New regulations for multispecific antibodies

Abstract

The present disclosure relates to multispecific antibodies comprising a binding domain that binds LAG-3 and a binding domain that binds PD-L1. Such multispecific antibodies have comparable, equal or higher potency than the combination of LAG-3 and PD-L1 reference antibodies. Methods are also provided for treating diseases, particularly diseases associated with a suppressed immune system, such as cancer, using the multispecific antibodies of the present disclosure. The present disclosure further relates to vectors and cells comprising nucleic acids encoding the heavy chain variable regions of the LAG-3 and PD-L1 binding domains.

Description

[Technical Field]

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

[0002] Despite numerous advances made in treating the disease and increasing knowledge of the molecular events that lead to cancer, cancer remains a leading cause of death worldwide. Traditionally, most cancer drug discoveries have focused on drugs that block essential cellular functions and kill dividing cells. However, in the case of advanced cancer, chemotherapy, no matter how aggressively applied, rarely results in a complete cure, even to the point where patients experience life-threatening side effects from treatment. In most cases, the tumor in the patient only stops growing or shrinks temporarily (known as remission), only to reappear, and sometimes even more rapidly, begin to grow again (known as relapse), becoming increasingly difficult to treat. In recent years, the focus of cancer drug development has shifted from broad-spectrum cytotoxic chemotherapy to less toxic targeted growth inhibitory therapies. Targeted therapy for advanced cancer has been clinically validated in leukemia and several other cancers. However, in most cancer types, targeted approaches have still proven insufficient to completely eliminate cancer in the majority of patients.

[0003] Cancer targeting has been achieved using a variety of different methods, including, for example, small molecules directed at signaling proteins on which cancer depends for survival and / or growth; vaccines containing tumor-specific proteins; cell therapies including antibodies that target tumors with immune cells and cytotoxic molecules that actively kill tumor cells; and disrupting signaling and / or (re)directing the host immune system to tumor cells.

[0004] A class of therapeutic antibody development is bispecific antibodies, which contain two different binding sites that bind to different antigens or different epitopes on the same antigen. Bispecific antibodies can be designed for several applications. Firstly, bispecific antibodies can offer higher tissue specificity than monospecific antibodies. Some tumor-associated antigens are expressed not only by tumor cells but also 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 the antibody 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, the involvement of immune cells in tumor cells and the 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 interesting 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 has its own challenges in producing effective therapeutic agents. Therefore, the need for the development of novel and effective LAG-3xPD-L1 bispecific antibodies remains. [Overview of the Initiative]

[0006] One of the purposes of this disclosure is to provide novel pharmaceutical agents for the treatment of human diseases, specifically cancer. This purpose is fulfilled by providing multispecific antibodies containing anti-human LAG-3 binding domains and anti-human PD-L1 binding domains.

[0007] In certain embodiments, the 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 at least equivalent, equal to, or higher potency than a combination of reference antibodies. In certain embodiments, the combination of reference antibodies includes a reference antibody (relatrimab analog 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 (atezolizumab analog 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.

[0008] In certain embodiments, the Disclosure further provides a multispecific antibody, or a variant thereof that maintains the binding specificity of an 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, the LAG-3 binding domain comprising a heavy chain variable region having the amino acid sequence shown in any one of SEQ ID NOs: 1-17 or SEQ ID NOs: 52-59, comprising heavy chain variable regions including heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3).

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

[0010] In certain embodiments, the disclosure further provides multispecific antibodies or variants thereof, or pharmaceutical compositions described herein, for use as pharmaceuticals.

[0011] In certain embodiments, the disclosure further provides multispecific antibodies or variants thereof, or pharmaceutical compositions described herein, for use in the treatment of diseases associated with a suppressed immune system. In certain embodiments, the disclosure provides multispecific antibodies or variants thereof, or pharmaceutical compositions described herein, for use in the treatment of cancer.

[0012] In certain embodiments, the 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 an effective amount of a multispecific antibody or variant thereof described herein, or a pharmaceutical composition, to an individual in need of such treatment.

[0013] In certain embodiments, the disclosure further 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.

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

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

[0016] In certain embodiments, the present disclosure provides a method for producing variants of the multispecific antibodies described herein, and variants obtained by such method. [Modes for carrying out the invention]

[0017] In certain embodiments, the Disclosure provides a multispecific antibody or a variant thereof comprising a binding domain that binds to LAG-3 and a binding domain that binds to PD-L1, wherein the antibody or variant has at least equivalent, equal to, or higher potency than a 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.

[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 IDs 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 that of CD4. The LAG-3 protein binds to the nonholomorphic region of 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 in both regulatory T cells (Tregs) and anerogenic T cells. LAG-3 can negatively regulate T cell proliferation, activation, and homeostasis.

[0019] PD-L1 is a type I transmembrane protein that plays a role in suppressing the immune response 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, as well as various leukemias and multiple myelomas (Bernstein et al., 2014, Thompson et al., 2005). PD-L1 is present in the cytoplasm and plasma membrane of cancer cells, but not all cancers or all cells within a tumor express PD-L1 (Dong et al., 2002). Multiple tumor microenvironment cells contribute to immunosuppression by upregulating PD-L1 expression. This effect is called "adaptive immune resistance" because the tumor protects itself 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 cancer genes, and this mechanism is known as innate 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). The expression of PD-L1 is induced by multiple pro-inflammatory 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] The multispecific antibodies according to the present disclosure are antibodies that comprise at least two binding domains having specificity for at least two different targets or epitopes in any antibody format. In certain embodiments, the multispecific antibodies of the present disclosure are bispecific antibodies. In certain embodiments, the multispecific antibodies of the present disclosure comprise an Fc region or a portion thereof. In certain embodiments, the multispecific antibodies of the present disclosure are IgG1 antibodies.

[0021] "Variants" of the multispecific antibodies described herein include functional parts, derivatives, and / or analogs of the multispecific antibodies. Variants can be structural variants, including, but not limited to, antibody fragments such as Fab fragments or single-chain variable fragments (scFvs). Variants can be sequence variants. Variants can be both structural and sequence variants. Variants maintain the binding specificity of the antibody, but do not necessarily maintain the binding affinity.

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

[0023] In certain embodiments, the multispecific antibody or a variant thereof includes 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, the multispecific antibody or a variant thereof is monovalent with respect to binding to LAG-3 and / or monovalent with respect to binding to PD-L1. In certain embodiments, the multispecific antibody or a variant thereof is monovalent with respect to binding to both LAG-3 and PD-L1.

[0024] Determining whether a multispecific antibody or variant has an efficacy equal to, or the same as, or higher than that of a combination of reference antibodies can be done by measuring the efficacy of both the multispecific antibody and the combination of antibodies in the same type of study using the same research conditions. Thus, in certain embodiments, the efficacy of the multispecific antibody or a variant thereof is measured in the same type of study using the same research conditions.

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

[0026] In the context of this disclosure, “efficacy” means the functional activity of a multispecific antibody or its variant, which can be determined in in vitro or in vivo studies.

[0027] In certain embodiments, the efficacy of a multispecific antibody or its variant is determined in an in vivo study, preferably in an in vivo mouse model such as the HuNSG® mouse model having a human MDA-231 tumor. In certain embodiments, the efficacy of a multispecific antibody or its variant is determined by measuring tumor volume reduction in such an in vivo mouse study. Tumor volume reduction induced by the 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 range of 5 times the tumor volume reduction of the reference antibody combination, and includes deviations of 5, 4, 3, or 2 times, preferably 3 or 2 times, from the tumor volume reduction of the reference antibody combination.

[0029] In certain embodiments, efficacy is determined in in vitro studies such as blockade assays, or SEB assays, including but not limited to T cell activation assays, antigen recall assays, or MLR assays, including but not limited to SEB assays, including but not limited to SEB assays, including but not limited to SEB assays, such as those described in Example 2. In certain embodiments, the efficacy of a multispecific antibody or a combination of its variant and a reference antibody is determined by measuring their efficacy 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 efficacy 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. In summary, 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 a rate of 100,000 cells per well. Test and control IgG are added equimolarly in a 6-step semi-logarithmic titration, followed by the addition of Raji cells (25,000 per well). T cells are activated by adding partially purified staphylococcal enterotoxin D (ppSED, at a concentration that achieves the best response with the positive assay control, e.g., 16.6 ng / mL in Example 2 or 150 ng / mL in Example 5). After incubation at 37°C for 6 hours, luciferase reporter gene activity is determined.

[0030] In certain embodiments, equivalent potency is the potency in blocking ligand or receptor binding to LAG-3 and / or PD-L1, preferably LAG-3 and PD-L1, which is within a range of 5 times 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, and includes deviations of 5, 4, 3, or 2, preferably 3 or 2, 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 includes an analogue of the anti-LAG-3 antibody relatrimab and an analogue of the anti-PD-L1 antibody atezolizumab. The relatrimab analogue has the same heavy chain variable region sequence (SEQ ID NO: 27) as relatrimab. The relatrimab analogue antibody has the same light chain variable region sequence (SEQ ID NO: 28) as relatrimab. Atezolizumab analogs have the same heavy chain variable region sequence (SEQ ID NO: 25) as atezolizumab. Atezolizumab analog antibodies have the same light chain variable region sequence (SEQ ID NO: 26) as atezolizumab. Reference antibodies are preferably produced using the same production method as the multispecific antibody or its variant. Preferably, the LAG-3 and PD-L1 binding domains of the combination of the multispecific antibody or its variant and the reference antibody of this disclosure are used at equimolar concentrations; that is, if the multispecific antibody or its variant is used at 100 μg / mL, the combination of reference antibodies includes 50 μg / mL of LAG-3 binding reference antibody and 50 μg / mL of PD-L1 binding reference antibody.

[0031] In certain embodiments, the Disclosure relates to a multispecific antibody, or a variant thereof that maintains the binding specificity of an antibody, comprising 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.

[0032] In certain embodiments, the LAG-3 binding domain of the multispecific antibody or variant thereof of this disclosure includes a heavy chain variable region, which comprises one heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3) from among the heavy chain variable regions having the amino acid sequences shown in SEQ ID NOs. 1-17 or SEQ ID NOs. 52-59. HCDRs by Kabat are shown in bold and underlined in the list of sequences provided herein. CDR sequences are numbered using the Kabat numbering scheme (Kabat et al., J. Biol. Chem. 252:6609-6616 (1977), and / or Kabat et al., USDept. 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 According to 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 This includes, but is not limited to, those described in Res.25:206-211 (1997), and can be defined using different methods.

[0033] Each of these numbering schemes defines the CDR based on the predicted contribution of amino acid residues within the heavy or light chain variable region to antigen binding. Therefore, the CDR of the binding domain of the present disclosure can be identified using each of the methods for identifying the CDR. In certain embodiments, the heavy chain CDR of the binding domain of the present disclosure is by Kabat, Chothia, or IMGT. In certain embodiments, the heavy chain CDR of the binding domain of the present disclosure is by Kabat. In certain embodiments, the heavy chain CDR of the binding domain of the present disclosure is by Chothia. In certain embodiments, the heavy chain CDR of the binding domain of the present disclosure is by IMGT.

[0034] In certain embodiments, the LAG-3 binding domain of a multispecific antibody or its variant is - 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 SEQ ID NO: 1, - 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. 2, - 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. 3, - 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. 4, -The heavy chain variable region having the amino acid sequence shown in Sequence ID No. 5, heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), - 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. 6, - 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. 7, - 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. 8, -The heavy chain variable region having the amino acid sequence shown in Sequence ID No. 9, heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), - 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 SEQ ID NO: 10, - 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. 11, -The heavy chain variable region having the amino acid sequence shown in Sequence ID No. 12, heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), -The heavy chain variable region having the amino acid sequence shown in Sequence ID No. 13, heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), - 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 SEQ ID NO: 14, - 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 SEQ ID NO: 15, - 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. 16, -The heavy chain variable region having the amino acid sequence shown in Sequence ID No. 17, heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), -The heavy chain variable region having the amino acid sequence shown in Sequence ID No. 52, heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), - 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, -The heavy chain variable region having the amino acid sequence shown in Sequence ID No. 54, heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), -The heavy chain variable region having the amino acid sequence shown in Sequence ID No. 55, heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), - 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, -The heavy chain variable region having the amino acid sequence shown in Sequence ID No. 57, heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), - 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 -Includes a heavy chain variable region having the amino acid sequence shown in Sequence ID No. 59, including heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), Each HCDR may contain up to three, two, or one amino acid substitutions.

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

[0036] In certain embodiments, the LAG-3 binding domain of a multispecific antibody or its variant is - 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 SEQ ID NO: 1, - 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. 3, -The heavy chain variable region having the amino acid sequence shown in Sequence ID No. 5, heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), - 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. 6, - 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 SEQ ID NO: 15, - 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. 16, -The heavy chain variable region having the amino acid sequence shown in Sequence ID No. 17, heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), -The heavy chain variable region having the amino acid sequence shown in Sequence ID No. 52, heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), - 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, -The heavy chain variable region having the amino acid sequence shown in Sequence ID No. 54, heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), -The heavy chain variable region having the amino acid sequence shown in Sequence ID No. 55, heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), - 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, -The heavy chain variable region having the amino acid sequence shown in Sequence ID No. 57, heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), - 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 -Includes 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, where each HCDR may contain up to 3, 2, or 1 amino acid substitutions.

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

[0038] In certain embodiments, the LAG-3 binding domain of a multispecific antibody or its variant is -The heavy chain variable region having the amino acid sequence shown in Sequence ID No. 5, heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), -The heavy chain variable region having the amino acid sequence shown in Sequence ID No. 17, heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), -The heavy chain variable region having the amino acid sequence shown in Sequence ID No. 52, heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), - 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, -The heavy chain variable region having the amino acid sequence shown in Sequence ID No. 54, heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), -The heavy chain variable region having the amino acid sequence shown in Sequence ID No. 55, heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), - 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, -The heavy chain variable region having the amino acid sequence shown in Sequence ID No. 57, heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), - 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 -Includes a heavy chain variable region having the amino acid sequence shown in Sequence ID No. 59, including heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), Each HCDR may contain up to three, two, or one amino acid substitutions.

