Multispecific antibodies

Abstract

The problem to be solved by the present invention is to provide new pharmaceutical agents for the treatment of human diseases, in particular for the treatment of cancer.SOLUTION: The present invention relates to multispecific antibodies comprising a binding domain that binds to LAG-3 and a binding domain that binds to PD - L1. Such multispecific antibodies have comparable or equal or higher potency than the combination of LAG-3 and PD - L1 reference antibodies. Also provided are methods for treating diseases, particularly diseases associated with a suppressed immune system such as cancer, using the multispecific antibodies of the present disclosure. It further relates to vectors and cells comprising nucleic acids encoding a heavy chain variable region of LAG-3 and a PD - L1 binding domain.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present disclosure relates to the field of antibodies. Specifically, the present disclosure relates to the field of therapeutic antibodies for the treatment of diseases involving abnormal cells. More specifically, the present disclosure relates to novel multispecific antibodies and variants thereof that bind to proteins of the LAG-3 and B7 family, specifically PD-L1. [Background technology]

[0002] Cancer remains a leading cause of death worldwide, despite numerous advances in disease treatment and increasing knowledge about the molecular events that lead to cancer. Traditionally, most cancer drug discovery has focused on agents that block essential cellular functions and kill dividing cells. However, in the case of advanced cancer, no matter how aggressively applied, chemotherapy rarely results in a complete cure, even to the point where patients experience life-threatening side effects from treatment. In most cases, tumors in patients only stop growing or temporarily shrink (regression), only to begin growing again, sometimes more rapidly (recurrence), becoming increasingly difficult to treat. In recent years, the focus of cancer drug development has shifted from broad-spectrum cytotoxic chemotherapy to targeted cytostatic therapies with reduced toxicity. Treatment of advanced cancer with targeted therapy has been clinically tested in leukemia and several other cancers. However, in most carcinomas, targeted approaches have yet to prove sufficiently effective to completely eradicate cancer in most patients.

[0003] Cancer targeting has been achieved using a variety of different methods including, for example, small molecules directed against signaling proteins that cancers depend on for survival and / or growth, vaccines with tumor-specific proteins, cell therapies involving antibodies that target immune cells and cytotoxic molecules to tumors that actively kill tumor cells, disrupting signal transduction and / or (re)directing the host's immune system to tumor cells.

[0004] A developing class of therapeutic antibodies is bispecific antibodies, which contain two distinct binding sites that bind to different antigens or different epitopes on the same antigen. Bispecific antibodies can be designed for several applications. First, bispecific antibodies can provide greater tissue specificity than monospecific antibodies. Some tumor-associated antigens are not only (over)expressed by tumor cells but also expressed on normal, healthy cells. Bispecific antibodies directed against two different tumor-associated antigens involved in a particular type of cancer can specifically target the antibody to the tumor site, where it induces tumor cell death, thereby preventing binding to non-tumor cells that express only one of the antigens and thus reducing off-site toxicity. Other mechanisms of action include, for example, immune cell engagement with tumor cells and disruption of two signaling pathways required for tumor growth.

[0005] For example, immune checkpoint proteins such as PD-1, PD-L1, CTLA-4, LAG-3, and TIM-3 are attractive targets for antibody therapy. To date, numerous monospecific antibodies targeting LAG-3 or PD-L1, as well as certain bispecific antibodies that bind to both LAG-3 and PD-L1, have been described. However, each of these bispecific antibodies presents its own challenges in producing effective therapeutics. Therefore, there remains a need for the development of novel, effective LAG-3xPD-L1 bispecific antibodies. Summary of the Invention

[0006] One of the objectives of the present disclosure is to provide new pharmaceutical agents for the treatment of human diseases, particularly cancer. This objective is met by the provision of a multispecific antibody comprising an anti-human LAG-3 binding domain and an anti-human PD-L1 binding domain.

[0007] In certain embodiments, the present disclosure provides a multispecific antibody, or a variant thereof that maintains the binding specificity of the antibody, comprising a binding domain that binds to LAG-3 and a binding domain that binds to PD-L1, wherein the antibody or variant has potency at least comparable to, or equal to, or greater than that of a reference antibody combination. In certain embodiments, the reference antibody combination includes a reference antibody (relatolimab analog antibody) comprising two heavy chain variable regions having the amino acid sequence set forth in SEQ ID NO: 27 and two light chain variable regions having the amino acid sequence set forth in SEQ ID NO: 28, and a reference antibody (atezolizumab analog antibody) comprising two heavy chain variable regions having the amino acid sequence set forth in SEQ ID NO: 25 and two light chain variable regions having the amino acid sequence set forth in SEQ ID NO: 26.

[0008] In certain embodiments, the present disclosure further provides a multispecific antibody, or a variant thereof that maintains the binding specificity of the antibody, wherein the antibody or variant thereof comprises a binding domain that specifically binds to the extracellular domain of LAG-3 and a binding domain that specifically binds to the extracellular domain of a B7 family protein, and the LAG-3 binding domain comprises a heavy chain variable region comprising heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3) of the heavy chain variable region having the amino acid sequence set forth in any one of SEQ ID NOs: 1-17 or 52-59.

[0009] In certain embodiments, the present disclosure further provides a pharmaceutical composition comprising an effective amount of a multispecific antibody or variant thereof described herein.

[0010] In certain embodiments, the present disclosure further provides a multispecific antibody or variant thereof, or pharmaceutical composition described herein, for use as a medicament.

[0011] In certain embodiments, the present disclosure further provides a multispecific antibody or variant thereof, or pharmaceutical composition described herein for use in the treatment of a disease associated with a suppressed immune system, hi certain embodiments, the present disclosure provides a multispecific antibody or variant thereof, or pharmaceutical composition described herein for use in the treatment of cancer.

[0012] In certain embodiments, the present disclosure further provides a method for treating (a) a disease, or (b) a disease associated with a suppressed immune system, or c) cancer, comprising administering to an individual in need thereof an effective amount of a multispecific antibody or variant thereof, or pharmaceutical composition as described herein.

[0013] In certain embodiments, the present disclosure further provides a vector comprising a nucleic acid sequence encoding the heavy chain variable region of a LAG-3 binding domain described herein, and a nucleic acid sequence encoding the heavy chain variable region of a PD-L1 binding domain described herein.

[0014] In certain embodiments, the present disclosure further provides a cell comprising a nucleic acid sequence encoding the heavy chain variable region of a LAG-3 binding domain described herein, and a nucleic acid sequence encoding the heavy chain variable region of a PD-L1 binding domain described herein.

[0015] In certain embodiments, the present disclosure further provides cells that produce the multispecific antibodies or variants thereof described herein.

[0016] In certain embodiments, the present disclosure provides methods for producing variants of the multispecific antibodies described herein, and the variants obtained by such methods. DETAILED DESCRIPTION OF THE INVENTION

[0017] In certain embodiments, the present disclosure provides a multispecific antibody or variant thereof that maintains the binding specificity of the antibody, wherein the antibody or variant has potency at least comparable to, or equal to, or greater than a reference antibody combination, wherein the reference antibody combination comprises a reference antibody comprising two heavy chain variable regions having the amino acid sequence set forth in SEQ ID NO: 27 and two light chain variable regions having the amino acid sequence set forth in SEQ ID NO: 28, and a reference antibody comprising two heavy chain variable regions having the amino acid sequence set forth in SEQ ID NO: 25 and two light chain variable regions having the amino acid sequence set forth in SEQ ID NO: 26.

[0018] LAG-3 is known by many different names, including lymphocyte activation 3, lymphocyte activation gene 3, CD223 antigen, protein FDC, CD223, LAG-3, or FDC. The external identifiers for LAG3 are HGNC:6476, Entrez Gene:3902, Ensembl:ENSG00000089692, OMIM:153337, and UniProtKB:P18627. LAG-3 is closely related to CD4. LAG-3 is located on human chromosome 12 (12p13.32) adjacent to the CD4 gene, and its sequence is approximately 20% identical to CD4. The LAG-3 protein binds to the nonholomorphic region of the major histocompatibility complex 2 (MHC class II) with higher affinity than CD4. LAG-3 is one of several immune checkpoint receptors that are coordinately upregulated on both regulatory T cells (Tregs) and anergic T cells, and can negatively regulate T cell proliferation, activation, and homeostasis.

[0019] PD-L1 is a type 1 transmembrane protein that plays a role in suppressing immune responses during certain events, such as pregnancy, tissue allografts, autoimmune diseases, and other disease states, such as hepatitis. PD-L1 is expressed in various types of cancer, particularly NSCLC (Boland et al., 2013; Velcheti et al., 2014), melanoma, renal cell carcinoma, gastric cancer, hepatocellular carcinoma, and various leukemias and multiple myeloma (Bernstein et al., 2014; Thompson et al., 2005). PD-L1 is present in the cytoplasm and plasma membrane of cancer cells, although not all cancers or cells within tumors express PD-L1 (Dong et al., 2002). Multiple tumor microenvironment cells contribute to immune suppression by upregulating PD-L1 expression. This effect is called "adaptive immune resistance" because tumors protect themselves by inducing PD-L1 in response to IFN-γ produced by activated T cells (Sharma et al., 2017). PD-L1 can also be regulated by oncogenes, a mechanism known as intrinsic immune resistance (Akbay et al., 2013). Within the tumor microenvironment, PD-L1 is also expressed on myeloid cells and activated T cells (Tumeh et al., 2014). PD-L1 expression is induced by multiple proinflammatory molecules, including type I and type II IFN-γ, TNF-α, LPS, GM-CSF, and VEGF, as well as the cytokines IL-10 and IL-4, with IFN-γ being the most potent inducer (Sznol and Chen, 2013).

[0020] A multispecific antibody according to the present disclosure is an antibody comprising at least two binding domains with specificity for at least two different targets or epitopes, in any antibody format. In certain embodiments, a multispecific antibody of the present disclosure is a bispecific antibody. In certain embodiments, a multispecific antibody of the present disclosure comprises an Fc region or a portion thereof. In certain embodiments, a multispecific antibody of the present disclosure is an IgG1 antibody.

[0021] "Variant" of a multispecific antibody described herein includes functional portions, derivatives, and / or analogs of a multispecific antibody. Variants may be structural variants, including, but not limited to, antibody fragments such as Fab fragments or single-chain variable fragments (scFv). Variants may be sequence variants. Variants may be structural and sequence variants. Variants maintain the binding specificity, but not necessarily the binding affinity, of the antibody.

[0022] In certain embodiments, the LAG-3 and / or PD-L1 binding domain comprises at least a heavy chain variable region and a light chain variable region. The light chain variable region may be any suitable light chain variable region further described herein. In certain embodiments, the light chain variable region is preferably that of a light chain that can pair with multiple heavy chains having different epitope specificities. Such light chains are also referred to in the art as "common light chains."

[0023] In certain embodiments, a multispecific antibody or variant thereof comprises a single binding domain that binds to LAG-3 and / or a single binding domain that binds to PD-L1. In other words, in certain embodiments, a multispecific antibody or variant thereof is monovalent for binding to LAG-3 and / or monovalent for binding to PD-L1. In certain embodiments, a multispecific antibody or variant thereof is monovalent for binding to both LAG-3 and PD-L1.

[0024] Determining whether a multispecific antibody or variant has comparable, equal or greater potency than a reference antibody combination can be done by measuring the potency of both the multispecific antibody and the antibody combination in the same type of study using the same study conditions. Thus, in certain embodiments, the potency of a multispecific antibody or variant thereof is measured in the same type of study using the same study conditions.

[0025] In certain embodiments, at least equivalent potency is potency within a 5-fold range of the potency of the reference antibody combination, including a 5-, 4-, 3-, or 2-fold, preferably a 3- or 2-fold deviation from the potency of the reference antibody combination.

[0026] In the context of the present disclosure, "potency" refers to the functional activity of a multispecific antibody or variant thereof, which can be determined in in vitro or in vivo studies.

[0027] In certain embodiments, the efficacy of a multispecific antibody or variant thereof is determined in an in vivo study, preferably in an in vivo mouse model, such as, for example, the HuNSG™ mouse model bearing human MDA-231 tumors. In certain embodiments, the efficacy of a multispecific antibody or variant thereof is determined by measuring tumor volume reduction in such an in vivo mouse study. Tumor volume reduction induced by a multispecific antibody provided herein is determined by the method described in Example 4.

[0028] In certain embodiments, equivalent efficacy is a tumor volume reduction within a 5-fold range of the tumor volume reduction of the reference antibody combination, including a 5-, 4-, 3-, or 2-fold, preferably a 3- or 2-fold deviation from the tumor volume reduction of the reference antibody combination.

[0029] In certain embodiments, potency is determined in in vitro studies such as, for example, a blocking assay, including but not limited to, a PD-1 / LAG-3 or PD-L1 / LAG-3 reporter assay, preferably, a PD-1 / LAG-3 reporter assay, e.g., as described in Example 2, or an SEB assay, preferably, including but not limited to, a T cell activation assay, an antigen recall assay, or an MLR assay, e.g., as described in Example 2. In certain embodiments, the potency of the combination of a multispecific antibody or variant thereof and a reference antibody is determined by measuring their potency in blocking ligand or receptor binding to LAG-3 and / or PD-L1, preferably, ligand or receptor binding to LAG-3 and PD-L1. The potency of the multispecific antibodies provided herein in blocking ligand or receptor binding to LAG-3 and / or PD-L1 is measured by the method described in Example 2. Briefly, the PD-1 / LAG-3 reporter assay is performed using PD-1- and LAG-3-expressing Jurkat T cells as effector cells and PD-L1-expressing Raji cells as target cells. PD-1 and LAG-3 effector cells are prepared and plated at 100,000 cells per well. Test and control IgGs are added in equimolar amounts in a six-step semi-logarithmic titration, followed by the addition of Raji cells (25,000 per well). T cells are activated by the addition of partially purified Staphylococcal enterotoxin D (ppSED, using the concentration that achieves the highest response as a positive assay control, e.g., 16.6 ng / mL in Example 2 or 150 ng / mL in Example 5). After 6 hours of incubation at 37°C, luciferase reporter gene activity is determined.

[0030] In certain embodiments, the equivalent potency is potency in blocking ligand or receptor binding to LAG-3 and / or PD-L1, preferably LAG-3 and PD-L1, that is within a 5-fold range of the potency of the reference antibody combination in blocking ligand or receptor binding to LAG-3 and / or PD-L1, preferably LAG-3 and PD-L1, including a 5-, 4-, 3-, or 2-fold, preferably a 3- or 2-fold deviation from the potency of the reference antibody combination in blocking ligand or receptor binding to LAG-3 and / or PD-L1, preferably LAG-3 and PD-L1. In certain embodiments, the reference antibody combination comprises an analog of the anti-LAG-3 antibody leratolimab and an analog of the anti-PD-L1 antibody atezolizumab. The leratolimab analog has the same heavy chain variable region sequence as leratolimab (SEQ ID NO: 27). The leratolimab analog antibody has the same light chain variable region sequence as leratolimab (SEQ ID NO: 28). The atezolizumab analog has the same heavy chain variable region sequence as atezolizumab (SEQ ID NO: 25). The atezolizumab analog antibody has the same light chain variable region sequence as atezolizumab (SEQ ID NO: 26). The reference antibody is preferably produced using the same production method as the multispecific antibody or variant thereof. Preferably, the LAG-3 and PD-L1 binding domains of the multispecific antibody or variant thereof and reference antibody combination of the present disclosure are used at equimolar concentrations, i.e., if the multispecific antibody or variant thereof is used at 100 μg / mL, the reference antibody combination contains 50 μg / mL of the LAG-3-binding reference antibody and 50 μg / mL of the PD-L1-binding reference antibody.

[0031] In certain embodiments, the present disclosure provides a multispecific antibody, or a variant thereof that maintains the binding specificity of the antibody, wherein the antibody or variant thereof comprises a binding domain that specifically binds to the extracellular domain of LAG-3 and a binding domain that specifically binds to the extracellular domain of a protein of the B7 family.

[0032] In certain embodiments, the LAG-3 binding domain of a multispecific antibody or variant thereof of the present disclosure comprises a heavy chain variable region comprising a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) of one of the heavy chain variable regions having the amino acid sequence set forth in SEQ ID NOs: 1 to 17 or 52 to 59. HCDRs according to Kabat are shown in bold and underlined in the sequence listings provided herein. CDR sequences may be numbered according to the Kabat numbering scheme (Kabat et al., J. Biol. Chem. 252:6609-6616 (1977), and / or Kabat et al., US Department of Health and Human Services, "Sequences of proteins of immunological interest" (1991)), the Chothia numbering scheme (Chothia et al., J. Mol. Biol. 196:901-917 (1987), Chothia et al., Nature 342:877-883, 1989, and / or Al-Lazikani B. et al., J. Mol. Biol., 273:927-948 (1997)), the Honegger and Plukthun numbering system (Honegger and Pluckthun, J. Mol. Biol., 309:657-670 (2001)), MacCallum's numbering system (MacCallum et al., J. Mol. Biol., 262:732-745 (1996), and / or Abhinandan and Martin, Mol. Immunol., 45:3832-3839 (2008)), Lefranc's numbering system (Lefranc MP et al., Dev. Comp. Immunol., 27:55-77 (2003), and / or Honegger and Pluckthun, J. Mol. Biol., 309:657-670 (2001)), or IMGT (Giudicelli et al., Nucleic Acids The term "antibody" may be defined using different methods, including, but not limited to, by the method discussed in J. Immunol. 2002, 2003, 2004, 2005, 2006, 2007, 2008, 2009, 2010, 2011, 2012, 2013, 2014, 2015, 2016, 2017,

[0033] Each of these numbering schemes defines CDRs based on the predicted contribution of amino acid residues within the heavy or light chain variable region to antigen binding. Thus, each method for identifying CDRs can be used to identify the CDRs of a binding domain of the present disclosure. In certain embodiments, the heavy chain CDRs of a binding domain of the present disclosure are according to Kabat, Chothia, or IMGT. In certain embodiments, the heavy chain CDRs of a binding domain of the present disclosure are according to Kabat. In certain embodiments, the heavy chain CDRs of a binding domain of the present disclosure are according to Chothia. In certain embodiments, the heavy chain CDRs of a binding domain of the present disclosure are according to IMGT.

[0034] In certain embodiments, the LAG-3 binding domain of the multispecific antibody or variant thereof comprises: - a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) of a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 1; - a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) of a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 2; - a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) of a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 3; - a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) of a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 4; - a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) of a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 5; - a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) of a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 6; - a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) of a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 7; - a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) of a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 8; - a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) of a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 9; - a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) of a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 10; - a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) of a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 11; - a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) of a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 12; - a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) of a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 13; - a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) of a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 14; - a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) of a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 15; - a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) of a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 16; - a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) of a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 17; - a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) of a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 52; - a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) of a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 53; - a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) of a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 54; - a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) of a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 55; - a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) of a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 56; - a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) of a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 57; - a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) of a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 58, or a heavy chain variable region comprising a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) having the amino acid sequence set forth in SEQ ID NO: 59; Each of the HCDRs may contain up to three, two, or one amino acid substitutions.

[0035] In certain embodiments, the LAG-3 binding domain of the multispecific antibody or variant thereof comprises: - heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3) having the amino acid sequences set forth in SEQ ID NO: 60, SEQ ID NO: 61, and SEQ ID NO: 62, respectively; - heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3) having the amino acid sequences set forth in SEQ ID NO: 60, SEQ ID NO: 63, and SEQ ID NO: 62, respectively; - heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3) having the amino acid sequences set forth in SEQ ID NO: 64, SEQ ID NO: 65, and SEQ ID NO: 66, respectively; - heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3) having the amino acid sequences set forth in SEQ ID NO: 67, SEQ ID NO: 68, and SEQ ID NO: 69, respectively; - heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3) having the amino acid sequences set forth in SEQ ID NO: 70, SEQ ID NO: 71, and SEQ ID NO: 72, respectively; - heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3) having the amino acid sequences set forth in SEQ ID NO: 73, SEQ ID NO: 74, and SEQ ID NO: 75, respectively; - heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3) having the amino acid sequences set forth in SEQ ID NO: 76, SEQ ID NO: 77, and SEQ ID NO: 78, respectively; - heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3) having the amino acid sequences set forth in SEQ ID NO: 79, SEQ ID NO: 80, and SEQ ID NO: 81, respectively; - heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3) having the amino acid sequences set forth in SEQ ID NO: 82, SEQ ID NO: 83, and SEQ ID NO: 84, respectively; - heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3) having the amino acid sequences set forth in SEQ ID NO: 85, SEQ ID NO: 86, and SEQ ID NO: 87, respectively; heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), the amino acid sequences set forth in SEQ ID NO: 88, SEQ ID NO: 89, and SEQ ID NO: 90, respectively; - heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3) having the amino acid sequences set forth in SEQ ID NO: 76, SEQ ID NO: 91, and SEQ ID NO: 92, respectively; - heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3) having the amino acid sequences set forth in SEQ ID NO: 76, SEQ ID NO: 91, and SEQ ID NO: 93, respectively; - heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3) having the amino acid sequences set forth in SEQ ID NO: 94, SEQ ID NO: 95, and SEQ ID NO: 96, respectively; - heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3) having the amino acid sequences set forth in SEQ ID NO: 97, SEQ ID NO: 98, and SEQ ID NO: 99, respectively; - heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3) having the amino acid sequences set forth in SEQ ID NO: 97, SEQ ID NO: 98, and SEQ ID NO: 100, respectively; - heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3) having the amino acid sequences set forth in SEQ ID NO: 64, SEQ ID NO: 65, and SEQ ID NO: 66, respectively; - heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3) having the amino acid sequences set forth in SEQ ID NO: 70, SEQ ID NO: 71, and SEQ ID NO: 118, respectively; - heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3) having the amino acid sequences set forth in SEQ ID NO: 64, SEQ ID NO: 65, and SEQ ID NO: 119, respectively; - heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3) having the amino acid sequences set forth in SEQ ID NO: 64, SEQ ID NO: 65, and SEQ ID NO: 120, respectively; - heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3) having the amino acid sequences set forth in SEQ ID NO: 64, SEQ ID NO: 65, and SEQ ID NO: 121, respectively; - heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3) having the amino acid sequences set forth in SEQ ID NO: 64, SEQ ID NO: 65, and SEQ ID NO: 122, respectively; - heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3) having the amino acid sequences set forth in SEQ ID NO: 64, SEQ ID NO: 65, and SEQ ID NO: 123, respectively; - a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) having the amino acid sequences set forth in SEQ ID NO: 64, SEQ ID NO: 65, and SEQ ID NO: 124, respectively; or - a heavy chain variable region comprising a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) having the amino acid sequences set forth in SEQ ID NO: 64, SEQ ID NO: 65, and SEQ ID NO: 125, respectively; Each of the HCDRs may contain up to three, two, or one amino acid substitutions.

