RU2784388C2 - Binding molecules, binding pd-l1 and lag-3 - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: antibody molecule binding to programmed cell death protein ligand-1 (hereinafter – PD-L1) and lymphocyte activation genome 3 (hereinafter – LAG-3) are proposed. The antibody molecule contains a site of binding PD-L1 based on CDR and a site of binding LAG-3, which is located in loops AB and EF of CH3-domain of the antibody molecule. The invention also relates to nucleic acid encoding the antibody, an expression vector and a recombinant host cell, and a method for the production of the antibody. In addition, a pharmaceutical composition including the specified antibody is proposed.

EFFECT: antibody molecules, according to the present invention, can be used in the treatment of cancer.

36 cl, 16 dwg, 17 tbl, 6 ex

Description

Related Applications

This application is related to US 62/352482, filed June 20, 2016, the contents of which are incorporated herein by reference in their entirety.

Field of invention

The present invention relates to antibody molecules that bind to programmed cell death protein ligand-1 (PD-L1) and lymphocyte activation gene 3 (LAG-3). Antibody molecules preferably contain a CDR-based PD-L1 antigen binding site (antigen-binding site) and a CDR-based LAG-3 antigen binding site, which may be located in two or more structural loops of the CH3 domain of the antibody molecule. The antibody molecules of the present invention find use, for example, in the treatment of cancer.

State of the art

The lymphocyte activation gene-3 (LAG-3; CD223) is a member of the Ig superfamily and is genetically and structurally related to CD4 (albeit with only 20% sequence identity). Like CD4, LAG-3 binds to class II MHC molecules, but with a higher affinity than CD4 (K _D = 60 nM). LAG-3 is expressed on activated T cells, NK cells, pDCs, B cells, γδ T cells, and is involved in immunosuppression, in particular, due to sustained intensive expression on a certain proportion of regulatory T cells (Treg) (Liang et al, 2008).

The LAG-3 gene is located on the 12th human chromosome, next to the CD4 gene, and includes 8 exons. There are five alternative transcripts, two of which form protein products: a full-length transmembrane protein and a soluble monomeric form resulting from alternative splicing. The full length transcript encodes a 70 kDa 525 amino acid protein with functional activity, while the soluble form does not appear to bind MHC class II molecules and its function is unknown. The full-length human LAG-3 protein has 93% sequence identity to Macaca fascicularis (cynomolgus macaque) LAG-3 and 70% sequence identity to Mus musculus (house mouse) LAG-3.

LAG-3 is a transmembrane protein with four extracellular Ig-like domains (D1-D4) and a cytoplasmic fragment responsible for the LAG-3 signaling pathway. The cytoplasmic domain contains an EP motif (glutamic acid/proline) binding to LAG-3-associated protein (LAP), as well as a KIEELE motif, which is considered necessary for the modulation of T-cell function by LAG-3. Reports on the role of the EP motif indicate that it may be responsible for transporting LAG-3 to the T cell membrane (Bae et al, 2014) or directly modulating further STAT5 signaling during T cell activation (Durham et al, 2014) , or possibly for both of these processes.

The mechanism of T cell immunosuppression by LAG-3 is believed to be mediated by LAG-3 cross-linking on activated T cells, resulting in decreased calcium flux and IL-2 release upon T cell activation (Huard et al, 1997) . On antigen-presenting cells (APCs), binding of MHC II molecules by LAG-3-positive regulatory T cells causes a decrease in IL-12 secretion and suppression of CD86 function (Liang et al, 2008), a “secondary signal” of activation, leading to insensitivity of T -cells due to improper activation and/or reduced APC antigen presentation. LAG-3 knockout mouse models are viable and show only mild lymphohyperproliferation (Workman et al, 2003), indicating that LAG-3 acts as a mild brake on the immune system.

It is hypothesized that this suppressive interaction between LAG-3 and MHC class II also occurs between Tregs and CD4-positive T cells (Sega et al, 2014). Tregs suppress the immune response by releasing suppressive cytokines (eg, IL-10 and TGFβ), manipulating the inflammatory mechanism (eg, adenosine catabolized by CD73), regulating APC maturation, or by direct interaction between regulatory T cells and effector T cells. There is evidence that human Tregs positive for MHC class II have improved suppressive functions compared to Tregs negative for MHC class II (Baecher-Allen et al, 2006) and actively suppress the immune response through direct interaction with LAG-3 expressed on effector T cells. Although LAG-3-negative Tregs can suppress normal T cell proliferation, LAG-3-negative CD4 and CD8 T cells are resistant to Treg immunosuppression. This process has been described to occur between T cells through a process known as trogocytosis (Sega et al, 2014), in which Tregs not only prevent APC maturation, but also produce class II MHC by suppressing primed LAG-3-positive CD4 T -cells.

Expression of LAG-3 is also a marker of antigenic restimulation. In cancer, T cells often develop a “wasted” phenotype involving the expression of immunosuppressive agents such as PD-1, CTLA-4, TIM-3, and LAG-3 (Wherry et al, 2011), in which the cells as a whole cannot properly proliferate and secrete chemokines in response to an antigen. Inhibition of these immunosuppressive agents lowers the immune threshold and (re)activates the proper antitumor T cell response. This was confirmed in preclinical models using anti-LAG-3, CTLA-4 and PD-1 antagonist antibodies, in which a reduction in tumor burden was observed. Inhibition of LAG-3 by antagonistic antibodies is believed to reactivate the immune response in the tumor microenvironment, where LAG-3 expression on CD4-positive T cells and CD8-positive T cells is associated with a depleted phenotype, and LAG-3 expression on Treg is associated with potency. immunosuppression. Antibodies that block LAG-3 increase the proliferation of effector T cells, cytokine production, cytotoxicity, and also reduce the suppressor activity of Treg, which leads to a weakening of tumor growth.

In human tumors, LAG-3 overexpression has been found on tumor-infiltrating lymphocytes (TILs) from clear cell carcinomas and other human tumors, such as melanomas and lymphomas (Demeure et al, 2001; Wolchock et al, 2013). Importantly, LAG-3 also correlates with T cell dysfunction in patients with chronic viral infection (Workman et al, 2005) and cancer (Workman et al, 2003). In addition, LAG-3 has been identified as a surface marker for tumor-infiltrating Tregs in various types of cancer (Camisachi et al. 2010; Gandhi et al, 2006).

Monoclonal antibodies to human LAG-3 are being clinically developed to eliminate immunosuppression and potentially enhance antigen presentation in cancer (solid and hematological malignancies).

LAG-525 and IMP-701 (Novartis AG) are human anti-LAG-3 antibodies; their trials have progressed to Phase II and Phase I clinical trials, respectively, in kidney cancer (clear cell carcinoma); non-small cell lung cancer (NSCLC); nasopharyngeal cancer; cancer of the colon and rectum; melanoma; gastric cancer and adenocarcinoma of the gastroesophageal junction.