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

[0040] In a particular embodiment, the LAG-3 binding domain of a multispecific antibody or its variant includes 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 SEQ ID NO: 5. Each HCDR may contain up to three, two, or one amino acid substitutions.

[0041] In a particular embodiment, the LAG-3 binding domain of a multispecific antibody or its variant includes 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 SEQ ID NO: 52. Each HCDR may contain up to three, two, or one amino acid substitutions.

[0042] In a particular embodiment, the LAG-3 binding domain of a multispecific antibody or its variant includes a heavy chain variable region comprising heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), each having the amino acid sequences shown in SEQ ID NO: 70, SEQ ID NO: 71, and SEQ ID NO: 72, respectively. Each HCDR may contain up to three, two, or one amino acid substitutions.

[0043] In a particular embodiment, the LAG-3 binding domain of a multispecific antibody or its variant includes a heavy chain variable region comprising heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), respectively, having the amino acid sequences shown in SEQ ID NO: 70, SEQ ID NO: 71, and SEQ ID NO: 118. Each HCDR may contain up to three, two, or one amino acid substitutions.

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

[0045] In certain embodiments, the LAG-3 binding domain of a multispecific antibody or its variant is -Heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), respectively, having the amino acid sequences shown in SEQ ID NO: 60, SEQ ID NO: 61, and SEQ ID NO: 62. -Heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), respectively, having the amino acid sequences shown in SEQ ID NO: 60, SEQ ID NO: 63, and SEQ ID NO: 62. -Heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), respectively, having the amino acid sequences shown in SEQ ID NO: 64, SEQ ID NO: 65, and SEQ ID NO: 66. -Heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), respectively, having the amino acid sequences shown in SEQ ID NO: 67, SEQ ID NO: 68, and SEQ ID NO: 69. -Heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), respectively, having the amino acid sequences shown in SEQ ID NO: 70, SEQ ID NO: 71, and SEQ ID NO: 72. -Heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), respectively, having the amino acid sequences shown in SEQ ID NOs. 73, 74, and 75. -Heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), respectively, having the amino acid sequences shown in SEQ ID NO: 76, SEQ ID NO: 77, and SEQ ID NO: 78. -Heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), respectively, having the amino acid sequences shown in SEQ ID NO: 79, SEQ ID NO: 80, and SEQ ID NO: 81. -Heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), respectively, having the amino acid sequences shown in SEQ ID NO: 82, SEQ ID NO: 83, and SEQ ID NO: 84. -Heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), respectively, having the amino acid sequences shown in SEQ ID NO: 85, SEQ ID NO: 86, and SEQ ID NO: 87. -Heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), respectively, having the amino acid sequences shown in SEQ ID NO: 88, SEQ ID NO: 89, and SEQ ID NO: 90. -Heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), respectively, having the amino acid sequences shown in SEQ ID NO: 76, SEQ ID NO: 91, and SEQ ID NO: 92. -Heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), respectively, having the amino acid sequences shown in SEQ ID NO: 76, SEQ ID NO: 91, and SEQ ID NO: 93. -Heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), respectively, having the amino acid sequences shown in SEQ ID NO: 94, SEQ ID NO: 95, and SEQ ID NO: 96. -Heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), respectively, having the amino acid sequences shown in SEQ ID NO: 97, SEQ ID NO: 98, and SEQ ID NO: 99. -Heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), respectively, having the amino acid sequences shown in SEQ ID NO: 97, SEQ ID NO: 98, and SEQ ID NO: 100. -Heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), respectively, having the amino acid sequences shown in SEQ ID NO: 64, SEQ ID NO: 65, and SEQ ID NO: 66. -Heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), respectively, having the amino acid sequences shown in SEQ ID NO: 70, SEQ ID NO: 71, and SEQ ID NO: 118. -Heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), respectively, having the amino acid sequences shown in SEQ ID NO: 64, SEQ ID NO: 65, and SEQ ID NO: 119. -Heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), respectively, having the amino acid sequences shown in SEQ ID NO: 64, SEQ ID NO: 65, and SEQ ID NO: 120. -Heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), respectively, having the amino acid sequences shown in SEQ ID NO: 64, SEQ ID NO: 65, and SEQ ID NO: 121. -Heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), respectively, having the amino acid sequences shown in SEQ ID NO: 64, SEQ ID NO: 65, and SEQ ID NO: 122. -Heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), respectively, having the amino acid sequences shown in SEQ ID NO: 64, SEQ ID NO: 65, and SEQ ID NO: 123. - Heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), respectively, having the amino acid sequences shown in SEQ ID NO: 64, SEQ ID NO: 65, and SEQ ID NO: 124, or -Each includes a heavy chain variable region comprising heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), having the amino acid sequences shown 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 a multispecific antibody or its variant is - 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 SEQ ID NO: 1, - 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. 3, -The heavy chain variable region having the amino acid sequence shown in Sequence ID No. 5, heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), - 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. 6, - 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 SEQ ID NO: 15, - 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. 16, -The heavy chain variable region having the amino acid sequence shown in Sequence ID No. 17, heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), -The heavy chain variable region having the amino acid sequence shown in Sequence ID No. 52, heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), - 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, -The heavy chain variable region having the amino acid sequence shown in Sequence ID No. 54, heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), -The heavy chain variable region having the amino acid sequence shown in Sequence ID No. 55, heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), - 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, -The heavy chain variable region having the amino acid sequence shown in Sequence ID No. 57, heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), - 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 -Includes a heavy chain variable region having the amino acid sequence shown in Sequence ID No. 59, comprising heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3).

[0047] In certain embodiments, the LAG-3 binding domain of a multispecific antibody or its variant is -Heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), respectively, having the amino acid sequences shown in SEQ ID NO: 60, SEQ ID NO: 61, and SEQ ID NO: 62. -Heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), respectively, having the amino acid sequences shown in SEQ ID NO: 64, SEQ ID NO: 65, and SEQ ID NO: 66. -Heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), respectively, having the amino acid sequences shown in SEQ ID NO: 70, SEQ ID NO: 71, and SEQ ID NO: 72. -Heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), respectively, having the amino acid sequences shown in SEQ ID NOs. 73, 74, and 75. -Heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), respectively, having the amino acid sequences shown in SEQ ID NO: 97, SEQ ID NO: 98, and SEQ ID NO: 99. -Heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), respectively, having the amino acid sequences shown in SEQ ID NO: 97, SEQ ID NO: 98, and SEQ ID NO: 100. -Heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), respectively, having the amino acid sequences shown in SEQ ID NO: 64, SEQ ID NO: 65, and SEQ ID NO: 66. -Heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), respectively, having the amino acid sequences shown in SEQ ID NO: 70, SEQ ID NO: 71, and SEQ ID NO: 118. -Heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), respectively, having the amino acid sequences shown in SEQ ID NO: 64, SEQ ID NO: 65, and SEQ ID NO: 119. -Heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), respectively, having the amino acid sequences shown in SEQ ID NO: 64, SEQ ID NO: 65, and SEQ ID NO: 120. -Heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), respectively, having the amino acid sequences shown in SEQ ID NO: 64, SEQ ID NO: 65, and SEQ ID NO: 121. -Heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), respectively, having the amino acid sequences shown in SEQ ID NO: 64, SEQ ID NO: 65, and SEQ ID NO: 122. -Heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), respectively, having the amino acid sequences shown in SEQ ID NO: 64, SEQ ID NO: 65, and SEQ ID NO: 123. - Heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), respectively, having the amino acid sequences shown in SEQ ID NO: 64, SEQ ID NO: 65, and SEQ ID NO: 124, or -Each includes a heavy chain variable region comprising heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), having the amino acid sequences shown 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 a multispecific antibody or its variant is -The heavy chain variable region having the amino acid sequence shown in Sequence ID No. 5, heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), -The heavy chain variable region having the amino acid sequence shown in Sequence ID No. 17, heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), -The heavy chain variable region having the amino acid sequence shown in Sequence ID No. 52, heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), - 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, -The heavy chain variable region having the amino acid sequence shown in Sequence ID No. 54, heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), -The heavy chain variable region having the amino acid sequence shown in Sequence ID No. 55, heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), - 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, -The heavy chain variable region having the amino acid sequence shown in Sequence ID No. 57, heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), - 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 -Includes a heavy chain variable region having the amino acid sequence shown in Sequence ID No. 59, comprising heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3).

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

[0050] In a particular embodiment, the LAG-3 binding domain of a multispecific antibody or its variant includes 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 SEQ ID NO: 5.

[0051] In a particular embodiment, the LAG-3 binding domain of a multispecific antibody or its variant includes a heavy chain variable region comprising heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), each having the amino acid sequences shown in SEQ ID NO: 70, SEQ ID NO: 71, and SEQ ID NO: 72, respectively.

[0052] In a particular embodiment, the LAG-3 binding domain of a multispecific antibody or its variant includes 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 SEQ ID NO: 52.

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

[0054] In certain embodiments, the LAG-3 binding domain of a multispecific antibody or its variant has an amino acid sequence shown in any one of SEQ ID NOs: 1-17 or 52-59, or includes a heavy chain variable region 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 its variant comprises a LAG-3 binding domain variant, where each HCDR may contain up to three, two, or one amino acid substitutions. Such variants are expected to retain LAG-3 binding specificity.

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

[0057] In a particular embodiment, the anti-human LAG-3 binding domain is - An HCDR1 having the amino acid sequence SX1X2WS, wherein in the formula, X1 can be Y or F, X2 may be Y or S, HCDR1, and / or -HCDR2 having the amino acid sequence YIX1YSGX2TNX3NPX4LKX5, wherein in the formula, X1 can be Y or D, X2 can be S or T. X3 can be Y or F. X4 can be S or F. X5 may be S or I, HCDR2, and / or -HCDR3 having the amino acid sequence X1LLYKWNYVEGFDI, wherein in the formula, X1 may be D or H, and includes HCDR3.

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

[0059] In a particular embodiment, the anti-human LAG-3 binding domain is -HCDR1 having the amino acid sequence X1YX2X3H, wherein in the formula, X1 can be S, N, or R. X2 can be G or D, X3 may be M, T, or I, HCDR1, and / or -HCDR2 having the amino acid sequence VISYDGX1NKX2YX3DSVKG, wherein in the formula, X1 can be S or N, X2 can be Y, F, or H. X3 may be A, E, or V, HCDR2, and / or -HCDR3 having the amino acid sequence ERX1WDVFDI, wherein in the formula, X1 may be G or D, and includes HCDR3.

[0060] For example, preferred positions for introducing amino acid mutations include, but are not limited to, the first and / or third amino acids of HCDR1, the fifth and / or eighth amino acids of HCDR2, and / or the third amino acid of HCDR3. Kabat CDR sequences are shown in bold and underlined in the list of sequences provided herein.

[0061] In a particular embodiment, the anti-human LAG-3 binding domain is -HCDR1 having the amino acid sequence X1YX2MH, wherein in the formula, X1 can be S or N, X2 may be G or A, HCDR1, and / or -HCDR2 having the amino acid sequence VISYX1GSX2KYYADSVKG, wherein in the formula, X1 can be D or H, X2 may be N or D, HCDR2, and / or -HCDR3 having the amino acid sequence DGDNWDX1FDI, wherein in the formula, X1 may be V or A, and includes HCDR3.

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

[0063] In certain embodiments, the LAG-3 binding domain of this disclosure is - A heavy chain variable region having at least 80%, preferably 85%, more preferably 90%, or most preferably 95% sequence identity with respect to the amino acid sequence shown in SEQ ID NO: 1, and comprising the HCDR1, HCDR2, and HCDR3 amino acid sequences shown 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 with respect to the amino acid sequence shown in SEQ ID NO: 2, and comprising the HCDR1, HCDR2, and HCDR3 amino acid sequences shown 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 with respect to the amino acid sequence shown in SEQ ID NO: 3, and comprising the HCDR1, HCDR2, and HCDR3 amino acid sequences shown 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 with respect to the amino acid sequence shown in SEQ ID NO: 4, and comprising the HCDR1, HCDR2, and HCDR3 amino acid sequences shown 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 with respect to the amino acid sequence shown in SEQ ID NO: 5, and comprising the HCDR1, HCDR2, and HCDR3 amino acid sequences shown 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 with respect to the amino acid sequence shown in SEQ ID NO: 6, and comprising the HCDR1, HCDR2, and HCDR3 amino acid sequences shown 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 with respect to the amino acid sequence shown in SEQ ID NO: 7, and comprising the HCDR1, HCDR2, and HCDR3 amino acid sequences shown 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 with respect to the amino acid sequence shown in SEQ ID NO: 8, and comprising the HCDR1, HCDR2, and HCDR3 amino acid sequences shown 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 with respect to the amino acid sequence shown in SEQ ID NO: 9, and comprising the HCDR1, HCDR2, and HCDR3 amino acid sequences shown 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 with respect to the amino acid sequence shown in SEQ ID NO: 10, and comprising the HCDR1, HCDR2, and HCDR3 amino acid sequences shown 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 with respect to the amino acid sequence shown in SEQ ID NO: 11, and comprising the HCDR1, HCDR2, and HCDR3 amino acid sequences shown 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 with respect to the amino acid sequence shown in SEQ ID NO: 12, and comprising the HCDR1, HCDR2, and HCDR3 amino acid sequences shown 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 with respect to the amino acid sequence shown in SEQ ID NO: 13, and comprising the HCDR1, HCDR2, and HCDR3 amino acid sequences shown 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 with respect to the amino acid sequence shown in SEQ ID NO: 14, and comprising the HCDR1, HCDR2, and HCDR3 amino acid sequences shown 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 with respect to the amino acid sequence shown in SEQ ID NO: 15, and comprising the HCDR1, HCDR2, and HCDR3 amino acid sequences shown 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 with respect to the amino acid sequence shown in SEQ ID NO: 16, wherein each heavy chain variable region includes the HCDR1, HCDR2, and HCDR3 amino acid sequences shown 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 with respect to the amino acid sequence shown in SEQ ID NO: 17, and comprising the HCDR1, HCDR2, and HCDR3 amino acid sequences shown 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 with respect to the amino acid sequence shown in SEQ ID NO: 52, and comprising the HCDR1, HCDR2, and HCDR3 amino acid sequences shown 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 with respect to the amino acid sequence shown in SEQ ID NO: 53, and comprising the HCDR1, HCDR2, and HCDR3 amino acid sequences shown 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 with respect to the amino acid sequence shown in SEQ ID NO: 54, and comprising the HCDR1, HCDR2, and HCDR3 amino acid sequences shown 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 with respect to the amino acid sequence shown in SEQ ID NO: 55, and comprising the HCDR1, HCDR2, and HCDR3 amino acid sequences shown 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 with respect to the amino acid sequence shown in SEQ ID NO: 56, and comprising the HCDR1, HCDR2, and HCDR3 amino acid sequences shown 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 with respect to the amino acid sequence shown in SEQ ID NO: 57, and comprising the HCDR1, HCDR2, and HCDR3 amino acid sequences shown 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 with respect to the amino acid sequence shown in SEQ ID NO: 58, and comprising the HCDR1, HCDR2, and HCDR3 amino acid sequences shown 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 with respect to the amino acid sequence shown in SEQ ID NO: 59, comprising a heavy chain variable region containing the HCDR1, HCDR2, and HCDR3 amino acid sequences shown 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 its variant further comprises a light chain variable region. In certain embodiments, the LAG-3 binding domain of a multispecific antibody or its variant comprises a common light chain variable region. A preferred example of a light chain variable region is a light chain variable region comprising light chain CDR1 (LCDR1), light chain CDR2 (LCDR2), and light chain CDR3 (LCDR3), each having the amino acid sequences shown in SEQ ID NOs. 31, 32, and 33, respectively, where each LCDR may contain up to 3, 2, or 1 amino acid substitutions. In certain embodiments, a preferred light chain variable region is a light chain variable region comprising light chain CDR1 (LCDR1), light chain CDR2 (LCDR2), and light chain CDR3 (LCDR3), each having the amino acid sequences shown in SEQ ID NOs. 31, 32, and 33, respectively. In certain embodiments, such light chain variable regions may include light chain variable regions having the amino acid sequence shown in SEQ ID NO: 30, or having at least 80%, preferably 85%, more preferably 90%, or most preferably 95% sequence identity thereto. These LCDRs and / or light chain variable regions, including the light chain or light chain variable regions, are referred to in the art as VK1-39 / JK1. This 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 with respect to the amino acid sequence shown in SEQ ID NO: 30, wherein the light chain variable region comprises the LCDR1 amino acid sequence shown in SEQ ID NO: 31, the LCDR2 amino acid sequence shown in SEQ ID NO: 32, and the LCDR3 amino acid sequence shown in SEQ ID NO: 33.