[0036] In certain embodiments, the LAG-3 binding domain of the multispecific antibody or variant thereof comprises: - a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) of a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 1; - a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) of a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 3; - a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) of a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 5; - a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) of a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 6; - a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) of a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 15; - a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) of a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 16; - a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) of a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 17; - a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) of a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 52; - a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) of a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 53; - a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) of a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 54; - a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) of a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 55; - a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) of a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 56; - a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) of a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 57; - a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) of a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 58, or - a heavy chain variable region comprising a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) having the amino acid sequence set forth in SEQ ID NO: 59, wherein each of the HCDRs may comprise up to three, two, or one amino acid substitutions.

[0037] In certain embodiments, the LAG-3 binding domain of the multispecific antibody or variant thereof comprises: - heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3) having the amino acid sequences set forth in SEQ ID NO: 60, SEQ ID NO: 61, and SEQ ID NO: 62, respectively; - heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3) having the amino acid sequences set forth in SEQ ID NO: 64, SEQ ID NO: 65, and SEQ ID NO: 66, respectively; - heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3) having the amino acid sequences set forth in SEQ ID NO: 70, SEQ ID NO: 71, and SEQ ID NO: 72, respectively; - heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3) having the amino acid sequences set forth in SEQ ID NO: 73, SEQ ID NO: 74, and SEQ ID NO: 75, respectively; - heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3) having the amino acid sequences set forth in SEQ ID NO: 97, SEQ ID NO: 98, and SEQ ID NO: 99, respectively; - heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3) having the amino acid sequences set forth in SEQ ID NO: 97, SEQ ID NO: 98, and SEQ ID NO: 100, respectively; - heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3) having the amino acid sequences set forth in SEQ ID NO: 64, SEQ ID NO: 65, and SEQ ID NO: 66, respectively; - heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3) having the amino acid sequences set forth in SEQ ID NO: 70, SEQ ID NO: 71, and SEQ ID NO: 118, respectively; - heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3) having the amino acid sequences set forth in SEQ ID NO: 64, SEQ ID NO: 65, and SEQ ID NO: 119, respectively; - heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3) having the amino acid sequences set forth in SEQ ID NO: 64, SEQ ID NO: 65, and SEQ ID NO: 120, respectively; - heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3) having the amino acid sequences set forth in SEQ ID NO: 64, SEQ ID NO: 65, and SEQ ID NO: 121, respectively; - heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3) having the amino acid sequences set forth in SEQ ID NO: 64, SEQ ID NO: 65, and SEQ ID NO: 122, respectively; - heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3) having the amino acid sequences set forth in SEQ ID NO: 64, SEQ ID NO: 65, and SEQ ID NO: 123, respectively; - a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) having the amino acid sequences set forth in SEQ ID NO: 64, SEQ ID NO: 65, and SEQ ID NO: 124, respectively; or - a heavy chain variable region comprising a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) having the amino acid sequences set forth in SEQ ID NO: 64, SEQ ID NO: 65, and SEQ ID NO: 125, respectively; Each of the HCDRs may contain up to three, two, or one amino acid substitutions.

[0038] In certain embodiments, the LAG-3 binding domain of the multispecific antibody or variant thereof comprises: - a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) of a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 5; - a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) of a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 17; - a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) of a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 52; - a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) of a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 53; - a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) of a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 54; - a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) of a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 55; - a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) of a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 56; - a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) of a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 57; - a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) of a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 58, or a heavy chain variable region comprising a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) having the amino acid sequence set forth in SEQ ID NO: 59; Each of the HCDRs may contain up to three, two, or one amino acid substitutions.

[0039] In certain embodiments, the LAG-3 binding domain of the multispecific antibody or variant thereof comprises: - heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3) having the amino acid sequences set forth in SEQ ID NO: 70, SEQ ID NO: 71, and SEQ ID NO: 72, respectively; - heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3) having the amino acid sequences set forth in SEQ ID NO: 64, SEQ ID NO: 65, and SEQ ID NO: 66, respectively; - heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3) having the amino acid sequences set forth in SEQ ID NO: 70, SEQ ID NO: 71, and SEQ ID NO: 118, respectively; - heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3) having the amino acid sequences set forth in SEQ ID NO: 64, SEQ ID NO: 65, and SEQ ID NO: 119, respectively; - heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3) having the amino acid sequences set forth in SEQ ID NO: 64, SEQ ID NO: 65, and SEQ ID NO: 120, respectively; - heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3) having the amino acid sequences set forth in SEQ ID NO: 64, SEQ ID NO: 65, and SEQ ID NO: 121, respectively; - heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3) having the amino acid sequences set forth in SEQ ID NO: 64, SEQ ID NO: 65, and SEQ ID NO: 122, respectively; - heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3) having the amino acid sequences set forth in SEQ ID NO: 64, SEQ ID NO: 65, and SEQ ID NO: 123, respectively; - a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) having the amino acid sequences set forth in SEQ ID NO: 64, SEQ ID NO: 65, and SEQ ID NO: 124, respectively; or - a heavy chain variable region comprising a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) having the amino acid sequences set forth in SEQ ID NO: 64, SEQ ID NO: 65, and SEQ ID NO: 125, respectively; Each of the HCDRs may contain up to three, two, or one amino acid substitutions.

[0040] In certain embodiments, the LAG-3 binding domain of the multispecific antibody or variant thereof comprises a heavy chain variable region comprising heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3) of the heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 5, Each of the HCDRs may contain up to three, two, or one amino acid substitutions.

[0041] In certain embodiments, the LAG-3 binding domain of the multispecific antibody or variant thereof comprises a heavy chain variable region comprising heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3) of the heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 52, Each of the HCDRs may contain up to three, two, or one amino acid substitutions.

[0042] In certain embodiments, the LAG-3 binding domain of the multispecific antibody or variant thereof comprises a heavy chain variable region comprising a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) having the amino acid sequences set forth in SEQ ID NO: 70, SEQ ID NO: 71, and SEQ ID NO: 72, respectively; Each of the HCDRs may contain up to three, two, or one amino acid substitutions.

[0043] In certain embodiments, the LAG-3 binding domain of the multispecific antibody or variant thereof comprises a heavy chain variable region comprising a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) having the amino acid sequences set forth in SEQ ID NO: 70, SEQ ID NO: 71, and SEQ ID NO: 118, respectively; Each of the HCDRs may contain up to three, two, or one amino acid substitutions.

[0044] In certain embodiments, the LAG-3 binding domain of the multispecific antibody or variant thereof comprises: - a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) of a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 1; - a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) of a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 2; - a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) of a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 3; - a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) of a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 4; - a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) of a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 5; - a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) of a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 6; - a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) of a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 7; - a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) of a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 8; - a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) of a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 9; - a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) of a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 10; - a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) of a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 11; - a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) of a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 12; - a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) of a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 13; - a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) of a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 14; - a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) of a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 15; - a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) of a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 16; - a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) of a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 17; - a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) of a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 52; - a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) of a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 53; - a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) of a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 54; - a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) of a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 55; - a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) of a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 56; - a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) of a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 57; - a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) of a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 58, or - a heavy chain variable region comprising heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3) of the heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 59.

[0045] In certain embodiments, the LAG-3 binding domain of the multispecific antibody or variant thereof comprises: - heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3) having the amino acid sequences set forth in SEQ ID NO: 60, SEQ ID NO: 61, and SEQ ID NO: 62, respectively; - heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3) having the amino acid sequences set forth in SEQ ID NO: 60, SEQ ID NO: 63, and SEQ ID NO: 62, respectively; - heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3) having the amino acid sequences set forth in SEQ ID NO: 64, SEQ ID NO: 65, and SEQ ID NO: 66, respectively; - heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3) having the amino acid sequences set forth in SEQ ID NO: 67, SEQ ID NO: 68, and SEQ ID NO: 69, respectively; - heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3) having the amino acid sequences set forth in SEQ ID NO: 70, SEQ ID NO: 71, and SEQ ID NO: 72, respectively; - heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3) having the amino acid sequences set forth in SEQ ID NO: 73, SEQ ID NO: 74, and SEQ ID NO: 75, respectively; - heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3) having the amino acid sequences set forth in SEQ ID NO: 76, SEQ ID NO: 77, and SEQ ID NO: 78, respectively; - heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3) having the amino acid sequences set forth in SEQ ID NO: 79, SEQ ID NO: 80, and SEQ ID NO: 81, respectively; - heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3) having the amino acid sequences set forth in SEQ ID NO: 82, SEQ ID NO: 83, and SEQ ID NO: 84, respectively; - heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3) having the amino acid sequences set forth in SEQ ID NO: 85, SEQ ID NO: 86, and SEQ ID NO: 87, respectively; - heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3) having the amino acid sequences set forth in SEQ ID NO: 88, SEQ ID NO: 89, and SEQ ID NO: 90, respectively; - heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3) having the amino acid sequences set forth in SEQ ID NO: 76, SEQ ID NO: 91, and SEQ ID NO: 92, respectively; - heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3) having the amino acid sequences set forth in SEQ ID NO: 76, SEQ ID NO: 91, and SEQ ID NO: 93, respectively; - heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3) having the amino acid sequences set forth in SEQ ID NO: 94, SEQ ID NO: 95, and SEQ ID NO: 96, respectively; - heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3) having the amino acid sequences set forth in SEQ ID NO: 97, SEQ ID NO: 98, and SEQ ID NO: 99, respectively; - heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3) having the amino acid sequences set forth in SEQ ID NO: 97, SEQ ID NO: 98, and SEQ ID NO: 100, respectively; - heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3) having the amino acid sequences set forth in SEQ ID NO: 64, SEQ ID NO: 65, and SEQ ID NO: 66, respectively; - heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3) having the amino acid sequences set forth in SEQ ID NO: 70, SEQ ID NO: 71, and SEQ ID NO: 118, respectively; - heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3) having the amino acid sequences set forth in SEQ ID NO: 64, SEQ ID NO: 65, and SEQ ID NO: 119, respectively; - heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3) having the amino acid sequences set forth in SEQ ID NO: 64, SEQ ID NO: 65, and SEQ ID NO: 120, respectively; - heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3) having the amino acid sequences set forth in SEQ ID NO: 64, SEQ ID NO: 65, and SEQ ID NO: 121, respectively; - heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3) having the amino acid sequences set forth in SEQ ID NO: 64, SEQ ID NO: 65, and SEQ ID NO: 122, respectively; - heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3) having the amino acid sequences set forth in SEQ ID NO: 64, SEQ ID NO: 65, and SEQ ID NO: 123, respectively; - a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) having the amino acid sequences set forth in SEQ ID NO: 64, SEQ ID NO: 65, and SEQ ID NO: 124, respectively; or - comprise a heavy chain variable region comprising a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) having the amino acid sequences set forth in SEQ ID NO: 64, SEQ ID NO: 65, and SEQ ID NO: 125, respectively.

[0046] In certain embodiments, the LAG-3 binding domain of the multispecific antibody or variant thereof comprises: - a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) of a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 1; - a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) of a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 3; - a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) of a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 5; - a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) of a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 6; - a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) of a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 15; - a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) of a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 16; - a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) of a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 17; - a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) of a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 52; - a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) of a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 53; - a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) of a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 54; - a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) of a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 55; - a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) of a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 56; - a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) of a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 57; - a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) of a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 58, or - a heavy chain variable region comprising heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3) of the heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 59.

[0047] In certain embodiments, the LAG-3 binding domain of the multispecific antibody or variant thereof comprises: - heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3) having the amino acid sequences set forth in SEQ ID NO: 60, SEQ ID NO: 61, and SEQ ID NO: 62, respectively; - heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3) having the amino acid sequences set forth in SEQ ID NO: 64, SEQ ID NO: 65, and SEQ ID NO: 66, respectively; - heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3) having the amino acid sequences set forth in SEQ ID NO: 70, SEQ ID NO: 71, and SEQ ID NO: 72, respectively; - heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3) having the amino acid sequences set forth in SEQ ID NO: 73, SEQ ID NO: 74, and SEQ ID NO: 75, respectively; - heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3) having the amino acid sequences set forth in SEQ ID NO: 97, SEQ ID NO: 98, and SEQ ID NO: 99, respectively; - heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3) having the amino acid sequences set forth in SEQ ID NO: 97, SEQ ID NO: 98, and SEQ ID NO: 100, respectively; - heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3) having the amino acid sequences set forth in SEQ ID NO: 64, SEQ ID NO: 65, and SEQ ID NO: 66, respectively; - heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3) having the amino acid sequences set forth in SEQ ID NO: 70, SEQ ID NO: 71, and SEQ ID NO: 118, respectively; - heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3) having the amino acid sequences set forth in SEQ ID NO: 64, SEQ ID NO: 65, and SEQ ID NO: 119, respectively; - heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3) having the amino acid sequences set forth in SEQ ID NO: 64, SEQ ID NO: 65, and SEQ ID NO: 120, respectively; - heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3) having the amino acid sequences set forth in SEQ ID NO: 64, SEQ ID NO: 65, and SEQ ID NO: 121, respectively; - heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3) having the amino acid sequences set forth in SEQ ID NO: 64, SEQ ID NO: 65, and SEQ ID NO: 122, respectively; - heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3) having the amino acid sequences set forth in SEQ ID NO: 64, SEQ ID NO: 65, and SEQ ID NO: 123, respectively; - a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) having the amino acid sequences set forth in SEQ ID NO: 64, SEQ ID NO: 65, and SEQ ID NO: 124, respectively; or - comprise a heavy chain variable region comprising a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) having the amino acid sequences set forth in SEQ ID NO: 64, SEQ ID NO: 65, and SEQ ID NO: 125, respectively.

[0048] In certain embodiments, the LAG-3 binding domain of the multispecific antibody or variant thereof comprises: - a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) of a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 5; - a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) of a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 17; - a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) of a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 52; - a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) of a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 53; - a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) of a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 54; - a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) of a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 55; - a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) of a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 56; - a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) of a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 57; - a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) of a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 58, or - a heavy chain variable region comprising heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3) of the heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 59.

[0049] In certain embodiments, the LAG-3 binding domain of the multispecific antibody or variant thereof comprises: - heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3) having the amino acid sequences set forth in SEQ ID NO: 70, SEQ ID NO: 71, and SEQ ID NO: 72, respectively; - heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3) having the amino acid sequences set forth in SEQ ID NO: 64, SEQ ID NO: 65, and SEQ ID NO: 66, respectively; - heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3) having the amino acid sequences set forth in SEQ ID NO: 70, SEQ ID NO: 71, and SEQ ID NO: 118, respectively; - heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3) having the amino acid sequences set forth in SEQ ID NO: 64, SEQ ID NO: 65, and SEQ ID NO: 119, respectively; - heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3) having the amino acid sequences set forth in SEQ ID NO: 64, SEQ ID NO: 65, and SEQ ID NO: 120, respectively; - heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3) having the amino acid sequences set forth in SEQ ID NO: 64, SEQ ID NO: 65, and SEQ ID NO: 121, respectively; - heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3) having the amino acid sequences set forth in SEQ ID NO: 64, SEQ ID NO: 65, and SEQ ID NO: 122, respectively; - heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3) having the amino acid sequences set forth in SEQ ID NO: 64, SEQ ID NO: 65, and SEQ ID NO: 123, respectively; - a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) having the amino acid sequences set forth in SEQ ID NO: 64, SEQ ID NO: 65, and SEQ ID NO: 124, respectively; or - comprise a heavy chain variable region comprising a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) having the amino acid sequences set forth in SEQ ID NO: 64, SEQ ID NO: 65, and SEQ ID NO: 125, respectively.

[0050] In certain embodiments, the LAG-3 binding domain of a multispecific antibody or variant thereof comprises a heavy chain variable region comprising heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3) of the heavy chain variable region having the amino acid sequence set forth in SEQ ID NO:5.

[0051] In certain embodiments, the LAG-3 binding domain of a multispecific antibody or variant thereof comprises a heavy chain variable region comprising a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) having the amino acid sequences set forth in SEQ ID NO: 70, SEQ ID NO: 71, and SEQ ID NO: 72, respectively.

[0052] In certain embodiments, the LAG-3 binding domain of a multispecific antibody or variant thereof comprises a heavy chain variable region comprising heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3) of the heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 52.

[0053] In certain embodiments, the LAG-3 binding domain of a multispecific antibody or variant thereof comprises a heavy chain variable region comprising a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) having the amino acid sequences set forth in SEQ ID NO: 70, SEQ ID NO: 71, and SEQ ID NO: 118, respectively.

[0054] In certain embodiments, the LAG-3 binding domain of a multispecific antibody or variant thereof comprises a heavy chain variable region having the amino acid sequence set forth in any one of SEQ ID NOs: 1-17 or 52-59, or having at least 80%, preferably 85%, more preferably 90%, or most preferably 95% sequence identity thereto.

[0055] In certain embodiments, the LAG-3 binding domain of a multispecific antibody or variant thereof comprises a LAG-3 binding domain variant, in which each of the HCDRs may contain up to three, two, or one amino acid substitutions, and such variants are expected to retain LAG-3 binding specificity.

[0056] For example, suitable positions for introducing an amino acid mutation include, but are not limited to, the second and / or third amino acid of HCDR1, the third, seventh, tenth, thirteenth and / or sixteenth amino acids of HCDR2, and / or the first amino acid of HCDR3. CDR sequences according to Kabat are shown in bold and underlined in the sequence listings provided herein.

[0057] In certain embodiments, the anti-human LAG-3 binding domain comprises: - HCDR1 having the amino acid sequence SX1X2WS, wherein X1 can be Y or F; HCDR1, and / or X2, which may be Y or S - an HCDR2 having the amino acid sequence YIX1YSGX2TNX3NPX4LKX5, wherein X1 can be Y or D; X2 can be S or T; X3 can be Y or F; X4 can be S or F; HCDR2, and / or X5, which may be S or I an HCDR3 having the amino acid sequence X1LLYKWNYVEGFDI, wherein X1 comprises an HCDR3, which can be D or H.

[0058] For example, suitable positions for introducing an amino acid mutation include, but are not limited to, the first, third, and / or fourth amino acid of HCDR1, the seventh, tenth, and / or twelfth amino acid of HCDR2, and / or the third amino acid of HCDR3. CDR sequences according to Kabat are shown in bold and underlined in the sequence listings provided herein.

[0059] In certain embodiments, the anti-human LAG-3 binding domain comprises: HCDR1 having the amino acid sequence X1YX2X3H, wherein X1 can be S, N, or R; X2 can be G or D; HCDR1, and / or X3, which may be M, T, or I; - an HCDR2 having the amino acid sequence VISYDGX1NKX2YX3DSVKG, wherein X1 can be S or N; X2 can be Y, F, or H; HCDR2, and / or X3, which may be A, E, or V - an HCDR3 having the amino acid sequence ERX1WDVFDI, wherein X1 comprises an HCDR3, which can be G or D.

[0060] For example, suitable positions for introducing an amino acid mutation include, but are not limited to, the first and / or third amino acid of HCDR1, the fifth and / or eighth amino acid of HCDR2, and / or the third amino acid of HCDR3. CDR sequences according to Kabat are shown in bold and underlined in the sequence listings provided herein.

[0061] In certain embodiments, the anti-human LAG-3 binding domain comprises: HCDR1 having the amino acid sequence X1YX2MH, wherein X1 can be S or N; HCDR1, and / or X2, which may be G or A - HCDR2 having the amino acid sequence VISYX1GSX2KYYADSVKG, wherein X1 can be D or H; HCDR2, where X2 can be N or D, and / or - an HCDR3 having the amino acid sequence DGDNWDX1FDI, wherein X1 comprises an HCDR3, which can be V or A.

