
SEQ ID NO: 25: Polynucleotide encoding VL3 of hLAG-3 mAb 1 SEQ ID NO: 26: VL4 of hLAG-3 mAb 1
SEQ ID NO: 27: Polynucleotide encoding VL4 of hLAG-3 mAb 1 SEQ ID NO: 28: VL4 CDR1 of hLAG-3 mAb 1
SEQ ID NO: 29: VH of LAG-3 mAb 2
SEQ ID NO: 30: Polynucleotide encoding the VH of LAG-3 mAb 2 SEQ ID NO: 31: VH CDR1 of LAG-3 mAb 2
SEQ ID NO: 32: VH CDR2 of LAG-3 mAb 2
SEQ ID NO: 33: VH CDR3 of LAG-3 mAb 2
SEQ ID NO: 34: VL of LAG-3 mAb 2
SEQ ID NO: 35: Polynucleotide encoding the VL of LAG-3 mAb 2 SEQ ID NO: 36: VL CDR1 of LAG-3 mAb 2
SEQ ID NO: 37: VL CDR2 of LAG-3 mAb 2
SEQ ID NO: 38: VL CDR3 of LAG-3 mAb 2
SEQ ID NO: 39: VH of LAG-3 mAb 3
SEQ ID NO: 40: Polynucleotide encoding the VH of LAG-3 mAb 3 SEQ ID NO: 41: VH CDR1 of LAG-3 mAb 3
SEQ ID NO: 42: VH CDR2 of LAG-3 mAb 3
SEQ ID NO: 43: VH CDR3 of LAG-3 mAb 3
SEQ ID NO: 44: VL of LAG-3 mAb 3
SEQ ID NO: 45: Polynucleotide encoding the VL of LAG-3 mAb 3 SEQ ID NO: 46: VL CDR1 of LAG-3 mAb 3
SEQ ID NO: 47: VL CDR2 of LAG-3 mAb 3
SEQ ID NO: 48: VL CDR3 of LAG-3 mAb 3
SEQ ID NO: 49: VH of LAG-3 mAb 4
SEQ ID NO: 50: Polynucleotide encoding the VH of LAG-3 mAb 4 SEQ ID NO: 51: VH CDR1 of LAG-3 mAb 4
SEQ ID NO: 52: VH CDR2 of LAG-3 mAb 4
SEQ ID NO: 53: VH CDR3 of LAG-3 mAb 4
SEQ ID NO: 54: VL of LAG-3 mAb 4
SEQ ID NO: 55: Polynucleotide encoding the VL of LAG-3 mAb 4 SEQ ID NO: 56: VL CDR1 of LAG-3 mAb 4
SEQ ID NO: 57: VL CDR2 of LAG-3 mAb 4
SEQ ID NO: 58: VL CDR3 of LAG-3 mAb 4
SEQ ID NO: 59: VH of LAG-3 mAb 5
SEQ ID NO: 60: Polynucleotide encoding the VH of LAG-3 mAb 5 SEQ ID NO: 61: VH CDR1 of LAG-3 mAb 5
SEQ ID NO: 62: VH CDR2 of LAG-3 mAb 5
SEQ ID NO: 63: VH CDR3 of LAG-3 mAb 5
SEQ ID NO: 64: VL of LAG-3 mAb 5
SEQ ID NO: 65: Polynucleotide encoding the VL of LAG-3 mAb 5 SEQ ID NO: 66: VL CDR1 of LAG-3 mAb 5
SEQ ID NO: 67: VL CDR2 of LAG-3 mAb 5
SEQ ID NO: 68: VL CDR3 of LAG-3 mAb 5
SEQ ID NO: 69: VH of LAG-3 mAb 6
SEQ ID NO: 70: Polynucleotide encoding the VH of LAG-3 mAb 6 SEQ ID NO: 71: VH CDR1 of LAG-3 mAb 6
SEQ ID NO: 72: VH CDR2 of LAG-3 mAb 6
SEQ ID NO: 73: VH CDR3 of LAG-3 mAb 6
SEQ ID NO: 74: VL of LAG-3 mAb 6
SEQ ID NO: 75: Polynucleotide encoding the VL of LAG-3 mAb 6 SEQ ID NO: 76: VL CDR1 of LAG-3 mAb 6
SEQ ID NO: 77: VL CDR2 of LAG-3 mAb 6
SEQ ID NO: 78: VL CDR3 of LAG-3 mAb 6
SEQ ID NO: 79: VH-1 of hLAG-3 mAb 6
SEQ ID NO: 80: Polynucleotide encoding VH-1 of hLAG-3 mAb 6 SEQ ID NO: 81: VH-2 of hLAG-3 mAb 6
SEQ ID NO: 82: Polynucleotide encoding VH-2 of hLAG-3 mAb 6 SEQ ID NO: 83: VL-1 of hLAG-3 mAb 6
SEQ ID NO: 84: Polynucleotide encoding VL-1 of hLAG-3 mAb 6 SEQ ID NO: 85: VL-2 of hLAG-3 mAb 6
SEQ ID NO: 86: Polynucleotide encoding VL-2 of hLAG-3 mAb 6 SEQ ID NO: 87: VL-1/VL-2 CDRL1 of hLAG-3 mAb 6
SEQ ID NO: 88: Linker SEQ ID NO: 89: Cys linker 2
SEQ ID NO: 90: ABD linker SEQ ID NO: 91-95: Linker SEQ ID NO: 96-100: Heterodimerization promoting domain SEQ ID NO: 101: E-coil SEQ ID NO: 102: K-coil SEQ ID NO: 103: Modified E-coil SEQ ID NO: 104: Modified K coil SEQ ID NO: 105: ABD
SEQ ID NO: 106-108: Deimmunized ABD
SEQ ID NO: 109: Hinge linker SEQ ID NO: 110: Linker SEQ ID NO: 111: Cys linker SEQ ID NO: 112-113: Linker SEQ ID NO: 114: IgG1 hinge SEQ ID NO: 115: IgG2 hinge SEQ ID NO: 116: IgG4 hinge SEQ ID NO: 117: IgG4 hinge (S228P )
SEQ ID NO: 118: LCκ
Sequence number 119: LCλ
SEQ ID NO: 120: IgG1 CH1
SEQ ID NO: 121: IgG2 CH1
SEQ ID NO: 122: IgG4 CH1
SEQ ID NO: 123: IgG1 (AA); Xaa217 is lysine (K) or absent SEQ ID NO: 124: IgG1 Fc (AA/YTE); Xaa217 is lysine (K) or absent SEQ ID NO: 125: IgG4 Fc (YTE) ; Xaa217 is Lysine (K) or absent SEQ ID NO: 126: Knob Fc domain; IgG1 Fc universal; Xaa4 (X1) and Xaa5 (X2