[0066] The term "common light chain" in this invention refers to a light chain that can pair with multiple different heavy chains, i.e., heavy chains having different antigen or epitope binding specificities. Common light chains are particularly useful, for example, in the production 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 unaffected. For example, it is possible, within the scope of the definition of a common light chain as 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, changes in amino acids in regions that do not contribute to binding specificity, or only partially contribute, 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 be used. Examples of such common light chains include, but are not limited to, VK1-39 / JK5, ​​which includes a light chain variable region comprising light chain CDR1 (LCDR1), light chain CDR2 (LCDR2), and light chain CDR3 (LCDR3) having the amino acid sequence shown in SEQ ID NO: 34. LCDRs by IMGT are shown in bold and underlined. In certain embodiments, the light chain includes a light chain variable region comprising light chain CDR1 (LCDR1), light chain CDR2 (LCDR2), and light chain CDR3 (LCDR3) having the amino acid sequence shown in SEQ ID NO: 34, where each LCDR may contain up to three, two, or one amino acid substitutions. In certain embodiments, the light chain includes a light chain variable region having the amino acid sequence shown 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 includes a light chain variable region comprising light chain CDR1 (LCDR1), light chain CDR2 (LCDR2), and light chain CDR3 (LCDR3) having the amino acid sequence shown in SEQ ID NO: 35. LCDR by IMGT is shown in bold and underlined. In certain embodiments, the light chain includes a light chain variable region comprising light chain CDR1 (LCDR1), light chain CDR2 (LCDR2), and light chain CDR3 (LCDR3) having the amino acid sequence shown in SEQ ID NO: 35, and each LCDR may contain up to 3, 2, or 1 amino acid substitutions. In certain embodiments, the light chain includes a light chain variable region having the amino acid sequence shown 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 includes a light chain variable region comprising light chain CDR1 (LCDR1), light chain CDR2 (LCDR2), and light chain CDR3 (LCDR3) having the amino acid sequence shown in SEQ ID NO: 36. LCDR by IMGT is shown in bold and underlined.In certain embodiments, the light chain includes a light chain variable region comprising light chain CDR1 (LCDR1), light chain CDR2 (LCDR2), and light chain CDR3 (LCDR3) having the amino acid sequence shown in SEQ ID NO: 36, where each LCDR may contain up to 3, 2, or 1 amino acid substitutions. In certain embodiments, the light chain includes a light chain variable region having the amino acid sequence shown in SEQ ID NO: 36, or having at least 80%, preferably 85%, more preferably 90%, or most preferably 95% sequence identity thereto, where VL3-21 / JL3 includes a light chain variable region comprising light chain CDR1 (LCDR1), light chain CDR2 (LCDR2), and light chain CDR3 (LCDR3) having the amino acid sequence shown in SEQ ID NO: 37. LCDRs by IMGT are shown in bold and underlined. In certain embodiments, the light chain includes a light chain variable region comprising light chain CDR1 (LCDR1), light chain CDR2 (LCDR2), and light chain CDR3 (LCDR3), each having the amino acid sequence shown in SEQ ID NO: 37, wherein each LCDR may contain up to three, two, or one amino acid substitutions. In certain embodiments, the light chain includes a light chain variable region having the amino acid sequence shown 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 the immunoglobulin variable kappa 1-39 gene. This gene is also known as immunoglobulin variable kappa 1-39, IGKV139, IGKV1-39, and IgVκ1-39. The external ID for this gene is HGNC:5740, Entrez Gene:28930, and Ensembl:ENSG00000242371. The 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 the five J regions. The two preferred combined sequences are shown as VK1-39 / JK1 and VK1-39 / JK5, ​​with alternative names being IgVκ1-39_01 / IGJκ1_01 or IgVκ1-39_01 / IGJκ5_01 (named by the IMGT database World Wide Web on imgt.org). These names are illustrative and encompass allele variants of the gene segment.

[0069] VK3-15 is an abbreviation for the immunoglobulin variable kappa 3-15 gene. This gene is also known as immunoglobulin variable kappa 3-15, IGKV315, IGKV3-15, and IgVκ3-15. The external ID for this gene is HGNC:5816, Entrez Gene:28913, and Ensembl:ENSG00000244437. The 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 the five J regions. The preferred combined sequence is shown as VK3-15 / JK1, with the alternative name Vκ3-15_01 / IGJκ1_01 (named by the IMGT database World Wide Web on imgt.org). This name is illustrative and encompasses allele variants of the gene segment.

[0070] VK3-20 is an abbreviation for the immunoglobulin variable kappa 3-20 gene. This gene is also known as immunoglobulin variable kappa 3-20, IGKV320, IGKV3-20, and IgVκ3-20. The external ID for this gene is HGNC:5817, Entrez Gene:28912, and Ensembl:ENSG00000239951. The preferred amino acid sequence for VK3-20 is sequence number 40. This is the sequence of the V region. The V region can be combined with one of the five J regions. The preferred combined sequence is shown as VK3-20 / JK1, with the alternative name IgVκ3-20_01 / IGJκ1_01 (named by the IMGT database World Wide Web on imgt.org). This name is illustrative and encompasses allele variants of the gene segment.

[0071] VL3-21 is an abbreviation for the immunoglobulin variable lambda 3-21 gene. This gene is also known as immunoglobulin variable lambda 3-21, IGLV321, IGLV3-21, and IgVλ3-21. The external ID for this gene is HGNC:5905, Entrez Gene:28796, Ensembl:ENSG00000211662.2. The 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 the five J regions. The preferred combined sequence is shown as VL3-21 / JL3, with the alternative name IgVλ3-21 / IGJλ3 (named by the IMGT database World Wide Web on imgt.org). This name is illustrative and encompasses allele variants of the gene segment.

[0072] Furthermore, any light chain variable region of a LAG-3 antibody available in the art, or any other light chain variable region readily obtainable from, for example, an antibody display library, may be used, by exhibiting antigen-binding activity when paired with the LAG-3 binding domain of the present invention.

[0073] In certain embodiments, the LAG-3 binding domain of a multispecific antibody or its variant may further include CH1 and CL regions. Any CH1 domain, specifically a human CH1 domain, may be used. An example of a preferred CH1 domain is provided by the amino acid sequence provided as SEQ ID NO: 42. Any CL domain, specifically a human CL domain, may be used. An example of a preferred 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 includes 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.

[0075] The B7 family comprises numerous structurally related cell surface proteins that bind to receptors on lymphocytes that modulate the immune response. Lymphocyte activation is initiated by the involvement of cell surface, antigen-specific T cell receptors, or B cell receptors. Additional signals delivered simultaneously by B7 ligands further determine the immune response of these cells. These so-called "co-stimulatory" 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 the immune response, while binding to co-inhibitory receptors attenuates the immune response. Currently, the following members are 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. The effects of these members on regulating the immune response have been 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 potential 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. In certain embodiments, the B7 family protein is PD-L1.

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

[0078] In certain embodiments, the PD-L1 binding domain of the multispecific antibody or variant thereof of this disclosure includes a heavy chain variable region, which comprises one heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3) of the heavy chain variable regions having the amino acid sequences shown in SEQ ID NOs. HCDRs by Kabat are shown in bold and underlined in the list of sequences provided herein.

[0079] In a particular embodiment, the binding domain that binds to PD-L1 is - 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 SEQ ID NO: 20, -The heavy chain variable region having the amino acid sequence shown in Sequence ID No. 21, heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), -The heavy chain variable region having the amino acid sequence shown in Sequence ID No. 22, heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), - 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 SEQ ID NO: 23, or -Includes a heavy chain variable region having the amino acid sequence shown in Sequence ID No. 24, including heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), Each HCDR may contain up to three, two, or one amino acid substitutions.

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

[0081] In a particular embodiment, the binding domain that binds to PD-L1 is - 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 SEQ ID NO: 20, or -Includes a heavy chain variable region having the amino acid sequence shown in Sequence ID No. 24, including heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), Each HCDR may contain up to three, two, or one amino acid substitutions.

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

[0083] In a particular embodiment, the binding domain that binds to PD-L1 is -Includes a heavy chain variable region having the amino acid sequence shown in Sequence ID No. 20, which includes heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), Each HCDR may contain up to three, two, or one amino acid substitutions.

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

[0085] In a particular embodiment, the binding domain that binds to PD-L1 is - 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 SEQ ID NO: 20, -The heavy chain variable region having the amino acid sequence shown in Sequence ID No. 21, heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), -The heavy chain variable region having the amino acid sequence shown in Sequence ID No. 22, heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), 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 SEQ ID NO: 23, or -Includes 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 SEQ ID NO: 24.

[0086] In a particular embodiment, the binding domain that binds to PD-L1 is -Heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), respectively, having the amino acid sequences shown in SEQ ID NO: 107, SEQ ID NO: 108, and SEQ ID NO: 109. -Heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), respectively, having the amino acid sequences shown in SEQ ID NO: 110, SEQ ID NO: 111, and SEQ ID NO: 112. -Heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), respectively, having the amino acid sequences shown in SEQ ID NO: 113, SEQ ID NO: 114, and SEQ ID NO: 115. These are heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), respectively, having the amino acid sequences shown in SEQ ID NO: 107, SEQ ID NO: 116, and SEQ ID NO: 109. -Each includes a heavy chain variable region comprising heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), having the amino acid sequences shown in SEQ ID NO: 107, SEQ ID NO: 117, and SEQ ID NO: 109, respectively.

[0087] In a particular embodiment, the binding domain that binds to PD-L1 is - 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 SEQ ID NO: 20, or -Includes 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 SEQ ID NO: 24.

[0088] In a particular embodiment, the binding domain that binds to PD-L1 is - Heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), respectively, having the amino acid sequences shown in SEQ ID NO: 107, SEQ ID NO: 108, and SEQ ID NO: 109, or -Each includes a heavy chain variable region comprising heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), having the amino acid sequences shown in SEQ ID NO: 107, SEQ ID NO: 117, and SEQ ID NO: 109, respectively.

[0089] In a particular embodiment, the binding domain that binds to PD-L1 is -Includes 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 SEQ ID NO: 20.

[0090] In a particular embodiment, the binding domain that binds to PD-L1 is -Each includes a heavy chain variable region comprising heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), having the amino acid sequences shown 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 a variant thereof includes a heavy chain variable region having the amino acid sequence shown in any one of SEQ ID NOs: 20-24, or having 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 its variant comprises a PD-L1 binding domain variant, where each HCDR may contain up to three, two, or one amino acid substitutions. Such variants are expected to retain PD-L1 binding specificity.

[0093] In certain embodiments, the PD-L1 binding domain of a multispecific antibody or its variant also includes PD-L1 binding domain variants that include one or more mutations in the framework region in addition to mutations in the HCDR. In certain embodiments, the PD-L1 binding domain variant of a multispecific antibody or its variant does not include mutations in the CDR region but includes one or more mutations in the framework region. Such variants are expected to have at least 80%, preferably 85%, more preferably 90%, or most preferably 95% sequence identity with respect to the sequences disclosed herein and to retain PD-L1 binding specificity. Therefore, in certain embodiments, the PD-L1 binding domain of this disclosure is - A heavy chain variable region having at least 80%, preferably 85%, more preferably 90%, or most preferably 95% sequence identity with respect to the amino acid sequence shown in SEQ ID NO: 20, comprising HCDR1, HCDR2, and HCDR3 of the amino acid sequence shown 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 with respect to the amino acid sequence shown in SEQ ID NO: 21, comprising HCDR1, HCDR2, and HCDR3 of the amino acid sequence shown 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 with respect to the amino acid sequence shown in SEQ ID NO: 22, comprising HCDR1, HCDR2, and HCDR3 of the amino acid sequence shown 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 with respect to the amino acid sequence shown in SEQ ID NO: 23, and including HCDR1, HCDR2, and HCDR3 of the amino acid sequence shown 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 with respect to the amino acid sequence shown in SEQ ID NO: 24, and comprising a heavy chain variable region including HCDR1, HCDR2, and HCDR3 of the amino acid sequence shown in SEQ ID NO: 24.