[0062] In certain embodiments, the LAG-3 binding domain of a multispecific antibody or variant thereof also includes a LAG-3 binding domain variant that, in addition to mutations in the HCDRs, comprises one or more mutations in the framework regions. In certain embodiments, the LAG-3 binding domain variant of a multispecific antibody or variant thereof does not comprise mutations in the CDR regions but comprises one or more mutations in the framework regions. Such variants have at least 80%, preferably 85%, more preferably 90%, or most preferably 95% sequence identity to the sequences disclosed herein and are expected to retain LAG-3 binding specificity. Thus, in certain embodiments, the LAG-3 binding domain of the present disclosure comprises: a heavy chain variable region having at least 80%, preferably 85%, more preferably 90%, or most preferably 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1, wherein the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 of the amino acid sequence set forth in SEQ ID NO: 1; a heavy chain variable region having at least 80%, preferably 85%, more preferably 90%, or most preferably 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 2, wherein the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 of the amino acid sequence set forth in SEQ ID NO: 2; a heavy chain variable region having at least 80%, preferably 85%, more preferably 90%, or most preferably 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 3, wherein the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 of the amino acid sequence set forth in SEQ ID NO: 3; a heavy chain variable region having at least 80%, preferably 85%, more preferably 90%, or most preferably 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 4, wherein the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 of the amino acid sequence set forth in SEQ ID NO: 4; a heavy chain variable region having at least 80%, preferably 85%, more preferably 90%, or most preferably 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 5, wherein the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 of the amino acid sequence set forth in SEQ ID NO: 5; a heavy chain variable region having at least 80%, preferably 85%, more preferably 90%, or most preferably 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 6, wherein the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 of the amino acid sequence set forth in SEQ ID NO: 6; a heavy chain variable region having at least 80%, preferably 85%, more preferably 90%, or most preferably 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 7, wherein the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 of the amino acid sequence set forth in SEQ ID NO: 7; a heavy chain variable region having at least 80%, preferably 85%, more preferably 90%, or most preferably 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 8, wherein the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 of the amino acid sequence set forth in SEQ ID NO: 8; a heavy chain variable region having at least 80%, preferably 85%, more preferably 90%, or most preferably 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 9, wherein the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 of the amino acid sequence set forth in SEQ ID NO: 9; a heavy chain variable region having at least 80%, preferably 85%, more preferably 90%, or most preferably 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 10, wherein the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 of the amino acid sequence set forth in SEQ ID NO: 10; a heavy chain variable region having at least 80%, preferably 85%, more preferably 90%, or most preferably 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 11, wherein the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 of the amino acid sequence set forth in SEQ ID NO: 11; a heavy chain variable region having at least 80%, preferably 85%, more preferably 90%, or most preferably 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 12, wherein the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 of the amino acid sequence set forth in SEQ ID NO: 12; a heavy chain variable region having at least 80%, preferably 85%, more preferably 90%, or most preferably 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 13, wherein the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 of the amino acid sequence set forth in SEQ ID NO: 13; a heavy chain variable region having at least 80%, preferably 85%, more preferably 90%, or most preferably 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 14, wherein the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 of the amino acid sequence set forth in SEQ ID NO: 14; a heavy chain variable region having at least 80%, preferably 85%, more preferably 90%, or most preferably 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 15, wherein the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 of the amino acid sequence set forth in SEQ ID NO: 15; a heavy chain variable region having at least 80%, preferably 85%, more preferably 90%, or most preferably 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 16, wherein the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 of the amino acid sequence set forth in SEQ ID NO: 16; a heavy chain variable region having at least 80%, preferably 85%, more preferably 90%, or most preferably 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 17, wherein the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 of the amino acid sequence set forth in SEQ ID NO: 17; a heavy chain variable region having at least 80%, preferably 85%, more preferably 90%, or most preferably 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 52, wherein the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 of the amino acid sequence set forth in SEQ ID NO: 52; a heavy chain variable region having at least 80%, preferably 85%, more preferably 90%, or most preferably 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 53, wherein the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 of the amino acid sequence set forth in SEQ ID NO: 53; a heavy chain variable region having at least 80%, preferably 85%, more preferably 90%, or most preferably 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 54, wherein the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 of the amino acid sequence set forth in SEQ ID NO: 54; a heavy chain variable region having at least 80%, preferably 85%, more preferably 90%, or most preferably 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 55, wherein the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 of the amino acid sequence set forth in SEQ ID NO: 55; - a heavy chain variable region having at least 80%, preferably 85%, more preferably 90%, or most preferably 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 56, wherein the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 of the amino acid sequence set forth in SEQ ID NO: 56; - a heavy chain variable region having at least 80%, preferably 85%, more preferably 90%, or most preferably 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 57, wherein the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 of the amino acid sequence set forth in SEQ ID NO: 57; a heavy chain variable region having at least 80%, preferably 85%, more preferably 90%, or most preferably 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 58, wherein the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 of the amino acid sequence set forth in SEQ ID NO: 58; or - a heavy chain variable region having at least 80%, preferably 85%, more preferably 90%, or most preferably 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 59, wherein the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 of the amino acid sequence set forth in SEQ ID NO: 59.

[0063] In certain embodiments, the LAG-3 binding domain of the present disclosure comprises: a heavy chain variable region having at least 80%, preferably 85%, more preferably 90%, or most preferably 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1, wherein the heavy chain variable region comprises the HCDR1, HCDR2, and HCDR3 amino acid sequences set forth in SEQ ID NO: 60, SEQ ID NO: 61, and SEQ ID NO: 62, respectively; a heavy chain variable region having at least 80%, preferably 85%, more preferably 90%, or most preferably 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 2, wherein the heavy chain variable region comprises the HCDR1, HCDR2, and HCDR3 amino acid sequences set forth in SEQ ID NO: 60, SEQ ID NO: 63, and SEQ ID NO: 62, respectively; a heavy chain variable region having at least 80%, preferably 85%, more preferably 90%, or most preferably 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 3, wherein the heavy chain variable region comprises the HCDR1, HCDR2, and HCDR3 amino acid sequences set forth in SEQ ID NO: 64, SEQ ID NO: 65, and SEQ ID NO: 66, respectively; a heavy chain variable region having at least 80%, preferably 85%, more preferably 90%, or most preferably 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 4, wherein the heavy chain variable region comprises the HCDR1, HCDR2, and HCDR3 amino acid sequences set forth in SEQ ID NO: 67, SEQ ID NO: 68, and SEQ ID NO: 69, respectively; a heavy chain variable region having at least 80%, preferably 85%, more preferably 90%, or most preferably 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 5, wherein the heavy chain variable region comprises the HCDR1, HCDR2, and HCDR3 amino acid sequences set forth in SEQ ID NO: 70, SEQ ID NO: 71, and SEQ ID NO: 72, respectively; a heavy chain variable region having at least 80%, preferably 85%, more preferably 90%, or most preferably 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 6, wherein the heavy chain variable region comprises the HCDR1, HCDR2, and HCDR3 amino acid sequences set forth in SEQ ID NO: 73, SEQ ID NO: 74, and SEQ ID NO: 75, respectively; a heavy chain variable region having at least 80%, preferably 85%, more preferably 90%, or most preferably 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 7, wherein the heavy chain variable region comprises the HCDR1, HCDR2, and HCDR3 amino acid sequences set forth in SEQ ID NO: 76, SEQ ID NO: 77, and SEQ ID NO: 78, respectively; a heavy chain variable region having at least 80%, preferably 85%, more preferably 90%, or most preferably 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 8, wherein the heavy chain variable region comprises the HCDR1, HCDR2, and HCDR3 amino acid sequences set forth in SEQ ID NO: 79, SEQ ID NO: 80, and SEQ ID NO: 81, respectively; a heavy chain variable region having at least 80%, preferably 85%, more preferably 90%, or most preferably 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 9, wherein the heavy chain variable region comprises the HCDR1, HCDR2, and HCDR3 amino acid sequences set forth in SEQ ID NO: 82, SEQ ID NO: 83, and SEQ ID NO: 84, respectively; a heavy chain variable region having at least 80%, preferably 85%, more preferably 90%, or most preferably 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 10, wherein the heavy chain variable region comprises the HCDR1, HCDR2, and HCDR3 amino acid sequences set forth in SEQ ID NO: 85, SEQ ID NO: 86, and SEQ ID NO: 87, respectively; a heavy chain variable region having at least 80%, preferably 85%, more preferably 90%, or most preferably 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 11, wherein the heavy chain variable region comprises the HCDR1, HCDR2, and HCDR3 amino acid sequences set forth in SEQ ID NO: 88, SEQ ID NO: 89, and SEQ ID NO: 90, respectively; a heavy chain variable region having at least 80%, preferably 85%, more preferably 90%, or most preferably 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 12, wherein the heavy chain variable region comprises the HCDR1, HCDR2, and HCDR3 amino acid sequences set forth in SEQ ID NO: 76, SEQ ID NO: 91, and SEQ ID NO: 92, respectively; a heavy chain variable region having at least 80%, preferably 85%, more preferably 90%, or most preferably 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 13, wherein the heavy chain variable region comprises the HCDR1, HCDR2, and HCDR3 amino acid sequences set forth in SEQ ID NO: 76, SEQ ID NO: 91, and SEQ ID NO: 93, respectively; a heavy chain variable region having at least 80%, preferably 85%, more preferably 90%, or most preferably 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 14, wherein the heavy chain variable region comprises the HCDR1, HCDR2, and HCDR3 amino acid sequences set forth in SEQ ID NO: 94, SEQ ID NO: 95, and SEQ ID NO: 96, respectively; a heavy chain variable region having at least 80%, preferably 85%, more preferably 90%, or most preferably 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 15, wherein the heavy chain variable region comprises the HCDR1, HCDR2, and HCDR3 amino acid sequences set forth in SEQ ID NO: 97, SEQ ID NO: 98, and SEQ ID NO: 99, respectively; a heavy chain variable region having at least 80%, preferably 85%, more preferably 90%, or most preferably 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 16, wherein the heavy chain variable region comprises the HCDR1, HCDR2, and HCDR3 amino acid sequences set forth in SEQ ID NO: 97, SEQ ID NO: 98, and SEQ ID NO: 100, respectively; a heavy chain variable region having at least 80%, preferably 85%, more preferably 90%, or most preferably 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 17, wherein the heavy chain variable region comprises the HCDR1, HCDR2, and HCDR3 amino acid sequences set forth in SEQ ID NO: 64, SEQ ID NO: 65, and SEQ ID NO: 66, respectively; a heavy chain variable region having at least 80%, preferably 85%, more preferably 90%, or most preferably 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 52, wherein the heavy chain variable region comprises the HCDR1, HCDR2, and HCDR3 amino acid sequences set forth in SEQ ID NO: 70, SEQ ID NO: 71, and SEQ ID NO: 118, respectively; a heavy chain variable region having at least 80%, preferably 85%, more preferably 90%, or most preferably 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 53, wherein the heavy chain variable region comprises the HCDR1, HCDR2, and HCDR3 amino acid sequences set forth in SEQ ID NO: 64, SEQ ID NO: 65, and SEQ ID NO: 119, respectively; a heavy chain variable region having at least 80%, preferably 85%, more preferably 90%, or most preferably 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 54, wherein the heavy chain variable region comprises the HCDR1, HCDR2, and HCDR3 amino acid sequences set forth in SEQ ID NO: 64, SEQ ID NO: 65, and SEQ ID NO: 120, respectively; a heavy chain variable region having at least 80%, preferably 85%, more preferably 90%, or most preferably 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 55, wherein the heavy chain variable region comprises the HCDR1, HCDR2, and HCDR3 amino acid sequences set forth in SEQ ID NO: 64, SEQ ID NO: 65, and SEQ ID NO: 121, respectively; a heavy chain variable region having at least 80%, preferably 85%, more preferably 90%, or most preferably 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 56, wherein the heavy chain variable region comprises the HCDR1, HCDR2, and HCDR3 amino acid sequences set forth in SEQ ID NO: 64, SEQ ID NO: 65, and SEQ ID NO: 122, respectively; a heavy chain variable region having at least 80%, preferably 85%, more preferably 90%, or most preferably 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 57, wherein the heavy chain variable region comprises the HCDR1, HCDR2, and HCDR3 amino acid sequences set forth in SEQ ID NO: 64, SEQ ID NO: 65, and SEQ ID NO: 123, respectively; a heavy chain variable region having at least 80%, preferably 85%, more preferably 90%, or most preferably 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 58, and comprising the HCDR1, HCDR2, and HCDR3 amino acid sequences set forth in SEQ ID NO: 64, SEQ ID NO: 65, and SEQ ID NO: 124, respectively; or - a heavy chain variable region having at least 80%, preferably 85%, more preferably 90%, or most preferably 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 59, and comprising the HCDR1, HCDR2, and HCDR3 amino acid sequences set forth in SEQ ID NO: 64, SEQ ID NO: 65, and SEQ ID NO: 125, respectively.

[0064] In certain embodiments, the LAG-3 binding domain of a multispecific antibody or variant thereof further comprises a light chain variable region. In certain embodiments, the LAG-3 binding domain of a multispecific antibody or variant thereof comprises a common light chain variable region. An example of a suitable light chain variable region is a light chain variable region comprising a light chain CDR1 (LCDR1), a light chain CDR2 (LCDR2), and a light chain CDR3 (LCDR3) having the amino acid sequences set forth in SEQ ID NO: 31, SEQ ID NO: 32, and SEQ ID NO: 33, respectively, where each LCDR may comprise up to three, two, or one amino acid substitutions. In certain embodiments, a suitable light chain variable region is a light chain variable region comprising a light chain CDR1 (LCDR1), a light chain CDR2 (LCDR2), and a light chain CDR3 (LCDR3) having the amino acid sequences set forth in SEQ ID NO: 31, SEQ ID NO: 32, and SEQ ID NO: 33, respectively. In certain embodiments, such a light chain variable region may comprise a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 30, or having at least 80%, preferably 85%, more preferably 90%, or most preferably 95% sequence identity thereto. A light chain or light chain variable region comprising these LCDRs and / or light chain variable regions is a light chain referred to in the art as VK1-39 / JK1, which is a common light chain.

[0065] In certain embodiments, the LAG-3 binding domain of the present disclosure comprises a light chain variable region having at least 80%, preferably 85%, more preferably 90%, or most preferably 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 30, wherein the light chain variable region comprises an LCDR1 amino acid sequence set forth in SEQ ID NO: 31, an LCDR2 amino acid sequence set forth in SEQ ID NO: 32, and an LCDR3 amino acid sequence set forth in SEQ ID NO: 33.

[0066] The term "common light chain" according to the present invention refers to a light chain that can pair with multiple different heavy chains, i.e., heavy chains with different antigen or epitope binding specificities. Common light chains are particularly useful, for example, in the generation of bispecific antibodies, where antibody production is more efficient when both binding domains contain the same light chain. The term "common light chain" encompasses light chains that are identical or have some amino acid sequence differences, while the binding specificity of the full-length antibody is not affected. For example, it is possible, within the scope of the definition of a common light chain used herein, to prepare or find light chains that are not identical but are still functionally equivalent by introducing and testing conservative amino acid changes, such as amino acid changes in regions that do not contribute or only partially contribute to binding specificity when paired with a heavy chain.

[0067] Apart from the common light chains containing the LCDRs and / or light chain variable regions described above, other common light chains known in the art may also be used. Examples of such common light chains include, but are not limited to, VK1-39 / JK5, ​​which contains a light chain variable region comprising a light chain CDR1 (LCDR1), a light chain CDR2 (LCDR2), and a light chain CDR3 (LCDR3) of the light chain variable region having the amino acid sequence set forth in SEQ ID NO: 34. The LCDRs according to IMGT are shown in bold and underlined. In certain embodiments, the light chain contains a light chain variable region comprising a light chain CDR1 (LCDR1), a light chain CDR2 (LCDR2), and a light chain CDR3 (LCDR3) of the light chain variable region having the amino acid sequence set forth in SEQ ID NO: 34, and each of the LCDRs may contain up to three, two, or one amino acid substitutions. In certain embodiments, the light chain comprises a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 34, or having at least 80%, preferably 85%, more preferably 90%, or most preferably 95% sequence identity thereto, and VK3-15 / JK1 comprises a light chain variable region comprising a light chain CDR1 (LCDR1), a light chain CDR2 (LCDR2), and a light chain CDR3 (LCDR3) of a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 35. LCDRs according to IMGT are shown in bold and underlined. In certain embodiments, the light chain comprises a light chain variable region comprising a light chain CDR1 (LCDR1), a light chain CDR2 (LCDR2), and a light chain CDR3 (LCDR3) of a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 35, and each of the LCDRs may comprise up to three, two, or one amino acid substitutions. In certain embodiments, the light chain comprises a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 35, or having at least 80%, preferably 85%, more preferably 90%, or most preferably 95% sequence identity thereto, and VK3-20 / JK1 comprises a light chain variable region comprising a light chain CDR1 (LCDR1), a light chain CDR2 (LCDR2), and a light chain CDR3 (LCDR3) of the light chain variable region having the amino acid sequence set forth in SEQ ID NO: 36. The LCDRs according to IMGT are shown in bold and underlined.In certain embodiments, the light chain comprises a light chain variable region comprising light chain CDR1 (LCDR1), light chain CDR2 (LCDR2), and light chain CDR3 (LCDR3) of a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 36, wherein each LCDR may comprise up to three, two, or one amino acid substitution. In certain embodiments, the light chain comprises a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 36 or having at least 80%, preferably 85%, more preferably 90%, or most preferably 95% sequence identity thereto, and VL3-21 / JL3 comprises a light chain variable region comprising light chain CDR1 (LCDR1), light chain CDR2 (LCDR2), and light chain CDR3 (LCDR3) of a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 37. The LCDRs according to IMGT are shown in bold and underlined. In certain embodiments, the light chain comprises a light chain variable region comprising a light chain CDR1 (LCDR1), a light chain CDR2 (LCDR2), and a light chain CDR3 (LCDR3) of the light chain variable region having the amino acid sequence set forth in SEQ ID NO: 37, wherein each of the LCDRs may comprise up to three, two, or one amino acid substitutions. In certain embodiments, the light chain comprises a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 37, or having at least 80%, preferably 85%, more preferably 90%, or most preferably 95% sequence identity thereto.

[0068] VK1-39 is an abbreviation for immunoglobulin variable kappa 1-39 gene. This gene is also known as immunoglobulin kappa variable 1-39, IGKV139, IGKV1-39, IgVκ1-39. The external IDs for this gene are HGNC:5740, Entrez Gene:28930, Ensembl:ENSG00000242371. A preferred amino acid sequence for VK1-39 is given as SEQ ID NO:38. This is the sequence of the V region. The V region can be combined with one of five J regions. Two preferred combined sequences are designated VK1-39 / JK1 and VK1-39 / JK5, ​​with alternative names IgVκ1-39_01 / IGJκ1_01 or IgVκ1-39_01 / IGJκ5_01 (designations from the IMGT database world wide web at imgt.org). These designations are exemplary and encompass allelic variants of the gene segments.

[0069] VK3-15 is an abbreviation for immunoglobulin variable kappa 3-15 gene. This gene is also known as immunoglobulin kappa variable 3-15, IGKV315, IGKV3-15, and IgVκ3-15. The external identifiers for this gene are HGNC:5816, Entrez Gene:28913, and Ensembl:ENSG00000244437. A preferred amino acid sequence for VK3-15 is given as SEQ ID NO:39. This is the sequence of the V region. The V region can be combined with one of five J regions. A preferred combined sequence is designated VK3-15 / JK1, with an alternative name being Vκ3-15_01 / IGJκ1_01 (designation by the IMGT database World Wide Web at imgt.org). This designation is exemplary and encompasses allelic variants of the gene segment.

[0070] VK3-20 is an abbreviation for immunoglobulin variable kappa 3-20 gene. This gene is also known as immunoglobulin kappa variable 3-20, IGKV320, IGKV3-20, and IgVκ3-20. The external identifiers for this gene are HGNC:5817, Entrez Gene:28912, and Ensembl:ENSG00000239951. A preferred amino acid sequence for VK3-20 is SEQ ID NO:40. This is the sequence of the V region. The V region can be combined with one of five J regions. A preferred combined sequence is designated VK3-20 / JK1, with an alternative name being IgVκ3-20_01 / IGJκ1_01 (designation by the IMGT database World Wide Web at imgt.org). This designation is exemplary and encompasses allelic variants of the gene segment.

[0071] VL3-21 is an abbreviation for immunoglobulin variable lambda 3-21 gene. This gene is also known as immunoglobulin lambda variable 3-21, IGLV321, IGLV3-21, or IgVλ3-21. The external identifiers for this gene are HGNC:5905, Entrez Gene:28796, and Ensembl:ENSG00000211662.2. A preferred amino acid sequence for VL3-21 is given as SEQ ID NO:41. This is the sequence of the V region. The V region can be combined with one of five J regions. A preferred combined sequence is designated VL3-21 / JL3, with an alternative name being IgVλ3-21 / IGJλ3 (designation by the IMGT database World Wide Web at imgt.org). This designation is exemplary and encompasses allelic variants of the gene segment.

[0072] Furthermore, any light chain variable region of an LAG-3 antibody available in the art, or any other light chain variable region that exhibits antigen-binding activity when paired with the LAG-3 binding domain of the present invention and can be easily obtained, for example, from an antibody display library, may be used.

[0073] In certain embodiments, the LAG-3 binding domain of the multispecific antibody or variant thereof may further comprise a CH1 and a CL region. Any CH1 domain, particularly a human CH1 domain, may be used. An example of a suitable CH1 domain is provided by the amino acid sequence provided as SEQ ID NO: 42. Any CL domain, particularly a human CL, may be used. An example of a suitable CL domain is provided by the amino acid sequence provided as SEQ ID NO: 43.

[0074] In certain embodiments, the multispecific antibody or variant thereof comprises a binding domain that specifically binds to the extracellular domain of LAG-3 and a binding domain that specifically binds to the extracellular domain of a protein of the B7 family.

[0075] The B7 family includes a number of structurally related cell surface proteins that bind to receptors on lymphocytes to regulate immune responses. Lymphocyte activation is initiated by cell surface, antigen-specific T cell receptor or B cell receptor engagement. Additional signals simultaneously delivered by B7 ligands further determine the immune response of these cells. These so-called "costimulatory" or "co-inhibitory" signals are delivered by B7 family members via the CD28 family of receptors on lymphocytes. Binding of B7 family members to costimulatory receptors enhances immune responses, while binding to costimulatory receptors attenuates immune responses. The following members are currently considered to be part of this family: B7.1 (CD80), B7.2 (CD86), inducible costimulatory ligand (ICOS-L), programmed death-1 ligand (PD-L1), programmed death-2 ligand (PD-L2), B7-H3 (CD276), B7-H4, B7-H5, B7-H6, and B7-H7. B7 family members are expressed in lymphoid and non-lymphoid tissues. Their effects on regulating immune responses are demonstrated in the development of immunodeficiency and autoimmune diseases in mice with mutations in B7 family genes. Manipulation of signals delivered by B7 ligands has shown promise in the treatment of autoimmune, inflammatory, and cancer diseases.