Anti-LAG-3 antibody BMS-986016 (Bristol-Myers Squibb Company) is in phase I clinical trials in ovarian cancer; NSCLC; cancer of the colon and rectum; cervical cancer; melanoma; stomach cancer; bladder cancer; squamous cell carcinoma of the head and neck; clear cell carcinoma, as well as phase II studies in NSCLC; recurrent chronic lymphocytic leukemia (CLL); persistent chronic lymphocytic leukemia (CLL); melanoma; non-Hodgkin's lymphoma; Hodgkin's lymphoma; diffuse large B-cell lymphoma; slow growing lymphoma; mantle cell lymphoma; persistent multiple myeloma; and relapsing multiple myeloma as both monotherapy and a component of combination therapy.

Additional anti-LAG-3 antibodies are also in preclinical development.

Programmed cell death protein-1 (PD-1) and its ligands PD-L1 (CD274, B7-H1) and PD-L2 (B7-DC) mediate the transmission of inhibitory signals that regulate the balance between T-cell activation, tolerance, and pathology of the immune system. systems. PD-L1 is transiently expressed on all immune cells and some tumor cells.

PD-L1 is a type I transmembrane protein containing two Ig-like domains with an extracellular region, a transmembrane domain and a short cytoplasmic domain. The cytoplasmic domain does not contain a signal transduction motif, indicating the absence of signal transduction from PD-L1 when this ligand interacts with its receptor. It has a molecular weight of 40 kDa (290 amino acids) and is encoded by the CD274 gene on mouse chromosome 19 and human chromosome 9, respectively. PD-L1 is a member of the B7 protein family and has approximately 20% amino acid sequence identity to B7.1 and B7.2. Human PD-L1 has 70% and 93% amino acid sequence identity to mouse and cynomolgus PD-L1 orthologues, respectively.

PD-L1 binds to its PD-1 receptor with an affinity (K _D ) of 770 nM. PD-1 is expressed on activated T cells, B cells, and myeloid cells and modulates the activation or inhibition of the cellular immune response. Binding of PD-L1 to PD-1 provides inhibitory signal transduction, reduced cytokine production and T-cell proliferation. In addition, PD-L1 expression by cells may mediate protection from killing by cytotoxic T-lymphocytes (CTL) and is a regulatory mechanism that attenuates chronic immune responses during viral infections. Cancer, as a chronic pro-inflammatory disease, causes disruption of this immune system defense pathway by stimulating PD-L1 expression, leading to avoidance of the host's immune response. In the context of an active immune response, IFNγ also stimulates PD-L1 expression.

PD-L1 also mediates immunosuppression by interacting with another protein, B7.1 (also known as CD80), blocking its ability to mediate one of the secondary T cell activation signals via CD28. The significance of this specific interaction for tumor resistance to immune response in terms of PD-L1 expression on tumor cells and its interaction with B7.1 is still unclear.

PD-L1 expression has been demonstrated in a variety of solid tumors. Eighty-nine of 654 (14%) specimens studied in one study covering 19 tumors from different areas of the body were PD-L1 positive (incidence ≥5%). The maximum frequency of PD-L1-positive cells was observed in head and neck cancer (17/54; 31%), cervical cancer (10/34; 29%), unknown primary origin (CUP; 8/29; 28%), glioblastoma multiforme (GBM; 5/20; 25%), bladder (8/37; 21%), esophageal (16/80; 20%), triple negative (TN) breast cancer (6/33; 18%), and hepatocarcinoma (6/41; 15%) (Grosso et al, 2013). It has been shown that tumor-associated expression of PD-L1 confers tumor resistance to the immune response and possibly protects tumor cells from T-cell mediated apoptosis.

Therapies targeting PD-L1 have shown excellent results in in vivo studies in mice. In a B16 melanoma mouse model, treatment with PD-L1 antagonists in combination with the GVAX or FVAX vaccination strategies resulted in a significant effect on both survival (30 days for control animals versus 52 days for PD-L1 treatment) and percentage of animals. tumor-free (5%) at study completion (Curran et al, 2010). In addition, a PD-L1 antagonist therapeutic agent is being used to investigate the mechanism of immunosuppression in the P815 mouse model of mastoma. P815 cells introduced into mice normally initiate a powerful immune response that leads to their rejection. When PD-L1 is expressed on P815 cells, these cells avoid attack by the immune system, which, in turn, is neutralized by the introduction of antibodies against PD-L1 (Iwai et al, 2002). It appears that targeting the PD-1/PD-L1 axis in immunogenic human malignancies axis (Herbst et at, 2014) leads to a beneficial effect on survival by stimulating the anti-cancer immune response (Wolchock et al, 2013; Larkin et al, 2015).

Atezolizumab (MPDL3280A, RG7466, TECENTRIQ) is a humanized IgG1 antibody that binds PD-L1. It is used in clinical trials as a single agent and also in combination with other biological and/or small molecular weight therapeutic agents for the treatment of solid tumors, including colon and rectal cancer, breast cancer, non-small cell lung carcinoma, bladder cancer, and renal cell carcinoma. carcinomas. Treatment with atezolizumab results in an objective response rate (ORR) of 23% for NSCLC, 36% for melanoma, 33% for bladder cancer, 14% for renal cell carcinoma, and 13% for head and neck cancer (Herbst et al, 2014 ; Powles et al, 2014).

Avelumab (MSB0010718C) is a fully human IgG1 antibody that binds to PD-L1 and is being clinically studied in a range of cancers, including bladder cancer, gastric cancer, head and neck cancer, mesothelioma, non-small cell lung carcinoma, ovarian cancer, kidney cancer and Merkel cell carcinoma.

Durvalumumab (MEDI4736) is a human IgG1 antibody that binds to PD-L1 and is being tested in clinical trials alone or in combination with tremelimumab in non-small cell lung cancer, head and neck squamous cell carcinoma, bladder cancer, pancreatic cancer, and other biological and/or or small molecular weight agents in studies of additional solid malignant tumors, for example, gastric cancer, melanoma and inoperable hepatocellular carcinoma.

Additional anti-PD-L1 antibodies are being tested in clinical studies, including BMS-936559 and other antibodies in preclinical studies.

However, only a small number of LAG-3 therapeutics are currently in clinical trials, and none are approved for therapeutic use; thus, there remains a need to develop additional molecules that target LAG-3. Although several anti-PD-L1 therapies are currently being developed, current data indicate that monotherapy with PD-L1 antagonists results in a response in less than 50% of cancer patients. Thus, there remains a need in the art for additional molecules that can target LAG-3 and/or PD-L1 and which find use in the treatment of cancer.

The essence of the invention

Anti-PD-1 and anti-PD-L1 antibodies are predominantly involved in the violation of immune tolerance and activation of the antitumor immune response. LAG-3 is expressed on T cells after activation and is also expressed constitutively on depleted T cells, helping to keep these cells in a state of suppression. In addition, blockade of LAG-3 in combination with other known immunosuppressive molecules (i.e., PD-1, PD-L1) has been shown to provide a synergistic enhanced immune response in mouse tumor models (Woo et al, 2012). The present inventors hypothesized that therapeutics that simultaneously target both of these pathways would directly influence the mechanisms that promote and maintain T cell depletion. In addition, the authors of the present invention expect that the targeting of LAG-3 can suppress antigen presentation through the action of LAG-3-expressing regulatory T cells on APC; a published study documented CD86 suppression (Grosso et al, 2013). It is expected that blocking this interaction will maintain antigen presentation, while blocking the PD-L1 signaling pathway will break tolerance, leading to a significant antitumor response while simultaneously inhibiting both pathways.