[0094] In certain embodiments, the PD-L1 binding domain of the present disclosure is - A heavy chain variable region having at least 80%, preferably 85%, more preferably 90%, or most preferably 95% sequence identity with respect to the amino acid sequence shown in SEQ ID NO: 20, and comprising the HCDR1, HCDR2, and HCDR3 amino acid sequences shown 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 with respect to the amino acid sequence shown in SEQ ID NO: 21, and comprising the HCDR1, HCDR2, and HCDR3 amino acid sequences shown 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 with respect to the amino acid sequence shown in SEQ ID NO: 22, and comprising the HCDR1, HCDR2, and HCDR3 amino acid sequences shown 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 with respect to the amino acid sequence shown in SEQ ID NO: 23, and comprising the HCDR1, HCDR2, and HCDR3 amino acid sequences shown 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 with respect to the amino acid sequence shown in SEQ ID NO: 24, and comprising a heavy chain variable region containing the HCDR1, HCDR2, and HCDR3 amino acid sequences shown 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 a variant thereof further comprises a light chain variable region. Examples of preferred light chain variable regions are those described herein. Alternatively, any other light chain variable region readily available from an antibody display library, for example, by exhibiting antigen-binding activity when paired with the PD-L1 binding domain of the Disclosure, may be used. Preferably, the PD-L1 binding domain of the 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 its variant may further include CH1 and CL regions. Any CH1 domain, specifically a human CH1 domain, may be used. An example of a preferred CH1 domain is provided by the amino acid sequence provided as SEQ ID NO: 42. Any CL domain, specifically a human CL domain, may be used. An example of a preferred 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 PD-L1 binding domain disclosed herein to produce the multispecific antibodies or variants thereof of the Disclosure. In certain embodiments, the Disclosure provides the multispecific antibodies PB1-PB125 or variants thereof, as presented in Table 1. [Table 1]

[0098] In one embodiment, a multispecific antibody or its variant is - The LAG-3 binding domain of this disclosure, 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 shown in SEQ ID NO: 1, and -Includes the PD-L1 binding domain of this disclosure, comprising heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3) of a heavy chain variable region having the amino acid sequence shown in SEQ ID NOs. 20, 23, or 24.

[0099] In one embodiment, a multispecific antibody or its variant is - The LAG-3 binding domain of this disclosure, 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 shown in SEQ ID NO: 3, and -Includes the PD-L1 binding domain of this disclosure, comprising heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3) of a heavy chain variable region having the amino acid sequence shown in SEQ ID NOs. 20, 23, or 24.

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

[0101] In one embodiment, a multispecific antibody or its variant is - The LAG-3 binding domain of this disclosure, 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 shown in SEQ ID NO: 15, and -Includes the PD-L1 binding domain of this disclosure, comprising heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3) of a heavy chain variable region having the amino acid sequence shown in SEQ ID NOs. 20, 23, or 24.

[0102] In one embodiment, a multispecific antibody or its variant is - The LAG-3 binding domain of this disclosure, 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 shown in SEQ ID NO: 16, and -Includes the PD-L1 binding domain of this disclosure, comprising heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3) of a heavy chain variable region having the amino acid sequence shown in SEQ ID NOs. 20, 23, or 24.

[0103] In one embodiment, a multispecific antibody or its variant is - The LAG-3 binding domain of this disclosure, 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 shown in Sequence ID No. 17, and -Includes the PD-L1 binding domain of this disclosure, comprising heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3) of a heavy chain variable region having the amino acid sequence shown in SEQ ID NOs. 20, 23, or 24.

[0104] In one embodiment, a multispecific antibody or its variant is - The LAG-3 binding domain of this disclosure, 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 shown in Sequence ID No. 52, and -Includes the PD-L1 binding domain of this disclosure, comprising heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3) of a heavy chain variable region having the amino acid sequence shown in SEQ ID NOs. 20, 23, or 24.

[0105] In one embodiment, a multispecific antibody or its variant is - The LAG-3 binding domain of this disclosure, 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 shown in Sequence ID No. 53, and -Includes the PD-L1 binding domain of this disclosure, comprising heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3) of a heavy chain variable region having the amino acid sequence shown in SEQ ID NOs. 20, 23, or 24.

[0106] In one embodiment, a multispecific antibody or its variant is - The LAG-3 binding domain of this disclosure, 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 shown in Sequence ID No. 54, and -Includes the PD-L1 binding domain of this disclosure, comprising heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3) of a heavy chain variable region having the amino acid sequence shown in SEQ ID NOs. 20, 23, or 24.

[0107] In one embodiment, a multispecific antibody or its variant is - The LAG-3 binding domain of this disclosure, 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 shown in SEQ ID NO: 55, and -Includes the PD-L1 binding domain of this disclosure, comprising heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3) of a heavy chain variable region having the amino acid sequence shown in SEQ ID NOs. 20, 23, or 24.

[0108] In one embodiment, a multispecific antibody or its variant is - The LAG-3 binding domain of this disclosure, 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 shown in Sequence ID No. 56, and -Includes the PD-L1 binding domain of this disclosure, comprising heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3) of a heavy chain variable region having the amino acid sequence shown in SEQ ID NOs. 20, 23, or 24.

[0109] In one embodiment, a multispecific antibody or its variant is - The LAG-3 binding domain of this disclosure, 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 shown in Sequence ID No. 57, and -Includes the PD-L1 binding domain of this disclosure, comprising heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3) of a heavy chain variable region having the amino acid sequence shown in SEQ ID NOs. 20, 23, or 24.

[0110] In one embodiment, a multispecific antibody or its variant is - The LAG-3 binding domain of this disclosure, 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 shown in Sequence ID No. 58, and -Includes the PD-L1 binding domain of this disclosure, comprising heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3) of a heavy chain variable region having the amino acid sequence shown in SEQ ID NOs. 20, 23, or 24.

[0111] In one embodiment, a multispecific antibody or its variant is - The LAG-3 binding domain of this disclosure, 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 shown in SEQ ID NO: 59, and -Includes the PD-L1 binding domain of this disclosure, comprising heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3) of a heavy chain variable region having the amino acid sequence shown in SEQ ID NOs. 20, 23, or 24.

[0112] In one embodiment, a multispecific antibody or its variant is - The LAG-3 binding domain of this disclosure, 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 shown in Sequence ID No. 17, and - The PD-L1 binding domain of this disclosure comprises heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3) of a heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 24.

[0113] In one embodiment, a multispecific antibody or its variant is - The LAG-3 binding domain of this disclosure comprises heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), respectively, having the amino acid sequences shown in SEQ ID NO: 64, SEQ ID NO: 65, and SEQ ID NO: 66, and -Each comprises a PD-L1 binding domain of the present disclosure, which includes heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), having the amino acid sequences shown in SEQ ID NO: 107, SEQ ID NO: 117, and SEQ ID NO: 109, respectively.

[0114] In one embodiment, a multispecific antibody or its variant is - The LAG-3 binding domain of this disclosure, 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 shown in Sequence ID No. 53, and - The PD-L1 binding domain of this disclosure comprises heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3) of a heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 24.

[0115] In one embodiment, a multispecific antibody or its variant is - The LAG-3 binding domain of this disclosure comprises heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), respectively, having the amino acid sequences shown in SEQ ID NO: 64, SEQ ID NO: 65, and SEQ ID NO: 119, and -Each comprises a PD-L1 binding domain of the present disclosure, which includes heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), having the amino acid sequences shown in SEQ ID NO: 107, SEQ ID NO: 117, and SEQ ID NO: 109, respectively.

[0116] In one embodiment, a multispecific antibody or its variant is - The LAG-3 binding domain of this disclosure, 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 shown in Sequence ID No. 54, and - The PD-L1 binding domain of this disclosure comprises heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3) of a heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 24.

[0117] In one embodiment, a multispecific antibody or its variant is - The LAG-3 binding domain of this disclosure comprises heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), respectively, having the amino acid sequences shown in SEQ ID NO: 64, SEQ ID NO: 65, and SEQ ID NO: 120, and -Each comprises a PD-L1 binding domain of the present disclosure, which includes heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), having the amino acid sequences shown in SEQ ID NO: 107, SEQ ID NO: 117, and SEQ ID NO: 109, respectively.

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

[0119] In one embodiment, a multispecific antibody or its variant is - The LAG-3 binding domain of this disclosure comprises heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), respectively, having the amino acid sequences shown in SEQ ID NO: 64, SEQ ID NO: 65, and SEQ ID NO: 121, and -Each comprises a PD-L1 binding domain of the present disclosure, which includes heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), having the amino acid sequences shown in SEQ ID NO: 107, SEQ ID NO: 117, and SEQ ID NO: 109, respectively.

[0120] In one embodiment, a multispecific antibody or its variant is - The LAG-3 binding domain of this disclosure, 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 shown in Sequence ID No. 56, and - The PD-L1 binding domain of this disclosure comprises heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3) of a heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 24.

[0121] In one embodiment, a multispecific antibody or its variant is - The LAG-3 binding domain of this disclosure comprises heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), respectively, having the amino acid sequences shown in SEQ ID NO: 64, SEQ ID NO: 65, and SEQ ID NO: 122, and -Each comprises a PD-L1 binding domain of the present disclosure, which includes heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), having the amino acid sequences shown in SEQ ID NO: 107, SEQ ID NO: 117, and SEQ ID NO: 109, respectively.

[0122] In one embodiment, a multispecific antibody or its variant is - The LAG-3 binding domain of this disclosure, 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 shown in Sequence ID No. 57, and - The PD-L1 binding domain of this disclosure comprises heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3) of a heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 24.

[0123] In one embodiment, a multispecific antibody or its variant is - The LAG-3 binding domain of this disclosure comprises heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), respectively, having the amino acid sequences shown in SEQ ID NO: 64, SEQ ID NO: 65, and SEQ ID NO: 123, and -Each comprises a PD-L1 binding domain of the present disclosure, which includes heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), having the amino acid sequences shown in SEQ ID NO: 107, SEQ ID NO: 117, and SEQ ID NO: 109, respectively.

[0124] In one embodiment, a multispecific antibody or its variant is - The LAG-3 binding domain of this disclosure, 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 shown in Sequence ID No. 58, and - The PD-L1 binding domain of this disclosure comprises heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3) of a heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 24.

[0125] In one embodiment, a multispecific antibody or its variant is - The LAG-3 binding domain of this disclosure comprises heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), respectively, having the amino acid sequences shown in SEQ ID NO: 64, SEQ ID NO: 65, and SEQ ID NO: 124, and -Each comprises a PD-L1 binding domain of the present disclosure, which includes heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), having the amino acid sequences shown in SEQ ID NO: 107, SEQ ID NO: 117, and SEQ ID NO: 109, respectively.

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

[0127] In one embodiment, a multispecific antibody or its variant is - The LAG-3 binding domain of this disclosure comprises heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), respectively, having the amino acid sequences shown in SEQ ID NO: 64, SEQ ID NO: 65, and SEQ ID NO: 125, and -Each comprises a PD-L1 binding domain of the present disclosure, which includes heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), having the amino acid sequences shown in SEQ ID NO: 107, SEQ ID NO: 117, and SEQ ID NO: 109, respectively.

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

[0129] In one embodiment, a multispecific antibody or its variant is - The LAG-3 binding domain of this disclosure comprises heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), respectively, having the amino acid sequences shown in SEQ ID NO: 70, SEQ ID NO: 71, and SEQ ID NO: 72, and -Each comprises a PD-L1 binding domain of the present disclosure, which includes heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), having the amino acid sequences shown in SEQ ID NO: 107, SEQ ID NO: 108, and SEQ ID NO: 109, respectively.

[0130] In one embodiment, a multispecific antibody or its variant is - The LAG-3 binding domain of this disclosure, 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 shown in Sequence ID No. 52, and -Includes the PD-L1 binding domain of this disclosure, comprising heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3) of a heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 20.

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

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

[0133] In one embodiment, a multispecific antibody or its variant is - The LAG-3 binding domain of this disclosure, 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 shown in Sequence ID No. 53, and -Includes the PD-L1 binding domain of this disclosure, which comprises 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 SEQ ID NO: 24, The LAG-3 binding domain and the PD-L1 binding domain include a light chain CDR1 (LCDR1) having the amino acid sequence shown in SEQ ID NO: 31, a light chain CDR2 (LCDR2) having the amino acid sequence shown in SEQ ID NO: 32, and a light chain CDR3 (LCDR3) having the amino acid sequence shown in SEQ ID NO: 33.

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

[0135] In one embodiment, a multispecific antibody or its variant is - The LAG-3 binding domain of this disclosure, 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 shown in Sequence ID No. 54, and -Includes the PD-L1 binding domain of this disclosure, which comprises 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 SEQ ID NO: 24, The LAG-3 binding domain and the PD-L1 binding domain include a light chain CDR1 (LCDR1) having the amino acid sequence shown in SEQ ID NO: 31, a light chain CDR2 (LCDR2) having the amino acid sequence shown in SEQ ID NO: 32, and a light chain CDR3 (LCDR3) having the amino acid sequence shown in SEQ ID NO: 33.

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

[0137] In one embodiment, a multispecific antibody or its variant is - The LAG-3 binding domain of this disclosure, 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 shown in SEQ ID NO: 55, and -Includes the PD-L1 binding domain of this disclosure, which comprises 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 SEQ ID NO: 24, The LAG-3 binding domain and the PD-L1 binding domain include a light chain CDR1 (LCDR1) having the amino acid sequence shown in SEQ ID NO: 31, a light chain CDR2 (LCDR2) having the amino acid sequence shown in SEQ ID NO: 32, and a light chain CDR3 (LCDR3) having the amino acid sequence shown in SEQ ID NO: 33.