[0076] In certain embodiments, the B7 family protein is selected from the group consisting of PD-L1, PD-L2, CD80, CD86, B7-H4, TNFRSF14, and B7-H7, hi certain embodiments, the B7 family protein is PD-L1.

[0077] In certain embodiments, the variable domain that specifically binds to the extracellular domain of a protein of the B7 family blocks the binding of PD-L1 to its receptor, which in certain embodiments is PD-1 and / or CD80.

[0078] In certain embodiments, the PD-L1-binding domain of a multispecific antibody or variant thereof of the present disclosure comprises a heavy chain variable region comprising a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) of one of the heavy chain variable regions having the amino acid sequences set forth in SEQ ID NOs: 20 to 24. The HCDRs according to Kabat are shown in bold and underlined in the sequence listings provided herein.

[0079] In certain embodiments, the binding domain that binds to PD-L1 is - a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) of a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 20; - a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) of a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 21; - a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) of a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 22; - a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) of a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 23, or - a heavy chain variable region comprising a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) of the heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 24; Each of the HCDRs may contain up to three, two, or one amino acid substitutions.

[0080] In certain embodiments, the binding domain that binds to PD-L1 is - heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3) having the amino acid sequences set forth in SEQ ID NO: 107, SEQ ID NO: 108, and SEQ ID NO: 109, respectively; - heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3) having the amino acid sequences set forth in SEQ ID NO: 110, SEQ ID NO: 111, and SEQ ID NO: 112, respectively; - heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3) having the amino acid sequences set forth in SEQ ID NO: 113, SEQ ID NO: 114, and SEQ ID NO: 115, respectively; - a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) having the amino acid sequences set forth in SEQ ID NO: 107, SEQ ID NO: 116, and SEQ ID NO: 109, respectively; or - a heavy chain variable region comprising a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) having the amino acid sequences set forth in SEQ ID NO: 107, SEQ ID NO: 117, and SEQ ID NO: 109, respectively; Each of the HCDRs may contain up to three, two, or one amino acid substitutions.

[0081] In certain embodiments, the binding domain that binds to PD-L1 is - a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) of a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 20, or - a heavy chain variable region comprising a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) of the heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 24; Each of the HCDRs may contain up to three, two, or one amino acid substitutions.

[0082] In certain embodiments, the binding domain that binds to PD-L1 is - a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) having the amino acid sequences set forth in SEQ ID NO: 107, SEQ ID NO: 108, and SEQ ID NO: 109, respectively; or - a heavy chain variable region comprising a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) of the heavy chain variable region having the amino acid sequences set forth in SEQ ID NO: 107, SEQ ID NO: 117, and SEQ ID NO: 109, respectively; Each of the HCDRs may contain up to three, two, or one amino acid substitutions.

[0083] In certain embodiments, the binding domain that binds to PD-L1 is a heavy chain variable region comprising a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) having the amino acid sequence set forth in SEQ ID NO: 20; Each of the HCDRs may contain up to three, two, or one amino acid substitutions.

[0084] In certain embodiments, the binding domain that binds to PD-L1 is - a heavy chain variable region comprising a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) having the amino acid sequences set forth in SEQ ID NO: 107, SEQ ID NO: 108, and SEQ ID NO: 109, respectively; Each of the HCDRs may contain up to three, two, or one amino acid substitutions.

[0085] In certain embodiments, the binding domain that binds to PD-L1 is - a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) of a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 20; - a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) of a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 21; - a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) of a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 22; a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) of a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 23; or - a heavy chain variable region comprising heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3) of the heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 24.

[0086] In certain embodiments, the binding domain that binds to PD-L1 is - heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3) having the amino acid sequences set forth in SEQ ID NO: 107, SEQ ID NO: 108, and SEQ ID NO: 109, respectively; - heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3) having the amino acid sequences set forth in SEQ ID NO: 110, SEQ ID NO: 111, and SEQ ID NO: 112, respectively; - heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3) having the amino acid sequences set forth in SEQ ID NO: 113, SEQ ID NO: 114, and SEQ ID NO: 115, respectively; a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) having the amino acid sequences set forth in SEQ ID NO: 107, SEQ ID NO: 116, and SEQ ID NO: 109, respectively; or - comprise a heavy chain variable region comprising a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) having the amino acid sequences set forth in SEQ ID NO: 107, SEQ ID NO: 117, and SEQ ID NO: 109, respectively.

[0087] In certain embodiments, the binding domain that binds to PD-L1 is - a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) of a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 20, or - a heavy chain variable region comprising heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3) of the heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 24.

[0088] In certain embodiments, the binding domain that binds to PD-L1 is - a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) having the amino acid sequences set forth in SEQ ID NO: 107, SEQ ID NO: 108, and SEQ ID NO: 109, respectively; or - comprise a heavy chain variable region comprising a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) having the amino acid sequences set forth in SEQ ID NO: 107, SEQ ID NO: 117, and SEQ ID NO: 109, respectively.

[0089] In certain embodiments, the binding domain that binds to PD-L1 is - a heavy chain variable region comprising heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3) of the heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 20.

[0090] In certain embodiments, the binding domain that binds to PD-L1 is - a heavy chain variable region comprising a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) having the amino acid sequences set forth in SEQ ID NO: 107, SEQ ID NO: 108, and SEQ ID NO: 109, respectively.

[0091] In certain embodiments, the PD-L1 binding domain of a multispecific antibody or variant thereof comprises a heavy chain variable region having the amino acid sequence set forth in any one of SEQ ID NOs: 20-24, or at least 80%, preferably 85%, more preferably 90%, or most preferably 95% sequence identity thereto.

[0092] In certain embodiments, the PD-L1 binding domain of a multispecific antibody or variant thereof comprises a PD-L1 binding domain variant in which each of the HCDRs may contain up to three, two, or one amino acid substitutions, and such variants are expected to retain PD-L1 binding specificity.

[0093] In certain embodiments, the PD-L1-binding domain of a multispecific antibody or variant thereof also includes PD-L1-binding domain variants that, in addition to mutations in the HCDRs, contain one or more mutations in the framework regions. In certain embodiments, the PD-L1-binding domain variant of a multispecific antibody or variant thereof does not contain mutations in the CDR regions, but does contain one or more mutations in the framework regions. Such variants have at least 80%, preferably 85%, more preferably 90%, or most preferably 95% sequence identity to the sequences disclosed herein, and are expected to retain PD-L1-binding specificity. Thus, in certain embodiments, the PD-L1-binding domain of the present disclosure comprises: a heavy chain variable region having at least 80%, preferably 85%, more preferably 90%, or most preferably 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 20, wherein the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 of the amino acid sequence set forth in SEQ ID NO: 20; a heavy chain variable region having at least 80%, preferably 85%, more preferably 90%, or most preferably 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 21, wherein the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 of the amino acid sequence set forth in SEQ ID NO: 21; a heavy chain variable region having at least 80%, preferably 85%, more preferably 90%, or most preferably 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 22, wherein the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 of the amino acid sequence set forth in SEQ ID NO: 22; a heavy chain variable region having at least 80%, preferably 85%, more preferably 90%, or most preferably 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 23, wherein the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 of the amino acid sequence set forth in SEQ ID NO: 23; or - a heavy chain variable region having at least 80%, preferably 85%, more preferably 90%, or most preferably 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 24, wherein the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 of the amino acid sequence set forth in SEQ ID NO: 24.

[0094] In certain embodiments, the PD-L1 binding domain of the disclosure comprises: a heavy chain variable region having at least 80%, preferably 85%, more preferably 90%, or most preferably 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 20, wherein the heavy chain variable region comprises the HCDR1, HCDR2, and HCDR3 amino acid sequences set forth in SEQ ID NO: 107, SEQ ID NO: 108, and SEQ ID NO: 109, respectively; a heavy chain variable region having at least 80%, preferably 85%, more preferably 90%, or most preferably 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 21, wherein the heavy chain variable region comprises the HCDR1, HCDR2, and HCDR3 amino acid sequences set forth in SEQ ID NO: 110, SEQ ID NO: 111, and SEQ ID NO: 112, respectively; a heavy chain variable region having at least 80%, preferably 85%, more preferably 90%, or most preferably 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 22, wherein the heavy chain variable region comprises the HCDR1, HCDR2, and HCDR3 amino acid sequences set forth in SEQ ID NO: 113, SEQ ID NO: 114, and SEQ ID NO: 115, respectively; a heavy chain variable region having at least 80%, preferably 85%, more preferably 90%, or most preferably 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 23, and comprising the HCDR1, HCDR2, and HCDR3 amino acid sequences set forth in SEQ ID NO: 107, SEQ ID NO: 116, and SEQ ID NO: 109, respectively; or - a heavy chain variable region having at least 80%, preferably 85%, more preferably 90%, or most preferably 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 24, and comprising the HCDR1, HCDR2, and HCDR3 amino acid sequences set forth in SEQ ID NO: 107, SEQ ID NO: 117, and SEQ ID NO: 109, respectively.

[0095] In certain embodiments, the PD-L1-binding domain of a multispecific antibody or variant thereof further comprises a light chain variable region. Examples of suitable light chain variable regions are the light chain variable regions described herein. The light chain variable regions of PD-L1 antibodies available in the art may be used, or any other light chain variable region that exhibits antigen-binding activity when paired with a PD-L1-binding domain of the present disclosure and can be readily obtained, for example, from an antibody display library. Preferably, the PD-L1-binding domain of the present disclosure comprises the VK1-39 / JK1, VK1-39 / JK5, ​​VK3-15 / JK1, VK3-20 / JK1, or VL3-21 / JL3 light chain variable region.

[0096] In certain embodiments, the PD-L1 binding domain of a multispecific antibody or variant thereof may further comprise a CH1 and a CL region. Any CH1 domain, particularly a human CH1 domain, may be used. An example of a suitable CH1 domain is provided by the amino acid sequence provided as SEQ ID NO: 42. Any CL domain, particularly a human CL, may be used. An example of a suitable CL domain is provided by the amino acid sequence provided as SEQ ID NO: 43.

[0097] In certain embodiments, the LAG-3 binding domain disclosed herein can be combined with any of the PD-L1 binding domains disclosed herein to produce a multispecific antibody or variant thereof of the present disclosure. In certain embodiments, the present disclosure provides the multispecific antibody PB1-PB125, or a variant thereof, as presented in Table 1. [Table 1]

[0098] In one embodiment, the multispecific antibody or variant thereof comprises: - a LAG-3 binding domain of the present disclosure, comprising a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) of a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 1; and - comprises a PD-L1-binding domain of the disclosure, comprising a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) of a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 20, 23, or 24.

[0099] In one embodiment, the multispecific antibody or variant thereof comprises: - a LAG-3 binding domain of the present disclosure, comprising a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) of a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 3; and - comprises a PD-L1-binding domain of the disclosure, comprising a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) of a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 20, 23, or 24.

[0100] In one embodiment, the multispecific antibody or variant thereof comprises: - a LAG-3 binding domain of the present disclosure, comprising a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) of a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 5; and - comprises a PD-L1 binding domain of the disclosure comprising a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) of a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 20, 23, or 24. In one embodiment, the multispecific antibody or variant thereof comprises: - a LAG-3 binding domain of the present disclosure, comprising a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) of a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 6; and - comprises a PD-L1-binding domain of the disclosure, comprising a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) of a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 20, 23, or 24.

[0101] In one embodiment, the multispecific antibody or variant thereof comprises: - a LAG-3 binding domain of the present disclosure, comprising a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) of a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 15; and - comprises a PD-L1-binding domain of the disclosure, comprising a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) of a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 20, 23, or 24.

[0102] In one embodiment, the multispecific antibody or variant thereof comprises: - a LAG-3 binding domain of the present disclosure, comprising a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) of a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 16; and - comprises a PD-L1-binding domain of the disclosure, comprising a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) of a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 20, 23, or 24.

[0103] In one embodiment, the multispecific antibody or variant thereof comprises: - a LAG-3 binding domain of the present disclosure, comprising a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) of a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 17; and - comprises a PD-L1-binding domain of the disclosure, comprising a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) of a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 20, 23, or 24.

[0104] In one embodiment, the multispecific antibody or variant thereof comprises: - a LAG-3 binding domain of the present disclosure, comprising a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) of a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 52; and - comprises a PD-L1-binding domain of the disclosure, comprising a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) of a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 20, 23, or 24.

[0105] In one embodiment, the multispecific antibody or variant thereof comprises: - a LAG-3 binding domain of the present disclosure, comprising a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) of a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 53; and - comprises a PD-L1-binding domain of the disclosure, comprising a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) of a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 20, 23, or 24.

[0106] In one embodiment, the multispecific antibody or variant thereof comprises: - a LAG-3 binding domain of the present disclosure comprising a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) of a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 54; and - comprises a PD-L1-binding domain of the disclosure, comprising a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) of a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 20, 23, or 24.

[0107] In one embodiment, the multispecific antibody or variant thereof comprises: - a LAG-3 binding domain of the present disclosure comprising a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) of a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 55; and - comprises a PD-L1-binding domain of the disclosure, comprising a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) of a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 20, 23, or 24.

[0108] In one embodiment, the multispecific antibody or variant thereof comprises: - a LAG-3 binding domain of the present disclosure comprising a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) of a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 56; and - comprises a PD-L1-binding domain of the disclosure, comprising a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) of a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 20, 23, or 24.

[0109] In one embodiment, the multispecific antibody or variant thereof comprises: - a LAG-3 binding domain of the present disclosure comprising a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) of a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 57; and - comprises a PD-L1-binding domain of the disclosure, comprising a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) of a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 20, 23, or 24.

[0110] In one embodiment, the multispecific antibody or variant thereof comprises: - a LAG-3 binding domain of the present disclosure comprising a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) of a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 58; and - comprises a PD-L1-binding domain of the disclosure, comprising a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) of a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 20, 23, or 24.

[0111] In one embodiment, the multispecific antibody or variant thereof comprises: - a LAG-3 binding domain of the present disclosure, comprising a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) of a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 59; and - comprises a PD-L1-binding domain of the disclosure, comprising a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) of a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 20, 23, or 24.

[0112] In one embodiment, the multispecific antibody or variant thereof comprises: - a LAG-3 binding domain of the present disclosure, comprising a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) of a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 17; and - comprises a PD-L1-binding domain of the disclosure, comprising a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) of a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 24.

[0113] In one embodiment, the multispecific antibody or variant thereof comprises: a LAG-3 binding domain of the present disclosure comprising a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) having the amino acid sequences set forth in SEQ ID NO: 64, SEQ ID NO: 65, and SEQ ID NO: 66, respectively; and - comprise a PD-L1-binding domain of the disclosure, comprising a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) having the amino acid sequences set forth in SEQ ID NO: 107, SEQ ID NO: 117, and SEQ ID NO: 109, respectively.

[0114] In one embodiment, the multispecific antibody or variant thereof comprises: - a LAG-3 binding domain of the present disclosure, comprising a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) of a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 53; and - comprises a PD-L1-binding domain of the disclosure, comprising a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) of a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 24.

[0115] In one embodiment, the multispecific antibody or variant thereof comprises: - a LAG-3 binding domain of the present disclosure comprising a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) having the amino acid sequences set forth in SEQ ID NO: 64, SEQ ID NO: 65, and SEQ ID NO: 119, respectively; and - comprise a PD-L1-binding domain of the disclosure, comprising a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) having the amino acid sequences set forth in SEQ ID NO: 107, SEQ ID NO: 117, and SEQ ID NO: 109, respectively.

[0116] In one embodiment, the multispecific antibody or variant thereof comprises: - a LAG-3 binding domain of the present disclosure comprising a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) of a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 54; and - comprises a PD-L1-binding domain of the disclosure, comprising a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) of a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 24.

[0117] In one embodiment, the multispecific antibody or variant thereof comprises: - a LAG-3 binding domain of the present disclosure comprising a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) having the amino acid sequences set forth in SEQ ID NO: 64, SEQ ID NO: 65, and SEQ ID NO: 120, respectively; and - comprise a PD-L1-binding domain of the disclosure, comprising a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) having the amino acid sequences set forth in SEQ ID NO: 107, SEQ ID NO: 117, and SEQ ID NO: 109, respectively.

[0118] In one embodiment, the multispecific antibody or variant thereof comprises: - a LAG-3 binding domain of the present disclosure comprising a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) of a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 55; and - comprises a PD-L1-binding domain of the disclosure, comprising a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) of a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 24.

[0119] In one embodiment, the multispecific antibody or variant thereof comprises: - a LAG-3 binding domain of the present disclosure comprising a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) having the amino acid sequences set forth in SEQ ID NO: 64, SEQ ID NO: 65, and SEQ ID NO: 121, respectively; and - comprise a PD-L1-binding domain of the disclosure, comprising a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) having the amino acid sequences set forth in SEQ ID NO: 107, SEQ ID NO: 117, and SEQ ID NO: 109, respectively.

[0120] In one embodiment, the multispecific antibody or variant thereof comprises: - a LAG-3 binding domain of the present disclosure comprising a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) of a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 56; and - comprises a PD-L1-binding domain of the disclosure, comprising a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) of a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 24.

[0121] In one embodiment, the multispecific antibody or variant thereof comprises: a LAG-3 binding domain of the present disclosure comprising a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) having the amino acid sequences set forth in SEQ ID NO: 64, SEQ ID NO: 65, and SEQ ID NO: 122, respectively; and - comprise a PD-L1-binding domain of the disclosure, comprising a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) having the amino acid sequences set forth in SEQ ID NO: 107, SEQ ID NO: 117, and SEQ ID NO: 109, respectively.

[0122] In one embodiment, the multispecific antibody or variant thereof comprises: - a LAG-3 binding domain of the present disclosure comprising a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) of a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 57; and - comprises a PD-L1-binding domain of the disclosure, comprising a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) of a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 24.

[0123] In one embodiment, the multispecific antibody or variant thereof comprises: - a LAG-3 binding domain of the present disclosure comprising a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) having the amino acid sequences set forth in SEQ ID NO: 64, SEQ ID NO: 65, and SEQ ID NO: 123, respectively; and - comprise a PD-L1-binding domain of the disclosure, comprising a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) having the amino acid sequences set forth in SEQ ID NO: 107, SEQ ID NO: 117, and SEQ ID NO: 109, respectively.

[0124] In one embodiment, the multispecific antibody or variant thereof comprises: - a LAG-3 binding domain of the present disclosure comprising a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) of a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 58; and - comprises a PD-L1-binding domain of the disclosure, comprising a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) of a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 24.

[0125] In one embodiment, the multispecific antibody or variant thereof comprises: - a LAG-3 binding domain of the present disclosure comprising a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) having the amino acid sequences set forth in SEQ ID NO: 64, SEQ ID NO: 65, and SEQ ID NO: 124, respectively; and - comprise a PD-L1-binding domain of the disclosure, comprising a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) having the amino acid sequences set forth in SEQ ID NO: 107, SEQ ID NO: 117, and SEQ ID NO: 109, respectively.

[0126] In one embodiment, the multispecific antibody or variant thereof comprises: - a LAG-3 binding domain of the present disclosure, comprising a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) of a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 59; and - comprises a PD-L1-binding domain of the disclosure, comprising a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) of a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 24.

[0127] In one embodiment, the multispecific antibody or variant thereof comprises: - a LAG-3 binding domain of the present disclosure comprising a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) having the amino acid sequences set forth in SEQ ID NO: 64, SEQ ID NO: 65, and SEQ ID NO: 125, respectively; and - comprise a PD-L1-binding domain of the disclosure, comprising a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) having the amino acid sequences set forth in SEQ ID NO: 107, SEQ ID NO: 117, and SEQ ID NO: 109, respectively.

[0128] In one embodiment, the multispecific antibody or variant thereof comprises: - a LAG-3 binding domain of the present disclosure, comprising a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) of a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 5; and - comprises a PD-L1-binding domain of the disclosure, comprising a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) of a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 20.

[0129] In one embodiment, the multispecific antibody or variant thereof comprises: - a LAG-3 binding domain of the present disclosure comprising a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) having the amino acid sequences set forth in SEQ ID NO: 70, SEQ ID NO: 71, and SEQ ID NO: 72, respectively; and - comprise a PD-L1-binding domain of the disclosure comprising a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) having the amino acid sequences set forth in SEQ ID NO: 107, SEQ ID NO: 108, and SEQ ID NO: 109, respectively.

[0130] In one embodiment, the multispecific antibody or variant thereof comprises: - a LAG-3 binding domain of the present disclosure, comprising a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) of a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 52; and - comprises a PD-L1-binding domain of the disclosure, comprising a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) of a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 20.

[0131] In one embodiment, the multispecific antibody or variant thereof comprises: - a LAG-3 binding domain of the present disclosure comprising a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) having the amino acid sequences set forth in SEQ ID NO: 70, SEQ ID NO: 71, and SEQ ID NO: 118, respectively; and - comprises a PD-L1 binding domain of the disclosure comprising a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) having the amino acid sequences set forth in SEQ ID NO: 107, SEQ ID NO: 108, and SEQ ID NO: 109, respectively. In one embodiment, the multispecific antibody or variant thereof comprises: - a LAG-3 binding domain of the present disclosure, comprising a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) of a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 17; and - comprises a PD-L1-binding domain of the disclosure, comprising a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) of a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 24; The LAG-3-binding domain and PD-L1-binding domain comprise a light chain CDR1 (LCDR1) having the amino acid sequence set forth in SEQ ID NO: 31, a light chain CDR2 (LCDR2) having the amino acid sequence set forth in SEQ ID NO: 32, and a light chain CDR3 (LCDR3) having the amino acid sequence set forth in SEQ ID NO: 33.