The amount of published data on combinations of antibodies against LAG-3 and PD-L1 is limited, although there are some results of preclinical studies in syngeneic models of tumor and viral load in mice. In a mouse myeloma model, a combination of blocking antibodies against PD-L1 and LAG-3 was administered after low-dose whole-body irradiation and improved survival to over 80% (Jing et al, 2015). No signs of systemic or organ-specific autoimmunity were observed. LAG-3 and PD-1 knockout mice showed significantly improved multi-tumor transplant survival and eradication (Woo et al, 2012).

The authors of the present invention suggested that bispecific antibodies that bind to both LAG-3 and PD-L1 will have a number of advantages over the combination of monoclonal antibodies against these antigens, including:

1. Targeted Therapy

Activated T cells express LAG-3 in lymph nodes. One part of the LAG-3/PD-L1 bispecific antibody targets primed LAG-3 positive T cells in the lymph nodes, which then travel to the tumor site to transport the bispecific antibody. Once in the tumor microenvironment, T cells bearing the bispecific antibody can immediately interact with PD-L1 on tumor cells via their anti-PD-L1 moiety and block it. Accordingly, all T cells that have migrated to the tumor region will be resistant to the LAG-3 and PD-L1/PD-1 signaling pathways.

2. Bridge formation

Primed CD8-positive T cells contact tumor antigens in the tumor microenvironment, where they generate a response by killing tumor cells in the absence of suppressive signals. It is expected that bispecific antibodies will be superior to combinations of single therapeutic monoclonal antibodies in terms of maintaining and prolonging this contact between T cells and tumor cells. Signal strength in T cell activation is important and may be a key factor in antigen presentation in cancer (Engels et al, 2013); it is expected that the presence of a bispecific anti-LAG-3/PD-L1 antibody associated with targets on APCs or cancer cells will increase the time during which T cells can successfully recognize the antigen and become activated.

3. Localization

In areas of inflammation and ongoing immune response, PD-L1 expression is significantly upregulated due to localized release of IFN-γ. This is true for cancer target cells, tumor-associated macrophages (TAMs), or restimulation of T cell populations. It is expected that a bispecific antibody with antagonistic activity against PD-L1 and LAG-3 will be localized and concentrated in areas with maximum expression of PD-L1 in tumors, which will allow their part against LAG-3 to bind and prevent LAG-3- mediated T-cell suppression.

After an extensive screening and affinity maturation program, the present inventors were able to identify ten specific binding partners containing a LAG-3 specific binding site in the CH3 domain of the molecule. These molecules were shown to have high affinity for both human and cynomolgus monkey LAG-3. The high affinity for human LAG-3 is expected to be useful in the treatment of, for example, cancers containing LAG-3-expressing tumor-infiltrating lymphocytes (TILs) in human patients, while the high affinity for Javanese LAG-3 macaque comparable to an affinity for human LAG-3 would be useful in evaluating the properties of specific binding partners in cynomolgus macaque disease models. The rationale for this is that the results obtained are more likely to predict specific binding partner activity in human patients than for molecules with higher affinity variability for human and cynomolgus macaque LAG-3 when tested in cynomolgus monkey models.

In addition, specific binding partners were shown to be highly active in the T cell activation assay, which was expected to be predictive of improved performance in human patients due to enhanced LAG-3 inhibition.

The present inventors further combined these specific binding partners containing a binding site specific for LAG-3 in the CH3 domain of an antibody Fab domain containing a CDR-based PD-L1 antigen binding site to obtain bispecific antibody molecules, containing both LAG-3 and PD-L1 binding sites, which are expected to have the advantages described above. The present inventors have also generated surrogate variants of these mouse-derived antibody molecules that bind to mouse LAG-3 and mouse PD-L1; it was shown that they could significantly inhibit tumor growth in syngeneic mouse models of cancer. In particular, the use of these mouse-derived surrogate molecules has demonstrated a synergistic effect with respect to tumor growth suppression when an antibody molecule containing both LAG-3 and PD-L1 binding sites is administered to mice in tested models. Based on the similar mechanism of action of mouse and human LAG-3 and PD-L1 in the tumor environment, it is expected that mouse studies demonstrating efficacy in reducing tumor burden can be translated into a clinical therapeutic benefit for human cancer patients. In addition, based on these results, it is expected that the antibody molecules of the present invention will show a better effect in the treatment of cancer in human patients, in particular with respect to the suppression of tumor growth, than, for example, the introduction of two separate molecules that bind to LAG- 3 and PD-L1, respectively.

Thus, in a first aspect of the present invention, an antibody molecule that binds to both PD-L1 and LAG-3 is provided. More specifically, these antibodies contain:

(i) a CDR-based antigen-binding site for PD-L1; and

(ii) a LAG-3 antigen binding site located or constructed in two or more structural loops of the CH3 domain of the antibody molecule. The LAG-3 binding site preferably contains the amino acid sequences WDEPWGED (SEQ ID NO: 1) and PYDRWVWPDE. (SEQ ID NO: 3).

The antibody molecule preferably contains the amino acid sequence shown in SEQ ID NO: 1 in the AB loop and the amino acid sequence shown in SEQ ID NO: 3 in the EF loop of the CH3 domain.

Thus, in a first aspect of the present invention, there is provided an antibody molecule that binds to programmed cell death protein ligand-1 (PD-L1) and lymphocyte activation gene 3 (LAG-3). The antibody molecule preferably contains (i) a CDR-based antigen-binding site for PD-L1; and (ii) a LAG-3 antigen binding site located in the CH3 domain of the antibody molecule.

The LAG-3 binding site preferably contains the amino acid sequences WDEPWGED (SEQ ID NO: 1) and PYDRWVWPDE (SEQ ID NO: 3). The amino acid sequence WDEPWGED is preferably located in the first structural loop of the CH3 domain, and the amino acid sequence PYDRWVWPDE is preferably located in the second structural loop of the CH3 domain.

For example, the LAG-3 antigen binding site may be located in the structural loop region of the CH3 domain of the antibody molecule, wherein said structural loop region preferably contains two or more structural loops, and the LAG-3 binding site preferably contains the amino acid sequences WDEPWGED (SEQ ID NO: 1 ) and PYDRWVWPDE (SEQ ID NO: 3).

As a further example, a LAG-3 antigen binding site can be constructed in two or more structural loops of the CH3 domain of an antibody molecule, with the LAG-3 binding site preferably containing the amino acid sequences WDEPWGED (SEQ ID NO: 1) and PYDRWVWPDE (SEQ ID NO: 3).

As indicated above, the LAG-3 binding site sequences are preferably located in two or more structural loops of the CH3 domain of an antibody molecule. In a preferred embodiment, the LAG-3 antigen binding site comprises the amino acid sequence shown in SEQ ID NO: 1 in loop AB and the amino acid sequence shown in SEQ ID NO: 3 in loop EF of the CH3 domain.