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

[0139] In one embodiment, a multispecific antibody or its variant is - The LAG-3 binding domain of this disclosure, 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 shown in Sequence ID No. 56, and -Includes the PD-L1 binding domain of this disclosure, which comprises 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 SEQ ID NO: 24, The LAG-3 binding domain and the PD-L1 binding domain include a light chain CDR1 (LCDR1) having the amino acid sequence shown in SEQ ID NO: 31, a light chain CDR2 (LCDR2) having the amino acid sequence shown in SEQ ID NO: 32, and a light chain CDR3 (LCDR3) having the amino acid sequence shown in SEQ ID NO: 33.

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

[0141] In one embodiment, a multispecific antibody or its variant is - The LAG-3 binding domain of this disclosure, 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 shown in Sequence ID No. 57, and -Includes the PD-L1 binding domain of this disclosure, which comprises 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 SEQ ID NO: 24, The LAG-3 binding domain and the PD-L1 binding domain include a light chain CDR1 (LCDR1) having the amino acid sequence shown in SEQ ID NO: 31, a light chain CDR2 (LCDR2) having the amino acid sequence shown in SEQ ID NO: 32, and a light chain CDR3 (LCDR3) having the amino acid sequence shown in SEQ ID NO: 33.

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

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

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

[0145] In one embodiment, a multispecific antibody or its variant is - The LAG-3 binding domain of this disclosure, 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 shown in SEQ ID NO: 59, and -Includes the PD-L1 binding domain of this disclosure, which comprises 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 SEQ ID NO: 24, The LAG-3 binding domain and the PD-L1 binding domain include a light chain CDR1 (LCDR1) having the amino acid sequence shown in SEQ ID NO: 31, a light chain CDR2 (LCDR2) having the amino acid sequence shown in SEQ ID NO: 32, and a light chain CDR3 (LCDR3) having the amino acid sequence shown in SEQ ID NO: 33.

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

[0147] In one embodiment, a multispecific antibody or its variant is - The LAG-3 binding domain of this disclosure, 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 shown in SEQ ID NO: 5, and -Includes the PD-L1 binding domain of this disclosure, 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 shown in SEQ ID NO: 20, The LAG-3 binding domain and the PD-L1 binding domain include a light chain CDR1 (LCDR1) having the amino acid sequence shown in SEQ ID NO: 31, a light chain CDR2 (LCDR2) having the amino acid sequence shown in SEQ ID NO: 32, and a light chain CDR3 (LCDR3) having the amino acid sequence shown in SEQ ID NO: 33.

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

[0149] In one embodiment, a multispecific antibody or its variant is - The LAG-3 binding domain of this disclosure, 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 shown in Sequence ID No. 52, and -Includes the PD-L1 binding domain of this disclosure, 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 shown in SEQ ID NO: 20, The LAG-3 binding domain and the PD-L1 binding domain include a light chain CDR1 (LCDR1) having the amino acid sequence shown in SEQ ID NO: 31, a light chain CDR2 (LCDR2) having the amino acid sequence shown in SEQ ID NO: 32, and a light chain CDR3 (LCDR3) having the amino acid sequence shown in SEQ ID NO: 33.

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

[0151] In one embodiment, a multispecific antibody or its variant is - The LAG-3 binding domain of this disclosure, including a heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 17, and -Includes the PD-L1 binding domain of this disclosure, which includes a heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 24.

[0152] In one embodiment, a multispecific antibody or its variant is - The LAG-3 binding domain of this disclosure, including a heavy chain variable region having the amino acid sequence shown in Sequence ID No. 53, and -Includes the PD-L1 binding domain of this disclosure, which includes a heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 24.

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

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

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

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

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

[0158] In one embodiment, a multispecific antibody or its variant is - The LAG-3 binding domain of this disclosure, including a heavy chain variable region having the amino acid sequence shown in Sequence ID No. 59, and -Includes the PD-L1 binding domain of this disclosure, which includes a heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 24.

[0159] In one embodiment, a multispecific antibody or its variant is - The LAG-3 binding domain of this disclosure, which includes a heavy chain variable region having the amino acid sequence shown in Sequence ID No. 5, and -Includes the PD-L1 binding domain of this disclosure, which includes a heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 20.

[0160] In one embodiment, a multispecific antibody or its variant is - The LAG-3 binding domain of this disclosure, including a heavy chain variable region having the amino acid sequence shown in Sequence ID No. 52, and -Includes a PD-L1 binding domain of the present disclosure, which includes a heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 20. In one embodiment, a multispecific antibody or a variant thereof is - The LAG-3 binding domain of this disclosure, including a heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 17, and -Includes a PD-L1 binding domain of this disclosure, which includes a heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 24, The LAG-3 binding domain and the PD-L1 binding domain include a light chain variable region having the amino acid sequence shown in SEQ ID NO: 30.

[0161] In one embodiment, a multispecific antibody or its variant is - The LAG-3 binding domain of this disclosure, including a heavy chain variable region having the amino acid sequence shown in Sequence ID No. 53, and -Includes a PD-L1 binding domain of this disclosure, which includes a heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 24, The LAG-3 binding domain and the PD-L1 binding domain include a light chain variable region having the amino acid sequence shown in SEQ ID NO: 30.

[0162] In one embodiment, a multispecific antibody or its variant is - The LAG-3 binding domain of this disclosure, including a heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 54, and -Includes a PD-L1 binding domain of this disclosure, which includes a heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 24, The LAG-3 binding domain and the PD-L1 binding domain include a light chain variable region having the amino acid sequence shown in SEQ ID NO: 30.

[0163] In one embodiment, a multispecific antibody or its variant is - The LAG-3 binding domain of this disclosure, including a heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 55, and -Includes a PD-L1 binding domain of this disclosure, which includes a heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 24, The LAG-3 binding domain and the PD-L1 binding domain include a light chain variable region having the amino acid sequence shown in SEQ ID NO: 30.

[0164] In one embodiment, a multispecific antibody or its variant is - The LAG-3 binding domain of this disclosure, including a heavy chain variable region having the amino acid sequence shown in Sequence ID No. 56, and -Includes a PD-L1 binding domain of this disclosure, which includes a heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 24, The LAG-3 binding domain and the PD-L1 binding domain include a light chain variable region having the amino acid sequence shown in SEQ ID NO: 30.

[0165] In one embodiment, a multispecific antibody or its variant is - The LAG-3 binding domain of this disclosure, including a heavy chain variable region having the amino acid sequence shown in Sequence ID No. 57, and -Includes a PD-L1 binding domain of this disclosure, which includes a heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 24, The LAG-3 binding domain and the PD-L1 binding domain include a light chain variable region having the amino acid sequence shown in SEQ ID NO: 30.

[0166] In one embodiment, a multispecific antibody or its variant is - The LAG-3 binding domain of this disclosure, including a heavy chain variable region having the amino acid sequence shown in Sequence ID No. 58, and -Includes a PD-L1 binding domain of this disclosure, which includes a heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 24, The LAG-3 binding domain and the PD-L1 binding domain include a light chain variable region having the amino acid sequence shown in SEQ ID NO: 30.

[0167] In one embodiment, a multispecific antibody or its variant is - The LAG-3 binding domain of this disclosure, including a heavy chain variable region having the amino acid sequence shown in Sequence ID No. 59, and -Includes a PD-L1 binding domain of this disclosure, which includes a heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 24, The LAG-3 binding domain and the PD-L1 binding domain include a light chain variable region having the amino acid sequence shown in SEQ ID NO: 30.

[0168] In one embodiment, a multispecific antibody or its variant is - The LAG-3 binding domain of this disclosure, which includes a heavy chain variable region having the amino acid sequence shown in Sequence ID No. 5, and -Includes a PD-L1 binding domain of this disclosure, which includes a heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 20, The LAG-3 binding domain and the PD-L1 binding domain include a light chain variable region having the amino acid sequence shown in SEQ ID NO: 30.

[0169] In one embodiment, a multispecific antibody or its variant is - The LAG-3 binding domain of this disclosure, including a heavy chain variable region having the amino acid sequence shown in Sequence ID No. 52, and -Includes a PD-L1 binding domain of this disclosure, which includes a heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 20, The LAG-3 binding domain and the PD-L1 binding domain include a light chain variable region having the amino acid sequence shown in SEQ ID NO: 30.

[0170] The LAG-3xPD-L1 multispecific antibody or variant thereof of this disclosure targets two immune checkpoints in the trans state: 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 the cis state by binding to upregulated PD-L1 on depleted T effector cells in the tumor microenvironment. In this way, the LAG-3xPD-L1 multispecific antibody or variant thereof of this disclosure prevents T cell inhibitory signaling and enhances tumor immunity.

[0171] In certain embodiments, the multispecific antibody or its variant has a binding affinity to 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 the SPR described herein. The term "about" allows for a 10% deviation from the stated value.

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

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

[0174] In certain embodiments, the 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 shown in SEQ ID NO: 27 and two light chain variable regions having the amino acid sequence shown 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 shown in SEQ ID NO: 25 and two light chain variable regions having the amino acid sequence shown 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. Therefore, 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 one that uses 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 ​​of the LAG-3 and PD-L1 binding domains provided herein are obtained by the method described in Example 3. In summary, anti-huIgG Fcγ is covalently bound to the surface of the CM5 sensor chip using free amine chemistry. A multispecific antibody is injected into a flow cell at a concentration up to 20 nM at a rate of 30 μL / min for 2 minutes. Subsequently, antigens (0.6–20 nM) are flowed onto the surface of the CM5 sensor chip at a rate of 30 μL / min for 2 minutes. Sensegmentograms of the association and dissociation phases for different antigens are obtained. The affinity of individual Fab arms is determined using BIA evaluation software and curve fitting with a 1:1 interaction model (for monovalent interactions).

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

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

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

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

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

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

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

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

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

[0186] In certain embodiments, the binding affinity to cynomolgus monkey LAG-3 is measured in a bivalent bispecificity IgG format by both the LAG-3 binding domain of the multispecificity antibody and the reference anti-LAG-3 binding domain. The bivalent bispecificity IgG format may include, for example, the LAG-3 binding domain of the antibody disclosed or the reference antibody and a binding domain that binds to PD-L1 or another unrelated target. Thus, monovalent interactions with cynomolgus monkey LAG-3 are measured.

[0187] In certain embodiments, the binding affinity to cynomolgus monkey PD-L1 is measured in a bivalent bispecificity IgG format by both the PD-L1 binding domain of the multispecificity antibody and the reference PD-L1 binding domain. The bivalent bispecificity IgG format may include, for example, the PD-L1 binding domain of the antibody disclosed or the reference antibody and a binding domain that binds to LAG-3 or another unrelated target. Thus, monovalent interactions with cynomolgus monkey PD-L1 are measured.

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

[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 the combination of reference antibodies described herein.

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

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

[0192] This disclosure further provides cells comprising nucleic acid sequences encoding heavy chain variable regions of the LAG-3 binding domain described herein, and nucleic acid sequences encoding heavy chain variable regions of the PD-L1 binding domain described herein. In certain embodiments, the cells may further comprise nucleic acid sequences encoding CH1 regions, and preferably hinge, CH2, and CH3 regions. In certain embodiments, the cells may further comprise at least one polynucleotide sequence encoding light chain variable regions, and preferably CL regions. In certain embodiments, the light chain variable regions are light chain variable regions of a light chain that can pair with a plurality of heavy chains having different epitope specificities.

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

[0194] This specification further provides a method for producing the multispecific antibodies or variants thereof of the present disclosure, the method comprising culturing the cells described herein and recovering the multispecific antibodies or variants thereof from the cells or the supernatant.

[0195] This specification further provides a method for producing variants of the LAG-3 binding domain and / or PD-L1 binding domain of the present disclosure, the method being described herein - To generate sequence variants of the heavy chain variable region described herein, - This includes expressing the sequence variant or variant described herein, and the light chain variable region, in cells.

[0196] Methods for generating sequence variants are well known in the art. Random or targeted approaches can be taken in generating sequence variants, for example, with the aim of introducing 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, with respect to the large-scale production of binding domains, or multispecific antibodies containing such binding domains, the aim may be to introduce mutations that reduce development risk. Mutations that are unlikely to cause loss of binding specificity and / or affect binding affinity may 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 specific locations based on the frequency of the germline or natural repertoire.

[0197] Whether amino acid residues within the CDR and / or framework region can be substituted, for example, with conserved amino acid residues, and whether such substitution can be done without or substantially without loss of binding specificity and / or affinity, can be determined by methods well known in the art. Examples of 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). For example, computational methods have been developed that can predict the effects of amino acid mutations, such as those described in 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] This specification further provides multispecific antibodies comprising any variant LAG-3 and / or PD-L1 binding domain produced by the above method, pharmaceutical compositions comprising a multispecific antibody comprising either the variant LAG-3 and / or PD-L1 binding domain, nucleic acids encoding either of the variant binding domains, vectors and cells comprising the nucleic acid, and the use of a multispecific antibody comprising either the variant LAG-3 and / or PD-L1 binding domain, or a pharmaceutical composition, for the treatment of cancer.

[0199] Pharmaceutical compositions and methods In certain embodiments, the Disclosure provides a pharmaceutical composition comprising a multispecific antibody or variant thereof as described herein and a pharmaceutically acceptable carrier, diluent, or excipient.

[0200] In certain embodiments, this disclosure provides multispecific antibodies or variants thereof, or pharmaceutical compositions described herein, for use as pharmaceuticals.

[0201] In certain embodiments, the Disclosure provides multispecific antibodies or variants thereof, or pharmaceutical compositions thereof, for use in the treatment of diseases associated with a suppressed immune system, specifically, cancer.