[0132] In one embodiment, the multispecific antibody or variant thereof comprises: a LAG-3 binding domain of the present disclosure comprising a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) having the amino acid sequences set forth in SEQ ID NO: 64, SEQ ID NO: 65, and SEQ ID NO: 66, respectively; and - comprises a PD-L1-binding domain of the disclosure, comprising a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) having the amino acid sequences set forth in SEQ ID NO: 107, SEQ ID NO: 117, and SEQ ID NO: 109, respectively; The LAG-3-binding domain and PD-L1-binding domain comprise a light chain CDR1 (LCDR1) having the amino acid sequence set forth in SEQ ID NO: 31, a light chain CDR2 (LCDR2) having the amino acid sequence set forth in SEQ ID NO: 32, and a light chain CDR3 (LCDR3) having the amino acid sequence set forth in SEQ ID NO: 33.

[0133] In one embodiment, the multispecific antibody or variant thereof comprises: - a LAG-3 binding domain of the present disclosure, comprising a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) of a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 53; and - comprises a PD-L1-binding domain of the disclosure, comprising a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) of a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 24; The LAG-3-binding domain and PD-L1-binding domain comprise a light chain CDR1 (LCDR1) having the amino acid sequence set forth in SEQ ID NO: 31, a light chain CDR2 (LCDR2) having the amino acid sequence set forth in SEQ ID NO: 32, and a light chain CDR3 (LCDR3) having the amino acid sequence set forth in SEQ ID NO: 33.

[0134] In one embodiment, the multispecific antibody or variant thereof comprises: - a LAG-3 binding domain of the present disclosure comprising a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) having the amino acid sequences set forth in SEQ ID NO: 64, SEQ ID NO: 65, and SEQ ID NO: 119, respectively; and - comprises a PD-L1-binding domain of the disclosure, comprising a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) having the amino acid sequences set forth in SEQ ID NO: 107, SEQ ID NO: 117, and SEQ ID NO: 109, respectively; The LAG-3-binding domain and PD-L1-binding domain comprise a light chain CDR1 (LCDR1) having the amino acid sequence set forth in SEQ ID NO: 31, a light chain CDR2 (LCDR2) having the amino acid sequence set forth in SEQ ID NO: 32, and a light chain CDR3 (LCDR3) having the amino acid sequence set forth in SEQ ID NO: 33.

[0135] In one embodiment, the multispecific antibody or variant thereof comprises: - a LAG-3 binding domain of the present disclosure comprising a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) of a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 54; and - comprises a PD-L1-binding domain of the disclosure, comprising a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) of a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 24; The LAG-3-binding domain and PD-L1-binding domain comprise a light chain CDR1 (LCDR1) having the amino acid sequence set forth in SEQ ID NO: 31, a light chain CDR2 (LCDR2) having the amino acid sequence set forth in SEQ ID NO: 32, and a light chain CDR3 (LCDR3) having the amino acid sequence set forth in SEQ ID NO: 33.

[0136] In one embodiment, the multispecific antibody or variant thereof comprises: - a LAG-3 binding domain of the present disclosure comprising a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) having the amino acid sequences set forth in SEQ ID NO: 64, SEQ ID NO: 65, and SEQ ID NO: 120, respectively; and - comprises a PD-L1-binding domain of the disclosure, comprising a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) having the amino acid sequences set forth in SEQ ID NO: 107, SEQ ID NO: 117, and SEQ ID NO: 109, respectively; The LAG-3-binding domain and PD-L1-binding domain comprise a light chain CDR1 (LCDR1) having the amino acid sequence set forth in SEQ ID NO: 31, a light chain CDR2 (LCDR2) having the amino acid sequence set forth in SEQ ID NO: 32, and a light chain CDR3 (LCDR3) having the amino acid sequence set forth in SEQ ID NO: 33.

[0137] In one embodiment, the multispecific antibody or variant thereof comprises: - a LAG-3 binding domain of the present disclosure comprising a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) of a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 55; and - comprises a PD-L1-binding domain of the disclosure, comprising a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) of a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 24; The LAG-3-binding domain and PD-L1-binding domain comprise a light chain CDR1 (LCDR1) having the amino acid sequence set forth in SEQ ID NO: 31, a light chain CDR2 (LCDR2) having the amino acid sequence set forth in SEQ ID NO: 32, and a light chain CDR3 (LCDR3) having the amino acid sequence set forth in SEQ ID NO: 33.

[0138] In one embodiment, the multispecific antibody or variant thereof comprises: - a LAG-3 binding domain of the present disclosure comprising a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) having the amino acid sequences set forth in SEQ ID NO: 64, SEQ ID NO: 65, and SEQ ID NO: 121, respectively; and - comprises a PD-L1-binding domain of the disclosure, comprising a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) having the amino acid sequences set forth in SEQ ID NO: 107, SEQ ID NO: 117, and SEQ ID NO: 109, respectively; The LAG-3-binding domain and PD-L1-binding domain comprise a light chain CDR1 (LCDR1) having the amino acid sequence set forth in SEQ ID NO: 31, a light chain CDR2 (LCDR2) having the amino acid sequence set forth in SEQ ID NO: 32, and a light chain CDR3 (LCDR3) having the amino acid sequence set forth in SEQ ID NO: 33.

[0139] In one embodiment, the multispecific antibody or variant thereof comprises: - a LAG-3 binding domain of the present disclosure comprising a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) of a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 56; and - comprises a PD-L1-binding domain of the disclosure, comprising a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) of a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 24; The LAG-3-binding domain and PD-L1-binding domain comprise a light chain CDR1 (LCDR1) having the amino acid sequence set forth in SEQ ID NO: 31, a light chain CDR2 (LCDR2) having the amino acid sequence set forth in SEQ ID NO: 32, and a light chain CDR3 (LCDR3) having the amino acid sequence set forth in SEQ ID NO: 33.

[0140] In one embodiment, the multispecific antibody or variant thereof comprises: a LAG-3 binding domain of the present disclosure comprising a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) having the amino acid sequences set forth in SEQ ID NO: 64, SEQ ID NO: 65, and SEQ ID NO: 122, respectively; and - comprises a PD-L1-binding domain of the disclosure, comprising a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) having the amino acid sequences set forth in SEQ ID NO: 107, SEQ ID NO: 117, and SEQ ID NO: 109, respectively; The LAG-3-binding domain and PD-L1-binding domain comprise a light chain CDR1 (LCDR1) having the amino acid sequence set forth in SEQ ID NO: 31, a light chain CDR2 (LCDR2) having the amino acid sequence set forth in SEQ ID NO: 32, and a light chain CDR3 (LCDR3) having the amino acid sequence set forth in SEQ ID NO: 33.

[0141] In one embodiment, the multispecific antibody or variant thereof comprises: - a LAG-3 binding domain of the present disclosure comprising a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) of a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 57; and - comprises a PD-L1-binding domain of the disclosure, comprising a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) of a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 24; The LAG-3-binding domain and PD-L1-binding domain comprise a light chain CDR1 (LCDR1) having the amino acid sequence set forth in SEQ ID NO: 31, a light chain CDR2 (LCDR2) having the amino acid sequence set forth in SEQ ID NO: 32, and a light chain CDR3 (LCDR3) having the amino acid sequence set forth in SEQ ID NO: 33.

[0142] In one embodiment, the multispecific antibody or variant thereof comprises: - a LAG-3 binding domain of the present disclosure comprising a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) having the amino acid sequences set forth in SEQ ID NO: 64, SEQ ID NO: 65, and SEQ ID NO: 123, respectively; and - comprises a PD-L1-binding domain of the disclosure, comprising a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) having the amino acid sequences set forth in SEQ ID NO: 107, SEQ ID NO: 117, and SEQ ID NO: 109, respectively; The LAG-3-binding domain and PD-L1-binding domain comprise a light chain CDR1 (LCDR1) having the amino acid sequence set forth in SEQ ID NO: 31, a light chain CDR2 (LCDR2) having the amino acid sequence set forth in SEQ ID NO: 32, and a light chain CDR3 (LCDR3) having the amino acid sequence set forth in SEQ ID NO: 33.

[0143] In one embodiment, the multispecific antibody or variant thereof comprises: - a LAG-3 binding domain of the present disclosure comprising a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) of a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 58; and - comprises a PD-L1-binding domain of the disclosure, comprising a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) of a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 24; The LAG-3-binding domain and PD-L1-binding domain comprise a light chain CDR1 (LCDR1) having the amino acid sequence set forth in SEQ ID NO: 31, a light chain CDR2 (LCDR2) having the amino acid sequence set forth in SEQ ID NO: 32, and a light chain CDR3 (LCDR3) having the amino acid sequence set forth in SEQ ID NO: 33.

[0144] In one embodiment, the multispecific antibody or variant thereof comprises: - a LAG-3 binding domain of the present disclosure comprising a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) having the amino acid sequences set forth in SEQ ID NO: 64, SEQ ID NO: 65, and SEQ ID NO: 124, respectively; and - comprises a PD-L1-binding domain of the disclosure, comprising a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) having the amino acid sequences set forth in SEQ ID NO: 107, SEQ ID NO: 117, and SEQ ID NO: 109, respectively; The LAG-3-binding domain and PD-L1-binding domain comprise a light chain CDR1 (LCDR1) having the amino acid sequence set forth in SEQ ID NO: 31, a light chain CDR2 (LCDR2) having the amino acid sequence set forth in SEQ ID NO: 32, and a light chain CDR3 (LCDR3) having the amino acid sequence set forth in SEQ ID NO: 33.

[0145] In one embodiment, the multispecific antibody or variant thereof comprises: - a LAG-3 binding domain of the present disclosure, comprising a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) of a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 59; and - comprises a PD-L1-binding domain of the disclosure, comprising a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) of a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 24; The LAG-3-binding domain and PD-L1-binding domain comprise a light chain CDR1 (LCDR1) having the amino acid sequence set forth in SEQ ID NO: 31, a light chain CDR2 (LCDR2) having the amino acid sequence set forth in SEQ ID NO: 32, and a light chain CDR3 (LCDR3) having the amino acid sequence set forth in SEQ ID NO: 33.

[0146] In one embodiment, the multispecific antibody or variant thereof comprises: - a LAG-3 binding domain of the present disclosure comprising a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) having the amino acid sequences set forth in SEQ ID NO: 64, SEQ ID NO: 65, and SEQ ID NO: 125, respectively; and - comprises a PD-L1-binding domain of the disclosure, comprising a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) having the amino acid sequences set forth in SEQ ID NO: 107, SEQ ID NO: 117, and SEQ ID NO: 109, respectively; The LAG-3-binding domain and PD-L1-binding domain comprise a light chain CDR1 (LCDR1) having the amino acid sequence set forth in SEQ ID NO: 31, a light chain CDR2 (LCDR2) having the amino acid sequence set forth in SEQ ID NO: 32, and a light chain CDR3 (LCDR3) having the amino acid sequence set forth in SEQ ID NO: 33.

[0147] In one embodiment, the multispecific antibody or variant thereof comprises: - a LAG-3 binding domain of the present disclosure, comprising a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) of a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 5; and - comprises a PD-L1-binding domain of the disclosure, comprising a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) of a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 20; The LAG-3-binding domain and PD-L1-binding domain comprise a light chain CDR1 (LCDR1) having the amino acid sequence set forth in SEQ ID NO: 31, a light chain CDR2 (LCDR2) having the amino acid sequence set forth in SEQ ID NO: 32, and a light chain CDR3 (LCDR3) having the amino acid sequence set forth in SEQ ID NO: 33.

[0148] In one embodiment, the multispecific antibody or variant thereof comprises: - a LAG-3 binding domain of the present disclosure comprising a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) having the amino acid sequences set forth in SEQ ID NO: 70, SEQ ID NO: 71, and SEQ ID NO: 72, respectively; and - comprises a PD-L1-binding domain of the disclosure, comprising a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) having the amino acid sequences set forth in SEQ ID NO: 107, SEQ ID NO: 108, and SEQ ID NO: 109, respectively; The LAG-3-binding domain and PD-L1-binding domain comprise a light chain CDR1 (LCDR1) having the amino acid sequence set forth in SEQ ID NO: 31, a light chain CDR2 (LCDR2) having the amino acid sequence set forth in SEQ ID NO: 32, and a light chain CDR3 (LCDR3) having the amino acid sequence set forth in SEQ ID NO: 33.

[0149] In one embodiment, the multispecific antibody or variant thereof comprises: - a LAG-3 binding domain of the present disclosure, comprising a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) of a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 52; and - comprises a PD-L1-binding domain of the disclosure, comprising a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) of a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 20; The LAG-3-binding domain and PD-L1-binding domain comprise a light chain CDR1 (LCDR1) having the amino acid sequence set forth in SEQ ID NO: 31, a light chain CDR2 (LCDR2) having the amino acid sequence set forth in SEQ ID NO: 32, and a light chain CDR3 (LCDR3) having the amino acid sequence set forth in SEQ ID NO: 33.

[0150] In one embodiment, the multispecific antibody or variant thereof comprises: - a LAG-3 binding domain of the present disclosure comprising a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) having the amino acid sequences set forth in SEQ ID NO: 70, SEQ ID NO: 71, and SEQ ID NO: 118, respectively; and - comprises a PD-L1-binding domain of the disclosure, comprising a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) having the amino acid sequences set forth in SEQ ID NO: 107, SEQ ID NO: 108, and SEQ ID NO: 109, respectively; The LAG-3-binding domain and PD-L1-binding domain comprise a light chain CDR1 (LCDR1) having the amino acid sequence set forth in SEQ ID NO: 31, a light chain CDR2 (LCDR2) having the amino acid sequence set forth in SEQ ID NO: 32, and a light chain CDR3 (LCDR3) having the amino acid sequence set forth in SEQ ID NO: 33.

[0151] In one embodiment, the multispecific antibody or variant thereof comprises: a LAG-3 binding domain of the present disclosure comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 17, and - comprises a PD-L1-binding domain of the disclosure comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 24.

[0152] In one embodiment, the multispecific antibody or variant thereof comprises: - a LAG-3 binding domain of the present disclosure comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 53, and - comprises a PD-L1-binding domain of the disclosure, comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 24.

[0153] In one embodiment, the multispecific antibody or variant thereof comprises: a LAG-3 binding domain of the present disclosure comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 54, and - comprises a PD-L1-binding domain of the disclosure, comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 24.

[0154] In one embodiment, the multispecific antibody or variant thereof comprises: a LAG-3 binding domain of the present disclosure comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 55, and - comprises a PD-L1-binding domain of the disclosure, comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 24.

[0155] In one embodiment, the multispecific antibody or variant thereof comprises: a LAG-3 binding domain of the present disclosure comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 56, and - comprises a PD-L1-binding domain of the disclosure, comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 24.

[0156] In one embodiment, the multispecific antibody or variant thereof comprises: a LAG-3 binding domain of the present disclosure comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 57, and - comprises a PD-L1-binding domain of the disclosure, comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 24.

[0157] In one embodiment, the multispecific antibody or variant thereof comprises: - a LAG-3 binding domain of the present disclosure comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 58, and - comprises a PD-L1-binding domain of the disclosure, comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 24.

[0158] In one embodiment, the multispecific antibody or variant thereof comprises: a LAG-3 binding domain of the present disclosure comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 59, and - comprises a PD-L1-binding domain of the disclosure, comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 24.

[0159] In one embodiment, the multispecific antibody or variant thereof comprises: a LAG-3 binding domain of the present disclosure comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 5, and - comprises a PD-L1-binding domain of the disclosure comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 20.

[0160] In one embodiment, the multispecific antibody or variant thereof comprises: a LAG-3 binding domain of the present disclosure comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 52, and - comprises a PD-L1 binding domain of the disclosure comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 20. In one embodiment, the multispecific antibody or variant thereof comprises: a LAG-3 binding domain of the present disclosure comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 17, and - comprises a PD-L1-binding domain of the disclosure, comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 24; The LAG-3 binding domain and the PD-L1 binding domain comprise a light chain variable region having the amino acid sequence set forth in SEQ ID NO:30.

[0161] In one embodiment, the multispecific antibody or variant thereof comprises: - a LAG-3 binding domain of the present disclosure comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 53, and - comprises a PD-L1-binding domain of the disclosure, comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 24; The LAG-3 binding domain and the PD-L1 binding domain comprise a light chain variable region having the amino acid sequence set forth in SEQ ID NO:30.

[0162] In one embodiment, the multispecific antibody or variant thereof comprises: a LAG-3 binding domain of the present disclosure comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 54, and - comprises a PD-L1-binding domain of the disclosure, comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 24; The LAG-3 binding domain and the PD-L1 binding domain comprise a light chain variable region having the amino acid sequence set forth in SEQ ID NO:30.

[0163] In one embodiment, the multispecific antibody or variant thereof comprises: a LAG-3 binding domain of the present disclosure comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 55, and - comprises a PD-L1-binding domain of the disclosure, comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 24; The LAG-3 binding domain and the PD-L1 binding domain comprise a light chain variable region having the amino acid sequence set forth in SEQ ID NO:30.

[0164] In one embodiment, the multispecific antibody or variant thereof comprises: a LAG-3 binding domain of the present disclosure comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 56, and - comprises a PD-L1-binding domain of the disclosure, comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 24; The LAG-3 binding domain and the PD-L1 binding domain comprise a light chain variable region having the amino acid sequence set forth in SEQ ID NO:30.

[0165] In one embodiment, the multispecific antibody or variant thereof comprises: a LAG-3 binding domain of the present disclosure comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 57, and - comprises a PD-L1-binding domain of the disclosure, comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 24; The LAG-3 binding domain and the PD-L1 binding domain comprise a light chain variable region having the amino acid sequence set forth in SEQ ID NO:30.

[0166] In one embodiment, the multispecific antibody or variant thereof comprises: - a LAG-3 binding domain of the present disclosure comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 58, and - comprises a PD-L1-binding domain of the disclosure, comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 24; The LAG-3 binding domain and the PD-L1 binding domain comprise a light chain variable region having the amino acid sequence set forth in SEQ ID NO:30.

[0167] In one embodiment, the multispecific antibody or variant thereof comprises: a LAG-3 binding domain of the present disclosure comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 59, and - comprises a PD-L1-binding domain of the disclosure, comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 24; The LAG-3 binding domain and the PD-L1 binding domain comprise a light chain variable region having the amino acid sequence set forth in SEQ ID NO:30.

[0168] In one embodiment, the multispecific antibody or variant thereof comprises: a LAG-3 binding domain of the present disclosure comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 5, and - comprises a PD-L1-binding domain of the disclosure, comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 20; The LAG-3 binding domain and the PD-L1 binding domain comprise a light chain variable region having the amino acid sequence set forth in SEQ ID NO:30.

[0169] In one embodiment, the multispecific antibody or variant thereof comprises: a LAG-3 binding domain of the present disclosure comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 52, and - comprises a PD-L1-binding domain of the disclosure, comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 20; The LAG-3 binding domain and the PD-L1 binding domain comprise a light chain variable region having the amino acid sequence set forth in SEQ ID NO:30.

[0170] The LAG-3xPD-L1 multispecific antibody or variant thereof of the present disclosure targets two immune checkpoints in trans: it binds to PD-L1 expressed on tumor cells or antigen-presenting cells, and simultaneously binds to LAG-3 expressed on T cells. It can also function in cis by binding to upregulated PD-L1 on exhausted T effector cells in the tumor microenvironment. In this way, the LAG-3xPD-L1 multispecific antibody or variant thereof of the present disclosure prevents T cell inhibitory signaling and improves tumor immunity.

[0171] In certain embodiments, the multispecific antibody or variant thereof has a binding affinity for human LAG-3 in the range of about 1-2 nM, specifically in the range of about 1.45-1.93 nM, as measured by SPR as described herein. The term "about" allows for a 10% deviation from the stated value.

[0172] In certain embodiments, the multispecific antibody or variant thereof has a binding affinity for human LAG-3 in the range of 1-2 nM, specifically in the range of 1.45-1.93 nM, as measured by SPR as described herein.

[0173] In certain embodiments, the multispecific antibody or variant thereof has a binding affinity for human PD-L1 in the range of 0.1-0.5 nM, specifically in the range of 0.17-0.41 nM, as measured by SPR as described herein.

[0174] In certain embodiments, the present disclosure provides a multispecific antibody comprising a binding domain that binds to LAG-3 and a binding domain that binds to PD-L1, or a variant thereof that maintains the binding specificity of the antibody, wherein the antibody or variant has a higher affinity for cynomolgus monkey LAG-3 than a reference antibody comprising two heavy chain variable regions having the amino acid sequence set forth in SEQ ID NO: 27 and two light chain variable regions having the amino acid sequence set forth in SEQ ID NO: 28, and has a higher affinity for cynomolgus monkey PD-L1 than a reference antibody comprising two heavy chain variable regions having the amino acid sequence set forth in SEQ ID NO: 25 and two light chain variable regions having the amino acid sequence set forth in SEQ ID NO: 26.

[0175] Determining whether a multispecific antibody or its variant has a higher binding affinity than a reference antibody can be done by measuring the binding affinity of both the multispecific antibody or its variant and the reference antibody in the same type of assay using the same assay conditions. Thus, in certain embodiments, the binding affinity of the multispecific antibody or its variant and the binding affinity of the reference antibody are measured in the same type of assay using the same assay conditions. In certain embodiments, the assay is an assay using surface plasmon resonance (SPR). SPR is an assay that uses surface plasmon resonance (SPR) to measure binding affinity, such as the Biacore® biosensor system or Solution Equilibrium Titration (SET) (see Friguet B et al. (1985) J. Immunol Methods; 77(2):305-319, and Hanel C et al. (2005) Anal Biochem; 339(1):182-184).