The amino acid sequence shown in SEQ ID NO: 1 is preferably located in the region of residues 11-18 of the CH3 domain; and/or the amino acid sequence shown in SEQ ID NO: 3 is preferably located in the region of residues 92-101 of the CH3 domain; with the numbering of amino acid residues following the ImMunoGeneTics IMGT numbering scheme).

The LAG-3 antigen binding site of the antibody molecule may further comprise one of the following sequences, preferably in the CD loop of the CH3 domain of the antibody molecule:

(i) SNGQPENNY (SEQ ID NOS 2, 8 and 18);

(ii) SNGQPEDNY (SEQ ID NO: 13);

(iii) SNGYPEIEF (SEQ ID NO: 23);

(iv) SNGIPEWNY (SEQ ID NO: 28);

(v) SNGYAEYNY (SEQ ID NO: 33);

(vi) SNGYKEENY (SEQ ID NO: 38);

(vii) SNGVPELNV (SEQ ID NO: 43); or

(viii) SNGYQEDNY (SEQ ID NO: 48).

The LAG-3 antigen binding site of the antibody molecule further preferably comprises one of the following sequences, preferably in the CD loop of the CH3 domain of the antibody molecule: the amino acid sequence shown in SEQ ID NO: 2, 28 or 38 in the CD loop of the CH3 domain. More preferably, the LAG-3 antigen binding site of the antibody molecule contains the amino acid sequence shown in SEQ ID NO: 2 in the CD loop of the CH3 domain.

The amino acid sequence shown in SEQ ID NOs: 2, 8, 13, 18, 23, 28, 33, 38, 43, or 48 is preferably located in the region of residues 43-78 of the CH3 domain of the antibody molecule, with residue numbering following the IMGT numbering scheme .

The sequence of the CH3 domain of an antibody molecule, with the exception of the sequences of the LAG-3 antigen binding site, is not particularly limited. The CH3 domain is preferably a human immunoglobulin G domain, eg the CH3 domain of human IgG1, IgG2, IgG3 or IgG4, most preferably the CH3 domain of human IgG1. The sequences of the CH3 domains of human IgG1, IgG2, IgG3, or IgG4 are known in the art.

In a preferred embodiment, the antibody molecule contains the CH3 domain shown in SEQ ID NO: 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50, more preferably the CH3 domain shown in SEQ ID NO: 5, 30 or 40, most preferably the CH3 domain shown in SEQ ID NO: 5. Alternatively, the antibody molecule may contain a CH3 domain with an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with respect to to SEQ ID NO: 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50, preferably SEQ ID NO: 5, 30 or 40, more preferably SEQ ID NO: 5.

The antibody molecule may further comprise a CH2 domain. The CH2 domain is preferably located at the N-terminus of the CH3 domain, as in the case of the human IgG molecule. The CH2 domain of the antibody molecule is preferably the CH2 domain of human IgG1, IgG2, IgG3 or IgG4, more preferably the CH2 domain of human IgG1. Human IgG domain sequences are known in the art. In a preferred embodiment, the antibody molecule contains an IgG CH2 domain with the sequence shown in SEQ ID NO: 53 or SEQ ID NO: 54, or a CH2 domain with an amino acid sequence having at least 70%, at least 75%, of at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with respect to to SEQ ID NO: 53 or SEQ ID NO: 54.

In a preferred embodiment, the antibody molecule contains the sequence shown in SEQ ID NOs: 6, 7, 11, 12, 16, 17, 21, 22, 26, 27, 31, 32, 36, 37, 41, 42, 46, 47 , 51 or 52, or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96% , at least 97%, at least 98%, or at least 99% sequence identity relative to the sequence shown in SEQ ID NO: 6, 7, 11, 12, 16, 17, 21, 22, 26, 27 , 31, 32, 36, 37, 41, 42, 46, 47, 51 or 52. More preferably, the antibody molecule contains the sequence shown in SEQ ID NO: 6, 7, 31, 32, 41 or 42, or the sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity ovality with respect to the sequence shown in SEQ ID NO: 6, 7, 31, 32, 41 or 42. Even more preferably, the antibody molecule contains the sequence shown in SEQ ID NO: 6 or 7, or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity with respect to the sequence shown in SEQ ID NO: 6 or 7.

In a preferred embodiment, the antibody molecule is a human immunoglobulin G molecule, eg a human IgG1, IgG2, IgG3 or IgG4 molecule, more preferably a human IgG1 molecule. The sequences of human immunoglobulin G molecules are known in the art, and the insertion of a CH3 domain or a CH3 domain sequence described herein into such a molecule would not be difficult for one skilled in the art.

The antibody molecule preferably contains the CDRs of the VH and/or VL domains shown in SEQ ID NOs: 92 and 93. Methods for determining the sequence of CDRs in a given VH or VL domain are known in the art and include the Kabat and IMGT numbering systems. More preferably, the antibody molecule preferably contains one or more, two or more, three or more, four or more, five or more, or all six complementarity determining regions set forth in SEQ ID NOs 86-91. The antibody molecule preferably contains the VH and/or VL domains shown in SEQ ID NOs: 92 and 93, respectively.

In a preferred embodiment, the antibody molecule comprises a heavy chain sequence as set forth in any one of SEQ ID NOs: 94-113 or a heavy chain with an amino acid sequence having at least 70%, at least 75%, at least 80% of at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any of SEQ ID NO : 94-113, provided that the VH domain of the heavy chain sequence remains unchanged. More preferably, the antibody molecule contains a heavy chain sequence shown in any of SEQ ID NOs: 94, 95, 104, 105, 108, and 109, or a heavy chain with an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to to any of SEQ ID NOs: 94, 95, 104, 105, 108 and 109, provided that the VH domain of the heavy chain sequence remains unchanged. Even more preferably, the antibody molecule contains the heavy chain sequence shown in SEQ ID NO: 94 or 95, or a heavy chain with an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 94 or 95 , provided that the VH domain of the heavy chain sequence remains unchanged.

In a preferred embodiment, the antibody molecule additionally or alternatively comprises a light chain sequence as set forth in SEQ ID NO: 116 or a light chain with an amino acid sequence having at least 70%, at least 75%, at least 80% at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 116, provided that the VL domain of the light chain sequence remains unchanged.

The antibody molecule can preferably simultaneously bind to PD-L1 and LAG-3. PD-L1 and LAG-3 can be, for example, on two different cells. Regardless of theory, it is believed that the results of cross-linking between cells and the internalization of PD-L1 and/or LAG-3 make them unavailable for stimulation.

The present inventors have shown that an antibody molecule comprising (i) an antigen-binding site based on the CDR for PD-L1; and (ii) the LAG-3 antigen binding site located in the CH3 domain of the antibody molecule of the present invention, FS18-7-9/84G09, was able to mediate complement dependent cytotoxicity (CDC) of PD-L1 expressing cells, but not cells expressing LAG-3, even if there is a mixture of cells expressing LAG-3 and PD-L1 in the sample. This property is expected to be useful where, as in the case of FS18-7-9/84G09, the target of the CDR-based antigen-binding site is the tumor cell and the binding site located in the constant domain of the antibody molecule targets the immune cell, so that the immune cell will be protected from CSC mediated by binding to the antibody molecule, while the tumor cell will be subject to CSC.