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

[0203] In certain embodiments, the Disclosure provides a method for treating a disease associated with a suppressed immune system, specifically cancer, comprising administering an effective amount of a multispecific antibody or variant thereof described herein, or a pharmaceutical composition, to an individual in need of such treatment.

[0204] As used herein, the terms “individual,” “subject,” and “patient” are interchangeable and refer to mammals such as humans, mice, rats, hamsters, guinea pigs, rabbits, cats, dogs, monkeys, cattle, horses, and pigs (for example, patients such as human patients with cancer).

[0205] As used herein, the terms “to treat,” “to treat,” and “treatment” refer to any type of intervention or process performed on a subject or administered to a subject with an active agent or combination of active agents for the purpose of curing or improving a disease or its symptoms. This includes reversing, alleviating, improving, suppressing or delaying symptoms, complications, conditions, or biochemical signs associated with the disease, and preventing the onset, progression, occurrence, worsening, or recurrence of symptoms, complications, conditions, or biochemical signs associated with the disease.

[0206] As used herein, “effective treatment” or “positive treatment response” means a treatment that results in a beneficial effect, for example, improvement of at least one symptom of a disease or disorder, such as cancer. A beneficial effect can take the form of improvement above baseline, including improvement beyond measurements or observations made before initiating treatment according to the method. For example, a beneficial effect can take the form of delaying, stabilizing, stopping, or reversing the progression of cancer in a subject at any clinical stage, as demonstrated by a reduction or elimination of clinical or diagnostic symptoms of the disease, or cancer markers. An effective treatment can, for example, reduce tumor size, reduce the presence of circulating tumor cells, reduce or prevent tumor metastasis, delay or stop tumor growth, and / or prevent or delay tumor recurrence or relapse.

[0207] The terms “therapeutic dose” or “effective dose” refer to the amount of an agent or combination of agents that provides a desired biological, therapeutic, and / or preventive outcome. The outcome may be reduction, improvement, remission, lessening, delay, and / or mitigation of one or more signs, symptoms, or causes of a disease, or any other desired change in the biological system. In some embodiments, the therapeutic dose is sufficient to delay tumor development. In some embodiments, the therapeutic dose is sufficient to prevent or delay tumor recurrence.

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

[0209] The effective dose may vary depending on factors such as the disease state, age, sex, and weight of the individual being treated, as well as the ability of the drug or drug combination to induce the desired response in the individual.

[0210] An effective dose can be administered in one or more doses.

[0211] The therapeutic dose also includes the amount that balances any toxic or adverse effects of the drug or combination of drugs with the therapeutically beneficial effects.

[0212] The term "drug" refers to a therapeutically active substance, in this case, the multispecific antibodies or their variants as disclosed herein, or the pharmaceutical compositions as disclosed herein.

[0213] General terminology As used herein, “includes” and its conjugations are used in their non-restrictive sense, meaning that the item preceding the word is included, but items not specifically mentioned are not excluded.

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

[0215] No reference in this specification to patent documents or other matters shall be deemed to be an admission that such documents or matters were known, or that the information contained herein was part of the common general knowledge as of the priority date of any of the claims.

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

[0217] In this specification, unless otherwise specified, amino acid positions assigned to the CDR and framework within the variable region of an antibody or antibody fragment are designated by Kabat numbering (see Sequences of Proteins of Immunological Interest (National Institute of Health, Bethesda, Md., 1987 and 1991)). Amino acids within the constant region are indicated by the EU numbering system.

[0218] The acceptance number is primarily provided to offer a further method for target identification, and the actual sequence of the bound protein may change due to mutations in coding genes, such as mutations that occur in certain cancers. The antigen-binding site binds to antigens and their various variants, such as those expressed by certain antigen-positive immune cells or tumor cells.

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

[0220] HGNC is an abbreviation for the HUGO Gene Nomenclature Committee. The number following the abbreviation is an access number that allows information about the gene and the protein encoded by that gene to be retrieved from the HGNC database. Entrez Gene provides an access number or gene ID that allows information about the gene or the protein encoded by that gene to be retrieved from the NCBI (National Center for Biotechnology Information) database. Ensemble provides an access number that allows information about the gene or the protein encoded by that gene to be obtained from the Ensemble database. Ensemble is a joint 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 drawing]

[0221] In the figure, 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 sequence of both binding domains. Each binding domain of the monospecific antibody includes a light chain. In the examples used to illustrate the disclosure but not intended to limit the disclosure in any way, each binding domain of the monospecific antibody includes a light chain variable region having the amino acid sequence shown in SEQ ID NO: 30, and a light chain constant region having the amino acid sequence shown in SEQ ID NO: 43. The monospecific antibody is preferably an IgG1 antibody containing CH1, hinge, CH2, and CH3. In the examples used to illustrate the disclosure but not intended to limit the disclosure in any way, the monospecific antibody is screened in IgG1 format, and the LAG-3 and PD-L1 binding heavy chains include CH1 having the amino acid sequence shown in SEQ ID NO: 42, CH2 having the amino acid sequence shown in SEQ ID NO: 45, and CH3 having the amino acid sequence shown in SEQ ID NO: 47.

[0222] Bispecific antibodies are indicated in the format SEQ ID NO: AxSEQ ID NO: B, where both SEQ ID NOs A and B refer to heavy chain variable sequences. Each binding domain of a bispecific antibody contains a light chain. In examples used to illustrate the disclosure, but not intended to limit the disclosure in any way, each binding domain of a bispecific antibody contains a light chain variable region having the amino acid sequence shown in SEQ ID NO: 30, and a light chain constant region having the amino acid sequence shown in SEQ ID NO: 43. The bispecific antibody is an IgG1 antibody containing 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, a bispecific antibody is screened in IgG1 format, the PD-L1-binding heavy chain comprises CH1 having the amino acid sequence shown in SEQ ID NO: 42, CH2 which may have the amino acid sequence shown in SEQ ID NO: 45, and CH3 which may have the amino acid sequence shown in SEQ ID NO: 50, and the LAG-3-binding heavy chain comprises CH1 having the amino acid sequence shown in SEQ ID NO: 42, CH2 which has the amino acid sequence shown in SEQ ID NO: 45, and CH3 which has the amino acid sequence shown 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, the bispecific antibody was screened in IgG1 format, the PD-L1-binding heavy chain comprising CH1 having the amino acid sequence shown in SEQ ID NO: 42, CH2 having the amino acid sequence shown in SEQ ID NO: 45, and CH3 having the amino acid sequence shown in SEQ ID NO: 50, and the LAG-3-binding heavy chain comprising CH1 having the amino acid sequence shown in SEQ ID NO: 42, CH2 having the amino acid sequence shown in SEQ ID NO: 45, and CH3 having the amino acid sequence shown 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 present disclosure in any way, bispecific antibodies were screened in IgG1 format.

[0226] With respect to Sequence ID No. 17xSequence ID No. 24, the PD-L1 binding heavy chain includes CH1 having the amino acid sequence shown in Sequence ID No. 42, CH2 having the amino acid sequence shown in Sequence ID No. 45, and CH3 having the amino acid sequence shown in Sequence ID No. 50, and the LAG-3 binding heavy chain includes CH1 having the amino acid sequence shown in Sequence ID No. 42, CH2 having the amino acid sequence shown in Sequence ID No. 45, and CH3 having the amino acid sequence shown in Sequence ID No. 51.

[0227] With respect to SEQ ID NOs. 5xSEQ ID NOs. 20, the PD-L1 binding heavy chain may include CH1 having the amino acid sequence shown in SEQ ID NOs. 42, CH2 having the amino acid sequence shown in SEQ ID NOs. 45, and CH3 having the amino acid sequence shown in SEQ ID NOs. 51. The LAG-3 binding heavy chain may include CH1 having the amino acid sequence shown in SEQ ID NOs. 42, CH2 having the amino acid sequence shown in SEQ ID NOs. 45, and CH3 having the amino acid sequence shown in SEQ ID NOs. 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, the bispecific antibody is screened in IgG format, and the PD-L1 binding heavy chain comprises CH1 having the amino acid sequence shown in SEQ ID NO: 42, CH2 having the amino acid sequence shown in SEQ ID NO: 45, and CH3 having the amino acid sequence shown in SEQ ID NO: 50 or 51, and the LAG-3 binding heavy chain comprises CH1 having the amino acid sequence shown in SEQ ID NO: 42, CH2 having the amino acid sequence shown in SEQ ID NO: 45, and CH3 having the amino acid sequence shown in SEQ ID NO: 51 or 50, as shown in Table 10. The bivalent monospecific relatrimab 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 relatrimab analog antibody contains two LAG-3 binding domains. A bivalent, monospecific atezolizumab analog antibody contains two PD-L1 binding domains. Each binding domain of the analog antibody contains a light chain. The combination of relatrimab and atezolizumab analog is indicated 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 relatrimab 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]The results of LAG-3 binding using FACS are shown. Bispecific antibodies containing heavy chain variable regions with the amino acid sequences shown in SEQ ID NOs: 1 and 23(a), SEQ ID NOs: 3 and 23(b), SEQ ID NOs: 5 and 23(c), SEQ ID NOs: 6 and 23(d), SEQ ID NOs: 15 and 23(e), and SEQ ID NOs: 16 and 23(f) were compared with bivalent monospecific antibodies containing their respective LAG-3 binding domains, a positive control relatrimab analog (SEQ ID NOs: 27 / 28), and a negative control antibody (SEQ ID NOs: 29 / 30). Each graph shows the results for binding to human and rhesus monkey LAG-3. The X-axis represents the antibody concentration in μg / mL. The Y-axis represents the binding level, expressed as mean fluorescence intensity (MFI). [Figure 1B] The results of PD-L1 binding using FACS are shown. Bispecific antibodies containing heavy chain variable regions with the amino acid sequences shown 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 were compared with a bivalent monospecific antibody containing a PD-L1 binding domain with the amino acid sequence shown in SEQ ID NO: 23, a positive control atezolizumab analog (SEQ ID NOs: 25 / 26), and a negative control antibody (SEQ ID NOs: 29 / 30). The upper graph shows the results for binding to human PD-L1, and the lower graph shows the results for binding to rhesus monkey PD-L1. The X-axis represents the antibody concentration in μg / mL. The Y-axis represents the binding level, expressed as mean fluorescence intensity (MFI). [Figure 2]The results from the PD-1 / LAG-3 reporter assay are shown. Bispecific antibodies containing heavy chain variable regions with the amino acid sequences shown 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 were compared with a positive control atezolizumab analog (SEQ ID NOs: 25 / 26), a positive control relatrimab analog (SEQ ID NOs: 27 / 28), a combination of atezolizumab and relatrimab analog (SEQ ID NOs: 25 / 26 + SEQ ID NOs: 27 / 28), and a negative control antibody (SEQ ID NOs: 29 / 30). The X-axis shows the antibody concentration in μg / mL. The Y-axis shows the induction factor. [Figure 3] The results from the SEB assay from Donor 1 (Figure 3A) and Donor 2 (Figure 3B) are shown. Figures 3A1 and 3B1 show a comparison of the induction ratios of IL-2 for bispecific antibodies containing heavy chain variable regions with the amino acid sequences shown 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 NOs. 25 / 26), a combination of atezolizumab and relatrimab analog (SEQ ID NOs. 25 / 26 + SEQ ID NOs. 27 / 28), and a negative control antibody (SEQ ID NOs. 29 / 30). Figures 3A2 and 3B2 show a comparison of the induction ratios of TNFa for the same antibodies. The X-axis represents the antibody concentration in μg / mL. The Y-axis represents the induction ratio of IL-2 or TNFa. The induction ratio for each antibody was calculated relative to a control well that did not contain IgG. [Figure 4] The binding affinity data for the two bispecific antibodies is shown. [Figure 5]The results for two bispecific antibodies in FACS binding, PD-1 / LAG-3 reporter, and SEB assays are shown. Figure 5A compares the binding of a bispecific antibody containing a heavy chain variable region with the amino acid sequences shown 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 relatrimab analog (SEQ ID NOs. 27 / 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 containing a heavy chain variable region with the amino acid sequences shown in SEQ ID NOs. 17 and 24, and SEQ ID NOs. 5 and 20, to human PD-L1 with the binding of a positive control atezolizumab analog (SEQ ID NOs. 25 / 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 antibody concentration in μg / mL. The Y-axis represents the binding level, expressed as mean fluorescence intensity (MFI). Figure 5E shows the results of the PD-1 / LAG-3 reporter assay, comparing bispecific antibodies containing heavy chain variable regions with the amino acid sequences shown in SEQ ID NOs: 17 and 24, and SEQ ID NOs: 5 and 20, with positive control atezolizumab analogs (SEQ ID NOs: 25 / 26) and relatrimab analogs (SEQ ID NOs: 27 / 28), combinations of relatrimab and atezolizumab analogs (SEQ ID NOs: 27 / 28 + SEQ ID NOs: 25 / 26), and negative control antibodies. Figure 5F shows the results of the SEB assay, comparing the induction ratios of IL-2 in four different donors with those of bispecific antibodies containing heavy chain variable regions with the amino acid sequences shown in SEQ ID NOs. 17 and 24, and SEQ ID NOs. 5 and 20, for positive control atezolizumab analogs (SEQ ID NOs. 25 / 26) and relatrimab analogs (SEQ ID NOs. 27 / 28), a combination of relatrimab and atezolizumab analogs (SEQ ID NOs. 27 / 28 + SEQ ID NOs. 25 / 26), and a negative control antibody. The four graphs represent the results from the four different donors. The X-axis shows the antibody concentration in μg / mL. The Y-axis shows the IL-2 concentration in pg / mL. [Figure 6]Data from in vivo studies are presented. The effect on tumor volume in hu-CD34 NSG® mice with MDA-MB-231 tumors was evaluated for bispecific antibodies containing heavy chain variable regions with amino acid sequences shown in SEQ ID NOs. 17 and 24, and SEQ ID NOs. 5 and 20, and compared with atezolizumab analogs (atezolizumab*), relatrimab analogs (relatrimab*), and combinations of relatrimab and atezolizumab analogs (relatrimab* + atezolizumab*). Figure 6A1 shows data from mice immunized with CD34+HSC from donor 1, Figure 6A2 shows data from mice immunized with CD34+HSC from donor 2, and Figure 6A3 shows data from mice immunized with CD34+HSC from donor 3. Figure 6A4 shows data from all combined donors. Figure 6B shows the results from the analysis of CD4+ and CD8+ T cell populations and regulatory T cell (Treg) populations in MDA-MB-231 tumor samples. The effect on the percentages 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 evaluated for bispecific antibodies containing heavy chain variable regions with the amino acid sequences shown in SEQ ID NOs. 17 and 24, and SEQ ID NOs. 5 and 20, and compared with atezolizumab analogs, relatrimab analogs, and combinations of relatrimab and atezolizumab analogs. [Figure 7] The results of binding assays using FACS are shown. Bispecific antibodies containing heavy chain variable regions with the amino acid sequences shown in Table 10 were tested together with RSV control antibodies (SEQ ID NO: 29 / SEQ ID NO: 30). The X-axis represents the antibody concentration in μg / mL. The Y-axis represents the induction ratio obtained by normalizing the mean MFI value to the mean MFI value of wells without the 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]The results from the PD-1 / LAG-3 reporter assay are shown. Bispecific antibodies containing heavy chain variable regions with the amino acid sequences shown in Table 10 were compared with a combination of atezolizumab and relatrimab 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 antibody concentration in μg / mL. The Y-axis shows the induction factor.