[0176] The binding affinity values ​​for the LAG-3 and PD-L1 binding domains provided herein are obtained by the method described in Example 3. Briefly, anti-huIgG Fcγ is covalently coupled to the surface of a CM5 sensor chip using free amine chemistry. Multispecific antibodies are injected into the flow cell at 30 μL / min at concentrations up to 20 nM for 2 minutes. Antigen (0.6-20 nM) is then flowed over the surface of the CM5 sensor chip at 30 μL / min for 2 minutes. Sensorgrams of the association and dissociation phases for different antigens are obtained. The affinity of each individual Fab arm is determined using BIA evaluation software and curve fitting using a 1:1 interaction model (for monovalent interactions).

[0177] In certain embodiments, binding affinity is measured with a LAG-3xPD-L1 multispecific antibody of the present disclosure in a bivalent, bispecific format and a reference anti-human LAG-3 antibody in a bivalent, monospecific IgG format. Thus, the binding affinity of the multispecific antibody to human LAG-3 represents a monovalent binding affinity.

[0178] In certain embodiments, binding affinity is measured with a LAG-3xPD-L1 multispecific antibody of the present disclosure in a bivalent, bispecific format and a reference anti-human PD-L1 antibody in a bivalent, monospecific IgG format. Thus, the binding affinity of the multispecific antibody to human PD-L1 represents a monovalent binding affinity.

[0179] In certain embodiments, the LAG-3 binding domain of a multispecific antibody or variant thereof has a binding affinity for cynomolgus monkey LAG-3 that is at least 10-fold, preferably 10-20-fold, higher than a reference anti-human LAG-3 binding domain described herein, as measured by SPR as described herein.

[0180] In certain embodiments, the LAG-3 binding domain of a multispecific antibody or variant thereof has a binding affinity for cynomolgus monkey LAG-3 that is 10-fold higher than a reference anti-human LAG-3 binding domain described herein, as measured by SPR as described herein.

[0181] In certain embodiments, the PD-L1 binding domain of the multispecific antibody or variant has a binding affinity for cynomolgus PD-L1 that is at least 10-fold higher, preferably 10-50-fold, particularly 10-40-fold, 10-30-fold, or 10-20-fold higher, than a reference anti-human PD-L1 binding domain described herein, as measured by SPR as described herein.

[0182] In certain embodiments, the LAG-3 binding domain of the multispecific antibody or variant has a binding affinity for cynomolgus monkey LAG-3 in the range of about 0.1-2 nM, particularly about 0.3-1.5 nM, about 0.35-1.5 nM, or about 0.4-1.2 nM, more particularly about 0.41-1.15 nM, as measured by SPR as described herein. The term "about" allows for a 10% deviation from the stated value.

[0183] In certain embodiments, the LAG-3 binding domain of the multispecific antibody or variant has a binding affinity for cynomolgus monkey LAG-3 in the range of 0.1-2 nM, particularly in the range of 0.3-1.5 nM, 0.35-1.5 nM, or 0.4-1.2 nM, more particularly in the range of 0.41-1.15 nM, as measured by SPR as described herein.

[0184] In certain embodiments, the PD-L1 binding domain of the multispecific antibody or variant has a binding affinity to cynomolgus PD-L1 in the range of about 0.05-1 nM, specifically about 0.05-0.5 nM, about 0.1-0.4 nM, or about 0.1-0.35 nM, more specifically about 0.15-0.34 nM, as measured by SPR as described herein. The term "about" allows for a 10% deviation from the stated value.

[0185] In certain embodiments, the PD-L1 binding domain of the multispecific antibody or variant has a binding affinity for cynomolgus PD-L1 in the range of 0.05-1 nM, specifically in the range of 0.05-0.5 nM, 0.1-0.4 nM, or 0.1-0.35 nM, more specifically in the range of 0.15-0.34 nM, as measured by SPR as described herein.

[0186] In certain embodiments, binding affinity to cynomolgus monkey LAG-3 is measured by both the LAG-3 binding domain of the multispecific antibody and a reference anti-LAG-3 binding domain in a bivalent, bispecific IgG format. The bivalent, bispecific IgG format may, for example, comprise a LAG-3 binding domain of the present disclosure or a reference antibody and a binding domain that binds to PD-L1 or another unrelated target. Thus, a monovalent interaction with cynomolgus monkey LAG-3 is measured.

[0187] In certain embodiments, binding affinity to cynomolgus PD-L1 is measured by both the PD-L1-binding domain of the multispecific antibody and a reference PD-L1-binding domain in a bivalent, bispecific IgG format. The bivalent, bispecific IgG format may, for example, comprise the PD-L1-binding domain of an antibody of the present disclosure or a reference antibody and a binding domain that binds to LAG-3 or another unrelated target. Thus, a monovalent interaction with cynomolgus PD-L1 is being measured.

[0188] In certain embodiments, the multispecific antibody or variant thereof is a CD4 +and / or CD8 + In certain embodiments, the multispecific antibody or variant thereof can enhance the proliferation of tumor-infiltrating T cells. + and / or CD8 + T cells, specifically, proliferating CD4 + and / or CD8 + The number of T cells can be increased by, for example, measuring CD4 T cells in tumor biopsies, as described, for example, in Example 4. + and / or CD8 + T cells, specifically, proliferating CD4 + and / or CD8 + It can be determined by measuring the number of T cells.

[0189] In certain embodiments, the multispecific antibody or variant thereof results in a decrease in the number of regulatory T cells in the tumor microenvironment compared to the number of regulatory T cells in response to a reference antibody combination described herein.

[0190] In certain embodiments, the multispecific antibody or variant thereof is a full length antibody, particularly a full length bispecific antibody, more particularly a full length bispecific IgG1 antibody.

[0191] In certain embodiments, the present disclosure provides a vector comprising a nucleic acid sequence encoding a heavy chain variable region of a LAG-3 binding domain described herein and a nucleic acid sequence encoding a heavy chain variable region of a PD-L1 binding domain described herein. In certain embodiments, the vector further comprises a nucleic acid sequence encoding a CH1 region, and preferably a hinge, CH2, and CH3 region. In certain embodiments, the vector further comprises at least one polynucleotide encoding a light chain variable region, and preferably a CL region. In certain embodiments, the light chain variable region is a light chain variable region of a light chain capable of pairing with multiple heavy chains having different epitope specificities.

[0192] The present disclosure further provides a cell comprising a nucleic acid sequence encoding a heavy chain variable region of a LAG-3-binding domain described herein and a nucleic acid sequence encoding a heavy chain variable region of a PD-L1-binding domain described herein. In certain embodiments, the cell may further comprise a nucleic acid sequence encoding a CH1 region, and preferably a hinge, CH2, and CH3 region. In certain embodiments, the cell may further comprise at least one polynucleotide sequence encoding a light chain variable region, and preferably a CL region. In certain embodiments, the light chain variable region is a light chain variable region of a light chain capable of pairing with multiple heavy chains having different epitope specificities.

[0193] The present disclosure also provides cells that produce the multispecific antibodies or variants thereof described herein. In certain embodiments, the cells are recombinant cells that have been transformed with the vectors described herein.

[0194] Further provided herein is a method for producing a multispecific antibody or variant thereof of the present disclosure, the method comprising culturing a cell as described herein and recovering the multispecific antibody or variant thereof from the cell or supernatant.

[0195] Further provided herein are methods for producing variants of the LAG-3-binding domain and / or PD-L1-binding domain of the disclosure, the methods comprising: - generating sequence variants of the heavy chain variable region described herein; - expressing in the cell a sequence variant or variants described herein and a light chain variable region.

[0196] Methods for generating sequence variants are well known in the art. The generation of sequence variants can be performed using a random or targeted approach, with the goal being to introduce mutations that are likely to increase or decrease binding affinity. Routine methods for affinity maturation of antibody binding domains are widely known in the art; see, for example, Tabasinezhad M. et al. Immunol Lett. 2019;212:106-113. Furthermore, the goal can be to introduce mutations that reduce developability risk in terms of large-scale production of binding domains or multispecific antibodies containing such binding domains. Mutations that are likely to not cause loss of binding specificity and / or not affect binding affinity can be introduced. Acceptable substitutions include, but are not limited to, substitutions that result in similar biophysical properties of the variant, such as isoleucine to leucine or valine, threonine to serine, arginine to lysine, aspartic acid to glutamic acid, and tryptophan to tyrosine. Mutations can be introduced at certain positions based on their frequency in the germline or natural repertoire.

[0197] Whether amino acid residues in the CDRs and / or framework regions can be substituted, for example, with conservative amino acid residues and whether they can be substituted without or substantially without loss of binding specificity and / or affinity can be determined by methods well known in the art. Exemplary experiments include, but are not limited to, alanine scanning (Cunningham BC, Wells JA. Science. 1989;244(4908):1081-5) and deep mutational scanning (Araya CL, Fowler DM. Trends Biotechnol. 2011;29(9):435-42). Computational methods have also been developed that can predict the effects of amino acid mutations, such as those described in, for example, Sruthi CK, Prakash M. PLoS One. 2020; 15(1): e0227621, Choi Y. et al. PLoS One. 2012; 7(10): e46688, and Munro D, Singh M. Bioinformatics. 2020; 36(22-23): 5322-9.

[0198] Further provided herein are multispecific antibodies comprising any of the variant LAG-3 and / or PD-L1 binding domains produced by the above methods, pharmaceutical compositions comprising multispecific antibodies comprising any of the variant LAG-3 and / or PD-L1 binding domains, nucleic acids encoding any of the variant binding domains, vectors and cells comprising the nucleic acids, and uses of multispecific antibodies comprising any of the variant LAG-3 and / or PD-L1 binding domains, or pharmaceutical compositions, for the treatment of cancer.

[0199] Pharmaceutical Compositions and Methods In certain embodiments, the present disclosure provides a pharmaceutical composition comprising a multispecific antibody or variant thereof described herein and a pharmaceutically acceptable carrier, diluent, or excipient.

[0200] In certain embodiments, the present disclosure provides a multispecific antibody or variant thereof, or pharmaceutical composition described herein, for use as a medicament.

[0201] In certain embodiments, the present disclosure provides a multispecific antibody or a variant thereof described herein, or a pharmaceutical composition described herein, for use in the treatment of a disease associated with a suppressed immune system, in particular cancer.

[0202] In certain embodiments, the present disclosure provides methods for treating a disease, comprising administering to an individual in need thereof an effective amount of a multispecific antibody or variant thereof, or pharmaceutical composition described herein.

[0203] In certain embodiments, the present disclosure provides methods for treating diseases associated with a suppressed immune system, in particular cancer, comprising administering to an individual in need thereof an effective amount of a multispecific antibody or variant thereof, or pharmaceutical composition as described herein.

[0204] As used herein, the terms "individual," "subject," and "patient" are used interchangeably and refer to a mammal (e.g., a patient, such as a human, mouse, rat, hamster, guinea pig, rabbit, cat, dog, monkey, cow, horse, pig, etc.) (e.g., a human patient with cancer).

[0205] As used herein, the terms "treat," "treating," and "treatment" refer to any type of intervention or process performed on a subject or administering to a subject an active agent or combination of active agents for the purpose of curing or ameliorating a disease or its symptoms, including reversing, alleviating, ameliorating, suppressing, or delaying symptoms, complications, conditions, or biochemical manifestations associated with a disease, as well as preventing the onset, progression, occurrence, severity, or recurrence of symptoms, complications, conditions, or biochemical manifestations associated with a disease.

[0206] As used herein, "effective treatment" or "positive therapeutic response" refers to treatment that results in a beneficial effect, e.g., an improvement in at least one symptom of a disease or disorder, e.g., cancer. A beneficial effect can take the form of an improvement over a baseline, including an improvement over measurements or observations made before initiating treatment according to the method. For example, a beneficial effect can take the form of slowing, stabilizing, halting, or reversing the progression of cancer in a subject at any clinical stage, as evidenced by a reduction or elimination of clinical or diagnostic symptoms of the disease or markers of cancer. An effective treatment can, for example, reduce tumor size, reduce the presence of circulating tumor cells, reduce or prevent tumor metastasis, slow or halt tumor growth, and / or prevent or delay tumor recurrence or relapse.

[0207] The term "therapeutic amount" or "effective amount" refers to an amount of an agent or combination of agents that provides a desired biological, therapeutic, and / or prophylactic result. That result can be reduction, amelioration, remission, lessening, delay, and / or alleviation of one or more of the signs, symptoms, or causes of a disease, or any other desired alteration of a biological system. In some embodiments, a therapeutic amount is an amount sufficient to delay tumor development. In some embodiments, a therapeutic amount is an amount sufficient to prevent or delay tumor recurrence.

[0208] An effective amount of the agent or composition may (i) reduce the number of cancer cells, (ii) reduce tumor size, (iii) reduce, slow, partially slow, and stop the invasion of cancer cells into peripheral organs, (iv) suppress tumor metastasis, (v) inhibit tumor growth, (vi) prevent or delay the onset and / or recurrence of tumors, and / or (vii) relieve to some extent one or more symptoms associated with cancer.

[0209] An effective amount may vary depending on factors such as the disease state, age, sex, and weight of the individual being treated, and the ability of the agent or agent combination to elicit a desired response in the individual.

[0210] An effective amount can be administered in one or more administrations.

[0211] A therapeutic amount also includes an amount that balances any toxic or detrimental effects of the agent or combination of agents with the therapeutically beneficial effects.

[0212] The term "drug" refers to a therapeutically active substance, in this case a multispecific antibody or variant thereof of the present disclosure, or a pharmaceutical composition of the present disclosure.

[0213] General terminology As used herein, "comprises" and its conjugations are used in their open-ended sense to mean that the items before the word are included, but not excluding items not specifically mentioned.

[0214] The articles "a" and "an" are used herein to refer to one or to more than one of the grammatical objects of the article. By way of example, "an element" means one or more elements.

[0215] The reference in this specification to a patent document or other matter shall not be deemed to be an admission that the document or matter was known or that the information contained therein was part of the common general knowledge at the priority date of any of the claims.

[0216] All patent and literature references cited herein are incorporated by reference in their entirety.

[0217] It should be noted that, unless otherwise specified, amino acid positions assigned to CDRs and frameworks in the variable regions of antibodies or antibody fragments are designated herein by Kabat numbering (see Sequences of Proteins of Immunological Interest (National Institute of Health, Bethesda, Md., 1987 and 1991)). Amino acids in the constant regions are designated by the EU numbering system.

[0218] The accession number is provided primarily to provide a means of further identifying the target, and the actual sequence of the bound protein may vary due to mutations in the encoding gene, such as those that occur in some cancers. The antigen-binding site binds to the antigen and its various variants, such as those expressed by some antigen-positive immune cells or tumor cells.

[0219] When a gene, protein is referred to herein, it preferably refers to the human form of that gene or protein. When a gene or protein is referred to herein, it refers to the native gene or protein as well as variants of that gene or protein that can be detected in tumors, cancers, etc., preferably human tumors, cancers, etc.

[0220] HGNC stands for HUGO Gene Nomenclature Committee. The number after the abbreviation is an accession number that allows information about the gene and the protein encoded by the gene to be retrieved from the HGNC database. Entrez Gene provides an accession number or gene ID that allows information about the gene or the protein encoded by the gene to be retrieved from the NCBI (National Center for Biotechnology Information) database. Ensemble provides an accession number that allows information about the gene or the protein encoded by the gene to be obtained from the Ensemble database. Ensembl is a collaborative project between EMBL-EBI and the Wellcome Trust Sanger Institute to develop a software system that generates and maintains automated annotations for selected eukaryotic genomes. [Brief explanation of the drawings]

[0221] In the figures, the bivalent monospecific antibody is shown in the format of SEQ ID NO: A, where SEQ ID NO: A refers to the heavy chain variable sequences of both binding domains. Each binding domain of the monospecific antibody comprises a light chain. In the example used to illustrate, but not intended to limit in any way, the present disclosure, each binding domain of the monospecific antibody comprises a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 30, and a light chain constant region having the amino acid sequence set forth in SEQ ID NO: 43. The monospecific antibody is preferably an IgG1 antibody comprising a CH1, hinge, CH2, and CH3. In the example used to illustrate, but not intended to limit in any way, the present disclosure, the monospecific antibody was screened in an IgG1 format, and the LAG-3 and PD-L1 binding heavy chain comprises a CH1 having the amino acid sequence set forth in SEQ ID NO: 42, a CH2 having the amino acid sequence set forth in SEQ ID NO: 45, and a CH3 having the amino acid sequence set forth in SEQ ID NO: 47.

[0222] Bispecific antibodies are represented in the format SEQ ID NO:AxSEQ ID NO:B, where both SEQ ID NO:A and B refer to heavy chain variable sequences. Each binding domain of a bispecific antibody comprises a light chain. In an example used to illustrate, but not intended to limit in any way, the present disclosure, each binding domain of a bispecific antibody comprises a light chain variable region having the amino acid sequence set forth in SEQ ID NO:30 and a light chain constant region having the amino acid sequence set forth in SEQ ID NO:43. The bispecific antibody is an IgG1 antibody comprising a CH1, hinge, CH2, and CH3.

[0223] In Example 1, which is used to illustrate the present disclosure but is not intended to limit the present disclosure in any way, bispecific antibodies were screened in an IgG1 format, the PD-L1-binding heavy chain comprises a CH1 having the amino acid sequence set forth in SEQ ID NO: 42, a CH2 which may have the amino acid sequence set forth in SEQ ID NO: 45, and a CH3 which may have the amino acid sequence set forth in SEQ ID NO: 50, and the LAG-3-binding heavy chain comprises a CH1 having the amino acid sequence set forth in SEQ ID NO: 42, a CH2 having the amino acid sequence set forth in SEQ ID NO: 45, and a CH3 having the amino acid sequence set forth in SEQ ID NO: 51.

[0224] In Example 2, which is used to illustrate the present disclosure but is not intended to limit the present disclosure in any way, bispecific antibodies were screened in an IgG1 format, and the PD-L1-binding heavy chain comprised a CH1 having the amino acid sequence set forth in SEQ ID NO: 42, a CH2 having the amino acid sequence set forth in SEQ ID NO: 45, and a CH3 having the amino acid sequence set forth in SEQ ID NO: 50, and the LAG-3-binding heavy chain comprised a CH1 having the amino acid sequence set forth in SEQ ID NO: 42, a CH2 having the amino acid sequence set forth in SEQ ID NO: 45, and a CH3 having the amino acid sequence set forth in SEQ ID NO: 51.

[0225] In Examples 3 and 4, which are used to illustrate the present disclosure but are not intended to limit the disclosure in any way, bispecific antibodies were screened in an IgG1 format.

[0226] With respect to SEQ ID NO:17×SEQ ID NO:24, the PD-L1-binding heavy chain comprises a CH1 having the amino acid sequence set forth in SEQ ID NO:42, a CH2 having the amino acid sequence set forth in SEQ ID NO:45, and a CH3 having the amino acid sequence set forth in SEQ ID NO:50, and the LAG-3-binding heavy chain comprises a CH1 having the amino acid sequence set forth in SEQ ID NO:42, a CH2 having the amino acid sequence set forth in SEQ ID NO:45, and a CH3 having the amino acid sequence set forth in SEQ ID NO:51.

[0227] With respect to SEQ ID NO:5×SEQ ID NO:20, the PD-L1-binding heavy chain may comprise a CH1 having the amino acid sequence set forth in SEQ ID NO:42, a CH2 having the amino acid sequence set forth in SEQ ID NO:45, and a CH3 having the amino acid sequence set forth in SEQ ID NO:51, and the LAG-3-binding heavy chain comprises a CH1 having the amino acid sequence set forth in SEQ ID NO:42, a CH2 having the amino acid sequence set forth in SEQ ID NO:45, and a CH3 having the amino acid sequence set forth in SEQ ID NO:50.

[0228] In Example 5, which is used to illustrate the present disclosure but is not intended to limit the present disclosure in any way, bispecific antibodies were screened in an IgG format, and the PD-L1-binding heavy chain comprised a CH1 having the amino acid sequence set forth in SEQ ID NO: 42, a CH2 having the amino acid sequence set forth in SEQ ID NO: 45, and a CH3 having the amino acid sequence set forth in SEQ ID NO: 50 or 51, and the LAG-3-binding heavy chain comprised a CH1 having the amino acid sequence set forth in SEQ ID NO: 42, a CH2 having the amino acid sequence set forth in SEQ ID NO: 45, and a CH3 having the amino acid sequence set forth in SEQ ID NO: 51 or 50, as shown in (Table 10). The bivalent, monospecific leratolimab analog antibody and the bivalent, monospecific atezolizumab analog antibody are shown in SEQ ID NO: A / SEQ ID NO: B format, where SEQ ID NO: A refers to the respective heavy chain sequence and SEQ ID NO: B refers to the respective light chain sequence. The bivalent, monospecific leratolimab analog antibody comprises two LAG-3-binding domains. Bivalent, monospecific atezolizumab analog antibodies contain two PD-L1-binding domains. Each binding domain of the analog antibody contains a light chain. The combination of leratolimab and atezolizumab analog is shown in the format SEQ ID NO:A / SEQ ID NO:B+SEQ ID NO:C / SEQ ID NO:D, where SEQ ID NO:A refers to the heavy chain sequence, SEQ ID NO:B refers to the light chain sequence of either leratolimab or the atezolizumab analog, SEQ ID NO:C refers to the heavy chain sequence, and SEQ ID NO:D refers to the light chain sequence of the other.