Thus, in a further embodiment, the present invention provides an antibody molecule that binds to a tumor antigen and an immune cell antigen, the antibody molecule comprising:

(i) a CDR-based antigen-binding site for a tumor antigen; and

(ii) an antigen-binding site for an immune cell antigen located in a constant domain,

preferably in the CH3 or CH2 domain, more preferably in the CH3 domain of the antibody molecule,

wherein the antibody molecule does not mediate or mediate to a significant extent the complement-dependent cytotoxicity of an immune cell containing said immune cell antigen when said immune cell is bound by the antibody molecule.

Said antibody molecule preferably mediates complement-dependent cytotoxicity of a tumor cell containing said tumor antigen when said tumor cell is bound by the antibody molecule.

Methods for measuring the CDC of an antibody molecule are known in the art and are described herein.

The present inventors have further shown that an antibody molecule comprising (i) an antigen-binding site based on the CDR for PD-L1; and (ii) the LAG-3 antigen binding site located in the CH3 domain of the antibody molecule of the present invention, FS18-7-9/84G09, was able to mediate weak antibody-dependent cellular cytotoxicity (ADCC) of cells expressing LAG-3 , compared with ADCC cells expressing PD-L1. Similarly, this property is expected to be useful where, as in the case of FS18-7-9/84G09, the target of the antigen-binding site based on the CDR is a tumor cell, and the target of the binding site located in the constant domain of the antibody molecule , is an immune cell, so that an immune cell will experience weaker ADCC compared to a tumor cell bound by the antibody.

As described herein, mutations are known in the art to reduce or eliminate the ADCC activity of antibody molecules. One such mutation is the LALA mutation described herein. Surprisingly, FS18-7-9/84G09 was found to have low ADCC activity against cells expressing LAG-3. If complete neutralization of ADCC activity is not required, this may be an advantage.

Thus, in a further embodiment, the present invention provides an antibody molecule that binds to a tumor antigen and an immune cell antigen, the antibody molecule comprising:

(i) a CDR-based antigen-binding site for a tumor antigen; and

(ii) an antigen-binding site for an immune cell antigen located in a constant domain,

preferably in the CH3 or CH2 domain, more preferably in the CH3 domain of the antibody molecule,

moreover, the antibody molecule causes a weakened ADCC in relation to immune cells containing the specified immune cell antigen, when said immune cells are bound by the antibody molecule, than in relation to tumor cells containing the specified tumor antigen, when said tumor cells are bound by the antibody molecule. The antibody molecule preferably does not or does not significantly mediate the ADCC of an immune cell containing said immune cell antigen when said immune cell is bound by the antibody molecule. The antibody molecule may also not mediate or not significantly mediate the complement-dependent cytotoxicity of an immune cell containing the specified immune cell antigen when said immune cell is bound by the antibody molecule, and/or may mediate the complement-dependent cytotoxicity of a tumor cell containing the specified tumor antigen, upon binding said tumor cell with an antibody molecule.

Methods for measuring the ADCC of an antibody molecule are known in the art and are described herein.

Various tumor antigens and immune cell antigens are known in the art. The tumor antigen and the immune cell antigen are preferably cell surface antigens. The immune cell antigen is preferably an antigen present on tumor-infiltrating lymphocytes.

The antigen-binding site for an immune cell antigen preferably contains one or more modifications in one or more of the structural loops of the constant domain of the antibody molecule, for example in the AB, CD or EF loop of the constant domain. For example, said attachment site may represent the LAG-3 attachment site described herein.

An antibody molecule of the present invention can be conjugated to an immune system modulator, a cytotoxic molecule, a radioactive isotope, or a detectable label. The immune system modulator may be a cytotoxic molecule, which is a cytokine.

The present invention provides a nucleic acid encoding an antibody molecule of the present invention, as well as a vector containing such nucleic acid.

In addition, a recombinant host cell containing a nucleic acid or a vector according to the present invention is provided. Such a recombinant host cell can be used to produce an antibody molecule. Thus, a method for producing an antibody molecule according to the present invention is also provided, said method comprising culturing a recombinant host cell under conditions for producing an antibody molecule. Said method may further include the step of isolating and/or purifying the antibody molecule.

It is expected that the antibody molecules of the present invention will find use in therapy, in particular therapy in humans, for example, in the treatment of cancer. Thus, the present invention also provides a pharmaceutical composition comprising an antibody molecule of the present invention and a pharmaceutically acceptable excipient.

The present invention also provides an antibody molecule of the present invention for use in a method of treating cancer in a patient. Also provided is a method for treating cancer in a patient, said method comprising administering to the patient a therapeutically effective amount of an antibody molecule of the present invention. Further provided is the use of an antibody molecule of the present invention for use in the manufacture of a medicament for the treatment of cancer in a patient. Treatment may further include administering a cancer vaccine and/or a chemotherapeutic agent to the patient.

Brief description of the drawings

Figure 1: A shows the sequence alignment of nine Fcabs detected after second affinity maturation, FS18-7-32; FS18-7-33; FS18-7-36; FS18-7-58; FS18-7-62; FS18-7-65; FS18-7-78; FS18-7-88; and FS18-7-95, vs. original Fcab, FS18-7-9. Figure B shows the percent sequence identity of each of these Fcabs with respect to the sequence of the original Fcab, FS18-7-9.

The figure 2 shows the results of the analysis of activation of T cells. Specifically, Figure 2 shows representative plots of IL-2 release, which is indicative of T-cell activation, for a panel of cell lines treated with mAb ² FS18-7-9/84G09 (Figure 2A-C) or FS18-7-62/84G09 or FS18-7-78/84G09 (figure 2D-F) and control antibodies. Figure 2A: When analyzed with LAG-3 and PD-L1, activation required inhibition of both targets (FS18-7-9/84G09LALA, FS18-7-9/4420LALA+84G09, 25F7+84G09LALA or 25F7+S1LALA). Figure 2B: When analyzed with LAG-3, activation required inhibition of LAG-3 only (FS18-7-9/84G09LALA, FS18-7-9/4420LALA+84G09, 25F7+84G09LALA, 25F7+S1LALA, 25F7, FS18-7- 9/4420 LALA). Figure 2C: In the PD-L1 assay, activation required PD-L1 inhibition (FS18-7-9/84G09LALA, FS18-7-9/4420LALA+84G09, 25F7+84G09LALA or 25F7+S1LALA). Figure 2D: When analyzed with LAG-3 and PD-L1, activation required inhibition of both targets (FS18-7-62/84G09LALA, FS18-7-78/84G09LALA, FS18-7-62/4420LALA+84G09, FS18-7- 78/4420LALA+84G09, 25F7+84G09LALA or 25F7+S1LALA). Figure E: When analyzed with LAG-3 alone, activation required LAG-3 inhibition (FS18-7-62/84G09LALA, FS18-7-62/4420LALA, FS18-7-78/84G09LALA, FS18-7-78/4420LALA, FS18-7-62/4420LALA+84G09, FS18-7-78/4420LALA+84G09, 25F7+84G09LALA or 25F7+S1LALA). Figure 2F: When assayed with PD-L1 alone, activation required PD-L1 inhibition (FS18-7-62/84G09LALA, FS18-7-78/84G09LALA, FS18-7-62/4420LALA+84G09, FS18-7-78/ 4420LALA+84G09, 25F7+84G09LALA or 25F7+S1LALA).