[0230] The following embodiments are illustrative of the disclosure but are not intended to limit it in any way. [Examples]

[0231] Example 1 - Production and identification of LAG-3xPD-L1 bispecific antibody Anti-human LAG-3 binding domains can be obtained by methods known in the art, such as those described in WO2019 / 009728. Large panels of heavy chain variable regions were obtained by immunizing transgenic mice (MeMo® mice) containing a common 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, such as those described in WO2018 / 056821 and WO2019 / 009726. Large panels of heavy chain variable regions were obtained by immunizing transgenic mice (MeMo® mice) containing a common IGKV1-39 light chain with human PD-L1 antigen moieties, including the use of different forms of DNA, protein, and cell-based antigen delivery.

[0233] Numerous anti-human LAG-3 Fabs were selected, and their heavy chain variable regions were used for the production of bispecific anti-LAG-3x anti-PD-L1 antibodies. The amino acid sequences of these heavy chain variable regions are shown 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 showed functional activity in LAG-3 reporter assays (data not shown). Two additional Fabs were selected, and their heavy chain variable regions contained the amino acid sequences shown 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 show functional activity in the LAG-3 reporter and were selected for the production of negative control bispecific antibodies.

[0234] Numerous anti-human PD-L1 Fabs were selected, and their heavy chain variable regions were used for the production of bispecific antibodies. The amino acid sequences of these heavy chain variable regions are shown 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 determined by a bispecific IgG format containing one PD-L1 binding Fab and one other Fab (binding tetanus toxoid (TT) or anti-PD-1) when measured using SPR. Therefore, the binding affinity for monovalent interactions with PD-L1 is measured.

[0235] The affinity of the anti-PD-L1 Fab arm for PD-L1 was determined using surface plasmon resonance (SPR). Affinity was measured in a bispecific IgG format with only one arm specific to PD-L1. Surface plasmon resonance (SPR) using a BIAcore T100 was used to determine the kinetics of anti-PD-L1 Fab arm binding to the antigen. Recombinant, purified, Fc-tagged human PD-L1 (R&D Systems, cat.nr.156-B7-100) was bound to flow cell (FC)2 of a CM5 sensor tip to approximately 200 resonance units (RUs) 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 was used as a blank for subtraction, activated, and then directly inactivated using ethanolamine). Next, bispecific IgG consisting of anti-PD-L1 Fab arms and unrelated Fab arms was run on surfaces FC1 and FC2 at different concentrations (100 nM and sequential 2-fold and 6-fold dilutions in HBS) in a kinetic run at 30 μL / min. Regeneration was performed using pulses 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. Very similar results were obtained in the different measurements, highlighting their validity. All measurements were performed at 25°C.

[0236] The binding affinity is shown in Table 2. [Table 2]

[0237] Bispecific IgG antibodies were 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. CH3 engineering techniques, as described in WO2013 / 157954 and WO2013 / 157953, were used to ensure efficient heterodimerization and the formation of bispecific antibodies. Both vectors further encode a common light chain containing the IGKV1-39 / Jk1 light chain variable region. Cell transfection, cell culture, and antibody collection 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 antibodies were compared with analogues of the anti-LAG-3 antibody relatrimab (relatrimab analogues) containing 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 analogues of the anti-PD-L1 antibody atezolizumab (atezolizumab analogues) containing 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 sequence 26, or combinations thereof.

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

[0240] We selected a number of bispecific antibodies for further screening, including bispecific antibodies containing the following combinations of heavy chain variable regions: [Table 3]

[0241] Bispecific antibodies were produced in Fc-silencing IgG1 format and purified by protein A and gel filtration. Potential homodimer impurities were removed by CIEX. LAG-3 homodimer formation was mitigated by the use of CH3 engineering techniques, reducing its potential impact on functional assays. Monospecific parental antibodies were included in screening assays to compare the activity of the bispecific antibodies to their parental IgG and to determine the sensitivity of functional assays against PD-L1 and LAG-3 monotargeting agents. Monospecific parental antibodies were produced and characterized in a similar manner to the bispecific antibodies.

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

[0243] Bispecific antibodies and parental antibodies were analyzed by an 8-step 5x titration starting at 50 μg / mL. Goat anti-human PE antibody (Invitrogen, H10104) was used as a secondary antibody at a concentration of 3 μg / mL. Relatrimab analog was used as a positive control in all plates containing LAG-3 expressing cells, while atezolizumab analog was used as a positive control in plates containing PD-L1 expressing cells. A 6-step 5x titration of RSV control antibody starting at 50 μg / mL was included as a negative control in all plates. Two wells were used as additional controls, containing only secondary antibodies and unstained cells. As a negative control, IgG was tested at only two maximum concentrations (50 and 10 μg / mL), including cells that do not express the target, 293FF, and CHO-K1 cells.

[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 together with the positive control antibody, the negative control antibody, and its parent LAG-3 binding antibody.

[0245] The positive control antibody relatrimab analog showed inconsistent binding to human LAG-3 across the three plates used in this assay: the degree of binding on the plate containing the LAG-3 arm, including SEQ ID NO: 16, was significantly lower than that observed on the other plates. EC50 values ​​were higher for the bispecific antibody (monovalent to LAG-3) than for the monospecific parent antibody (bivalent to LAG-3). The LAG-3 arms containing SEQ ID NOs: 3 and 5 showed the highest degree of 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 together with the positive control antibody, negative control antibody, and parental PD-L1 binding antibody.

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

[0248] PD-1 / LAG-3 Reporter Assay A PD-1 / LAG-3 reporter assay was 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 a concentration of 100,000 cells per well. Antibody solution was added, followed by Raji cells (25,000 cells per well). T cells were activated by adding partially purified staphylococcal enterotoxin D (ppSED, final concentration 16.6 ng / mL). After incubation at 37°C for 6 hours, Bio-Glo reagent was added, and luciferase reporter gene activity was determined by measuring luminescence on an EnVision plate reader.

[0249] Bispecific IgG was tested in a six-step semi-logarithmic titration starting at 100 μg / mL (final concentration). IgG dilutions were prepared in assay medium at three times the final concentration. As a positive control, a six-step semi-logarithmic titration of a combination of atezolizumab analog and relatrimab analog at a maximum concentration of 50 μg / mL + 50 μg / mL was included in each plate. Atezolizumab analog and relatrimab analog were also tested as monoassets at a starting concentration of 100 μg / mL. As a negative control on each plate, a four-step semi-logarithmic titration of RSV control antibody starting at 100 μg / mL was used. Two wells were left without IgG as a control for baseline activity.

[0250] The results are shown in Figure 2. Bispecific antibodies were compared with each other, with atezolizumab analogs, relatrimab analogs, and combinations of relatrimab analogs and atezolizumab analogs. The area under the curve (AUC) of the antibody response was determined using GraphPad Prism software version 7.02. The AUC is expressed in comparison to the AUC of the positive control.

[0251] Table 5 shows an overview of AUC. [Table 6]

[0252] SEB assay Bispecific antibodies and parental antibodies were analyzed by SEB assay using cells from two donors, PBMC donor 1 and PBMC donor 2. First, PBMCs from these donors were shown to respond to the relatrimab analog of the anti-LAG-3 reference antibody and the atezolizumab analog of the anti-PD-L1 antibody (data not shown). In short, the IgG dilution series was 2x10⁻¹⁰ 5 PBMC cells were treated with 2 μg / mL SEB (final concentration), followed by the addition of IgG. IgG was analyzed in a 7-fold dilution series of 6 steps, starting at 50 μg / mL (final concentration). As a positive control, each plate included a 5-fold titration of a combination of atezolizumab analog and relatrimab analog, starting at 25 μg / mL + 25 μg / mL. As a negative control on each plate, a 4-step semi-logarithmic titration of RSV control antibody, starting at 50 μg / mL, was used. Two wells were left without IgG as a control for baseline activity. PBMCs were cultured for 3 days, after which IL-2 and TNFα levels were measured in the supernatant using 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] The standard curves for cytokine measurement using Luminex were as expected, and the 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 performed as expected for both IL-2 and TNFα readout. The activity of the atezolizumab and relatrimab analog combination was higher for IL-2 readout than that of the atezolizumab analog alone. For both IL-2 and TNFα readout, the LAG-3xPD-L1 bispecific antibody was at least as potent as the atezolizumab and relatrimab analog combination.

[0256] In the second assay (PBMC donor 2), the positive and negative controls performed as expected for IL-2. However, the degree of TNFα induction in the positive control was considerably lower. To reiterate, the activity of the atezolizumab and relatrimab analog combination was higher for IL-2 readout than that of the atezolizumab analog alone. For both readouts, the LAG-3xPD-L1 bispecific antibody was at least as potent as the atezolizumab and relatrimab analog combination. 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 shown in SEQ ID NOs. 5, 15, and 16 showed similar activity to each other and were at least as potent as the atezolizumab and relatrimab analog combination.

[0257] Example 3 - Binding affinity of bispecific antibodies containing further LAG-3 and PD-L1 heavy chain variable regions The binding affinity of a bispecific antibody containing a LAG-3 binding domain having a heavy chain variable region with the amino acid sequence shown in SEQ ID NO: 5 and a PD-L1 binding domain having a heavy chain variable region with the amino acid sequence shown in SEQ ID NO: 20, and a bispecific antibody containing a LAG-3 binding domain having a heavy chain variable region with the amino acid sequence shown in SEQ ID NO: 17 and a PD-L1 binding domain having a heavy chain variable region with the amino acid sequence shown in SEQ ID NO: 24, was determined using SPR technology with 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 bound 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 by NHS / EDC (as recommended by the manufacturer). The bispecific antibody was then injected into a flow cell at a concentration up to 20 nM at a rate of 30 μL / min for 2 minutes. Subsequently, 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 flowed onto the surface of the CM5 sensor chip at 30 μL / min for 2 minutes. Thus, sensorgrams of the association and dissociation phases for different antigens are obtained. The affinity of individual Fab arms 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. The bispecific antibody containing a LAG-3 binding domain with a heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 5 and a PD-L1 binding domain having a heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 20, and the bispecific antibody containing a LAG-3 binding domain with a heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 17 and a PD-L1 binding domain having a heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 24, had a lower binding affinity to human LAG-3 and a higher binding affinity to cynomolgus monkey LAG-3 compared to the relatrimab analog. The bispecific antibody had a similar binding affinity to human PD-L1 and a higher binding affinity to cynomolgus monkey PD-L1 compared to the atezolizumab analog.

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

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

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

[0263] Female hu-CD34 NSG(trademark) mice (JAX stock #705557) that had been transplanted with human CD34+ cells and, 14 weeks after transplantation, possessed more than 25% human CD45+ cells in their peripheral blood within 24 weeks were used. CD34 cells were obtained from three independent donors. + A cohort of hu-CD34 NSG(trademark) mice transplanted with cells was used.

[0264] 120 HSC-NSG mice (72 + surplus) were given 5x10 6Individual MDA-MB-231 human mammary gland cancer cells were transplanted, resuspended in PBS with Matrigel in a 1:1 ratio, and transplanted into breast fat pads. HSC-NSG mice were then treated with human CD34 from umbilical cord blood. + Hematopoietic stem cell (HSC) transplanted immunodeficiency NOD.Cg-Prkdc scid 1l2rg tm1Wjl This is a / SzJ(NSG(trademark)) mouse, which undergoes multilineal differentiation into all major immune cell types.

[0265] Tumor volume (TV) is 50-150 mm 3 In this case, 72 mice were registered 15 days after tumor dissemination. Tumor volume for registration was measured the day before (-1 day). Medication was started on the day of registration, with day 0 of treatment designated. Each treatment arm (Table 1) had mice from 3 HSC donors, with 4 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 final day + and CD8 + For flow cytometry analysis of T cell and regulatory T cell (Treg) populations, terminal tumors were isolated 24 hours after the final dose.

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

[0268] The tumor growth profile is shown in Table 9. [Table 10]

[0269] Figure 6B4 shows that PD-L1 and LAG-3xPD-L1 therapy promotes the proliferation of CD8 + This indicates an increase in the percentage of LAG-3, PD-L1, and LAG-3xPD-L1 therapy. + The percentage of T cells is increased (Figure 6B5). Figure 6B3 shows that treatment with a bispecific antibody results in a lower percentage of Tregs than treatment with a combination of relatrimab and atezolizumab analogs.

[0270] Example 5: Further screening of LAG-3xPD-L1 bispecific antibodies Basically, as described in Example 2, we screened for the bispecific antibodies listed in Table 10. [Table 11]

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

[0272] Bispecific antibodies were analyzed by 8-step 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 a secondary antibody at a concentration of 3 μg / mL. RSV control antibody was included in all plates as a negative control in 8-step 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, containing only secondary antibodies and unstained cells. As negative controls, IgG was tested at three maximum concentrations: 100, 25, and 6.3 μg / mL on 293FF cells and 25, 6.3, and 1.6 μg / mL on CHO-K1 cells, including cells that do not express the target, 293FF, and CHO-K1 cells.