[0229] [Figure 1A]

[0049] Figure 1 shows the results of LAG-3 binding using FACS. Bispecific antibodies comprising heavy chain variable regions having the amino acid sequences set forth in SEQ ID NO:1 and SEQ ID NO:23(a), SEQ ID NO:3 and SEQ ID NO:23(b), SEQ ID NO:5 and SEQ ID NO:23(c), SEQ ID NO:6 and SEQ ID NO:23(d), SEQ ID NO:15 and SEQ ID NO:23(e), and SEQ ID NO:16 and SEQ ID NO:23(f) were compared with bivalent monospecific antibodies comprising the respective LAG-3 binding domains, a positive control leratolimab analog (SEQ ID NO:27 / SEQ ID NO:28), and a negative control antibody (SEQ ID NO:29 / SEQ ID NO:30). Each graph shows the results for binding to human and rhesus monkey LAG-3. The X-axis shows the concentration of antibody in μg / mL. The Y-axis shows the binding level, expressed as mean fluorescence intensity (MFI). [Figure 1B] Figure 1 shows PD-L1 binding results using FACS. Bispecific antibodies comprising heavy chain variable regions having the amino acid sequences set forth in SEQ ID NO:1 and SEQ ID NO:23, SEQ ID NO:3 and SEQ ID NO:23, SEQ ID NO:5 and SEQ ID NO:23, SEQ ID NO:6 and SEQ ID NO:23, SEQ ID NO:15 and SEQ ID NO:23, and SEQ ID NO:16 and SEQ ID NO:23 were compared with a bivalent monospecific antibody comprising a PD-L1-binding domain having the amino acid sequence set forth in SEQ ID NO:23, a positive control atezolizumab analogue (SEQ ID NO:25 / SEQ ID NO:26), and a negative control antibody (SEQ ID NO:29 / SEQ ID NO:30). The top graph shows the results for binding to human PD-L1, and the bottom graph shows the results for binding to rhesus monkey PD-L1. The X-axis shows the concentration of antibody in μg / mL. The Y-axis shows the level of binding, expressed as mean fluorescence intensity (MFI). [Figure 2]

[0023] Figure 1 shows results from a PD-1 / LAG-3 reporter assay. Bispecific antibodies comprising heavy chain variable regions having the amino acid sequences set forth in SEQ ID NO:1 and SEQ ID NO:23, SEQ ID NO:3 and SEQ ID NO:23, SEQ ID NO:5 and SEQ ID NO:23, SEQ ID NO:6 and SEQ ID NO:23, SEQ ID NO:15 and SEQ ID NO:23, and SEQ ID NO:16 and SEQ ID NO:23 were compared to a positive control atezolizumab analog (SEQ ID NO:25 / SEQ ID NO:26), a positive control leratolimab analog (SEQ ID NO:27 / SEQ ID NO:28), a combination of atezolizumab and leratolimab analog (SEQ ID NO:25 / SEQ ID NO:26 + SEQ ID NO:27 / SEQ ID NO:28), and a negative control antibody (SEQ ID NO:29 / SEQ ID NO:30). The X-axis indicates antibody concentration in μg / mL. The Y-axis indicates fold induction. [Figure 3] Results from SEB assays from donor 1 (FIG. 3A) and donor 2 (FIG. 3B) are shown. Figures 3A1 and 3B1 show a comparison of the fold induction of IL-2 of bispecific antibodies comprising heavy chain variable regions having the amino acid sequences set forth in SEQ ID NOs: 1 and 23, SEQ ID NOs: 3 and 23, SEQ ID NOs: 5 and 23, SEQ ID NOs: 6 and 23, SEQ ID NOs: 15 and 23, and SEQ ID NOs: 16 and 23, with a positive control atezolizumab analog (SEQ ID NO: 25 / SEQ ID NO: 26), a combination of atezolizumab and leratolimab analogs (SEQ ID NO: 25 / SEQ ID NO: 26 + SEQ ID NO: 27 / SEQ ID NO: 28), and a negative control antibody (SEQ ID NO: 29 / SEQ ID NO: 30). Figures 3A2 and 3B2 show a comparison of the fold induction of TNFα of the same antibodies. The X-axis shows the concentration of antibody in μg / mL. The Y-axis shows the fold induction of IL-2 or TNFα. The fold induction of each antibody was calculated relative to control wells containing no IgG. [Figure 4] Binding affinity data for two bispecific antibodies is shown. [Figure 5]Figure 5 shows the results of two bispecific antibodies in FACS binding, PD-1 / LAG-3 reporter, and SEB assays. Figure 5A compares the binding of a bispecific antibody comprising heavy chain variable regions having the amino acid sequences set forth in SEQ ID NOs: 17 and 24, and SEQ ID NOs: 5 and 20, to human LAG-3 with the binding of a positive control leratolimab analog (SEQ ID NO: 27 / SEQ ID NO: 28) and a negative control antibody. Figure 5B compares the binding of the same antibody to rhesus monkey LAG-3. Figure 5C compares the binding of a bispecific antibody comprising heavy chain variable regions having the amino acid sequences set forth in SEQ ID NOs: 17 and 24, and SEQ ID NOs: 5 and 20, to the binding of a positive control atezolizumab analog (SEQ ID NO: 25 / SEQ ID NO: 26) and a negative control antibody. Figure 5D compares the binding of the same antibody to rhesus monkey PD-L1. The X-axis represents the concentration of antibody in μg / mL. The Y-axis represents the level of binding, expressed as mean fluorescence intensity (MFI). Figure 5E shows the results of a PD-1 / LAG-3 reporter assay comparing bispecific antibodies comprising heavy chain variable regions having the amino acid sequences set forth in SEQ ID NO:17 and SEQ ID NO:24, and SEQ ID NO:5 and SEQ ID NO:20, with positive control atezolizumab analogs (SEQ ID NO:25 / SEQ ID NO:26) and leratolimab analogs (SEQ ID NO:27 / SEQ ID NO:28), a combination of leratolimab and atezolizumab analogs (SEQ ID NO:27 / SEQ ID NO:28 + SEQ ID NO:25 / SEQ ID NO:26), and a negative control antibody. FIG. 5F shows the results of an SEB assay comparing the fold induction of IL-2 of a bispecific antibody comprising heavy chain variable regions having the amino acid sequences set forth in SEQ ID NO: 17 and SEQ ID NO: 24, and SEQ ID NO: 5 and SEQ ID NO: 20, to the fold induction of positive controls atezolizumab analogs (SEQ ID NO: 25 / SEQ ID NO: 26) and leratolimab analogs (SEQ ID NO: 27 / SEQ ID NO: 28), a combination of leratolimab and atezolizumab analogs (SEQ ID NO: 27 / SEQ ID NO: 28 + SEQ ID NO: 25 / SEQ ID NO: 26), and a negative control antibody in four different donors. The four graphs represent the results of four different donors. The X-axis shows the concentration of antibody in μg / mL. The Y-axis shows the concentration of IL-2 in pg / mL. [Figure 6]Figure 6A shows data from an in vivo study. The effect on tumor volume in hu-CD34 NSG™ mice bearing MDA-MB-231 tumors was evaluated for bispecific antibodies comprising heavy chain variable regions having the amino acid sequences set forth in SEQ ID NOs: 17 and 24, and SEQ ID NOs: 5 and 20, and compared to an atezolizumab analog (atezolizumab*), a leratolimab analog (leratolimab*), and a combination of leratolimab and an atezolizumab analog (leratolimab* + atezolizumab*). Figure 6A1 shows data from mice immunized with CD34+ HSCs from donor 1, Figure 6A2 shows data from mice immunized with CD34+ HSCs from donor 2, and Figure 6A3 shows data from mice immunized with CD34+ HSCs from donor 3. Figure 6A4 shows data from all donors combined. Figure 6B shows results from an analysis of CD4+ and CD8+ T cell and regulatory T cell (Treg) populations in MDA-MB-231 tumor samples. The effect on the percentage of CD3+ T cells (Figure 6B1), CD8+ T cells (Figure 6B2), FOXP3+ regulatory T cells (Figure 6B3), proliferating CD8+ T cells (Figure 6B4), and proliferating CD4+ T cells (Figure 6B5) was assessed for bispecific antibodies comprising heavy chain variable regions having the amino acid sequences set forth in SEQ ID NOs: 17 and 24, and SEQ ID NOs: 5 and 20, and compared to an atezolizumab analog, a leratolimab analog, and a combination of leratolimab and atezolizumab analog. [Figure 7] Figure 7 shows the results of a binding assay using FACS. Bispecific antibodies comprising heavy chain variable regions having the amino acid sequences shown in Table 10 were tested alongside RSV control antibodies (SEQ ID NO: 29 / SEQ ID NO: 30). The X-axis shows the antibody concentration in μg / mL. The Y-axis shows the fold induction obtained by normalizing the mean MFI value to the mean MFI value of wells without antibody. Figure 7A shows the results of binding to human LAG-3. Figure 7B shows the results of binding to rhesus monkey LAG-3. Figure 7C shows the results of binding to human PD-L1. [Figure 8]

[0023] Figure 1 shows results from a PD-1 / LAG-3 reporter assay. A bispecific antibody comprising a heavy chain variable region having the amino acid sequence shown in Table 10 was compared to a combination of atezolizumab and leratolimab analogs (SEQ ID NO:25 / SEQ ID NO:26 + SEQ ID NO:27 / SEQ ID NO:28), and an RSV control antibody (SEQ ID NO:29 / SEQ ID NO:30). The X-axis shows the concentration of antibody in μg / mL. The Y-axis shows fold induction.

[0230] The following examples illustrate the present disclosure but are not intended to limit the disclosure in any way. [Example]

[0231] Example 1 - Generation and Characterization of LAG-3xPD-L1 Bispecific Antibodies Anti-human LAG-3 binding domains can be obtained by methods known in the art, for example, as described in WO 2019 / 009728. A large panel of heavy chain variable regions was obtained by immunizing transgenic mice (MeMo® mice) containing the consensus IGKV1-39 light chain with human LAG-3 antigen moieties, including the use of different forms of DNA, protein, and cell-based antigen delivery.

[0232] Anti-human PD-L1 binding domains can be obtained by methods known in the art, for example, as described in WO2018 / 056821 and WO2019 / 009726. A large panel of heavy chain variable regions was obtained by immunizing transgenic mice containing the consensus IGKV1-39 light chain (MeMo® mice) with human PD-L1 antigen moieties, including the use of different forms of DNA, protein, and cell-based antigen delivery.

[0233] A number of anti-human LAG-3 Fabs were selected, and their heavy chain variable regions were used to produce bispecific anti-LAG-3 x anti-PD-L1 antibodies. The amino acid sequences of these heavy chain variable regions are set forth in SEQ ID NOS: 1-16. Fabs containing these heavy chain variable regions bound to domain 1 or domain 2 of human LAG-3 and demonstrated functional activity in a LAG-3 reporter assay (data not shown). Two additional Fabs were selected, whose heavy chain variable regions comprise the amino acid sequences set forth in SEQ ID NOS: 18 and 19. Fabs containing these heavy chain variable regions bound to domains 3 and 4 of human LAG-3. These two Fabs did not demonstrate functional activity in the LAG-3 reporter assay and were selected for the production of negative control bispecific antibodies.

[0234] A number of anti-human PD-L1 Fabs were selected, and their heavy chain variable regions were used to generate bispecific antibodies. The amino acid sequences of these heavy chain variable regions are set forth in SEQ ID NOS: 21-23. Fabs containing these heavy chain variable regions block the binding of PD-L1 to PD-1 (data not shown). The heavy chain variable domains exhibit different affinities, as measured using SPR, as determined in a bispecific IgG format containing one PD-L1-binding Fab and one other Fab (conjugated tetanus toxoid (TT) or anti-PD-1). Thus, the binding affinity of a monovalent interaction with PD-L1 has been measured.

[0235] The affinity of the anti-PD-L1 Fab arms to PD-L1 was determined using surface plasmon resonance (SPR). Affinity was measured in a bispecific IgG format with only one arm specific for PD-L1. Surface plasmon resonance (SPR) was used to determine the binding kinetics of the anti-PD-L1 Fab arms to antigen using a BIAcore T100. Recombinant, purified, Fc-tagged human PD-L1 (R&D Systems, cat. nr. 156-B7-100) was coupled to flow cell (FC) 2 of a CM5 sensor chip to a level of approximately 200 resonance units (RU) using NHS / EDC chemistry at pH 5.0 (NaAc buffer), an antigen concentration of 2 μg / mL, and a flow rate of 10 μL / min (FC1 served as a blank for subtraction and was activated and then directly deactivated using ethanolamine). Bispecific IgG composed of an anti-PD-L1 Fab arm and an irrelevant Fab arm was then run on the FC1 and 2 surfaces at different concentrations (100 nM and serial 2-fold dilutions in HBS, 6 dilutions) in a kinetic run at 30 μL / min. Regeneration was performed using a pulse of 50 mM HCl in water (15 μL at a flow rate of 10 μL / min). The resulting sensorgrams were evaluated using BIA evaluation software to determine the kinetic association and dissociation rate constants. Several measurements were performed on different surfaces of different sizes over several days. The different measurements gave very similar results, highlighting their validity. All measurements were performed at 25°C.

[0236] The binding affinities are presented in Table 2. [Table 2]

[0237] The bispecific IgG antibody was generated by transient co-transfection of two plasmid vectors: one encoding an IgG heavy chain with a LAG-3-binding VH region and the other encoding an IgG heavy chain with a PD-L1-binding VH region. To ensure efficient heterodimerization and bispecific antibody formation, CH3 engineering technology was used as described in WO2013 / 157954 and WO2013 / 157953. Both vectors further encode a common light chain comprising the IGKV1-39 / Jk1 light chain variable region. Cell transfection, cell culture, and antibody harvesting and purification were performed by methods known in the art.

[0238] Example 2 - Screening of LAG-3xPD-L1 bispecific antibodies Reference and control antibodies The bispecific antibody was compared to an analog of the anti-LAG-3 antibody leratolimab (leratolimab analog), which comprises two heavy chain variable regions having the amino acid sequences set forth in SEQ ID NO: 27 and two light chain variable regions having the amino acid sequences set forth in SEQ ID NO: 28, and an analog of the anti-PD-L1 antibody atezolizumab (atezolizumab analog), which comprises two heavy chain variable regions having the amino acid sequences set forth in SEQ ID NO: 25 and two light chain variable regions having the amino acid sequences set forth in SEQ ID NO: 26, or combinations thereof.

[0239] An Fc-silenced anti-RSV antibody containing two heavy chain variable regions having the amino acid sequence set forth in SEQ ID NO: 29 and two light chain variable regions having the amino acid sequence set forth in SEQ ID NO: 30 was used as a negative control antibody (RSV control antibody).

[0240] A number of bispecific antibodies were selected for further screening, including bispecific antibodies comprising the following combinations of heavy chain variable regions: [Table 3]

[0241] Bispecific antibodies were produced in an Fc-silenced IgG1 format and purified by protein A and gel filtration. Potential homodimeric impurities were removed by CIEX. LAG-3 homodimer formation was mitigated by the use of CH3 engineering techniques, reducing potential effects in functional assays. Monospecific parent antibodies were included in screening assays to compare the activity of the bispecific antibodies to their parent IgGs and to determine the sensitivity of the functional assays to PD-L1 and LAG-3 single-targeting agents. Monospecific parent antibodies were produced and characterized in a similar manner to the bispecific antibodies.

[0242] antigen binding Bispecific antibody binding to LAG-3 and PD-L1 was analyzed by FACS using 293FF cell lines stably transfected with human and rhesus LAG-3, and CHO-K1 cell lines stably transfected with human and rhesus PD-L1.

[0243] Bispecific and parental antibodies were analyzed in eight 5-fold titrations starting at 50 μg / mL. Goat anti-human PE antibody (Invitrogen, H10104) was used as the secondary antibody at a concentration of 3 μg / mL. A leratolimab analog was used as a positive control in all plates containing LAG-3-expressing cells, while an atezolizumab analog was used as a positive control in plates containing PD-L1-expressing cells. Six 5-fold titrations starting at 50 μg / mL of an RSV control antibody were included on all plates as negative controls. Two wells were used for secondary antibody only and unstained cells as additional controls. Cells not expressing the target, 293FF and CHO-K1 cells, were included as negative controls, and IgG was tested only at the two highest concentrations (50 and 10 μg / mL).

[0244] The results for LAG-3 binding are shown in Figure 1A. EC50 values ​​were calculated using GraphPad Prism software version 7.02 (nonlinear regression, 3-parameter dose-response curve). Each bispecific antibody is plotted alongside a positive control antibody, a negative control antibody, and its parent LAG-3 binding antibody.

[0245] The positive control antibody leratolimab analog showed inconsistent binding to human LAG-3 across the three plates used in this assay: binding on the plate with the LAG-3 arm containing SEQ ID NO: 16 was much lower than that observed on the other plates. EC50 values ​​were higher for the bispecific antibody (monovalent for LAG-3) than for the monospecific parent antibody (bivalent for LAG-3). The LAG-3 arms containing SEQ ID NOs: 3 and 5 showed the highest binding to human LAG-3. A summary of EC50 values ​​is shown in Table 3. [Table 4]

[0246] The results for PD-L1 binding are shown in Figure 1B. EC50 values ​​were calculated using GraphPad Prism software version 7.02 (nonlinear regression, 3-parameter dose-response curve). The bispecific antibody is plotted alongside the positive control antibody, negative control antibody, and parental PD-L1-binding antibody.

[0247] All bispecific antibodies demonstrated binding to CHO cells expressing huPD-L1. The maximal effect at highest drug concentration (Emax) was higher for bispecific antibodies containing SEQ ID NO: 23, which are monovalent for PD-L1, than for the bivalent monospecific parent antibodies. Many bispecific antibodies containing a PD-L1 arm with SEQ ID NO: 23 demonstrated better or equivalent binding than atezolizumab analogues. A summary of EC50 values ​​is shown in Table 4. [Table 5]

[0248] PD-1 / LAG-3 reporter assay PD-1 / LAG-3 reporter assays were performed using PD-1- and LAG-3-expressing Jurkat T cells as effector cells and PD-L1-expressing Raji cells as target cells. PD-1 and LAG-3 effector cells were prepared and plated at 100,000 cells per well. Antibody solution was added, followed by Raji cells (25,000 per well). T cells were activated by adding partially purified Staphylococcal enterotoxin D (ppSED, final concentration 16.6 ng / mL). After 6 hours of incubation at 37°C, luciferase reporter gene activity was determined by adding Bio-Glo reagent and measuring luminescence on an EnVision plate reader.

[0249] The bispecific IgG was tested in a six-step semi-log titration starting at 100 μg / mL (final concentration). IgG dilutions were prepared at 3x the final concentration in assay medium. As a positive control, a six-step semi-log titration of a combination of atezolizumab analogs and leratolimab analogs with a top concentration of 50 μg / mL + 50 μg / mL was included on each plate. Atezolizumab analogs and leratolimab analogs were also tested as single agents at a starting concentration of 100 μg / mL. As a negative control on each plate, a four-step semi-log titration of an RSV control antibody starting at 100 μg / mL was used. Two wells were left without IgG to control for basal activity.

[0250] The results are shown in Figure 2. The bispecific antibodies were compared to each other, to an atezolizumab analog, to a leratolimab analog, and to a combination of a leratolimab analog and an atezolizumab analog. The area under the curve (AUC) of the antibody response was determined using GraphPad Prism software version 7.02. The AUC was expressed relative to the AUC of the positive control.

[0251] A summary of the AUCs is shown in Table 5. [Table 6]

[0252] SEB assay The bispecific and parental antibodies were analyzed in the SEB assay using cells from two donors, PBMC donor 1 and PBMC donor 2. PBMCs from these donors were initially shown to respond to the anti-LAG-3 reference antibody leratolimab analog and the anti-PD-L1 antibody atezolizumab analog (data not shown). Briefly, a dilution series of IgG was administered at 2x10 5 PBMC cells were added, followed by the addition of 2 μg / mL SEB (final concentration). IgG was analyzed in six 7-fold dilution series starting at 50 μg / mL (final concentration). As a positive control, a 5-fold titration of a combination of atezolizumab analog and leratolimab analog starting at 25 μg / mL + 25 μg / mL was included on each plate. As a negative control on each plate, a 4-step semi-logarithmic titration of an RSV control antibody starting at 50 μg / mL was used. As a control for basal activity, two wells were left without IgG. PBMCs were cultured for 3 days, after which IL-2 and TNFα levels were measured in the supernatant by Luminex.

[0253] The results are shown in Figure 3. The AUC of the antibody response was determined using GraphPad Prism software version 7.02.

[0254] Standard curves for cytokine measurements by Luminex were as expected, and MFI values ​​for all samples were within the range of the standard curves for IL-2 and TNFα (data not shown). IL-2 data from the SEB assay are shown in Figure 3A1 (donor 1) and Figure 3B1 (donor 2). Data for TNFα are provided in Figure 3A2 (donor 1) and Figure 3B2 (donor 2).

[0255] In the first assay (PBMC donor 1), the positive and negative controls were as expected for both the IL-2 and TNFα readouts. The activity of the combination of atezolizumab and a relatolimab analogue was greater than that of the atezolizumab analogue alone for the IL-2 readout. For both the IL-2 and TNFα readouts, the LAG-3xPD-L1 bispecific antibody was at least as potent as the combination of atezolizumab and a relatolimab analogue.

[0256] In the second assay (PBMC donor 2), the positive and negative controls were as expected for IL-2. However, the magnitude of TNFα induction for the positive control was significantly lower. Again, the activity of the combination of atezolizumab and a relatolimab analog was higher than that of the atezolizumab analog alone for the IL-2 readout. For both readouts, the LAG-3xPD-L1 bispecific antibody was at least as potent as the combination of atezolizumab and a relatolimab analog. The activity of the LAG-3xPD-L1 bispecific antibody correlated well between the two donors. Bispecific antibodies with LAG-3 arms having the amino acid sequences set forth in SEQ ID NO: 5, SEQ ID NO: 15, and SEQ ID NO: 16 exhibited similar activity to each other and were at least as potent as the combination of atezolizumab and a relatolimab analog.