Figure 3 shows that FS18-7-9/84G09 could induce T cell activation, as measured by IL-2 release, in the presence of both cLAG-3 + cPD-L1 and either cLAG-3 or cPD-L1 alone, indicating functional cross-reactivity with cynomolgus monkey antigens.

Figure 4 shows a representative plot of the SEB analysis. mAb ² LAG-3/PD-L1 and mAb ² LAG-3/4420 + 84G09LALA combination showed more pronounced activation than mAb 84G09LALA alone, while mAb ² LAG-3/4420 or mAb 4420 did not show significant activation.

Figure 5 shows the mass of end tumors at day 20 in the undeveloped syngeneic tumor model MC38. Mice treated with mAb ² LAG-3/PD-L1 (FS18-29/S1) had significantly smaller end tumors compared to mice treated with the combination of C9B7W and S1 reference mAbs.

Figure 6 shows growth curves in the MC38 undeveloped syngeneic tumor model. Mice treated with mAb ² LAG-3/PD-L1 (FS18-29/S1) had smaller tumors compared to mice treated with the combination of reference mAbs C9B7W and S1 or S1 alone. mAb ² LAG-3/4420 and reference mAb against LAG-3 had little effect on tumor growth.

Figure 7 shows the mass of end tumors at day 24 in an advanced syngeneic tumor model MC38. The anti-LAG-3/PD-L1 mAb ² (FS18-29/S1) suppressed tumor growth as effectively as the combination of the C9B7W and S1 reference antibodies. FS18-29/4420 alone had no significant effect on tumor growth, while S1 and C9B7W slightly affected the overall tumor growth.

Figure 8 shows tumor growth curves in an advanced syngeneic tumor model MC38. Tumor volume in mice treated with mAb ² LAG-3/PD-L1 (FS18-29/S1) was similar to that in mice treated with the combination of reference mAb C9B7W and S1. Mice treated with S1 or C9B7W alone showed tumors of intermediate volume, while mAb ² LAG-3/4420 treatment had no effect on tumor growth.

Figure 9 shows the mass of end tumors at day 20 in a CT26 undeveloped syngeneic tumor model. mAb ² LAG-3/PD-L1 (FS18-29/S1) suppressed tumor growth to a greater extent than the combination of reference antibodies C9B7W and S1.

Figure 10 shows tumor growth curves in a CT26 undeveloped syngeneic tumor model. A statistically significant difference between FS18-35/S1 and control IgG was demonstrated in the suppression of tumor growth. No such statistically significant difference was observed when using the combination of reference antibodies compared to the IgG control group.

Figure 11 shows the mass of end tumors at day 22 in the undeveloped syngeneic tumor model MC38 used to compare the effect of a LALA mutation in mAb ² on tumor growth inhibition. There were no statistically significant differences in the mass of end tumors in mice treated with mAb ² with and without the LALA mutation.

Figure 12 shows tumor growth curves in the MC38 undeveloped syngeneic tumor model used to compare the effect of mAb ² with and without a LALA mutation on tumor growth inhibition. There were no statistically significant differences in tumor growth curves in mice treated with mAb ² with and without the LALA mutation, but there was a trend towards increased tumor growth inhibition by molecules containing the LALA mutation.

Figure 13 shows the effect of mAb ² treatment on T cell expression of LAG-3. Expression of LAG-3 on CD8 (A), CD4 (B), and FoxP3 (C) tumor-infiltrating lymphocytes (TIL) treated with mAb ² FS18-29/S1, FS18-29/4420, S1, FS18-29/4420 is shown and S1, or control antibody 4420, on days 19 and 23 after tumor inoculation, which corresponded to days 3 and 7 after the last mAb ² /antibody injection, respectively. LAG-3 expression decreased after treatment with mAb ² FS18-29/S1 on days 19 and 23. Animals treated with the combination of FS18-29/4420 and S1 also showed a decrease in LAG-3 expression, however, this effect was delayed until day 23, while FS18-29/4420 and S1, when administered alone, resulted in a slight decrease or no decrease in LAG-3 expression.

Figure 14 shows the percentage of lysis of Raji cells expressing PD-L1 and LAG-3 after treatment with various antibodies/mAbs ² using a CDC assay for lactate dehydrogenase (LDH) release. Figures A and B show CDC-mediated lysis of Raji cells expressing PD-L1 and LAG-3, respectively. Cells were incubated with anti-LAG-3 antibody 25F7, anti-PD-L1 antibody 84G09, LALA-mutated anti-PD-L1 antibody 84G09, anti-CD20 rituximab, mAb ² FS18-7-9/84G09, mAb ² FS18-7-9 /84G09 containing the LALA mutation, or rituximab containing the LALA mutation. The release of LDH was measured 4 hours after the complement treatment of the baby rabbit and expressed as a percentage of complete lysis. The concentration of antibody/mAb ² during processing is indicated on the x-axis. To enable comparison of the curves, the slope of all curves was set to a common value.

Figure 15 shows the percentage of dead Raji cells expressing PD-L1 and LAG-3 after various treatments using flow cytometry-based CSC analysis. A mixture of cells expressing PD-L1 and LAG-3 labeled with various fluorescent labels was incubated with control antibody 4420, rituximab (RIT) against CD20, a combination of anti-LAG-3 antibody 25F7 and anti-PD-L1 antibody 84G09 or mAb ² FS18- 7-9/84G09. The cells were then treated with baby rabbit complement and stained with a dye that stains only dead cells. The percentage of dead cells in the two cell populations was evaluated as a percentage of the total number of cells. The cell type (expressing PD-L1 or expressing LAG-3 as determined by different fluorescent labels) assessed after each treatment is indicated in Figure 15 after the name of the respective treatment, see e.g. 4420 PDL1, which refers to Raji cell viability assessment expressing PD-L1 after treatment with control antibody 4420. Treatment antibody/mAb ² concentration is indicated on the x-axis

Figure 16 shows the percent lysis (cytotoxicity) of Raji cells expressing PD-L1 and LAG-3 after different treatments using the ADCC assay for lactate dehydrogenase (LDH) release. Cells were treated with rituximab anti-CD20 antibody, 84G09 anti-PD-L1 antibody, mAb ² FS18-7-9/84G09, rituximab containing LALA mutation, mAb ² FS18-7-9/84G09 containing LALA mutation, 25F7 anti-LAG-3 antibody , the 84G09 anti-PD-L1 antibody containing the LALA mutation, the control antibody 4420, or the control antibody 4420 containing the LALA mutation. The treated cells were then co-incubated with primary NK cells and the specific release of LDH was measured as a percentage of complete target cell lysis. The concentration of antibody/mAb ² during processing is indicated on the x-axis.