[0273] Results for human and rhesus monkey LAG-3 binding are shown in Figures 7A and 7B. Results for PD-L1 binding are shown in Figure 7C. Induction ratios were obtained by normalizing the mean MFI value to the mean MFI value of the antibody-free well and plotted as a logarithmic function 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 together with the negative control antibody.

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

[0275] PD-1 / LAG-3 Reporter Assay Basically, the PD-1 / LAG-3 reporter assay was performed as described in Example 2. The use of staphylococcal enterotoxin D (ppSED) at a final concentration of 150 ng / mL was verified.

[0276] Bispecific IgG was tested by a six-step semi-logarithmic titration starting at 100 μg / mL (final concentration). IgG dilutions were prepared in assay medium at three times the final concentration. As a positive control, a six-step semi-logarithmic titration of a combination of atezolizumab analog and relatrimab analog with a peak concentration of 50 μg / mL + 50 μg / mL was included in each plate. As a negative control on each plate, a four-step semi-logarithmic titration of RSV control antibody starting at 100 μg / mL was used. Two wells were left without IgG as a control for baseline activity.

[0277] The results are shown in Figure 8. All LAG-3xPD-L1 bispecific antibodies showed activity compared to the combination of relatrimab and atezolizumab analogs. 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 in comparison to the AUC of the combination of relatrimab and atezolizumab analogs. [Table 13]

[0278] array Sequence ID 1 [ka] Sequence ID 2 [ka] Sequence ID 3 [ka] Sequence ID 4 [ka] Sequence ID 5 [ka] Sequence ID 6 [ka] Sequence ID 7 [ka] Sequence ID 8 [ka] Sequence ID 9 [ka] Sequence ID 10 [ka] Sequence ID 11 [ka] Sequence ID 12 [ka] Sequence ID 13 [ka] Sequence ID 14 [ka] Sequence ID 15 [ka] Sequence ID 16 [ka] Sequence ID 17 [ka] Sequence ID 18 [ka] Sequence ID 19 [ka] Sequence ID 20 [ka] Sequence ID 21 [ka] Sequence ID 22 [ka] Sequence ID 23 [ka] Sequence ID 24 [ka] Sequence ID 25 [ka] Sequence ID 26 [ka] Sequence ID 27 [ka] Sequence ID 28 [ka] Sequence ID 29 [ka] LCDR shown according to Sequence ID 30 IMGT [ka] Sequence ID 31: Light chain CDR1 IMGT [ka] Sequence ID 32: Light chain CDR2 IMGT [ka] Sequence ID 33: Light chain CDR3 IMGT [ka] LCDR shown according to Sequence ID 34 IMGT [ka] LCDR shown according to Sequence ID 35 IMGT [ka] LCDR shown according to Sequence ID 36 IMGT [ka] LCDR shown according to Sequence ID 37 IMGT [ka] Sequence ID 38 [ka] Sequence ID 39 [ka] Sequence ID 40 [ka] Sequence ID 41 [ka] Sequence ID 42 [ka] Sequence ID 43 [ka] Sequence ID 44 [ka] Sequence ID 45 [ka] Sequence ID 46 Sequence ID 47 [ka] Sequence ID 48 Sequence ID 49 Sequence ID 50 [ka] Sequence ID 51 [ka] Sequence ID 52 [ka] Sequence ID 53 [ka] Sequence ID 54 [ka] Sequence ID 55 [ka] Sequence ID 56 [ka] Sequence ID 57 [ka] Sequence ID 58 [ka] Sequence ID 59 [ka] Sequence ID 60 [ka] Sequence ID 61 [ka] Sequence ID 62 [ka] Sequence ID 63 [ka] Sequence ID 64 [ka] Sequence ID 65 [ka] Sequence ID 66 [ka] Sequence ID 67 [ka] Sequence ID 68 [ka] Sequence ID 69 [ka] Sequence ID 70 [ka] Sequence ID 71 [ka] Sequence ID 72 [ka] Sequence ID 73 [ka] Sequence ID 74 [ka] Sequence ID 75 [ka] Sequence ID 76 [ka] Sequence ID 77 [ka] Sequence ID 78 [ka] Sequence ID 79 [ka] Sequence ID 80 [ka] Sequence ID 81 [ka] Sequence ID 82 [ka] Sequence ID 83 [ka] Sequence ID 84 [ka] Sequence ID 85 [ka] Sequence ID 86 [ka] Sequence ID 87 [ka] Sequence ID 88 [ka] Sequence ID 89 [ka] Sequence ID 90 [ka] Sequence ID 91 [ka] Sequence ID 92 [ka] Sequence ID 93 [ka] Sequence ID 94 [ka] Sequence ID 95 [ka] Sequence ID 96 [ka] Sequence ID 97 [ka] Sequence ID 98 [ka] Sequence ID 99 [ka] Sequence ID 100 [ka] Sequence ID 101 [ka] Sequence ID 102 [ka] Sequence ID 103 [ka] Sequence ID 104 [ka] Sequence ID 105 [ka] Sequence ID 106 [ka] Sequence ID 107 [ka] Sequence ID 108 [ka] Sequence ID 109 [ka] Sequence ID 110 [ka] Sequence ID 111 [ka] Sequence ID 112 [ka] Sequence ID 113 [ka] Sequence ID 114 [ka] Sequence ID 115 [ka] Sequence ID 116 [ka] Sequence ID 117 [ka] Sequence ID 118 [ka] Sequence ID 119 [ka] Sequence ID 120 [ka] Sequence ID 121 [ka] Sequence ID 122 [ka] Sequence ID 123 [ka] Sequence ID 124 [ka] Sequence ID 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 PD-L1, 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, The PD-L1 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. 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, wherein the LAG-3 binding domain and the PD-L1 binding domain each include a light chain variable region comprising light chain CDR1 (LCDR1), light chain CDR2 (LCDR2), and light chain CDR3 (LCDR3), each having the amino acid sequences shown in SEQ ID NO: 31, SEQ ID NO: 32, and SEQ ID NO: 33, respectively.

2. The multispecific antibody or variant thereof according to claim 1, wherein the LAG-3 binding domain includes a heavy chain variable region having the amino acid sequence shown in any one of SEQ ID NOs. 52 to 59.

3. The multispecific antibody or variant thereof according to claim 1 or 2, wherein the LAG-3 binding domain includes a light chain variable region having the amino acid sequence shown in SEQ ID NO:

30.

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

5. The multispecific antibody or variant thereof according to any one of claims 1 to 4, wherein the antibody or variant is monovalent for binding to LAG-3.

6. A multispecific antibody or variant thereof according to any one of claims 1 to 5, wherein a variable domain that specifically binds to the extracellular domain of PD-L1 blocks the binding of PD-L1 to its receptor, and the receptor is PD-1 and / or CD80.

7. The multispecific antibody or variant thereof according to any one of claims 1 to 6, wherein the PD-L1 binding domain includes a heavy chain variable region having the amino acid sequence shown in any one of SEQ ID NOs: 20 to 24.

8. The multispecific antibody or variant thereof according to claim 7, wherein the PD-L1 binding domain includes a heavy chain variable region having the amino acid sequence shown in either SEQ ID NO: 20 or SEQ ID NO:

24.

9. The multispecific antibody or variant thereof according to any one of claims 1 to 8, wherein the PD-L1 binding domain includes a light chain variable region having the amino acid sequence shown in SEQ ID NO:

30.

10. The multispecific antibody or variant thereof according to any one of claims 1 to 9, wherein the PD-L1 binding domain further comprises CH1 and CL regions.

11. The multispecific antibody or variant thereof according to any one of claims 1 to 10, wherein the antibody or variant is monovalent for binding to PD-L1.

12. 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 PD-L1, The LAG-3 binding domain includes a heavy chain variable region having the amino acid sequence shown in Sequence ID No. 52, which includes heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3). The binding domain that binds to PD-L1 includes a heavy chain variable region having the amino acid sequence shown in Sequence ID No. 20, which includes heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3). A multispecific antibody or variant thereof, wherein the LAG-3 binding domain and the PD-L1 binding domain each include a light chain variable region comprising light chain CDR1 (LCDR1), light chain CDR2 (LCDR2), and light chain CDR3 (LCDR3), each having the amino acid sequences shown in SEQ ID NO: 31, SEQ ID NO: 32, and SEQ ID NO: 33, respectively.

13. The LAG-3 binding domain includes a heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 52, The PD-L1 binding domain includes a heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 20, The multispecific antibody or variant thereof according to claim 12, wherein the LAG-3 binding domain and the PD-L1 binding domain include a light chain variable region having the amino acid sequence shown in SEQ ID NO:

30.

14. 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 PD-L1, The LAG-3 binding domain includes a heavy chain variable region having the amino acid sequence shown in Sequence ID No. 52, which includes heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3). The binding domain that binds to PD-L1 includes a heavy chain variable region having the amino acid sequence shown in Sequence ID No. 24, which includes heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3). A multispecific antibody or variant thereof, wherein the LAG-3 binding domain and the PD-L1 binding domain each include a light chain variable region comprising light chain CDR1 (LCDR1), light chain CDR2 (LCDR2), and light chain CDR3 (LCDR3), each having the amino acid sequences shown in SEQ ID NO: 31, SEQ ID NO: 32, and SEQ ID NO: 33, respectively.

15. The LAG-3 binding domain includes a heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 52, The PD-L1 binding domain includes a heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 24, The multispecific antibody or variant thereof according to claim 14, wherein the LAG-3 binding domain and the PD-L1 binding domain include a light chain variable region having the amino acid sequence shown in SEQ ID NO:

30.

16. 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 PD-L1, 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. 53, 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. 54, 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. 55, 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. 56, 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. 57, 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. 58, 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. 59 Includes a heavy chain variable region, The binding domain that binds to PD-L1 includes a heavy chain variable region having the amino acid sequence shown in Sequence ID No. 20, which includes heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3). A multispecific antibody or variant thereof, wherein the LAG-3 binding domain and the PD-L1 binding domain each include a light chain variable region comprising light chain CDR1 (LCDR1), light chain CDR2 (LCDR2), and light chain CDR3 (LCDR3), each having the amino acid sequences shown in SEQ ID NO: 31, SEQ ID NO: 32, and SEQ ID NO: 33, respectively.

17. The LAG-3 binding domain includes a heavy chain variable region having the amino acid sequence shown in any one of SEQ ID NOs: 53 to 59. The PD-L1 binding domain includes a heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 20, The multispecific antibody or variant thereof according to claim 16, wherein the LAG-3 binding domain and the PD-L1 binding domain include a light chain variable region having the amino acid sequence shown in SEQ ID NO:

30.

18. 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 PD-L1, 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. 53, 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. 54, 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. 55, 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. 56, 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. 57, 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. 58, 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. 59 Includes a heavy chain variable region, The binding domain that binds to PD-L1 includes a heavy chain variable region having the amino acid sequence shown in Sequence ID No. 24, which includes heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3). A multispecific antibody or variant thereof, wherein the LAG-3 binding domain and the PD-L1 binding domain each include a light chain variable region comprising light chain CDR1 (LCDR1), light chain CDR2 (LCDR2), and light chain CDR3 (LCDR3), each having the amino acid sequences shown in SEQ ID NO: 31, SEQ ID NO: 32, and SEQ ID NO: 33, respectively.

19. The LAG-3 binding domain includes a heavy chain variable region having the amino acid sequence shown in any one of SEQ ID NOs: 53 to 59. The PD-L1 binding domain includes a heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 24, The multispecific antibody or variant thereof according to claim 18, wherein the LAG-3 binding domain and the PD-L1 binding domain include a light chain variable region having the amino acid sequence shown in SEQ ID NO:

30.

20. The multispecific antibody or variant thereof according to any one of claims 12 to 19, wherein the LAG-3 binding domain and the PD-L1 binding domain further comprise CH1 and CL regions.

21. The multispecific antibody or variant thereof according to any one of claims 12 to 20, wherein the antibody or variant thereof is monovalent for binding to LAG-3 and monovalent for binding to PD-L1.

22. The multispecific antibody or variant thereof according to any one of claims 1 to 21, wherein the antibody or variant thereof is a full-length antibody or a full-length bispecific antibody.

23. A pharmaceutical composition comprising a multispecific antibody or a variant thereof according to any one of claims 1 to 22, and a pharmaceutically acceptable carrier, diluent, or excipient.

24. A multispecific antibody or variant thereof according to any one of claims 1 to 22, or a pharmaceutical composition according to claim 23, for use as a pharmaceutical.

25. A multispecific antibody or variant thereof according to any one of claims 1 to 22, or a pharmaceutical composition according to claim 23, for use in the treatment of diseases associated with a suppressed immune system, or for use in the treatment of cancer.

26. A vector comprising a nucleic acid sequence encoding the heavy chain variable region of a LAG-3 binding domain as defined in any one of claims 1 to 5, a nucleic acid sequence encoding the heavy chain variable region of a PD-L1 binding domain as defined in claim 1 or any one of claims 7 to 11, and a nucleic acid sequence encoding the light chain variable region as defined in any one of claims 1, 3, or 9.

27. The vector according to claim 26, wherein the vector further comprises nucleic acid sequences encoding a CH1 region, a hinge, a CH2 region, and a CH3 region.

28. A cell comprising a nucleic acid sequence encoding the heavy chain variable region of a LAG-3 binding domain as defined in any one of claims 1 to 5, a nucleic acid sequence encoding the heavy chain variable region of a PD-L1 binding domain as defined in claim 1 or any one of claims 7 to 11, and a nucleic acid sequence encoding the light chain variable region as defined in any one of claims 1, 3, or 9.

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

30. Cells that produce a multispecific antibody or a variant thereof according to any one of claims 1 to 22, and which are transformed with a vector or combination of vectors according to any one of claims 26 to 27.

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