) are either L (wild type) or both A (decreased FcR binding); Xaa22 (X3), Xaa24 (X4) and Xaa26 (X5) are M, S and T (
Xaa136 (X6) and Xaa138 (X7) are respectively T and L (wild type) or W and L (knob); or S and A (hole); Xaa177 (X8) is Y (wild type) when Xaa136 (X6) is T and Xaa138 (X7) is L, or Xaa136 (X6) is W and When (X7) is L, it is Y (knob), or when Xaa136 (X6) is S and Xaa138 (X7) is A, it is V (hole); Xaa204 (X9) and Xaa205 (X10) are respectively N and H (wild type), or N and R (no protein A binding), or A and K (no protein A binding); Xaa217 (X11) is K or absent Sequence Number 129: VH of LAG-3 mAb A
SEQ ID NO: 130: VL of LAG-3 mAb A
SEQ ID NO: 131: VH of PD-1 mAb 1
SEQ ID NO: 132: VL of PD-1 mAb 1
SEQ ID NO: 134: VL of PD-1 mAb 2
SEQ ID NO: 135: VH of PD-1 mAb 3
SEQ ID NO: 136: VL of PD-1 mAb 3
SEQ ID NO: 137: VH of PD-1 mAb 4
SEQ ID NO: 138: VL of PD-1 mAb 4
SEQ ID NO: 139: IgG1 entire constant region (AA); Xaa330 is K or absent SEQ ID NO: 140: IgG4 entire constant region (P); Xaa327 is K or absent

Claims (18)

A LAG-3 binding molecule capable of binding to both human LAG-3 and cynomolgus LAG-3, comprising:
the LAG-3 binding molecule comprises an epitope binding domain of an antibody;
(i) the epitope binding domain comprises an antibody heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 79 and an antibody light chain variable domain comprising the amino acid sequence of SEQ ID NO: 83;
(ii) The LAG-3 binding molecule specifically binds to human LAG-3 with an equilibrium binding constant (K _D ) of 0.5 nM or less. 2. The LAG-3 binding molecule of claim 1, wherein said molecule is an antibody. 3. The LAG-3 binding molecule of claim 2, wherein the molecule is a chimeric or humanized antibody. LAG-3 binding molecule according to any one of claims 1 to 3, wherein said molecule is a bispecific binding molecule capable of simultaneously binding human LAG-3 and a second epitope. 5. The LAG-3 binding molecule according to claim 4, wherein the second epitope is an epitope of a molecule involved in regulating immune checkpoints present on the surface of immune cells. The second epitope is B7-H3, B7-H4, BTLA, CD40, CD40L, CD47, CD70, CD80, CD86, CD94, CD137, CD137L, CD226, CTLA-4, galectin-9, GITR, GITRL, HHLA2. , ICOS, ICOSL, KIR, LAG-3, LIGHT, MHC class I or II, NKG2a, NKG2d, OX40, OX40L, PD1H, PD-1, PD-L1, PD-L2, PVR, SIRPa, TCR, TIGIT, TIM 5. The LAG-3 binding molecule according to claim 4, which is an epitope of -3 or VISTA. 7. The LAG-3 binding molecule of claim 6, wherein the second epitope is an epitope of PD-1. The molecule is:
(a) a diabody, said diabody being a covalent complex comprising two or three or four different polypeptide chains; or (b) a trivalent binding molecule, said trivalent binding molecule LAG-3 binding molecule according to any one of claims 4 to 7, wherein is a covalent complex comprising three, four or five polypeptide chains. LAG-3 binding molecule according to any one of claims 1 to 8, wherein said molecule comprises an Fc region. The Fc region is a mutant Fc region, and the mutant Fc region is:
(a) one or more amino acid modifications that reduce the affinity of said variant Fc region for FcγR; and/or (b) one or more amino acid modifications that increase the serum half-life of said variant Fc region. 10. The LAG-3 binding molecule of claim 9, comprising: (a) The one or more amino acid modifications that reduce the affinity of the variant Fc region include substitutions:
(1) L234A; L235A; or (2) L234A and L235A;
including;