Detailed description of the invention

The present invention relates to antibody molecules that bind to both PD-L1 and LAG-3. More specifically, the antibody molecules of the present invention comprise a CDR-based PD-L1 antigen binding site and a LAG-3 antigen binding site located in the constant domain of the antibody molecule. The terms "PD-L1" and "LAG-3" may refer to human PD-L1 and LAG-3, mouse PD-L1 and LAG-3, and/or cynomolgus monkey PD-L1 and LAG-3 unless the context otherwise requires. . The terms "PD-L1" and "LAG-3" preferably refer to human PD-L1 and LAG-3 unless the context otherwise requires.

The term "antibody molecule" describes a natural or fully or partially synthesized immunoglobulin. The antibody molecule may be a human antibody or a humanized antibody.

The antibody molecule is preferably a monoclonal antibody molecule. Exemplary antibodies are immunoglobulin isotypes, eg, immunoglobulin G, and their isotype subclasses, eg, IgG1, IgG2, IgG3, and IgG4, and fragments thereof.

Thus, as used herein, the term "antibody molecule" includes antibody fragments, provided that such fragments contain a CDR-based PD-L1 antigen binding site and a LAG-3 antigen binding site located in the constant domain, e.g., CH1-, CH2 - or the CH3 domain, preferably the CH3 domain of the antibody molecule. Unless the context otherwise requires, the term "antibody molecule" in this document is equivalent to the term "antibody molecule or fragment thereof".

You can take monoclonal and other antibodies and use techniques of recombinant DNA technology to obtain other antibodies or chimeric molecules that retain the specificity of the original antibody. Such techniques may include inserting CDR sequences or variable regions and/or a constant domain containing a LAG-3 antigen binding site into another immunoglobulin. The introduction of the CDR of one immunoglobulin into another immunoglobulin is described, for example, in EP-A-184187, GB 2188638A or EP-A-239400. Similar techniques can be applied to the corresponding constant domain sequences. Alternatively, the hybridoma or other cell producing the antibody molecule may be subjected to genetic mutation or other changes that may or may not alter the binding specificity of the resulting antibodies.

Because antibodies can be modified in a variety of ways, the term "antibody molecule" should be considered to encompass antibody fragments, derivatives, functional equivalents, and homologues of antibodies, including any polypeptide containing an immunoglobulin binding domain, whether natural or fully or partially synthetic. Thus, the term includes chimeric molecules containing an immunoglobulin binding domain or equivalent attached to another polypeptide. Cloning and expression. chimeric antibodies are described in EP-A-0120694 and EP-A-0125023.

An example of an antibody fragment containing both CDR sequences and a CH3 domain is a mini-antibody containing an scFv fused to a CH3 domain (Hu et al. (1996), Cancer Res., 56(13):3055-61).

The antibody molecule of the present invention binds to PD-L1 and LAG-3. In this context, binding may refer to specific binding The term "specific" may refer to the situation in which an antibody molecule does not show significant binding to molecules other than its specific binding partner(s), in this case PD-L1 and LAG-3 . The term "specific" is also applicable if the antibody molecule is specific for specific epitopes, for example epitopes on PD-L1 and LAG-3, which carry a range of antigens; in this case, the antibody molecule will be able to bind to various antigens that carry this epitope.

LAG-3 shares 40% sequence identity with CD4, its closest protein. The authors of the present invention tested Fcab FS18-7-9, which contains the amino acid sequence shown in SEQ ID NO: 1-3, for binding to CD4. It was shown that Fcab FS18-7-9 did not bind to CD4, which demonstrates the specificity of the binding of this molecule to LAG-3. Thus, in a preferred embodiment, the LAG-3 binding site of an antibody molecule of the present invention does not bind or does not show significant binding to CD4.

The antibody molecule of the present invention preferably contains a LAG-3 antigen binding site. The binding site of the LAG-3 antigen is located in the constant domain of the antibody molecule, for example the CH1-, CH2-, CH3- or CH4-domain. The binding site of the LAG-3 antigen is preferably located in the CH3 domain of the antibody molecule. The LAG-3 binding site preferably contains the amino acid sequences WDEPWGED (SEQ ID NO: 1) and PYDRWVWPDE. (SEQ ID NO: 3). These sequences were present in all major anti-LAG-3 Fcab clones identified by the present inventors after an intensive screening and characterization program described in the Examples section.

The amino acid sequences shown in SEQ ID NOs: 1 and 2 are preferably located in the structural loops of the constant domain of the antibody molecule. The introduction of sequences in the region of the structural loops of antibody constant domains in order to create new antigen-binding sites is described, for example, in applications WO 2006/072620 and WO 2009/132876.

Structural loops of antibody constant domains include loops AB, CD, and EF. Loops AB, CD, and EF in the CH3 domain are located in the region of residues 11–18, 43–78, and 92–101 of the CH3 domain; moreover, the numbering of amino acid residues corresponds to the ImMunoGeneTics (IMGT) numbering scheme. The amino acid sequence shown in SEQ ID NO: 1 is preferably located in the AB loop of the constant domain. The amino acid sequence shown in SEQ ID NO: 3 is preferably located in the EF loop of the constant domain. More preferably, the amino acid sequence shown in SEQ ID NO: 1 is located in the region of residues 11-18 of the CH3 domain; and/or the amino acid sequence shown in SEQ ID NO: 3 is preferably located in the region of residues 92-101 of the CH3 domain, the numbering of the amino acid residues following the IMGT numbering scheme.

In addition, the antibody molecule preferably contains the amino acid sequence shown in SEQ ID NO: 2, 8, 13, 18, 23, 28, 33, 38, 43 or 48, more preferably SEQ ID NO: 2, 28 or 38, more more preferably SEQ ID NO: 2, in the structural loop of the constant domain of the antibody molecule. The structural loop is preferably a CD structural loop and the constant domain is preferably a CH3 domain. The amino acid sequence shown in SEQ ID NOs: 2, 8, 13, 18, 23, 28, 33, 38, 43, or 48 is preferably located in the region of residues 43-78 of the CH3 domain, with amino acid residue numbering following the IMGT numbering scheme.

The antibody molecule of the present invention further comprises a glutamic acid (E) residue at position 36 and/or a tyrosine (Y) residue at position 85.2 of the CH3 domain (as shown in Figure 1A), with amino acid residue numbering following the IMGT numbering scheme. In particular, an antibody molecule containing the CD structural loop region shown in SEQ ID NO: 8 preferably contains a glutamic acid residue (E) at position 36 of the CH3 domain. Similarly, an antibody molecule containing the structural loop region shown in SEQ ID NO: 18 preferably contains a tyrosine (Y) residue at position 85.2 of the CH3 domain.

In a preferred embodiment, an antibody molecule of the present invention comprises a CH3 domain comprising or consisting of the sequence shown in SEQ ID NO: 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50, preferably a CH3 domain with the sequence given in SEQ ID NO: 5, 30 or 40, more preferably CH3 with the sequence given in SEQ ID NO: 5.