[0257] Example 3 - Binding affinity of bispecific antibodies comprising additional LAG-3 and PD-L1 heavy chain variable regions The binding affinities of a bispecific antibody comprising a LAG-3 binding domain having a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 5 and a PD-L1 binding domain having a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 20, and a bispecific antibody comprising a LAG-3 binding domain having a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 17 and a PD-L1 binding domain having a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 24 were determined by SPR technology using a BIAcore T200.

[0258] Bispecific antibody binding to four recombinant protein antigens supplied by Sino Biological (human LAG-3-His, Cat. nr. 16498-H08H; cynomolgus monkey LAG-3-His, Cat. nr. 90841-C08H; human PD-L1-His, Cat. nr. 10084-H08H; cynomolgus monkey PD-L1-His, Cat. nr. 90251-C08H) was determined in separate experiments. In these assays, anti-huIgG Fcγ (JIR, Cat. nr. 109-005-098) was covalently coupled to the surface of a CM5 sensor chip using free amine chemistry: the capture antibody was diluted to 40 μg / mL in kAc buffer and bound to the surface activated with NHS / EDC (per the manufacturer's recommendations). The bispecific antibody was then injected into the flow cell at concentrations up to 20 nM at 30 μL / min for 2 min. Antigens (0.6–20 nM) in 0.01 M HEPES, 0.5 M NaCl, 0.003 M EDTA, and 0.05% v / v surfactant P20 buffer are then flowed over the surface of a CM5 sensor chip at 30 μL / min for 2 min. Thus, sensorgrams of the association and dissociation phases for different antigens are obtained. The affinity of each individual Fab arm is determined using BIA evaluation software and curve fitting with a 1:1 interaction model (for monovalent interactions).

[0259] The results are shown in Figure 4. A bispecific antibody comprising a LAG-3 binding domain having a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO:5 and a PD-L1 binding domain having a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO:20, and a bispecific antibody comprising a LAG-3 binding domain having a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO:17 and a PD-L1 binding domain having a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO:24, have lower binding affinity to human LAG-3 and higher binding affinity to cynomolgus monkey LAG-3 compared to the leratolimab analog. The bispecific antibody has similar binding affinity to human PD-L1 and higher binding affinity to cynomolgus monkey PD-L1 compared to the atezolizumab analog.

[0260] Both bispecific antibodies simultaneously bind to human LAG-3 and human PD-L1 (data not shown).

[0261] These antibodies were screened in binding, PD-1 / LAG-3 reporter, and SEB assays to confirm their properties, as described in Example 2. The results are shown in Figure 5. Both bispecific antibodies bind to human and rhesus LAG-3 (Figures 5A and B) and human and rhesus PD-L1 (Figures 5C and D). Both bispecific antibodies show activity comparable to the combination of leratolimab and atezolizumab analogs in the reporter assay (Figure 5E) and SEB assay (Figure 5F). EC50 values ​​are provided in Tables 6 and 7. [Table 7] [Table 8]

[0262] Example 4 - In vivo studies The antitumor efficacy of the bispecific antibody compared to leratolimab and atezolizumab analogs was evaluated in a human stem cell-humanized NSG mouse model bearing orthotopic MDA-MB-231 tumors.

[0263] Female hu-CD34 NSG™ mice (JAX stock #705557) were used, which had been transplanted with human CD34+ cells and had >25% human CD45+ cells in their peripheral blood within 24 weeks, 14 weeks after transplantation. + A cohort of cell-transplanted hu-CD34 NSG™ mice was used.

[0264] 120 HSC-NSG mice (72 + surplus) were cultured at 5x10 6MDA-MB-231 human breast adenocarcinoma cells were transplanted into the mammary fat pad, resuspended in PBS with Matrigel at a 1:1 ratio. HSC-NSG mice were cultured with human CD34 cells from umbilical cord blood. + Immunodeficient NOD.Cg-Prkdc mice transplanted with hematopoietic stem cells (HSCs) scid 1l2rg tm1Wjl / SzJ(NSG™) mice, which undergo multilineage differentiation into all major immune cell types.

[0265] Tumor volume (TV) 50–150 mm 3 Seventy-two mice were enrolled 15 days after tumor inoculation, when tumor volume was 0. Tumor volumes were measured the day before enrollment (day -1). Dosing began on the day of enrollment, designated day 0 of treatment. Each treatment arm (Table 1) had mice from three HSC donors, four mice per donor (N=12). Tumor size and body weight were measured twice weekly. [Table 9]

[0266] CD4 in MDA-MB-231 tumor samples collected on the end date + and CD8 + Terminal tumors were isolated 24 hours after the final dose for flow cytometry analysis of T cell as well as regulatory T cell (Treg) populations.

[0267] The results are shown in Figure 6. Single and combination treatments with the reference anti-LAG-3 antibody leratolimab analog and the anti-PD-L1 antibody atezolizumab analog were ineffective. The reduction in tumor size in mice treated with the bispecific antibody comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 17, a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 24, and two light chain variable regions having the amino acid sequences set forth in SEQ ID NO: 30 is significant when data from all donors are combined (Figure 6A).

[0268] Tumor growth profiles are presented in Table 9. [Table 10]

[0269] Figure 6B4 shows that PD-L1 and LAG-3xPD-L1 treatments suppressed proliferation of CD8 + LAG-3, PD-L1, and LAG-3xPD-L1 treatments increased the percentage of proliferating CD4 + Increases the percentage of T cells (Figure 6B5). Figure 6B3 shows that treatment with the bispecific antibody results in a lower percentage of Tregs than treatment with the combination of leratolimab and atezolizumab analogs.

[0270] Example 5: Screening of further LAG-3xPD-L1 bispecific antibodies The bispecific antibodies listed in Table 10 were screened essentially as described in Example 2. [Table 11]

[0271] antigen binding Bispecific antibody binding to LAG-3 and PD-L1 was analyzed by FACS using 293FF cell lines stably transfected with human and rhesus LAG-3, and CHO-K1 cell lines stably transfected with human and rhesus PD-L1, as in Example 2.

[0272] Bispecific antibodies were analyzed in eight 4-fold titrations starting at 100 μg / mL on 293FF-huLAG-3 and 293FF-reLAG-3 cells and 25 μg / mL on CHO-K1-huPD-L1 cells. Goat anti-human PE antibody (Invitrogen, H10104) was used as the secondary antibody at a concentration of 3 μg / mL. An RSV control antibody was included in every plate as a negative control in eight 4-fold dilutions starting at 100 μg / mL on 293FF-huLAG-3 and 293FF-reLAG-3 cells and 25 μg / mL on CHO-K1-huPD-L1 cells. Two wells were used as additional controls: secondary antibody only and unstained cells. As negative controls, cells not expressing the target, 293FF and CHO-K1 cells, were included and IgG was tested at the three highest concentrations of 100, 25, and 6.3 μg / mL on 293FF cells and 25, 6.3, and 1.6 μg / mL on CHO-K1 cells.

[0273] Results for human and rhesus LAG-3 binding are shown in Figures 7A and 7B. Results for PD-L1 binding are shown in Figure 7C. Fold induction was obtained by normalizing the mean MFI values ​​to the mean MFI values ​​of wells without antibody and plotted as a function of the logarithm of antibody concentration. EC50 values ​​were calculated using GraphPad Prism software version 7.02 (nonlinear regression, 3-parameter dose-response curve). Each bispecific antibody is plotted alongside a negative control antibody.

[0274] All bispecific antibodies showed binding to human and rhesus LAG-3. All bispecific antibodies showed binding to CHO cells expressing huPD-L1. EC50 values ​​are provided in Table 11. [Table 12]

[0275] PD-1 / LAG-3 reporter assay The PD-1 / LAG-3 reporter assay was performed essentially as described in Example 2. Staphylococcal enterotoxin D (ppSED) was tested for use at a final concentration of 150 ng / mL.

[0276] Bispecific IgG was tested in a six-step semi-log titration starting at 100 μg / mL (final concentration). IgG dilutions were prepared in assay medium at 3x the final concentration. As a positive control, a six-step semi-log titration of a combination of atezolizumab analog and leratolimab analog with a top concentration of 50 μg / mL + 50 μg / mL was included on each plate. As a negative control on each plate, a four-step semi-log titration of an RSV control antibody starting at 100 μg / mL was used. As a control for basal activity, two wells were left without IgG.

[0277] The results are shown in Figure 8. All LAG-3xPD-L1 bispecific antibodies demonstrate activity relative to the combination of leratolimab and an atezolizumab analog. The EC50 and area under the curve (AUC) of the antibody response were determined using GraphPad Prism software version 7.02. The AUC is expressed relative to the AUC of the combination of leratolimab and an atezolizumab analog. [Table 13]

[0278] array SEQ ID NO: 1 [ka] SEQ ID NO: 2 [ka] SEQ ID NO: 3 [ka] SEQ ID NO:4 [ka] SEQ ID NO:5 [ka] SEQ ID NO:6 [ka] SEQ ID NO:7 [ka] SEQ ID NO:8 [ka] SEQ ID NO:9 [ka] SEQ ID NO: 10 [ka] SEQ ID NO: 11 [ka] SEQ ID NO: 12 [ka] SEQ ID NO: 13 [ka] SEQ ID NO: 14 [ka] SEQ ID NO: 15 [ka] SEQ ID NO: 16 [ka] SEQ ID NO: 17 [ka] SEQ ID NO: 18 [ka] SEQ ID NO: 19 [ka] SEQ ID NO: 20 [ka] SEQ ID NO: 21 [ka] SEQ ID NO: 22 [ka] SEQ ID NO: 23 [ka] SEQ ID NO: 24 [ka] SEQ ID NO: 25 [ka] SEQ ID NO: 26 [ka] SEQ ID NO: 27 [ka] SEQ ID NO: 28 [ka] SEQ ID NO: 29 [ka] LCDR shown according to SEQ ID NO: 30 IMGT [ka] SEQ ID NO: 31 Light chain CDR1 IMGT [ka] SEQ ID NO: 32 Light chain CDR2 IMGT [ka] SEQ ID NO: 33 Light chain CDR3 IMGT [ka] LCDR shown according to SEQ ID NO: 34 IMGT [ka] LCDR shown according to SEQ ID NO: 35 IMGT [ka] SEQ ID NO: 36 LCDR shown according to IMGT [ka] SEQ ID NO: 37 LCDR shown according to IMGT [ka] SEQ ID NO: 38 [ka] SEQ ID NO: 39 [ka] SEQ ID NO: 40 [ka] SEQ ID NO: 41 [ka] SEQ ID NO: 42 [ka] SEQ ID NO: 43 [ka] SEQ ID NO: 44 [ka] SEQ ID NO: 45 [ka] SEQ ID NO: 46 SEQ ID NO: 47 [ka] SEQ ID NO: 48 SEQ ID NO: 49 SEQ ID NO:50 [ka] SEQ ID NO:51 [ka] SEQ ID NO:52 [ka] SEQ ID NO:53 [ka] SEQ ID NO:54 [ka] SEQ ID NO: 55 [ka] SEQ ID NO:56 [ka] SEQ ID NO:57 [ka] SEQ ID NO:58 [ka] SEQ ID NO:59 [ka] SEQ ID NO: 60 [ka] SEQ ID NO: 61 [ka] SEQ ID NO: 62 [ka] SEQ ID NO: 63 [ka] SEQ ID NO: 64 [ka] SEQ ID NO: 65 [ka] SEQ ID NO: 66 [ka] SEQ ID NO: 67 [ka] SEQ ID NO: 68 [ka] SEQ ID NO: 69 [ka] SEQ ID NO: 70 [ka] SEQ ID NO:71 [ka] SEQ ID NO:72 [ka] SEQ ID NO: 73 [ka] SEQ ID NO:74 [ka] SEQ ID NO: 75 [ka] SEQ ID NO:76 [ka] SEQ ID NO:77 [ka] SEQ ID NO:78 [ka] SEQ ID NO:79 [ka] SEQ ID NO: 80 [ka] SEQ ID NO: 81 [ka] SEQ ID NO:82 [ka] SEQ ID NO: 83 [ka] SEQ ID NO:84 [ka] SEQ ID NO: 85 [ka] SEQ ID NO:86 [ka] SEQ ID NO:87 [ka] SEQ ID NO: 88 [ka] SEQ ID NO:89 [ka] SEQ ID NO: 90 [ka] SEQ ID NO: 91 [ka] SEQ ID NO:92 [ka] SEQ ID NO: 93 [ka] SEQ ID NO:94 [ka] SEQ ID NO: 95 [ka] SEQ ID NO:96 [ka] SEQ ID NO:97 [ka] SEQ ID NO: 98 [ka] SEQ ID NO: 99 [ka] SEQ ID NO: 100 [ka] SEQ ID NO: 101 [ka] SEQ ID NO: 102 [ka] SEQ ID NO: 103 [ka] SEQ ID NO: 104 [ka] SEQ ID NO: 105 [ka] SEQ ID NO: 106 [ka] SEQ ID NO: 107 [ka] SEQ ID NO: 108 [ka] SEQ ID NO: 109 [ka] SEQ ID NO: 110 [ka] SEQ ID NO: 111 [ka] SEQ ID NO: 112 [ka] SEQ ID NO: 113 [ka] SEQ ID NO: 114 [ka] SEQ ID NO: 115 [ka] SEQ ID NO: 116 [ka] SEQ ID NO: 117 [ka] SEQ ID NO: 118 [ka] SEQ ID NO: 119 [ka] SEQ ID NO: 120 [ka] SEQ ID NO: 121 [ka] SEQ ID NO: 122 [ka] SEQ ID NO: 123 [ka] SEQ ID NO: 124 [ka] SEQ ID NO: 125 [ka]

Claims

1. A multispecific antibody, or a variant thereof that maintains the binding specificity of the antibody, wherein the antibody or variant comprises a binding domain that specifically binds to the extracellular domain of LAG-3 and a binding domain that specifically binds to the extracellular domain of a B7 family protein, The LAG-3 binding domain is a) Heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3) of the heavy chain variable region having the amino acid sequence shown in Sequence ID No. 52, b) Heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3) of the heavy chain variable region having the amino acid sequence shown in Sequence ID No. 53, c) Heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3) of the heavy chain variable region having the amino acid sequence shown in Sequence ID No. 54, d) Heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3) of the heavy chain variable region having the amino acid sequence shown in Sequence ID No. 55, e) Heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3) of the heavy chain variable region having the amino acid sequence shown in Sequence ID No. 56, f) Heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3) of the heavy chain variable region having the amino acid sequence shown in Sequence ID No. 57, g) Heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3) of the heavy chain variable region having the amino acid sequence shown in Sequence ID No. 58, or h) A heavy chain variable region comprising heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3) having the amino acid sequence shown in Sequence ID No. 59, A multispecific antibody or variant thereof, wherein each of the HCDRs may contain up to three, two, or one amino acid substitutions.

2. The multispecific antibody or variant thereof according to claim 1, wherein the LAG-3 binding domain has an amino acid sequence shown in any one of SEQ ID NOs. 52 to 59, or includes a heavy chain variable region having at least 80%, preferably 85%, more preferably 90%, or most preferably 95% sequence identity thereto.

3. The multispecific antibody or variant thereof according to claim 1 or 2, wherein the LAG-3 binding domain further comprises a light chain variable region.

4. The multispecific antibody or variant thereof according to any one of claims 1 to 3, wherein the LAG-3 binding domain includes a light chain variable region having the amino acid sequence shown in SEQ ID NO:

30.

5. The multispecific antibody or variant thereof according to any one of claims 1 to 4, wherein the LAG-3 binding domain further comprises CH1 and CL regions.

6. The multispecific antibody or variant thereof according to any one of claims 1 to 5, wherein the antibody or variant is monovalent for binding to LAG-3.

7. A multispecific antibody or variant thereof according to any one of claims 1 to 6, wherein the protein of the B7 family is selected from the group consisting of PD-L1, PD-L2, CD80, CD86, B7-H4, TNFRSF14, and B7-H7.

8. A multispecific antibody or variant thereof according to any one of claims 1 to 7, wherein the protein of the B7 family is PD-L1.

9. A multispecific antibody or variant thereof according to any one of claims 1 to 8, wherein a variable domain that specifically binds to the extracellular domain of a B7 family protein blocks the binding of PD-L1 to its receptor.

10. The antibody or variant thereof according to claim 9, wherein the receptor is PD-1 and / or CD80.

11. The binding domain that binds to PD-L1 is a) Heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3) of the heavy chain variable region having the amino acid sequence shown in Sequence ID No. 20, b) Heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3) of the heavy chain variable region having the amino acid sequence shown in Sequence ID No. 21, c) Heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3) of the heavy chain variable region having the amino acid sequence shown in Sequence ID No. 22, d) Heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3) of the heavy chain variable region having the amino acid sequence shown in Sequence ID No. 23, or e) A heavy chain variable region comprising heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3) having the amino acid sequence shown in Sequence ID No. 24, A multispecific antibody or variant thereof according to any one of claims 8 to 10, wherein each of the HCDRs may contain up to three, two, or one amino acid substitutions.

12. The multispecific antibody or variant thereof according to any one of claims 8 to 11, wherein the PD-L1 binding domain has an amino acid sequence shown in any one of SEQ ID NOs: 20 to 24, or includes a heavy chain variable region having at least 80%, preferably 85%, more preferably 90%, or most preferably 95% sequence identity thereto.

13. The multispecific antibody or variant thereof according to any one of claims 8 to 12, wherein the PD-L1 binding domain further comprises a light chain variable region.

14. The multispecific antibody or variant thereof according to any one of claims 8 to 13, wherein the LAG-3 binding domain includes a light chain variable region having the amino acid sequence shown in SEQ ID NO:

30.

15. The multispecific antibody or variant thereof according to any one of claims 8 to 14, wherein the PD-L1 binding domain further comprises CH1 and CL regions.

16. The multispecific antibody or variant thereof according to any one of claims 8 to 15, wherein the antibody or variant is monovalent for binding to PD-L1.

17. The multispecific antibody or variant thereof according to any one of claims 1 to 16, wherein the antibody or variant has equivalent, equal to, or higher potency than the combination of reference antibodies, and the combination of reference antibodies comprises a reference antibody comprising two heavy chain variable regions having the amino acid sequence shown in SEQ ID NO: 27 and two light chain variable regions having the amino acid sequence shown in SEQ ID NO: 28, and a reference antibody comprising two heavy chain variable regions having the amino acid sequence shown in SEQ ID NO: 25 and two light chain variable regions having the amino acid sequence shown in SEQ ID NO:

26.

18. The multispecific antibody or variant thereof according to claim 17, wherein the efficacy is determined by measuring tumor volume reduction in an in vivo mouse study.

19. A multispecific antibody or variant thereof according to claim 17 or 18, wherein the equivalent efficacy includes a 5- to 2-fold, preferably 3- to 2-fold, deviation from the tumor volume reduction of the combination of reference antibodies.

20. The multispecific antibody or variant thereof according to claim 17, wherein the efficacy is determined by the efficacy in blocking ligand or receptor binding to LAG-3 and / or PD-L1.

21. The multispecific antibody or variant thereof according to claim 20, wherein the efficacy in blocking ligand binding to LAG-3 and / or PD-L1 is measured in a PD-1 / LAG-3 reporter assay.

22. The multispecific antibody or variant thereof according to claim 20 or 21, wherein the equivalent efficacy is within a range of five times the efficacy of the reference antibody combination in blocking ligand or receptor binding to LAG-3 and / or PD-L1, and includes a deviation of 5 to 2 times, preferably 3 to 2 times, from the LAG-3 and PD-L1 blocking activity of the reference antibody combination.

23. The multispecific antibody or variant thereof according to any one of claims 1 to 22, wherein the antibody or variant is a full-length antibody, specifically a full-length bispecific antibody.

24. A pharmaceutical composition comprising a multispecific antibody or a variant thereof according to any one of claims 1 to 23, and a pharmaceutically acceptable carrier, diluent, or excipient.

25. A multispecific antibody or variant thereof according to any one of claims 1 to 23, or a pharmaceutical composition according to claim 24, for use as a pharmaceutical.

26. A multispecific antibody or variant thereof according to any one of claims 1 to 23, or a pharmaceutical composition according to claim 24, for use in the treatment of diseases associated with a suppressed immune system.

27. A multispecific antibody or variant thereof according to any one of claims 1 to 23, or a pharmaceutical composition according to claim 24, for use in the treatment of cancer.

28. A vector comprising a nucleic acid sequence encoding the heavy chain variable region of the LAG-3 binding domain as defined in claim 1 or 2, and a nucleic acid sequence encoding the heavy chain variable region of the PD-L1 binding domain as defined in claim 11 or 12.

29. The vector according to claim 28, wherein the vector further comprises nucleic acid sequences encoding a CH1 region, and preferably a hinge, CH2, and CH3 region.

30. The vector according to claim 28 or 29, further comprising at least one nucleic acid sequence encoding a light chain variable region and preferably a CL region.

31. The vector according to claim 30, wherein the light chain variable region is a light chain variable region of a light chain that can pair with a plurality of heavy chains having different epitope specificities.

32. A cell comprising a nucleic acid sequence encoding the heavy chain variable region of the LAG-3 binding domain as defined in claim 1 or 2, and a nucleic acid sequence encoding the heavy chain variable region of the PD-L1 binding domain as defined in claim 11 or 12.

33. The cell according to claim 32, wherein the cell further comprises nucleic acid sequences encoding a CH1 region, and preferably hinge, CH2, and CH3 regions.

34. The cell according to claim 32 or 33, further comprising at least one nucleic acid sequence encoding a light chain variable region and preferably a CL region.

35. The cell according to claim 34, wherein the light chain variable region is a light chain variable region of a light chain that can pair with a plurality of heavy chains having different epitope specificities.

36. A cell that produces a multispecific antibody or a variant thereof according to any one of claims 1 to 23.

37. The cell according to claim 36, wherein the cell is a recombinant cell transformed with the vector described in any one of claims 28 to 31.

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