(b) one or more of said amino acid modifications that increase the serum half-life of said variant Fc region include the substitution:
(1) M252Y and S254T;
(2) M252Y and T256E;
(3) M252Y, S254T and T256E; or (4) K288D and H435K
including;
11. The LAG-3 binding molecule of claim 10, wherein the numbering is the EU index numbering according to Kabat. A pharmaceutical composition comprising a LAG-3 binding molecule according to any one of claims 1 to 11 and a pharmaceutically acceptable carrier. A LAG-3 binding molecule according to any one of claims 1 to 11 for use in stimulating a T cell-mediated immune response in a subject in need of stimulation of a T cell-mediated immune response, or Pharmaceutical composition according to claim 12. A LAG-3 binding molecule according to any one of claims 1 to 11 or a pharmaceutical composition according to claim 12 for use in treating diseases or conditions associated with a suppressed immune system. . The LAG-3 binding molecule according to any one of claims 1 to 11, or the pharmaceutical composition according to claim 12, wherein the disease or condition is cancer or an infectious disease. The diseases or conditions are: adrenal cancer; AIDS-related cancer; alveolar soft tissue sarcoma; astrocytic tumor; bladder cancer; bone cancer; brain and spinal cord cancer; metastatic brain tumor; breast cancer; carotid bulb tumor; cervical cancer; cartilage Sarcoma; chordoma; chromatic renal cell carcinoma; clear cell carcinoma; colorectal cancer; colorectal cancer; desmoplastic small round cell tumor; ependymoma; Ewing tumor; extraosseous myxoid chondrosarcoma; gallbladder or cholangiocarcinoma; digestive Organ cancer; gestational trophoblastic disease; germ cell tumor; head and neck cancer; hepatocellular carcinoma; pancreatic islet cell tumor; Kaposi's sarcoma; kidney cancer; leukemia; liposarcoma/malignant liposomal tumor; liver cancer; lymphoma; lung cancer; medulloblastoma ; melanoma; meningioma; multiple endocrine tumors; multiple myeloma; myelodysplastic syndrome; neuroblastoma; neuroendocrine tumor; ovarian cancer; pancreatic cancer; papillary thyroid cancer; parathyroid tumor; childhood cancer; Peripheral nerve sheath tumor; pheochromocytoma; pituitary tumor; prostate cancer; posterior uveal melanoma; renal metastatic cancer; rhabdoid tumor; rhabdomyosarcoma; sarcoma; skin cancer; soft tissue sarcoma; squamous cell carcinoma; gastric cancer; 16. The LAG-3 binding molecule or pharmaceutical composition according to claim 15, which is a cancer selected from the group consisting of synovial sarcoma; testicular cancer; thymic cancer; thymoma; metastatic thyroid cancer; and uterine cancer. The diseases or conditions are: colorectal cancer; hepatocellular carcinoma; glioma; kidney cancer; breast cancer; multiple myeloma; bladder cancer; neuroblastoma; sarcoma; non-Hodgkin's lymphoma; non-small cell lung cancer; ovarian cancer: Pancreatic cancer; rectal cancer; acute myeloid leukemia (AML); chronic myeloid leukemia (CML); acute B-lymphoblastic leukemia (B-ALL); chronic lymphocytic leukemia (CLL); hairy cell leukemia (HCL) ); including blastic plasmacytoid dendritic cell neoplasm (BPDCN); mantle cell leukemia (MCL); small lymphocytic lymphoma (SLL); mantle cell leukemia (MCL) and small lymphocytic lymphoma (SLL). 16. The LAG-3 binding molecule or pharmaceutical composition of claim 15, selected from the group consisting of non-Hodgkin's lymphoma (NHL); Hodgkin's lymphoma; systemic mastocytosis; and Burkitt's lymphoma. LAG-3 binding molecule according to any one of claims 1 to 11, wherein said molecule is detectably labeled and for use in detecting LAG-3.