An antibody molecule of the present invention may comprise a CH3 domain comprising or consisting of the sequence shown in SEQ ID NO: 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50, wherein the CH3 domain sequence further comprises a lysine residue (K) at the very C-terminus of the sequence shown in SEQ ID NO: 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50. For example, an antibody molecule of the present invention may contain CH3 -domain containing or consisting of the sequence shown in SEQ ID NO: 5 with a lysine residue at the very C-terminus of the sequence shown in SEQ ID NO: 5. The sequence of such a CH3 lomen would be:

GQPREPQVYTLPPSWDEPWGEDVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS DGSFFLYSKLTVPYDRWVWPDEFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 135)

In addition, the antibody molecule of the present invention may comprise the CH2 domain of an immunoglobulin G molecule, such as the CH2 domain of an IgG1, IgG2, IgG3 or IgG4 molecule. The antibody molecule of the present invention preferably contains the CH2 domain of an IgG1 molecule. The CH2 domain may contain the sequence shown in SEQ ID NO: 53.

The CH2 domain of an antibody molecule may contain a mutation that reduces or eliminates the binding of the CH2 domain to one or more Fcγ receptors, eg, FcγRI, FcγRIIa, FcγRIIb, FcγRIII, and/or complement. The CH2 domains of human IgG domains generally bind to Fcγ receptors or complement, and the present inventors contemplate that reduced binding to Fcγ receptors will attenuate antibody-dependent cell-mediated cytotoxicity (ADCC) and reduced binding to complement will attenuate complement -dependent cytotoxicity (CSC) of the antibody molecule. Mutations to reduce or eliminate binding of the CH2 domain to one or more of the Fcγ receptors and complement are known and include the “LALA mutation” described in Bruhns, et al. (2009) and Xu et al. (2000). Thus, an antibody molecule may contain a CH2 domain, wherein said CH2 domain contains alanine residues at positions 4 and 5 of the CH2 domain, and the numbering follows the IMGT numbering scheme. For example, an antibody molecule contains an IgG1 CH2 domain containing or consisting of the sequence shown in SEQ ID NO: 54.

The antibody molecule of the present invention contains an antigen-binding site based on the CDR for PD-L1. The term “CDR-based antigen-binding site” refers to the antigen-binding site of the variable region of an antibody molecule, which consists of six CDRs. Obtaining anti-PD-L1 antibody molecules and determining the CDR sequences of such antibody molecules is within the ability of those skilled in the art, and a large number of suitable techniques are known in the art.

The antibody molecule of the present invention preferably contains the HCDR3 of the 84G09 antibody. HCDR3 is known to play a role in determining the specificity of an antibody molecule (Segal et al., (1974), PNAS, 71:4298-4302; Amit et al., (1986), Science, 233:747-753; Chothia et al. , (1987), J. Mol. Biol., 196:901-917 Chothia et al., (1989), Nature, 342:877-883, Caton et al., (1990), J. Immunol., 144 :1965-1968; Sharon et al., (1990a), PNAS, 87:4814-4817; Sharon et al., (1990b), J. Immunol., 144:4863-4869; Kabat et al., (1991b) , J. Immunol., 147:1709-1719).

The antibody molecule may further comprise the HCDR1, HCDR2, LCDR1, LCDR2 and/or LCDR3 of the 84G09 antibody. It will not be difficult for one skilled in the art to determine the CDR sequences from the sequences of the VH and VL domains of the 84G09 antibody shown in SEQ ID NOs 92 and 93, respectively. CDR sequences can be determined, for example, according to the Kabat numbering scheme (Kabat, E.A. et al., (1991)) or IMGT.

The sequences of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of the 84G09 antibody according to the IMGT numbering scheme are given in SEQ ID NOs 86, 87, 88, 89, 90 and 91, respectively.

The sequences of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of the 84G09 antibody according to Kabat are shown in SEQ ID NOS 136, 137, 138, 139, 140 and 141, respectively.

In addition, the antibody may contain the VH and/or VL domain of the 84G09 antibody. The sequences of the VH and VL domains of the 84G09 antibody are shown in SEQ ID NOs 92 and 93, respectively.

In a preferred embodiment, an antibody molecule of the present invention comprises (i) a CDR-based PD-L1 antigen binding site comprising the sequences HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of the 84G09 antibody, and (ii) a LAG-3 antigen binding site, located in the CH3 domain of the antibody molecule, wherein the LAG-3 binding site contains the amino acid sequences shown in SEQ ID NOs 1 and 3 and an amino acid sequence selected from the group consisting of: SEQ ID NOs 2, 8, 13, 18, 23 , 28, 33, 38, 43 and 48.

More preferably, the antibody molecule of the present invention comprises (i) a CDR-based PD-L1 antigen binding site comprising the sequences HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of the 84G09 antibody, and (ii) a LAG-3 antigen binding site located in the CH3 domain of the antibody molecule, wherein the LAG-3 binding site contains the amino acid sequences shown in SEQ ID NOs 1 and 3 and an amino acid sequence selected from the group consisting of: SEQ ID NOs 2, 28 and 38.

Even more preferably, the antibody molecule of the present invention comprises (i) a CDR-based PD-L1 antigen binding site comprising the sequences HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of the 84G09 antibody, and (ii) a LAG-3 antigen binding site, located in the CH3 domain of the antibody molecule, and the LAG-3 binding site contains the amino acid sequences shown in SEQ ID NOs 1, 2 and 3.

In a preferred embodiment, an antibody molecule of the present invention comprises a VH domain and a VL domain containing or consisting of the sequence shown in SEQ ID NOs 93 and 93, respectively, and a CH3 domain containing or consisting of the sequence shown in SEQ ID NO: 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50, preferably a CH3 domain containing or consisting of the sequence shown in SEQ ID NO: 5, 30 or 40, more preferably CH3- a domain containing or consisting of the sequence shown in SEQ ID NO: 5.

In a further preferred embodiment, the antibody molecule comprises a heavy chain containing or consisting of the sequence shown in SEQ ID NO: 93-113 and a light chain containing or consisting of the sequence shown in SEQ ID NO: 116. More preferably, the antibody molecule contains a heavy chain containing or consisting of the sequence shown in SEQ ID NOS: 94, 95, 104, 105, 108 and 109, and a light chain containing or consisting of the sequence shown in SEQ ID NO: 116. Most preferably, the molecule the antibody contains a heavy chain containing or consisting of the sequence shown in SEQ ID NO: 94 or 95, and a light chain containing or consisting of the sequence shown in SEQ ID NO: 116.

Antibody molecules of the present invention may also contain the sequence variants of the structural loop, CH3 domain, CH2 domain, CH2 and CH3 domain, light chain or heavy chain described herein, provided that the sequences of the VL and VH domains light and heavy chains, respectively, remain unchanged. Suitable variants can be obtained through sequence alteration or mutation methods and screening. In a preferred embodiment, an antibody molecule comprising one or more of the variant sequences retains one or more of the functional characteristics of the parent antibody molecule, such as binding specificity and/or binding affinity for LAG-3 and PD-L1. For example, an antibody molecule containing one or more of the variant sequences preferentially binds to LAG-3 with the same or higher affinity than the (original) antibody molecule. The original antibody molecule is an antibody molecule that does not contain amino acid(s) substitution(s), deletion(s) and/or insertion(s) introduced into the variant antibody molecule.

For example, an antibody molecule of the present invention may comprise a structural loop, CH3 domain, CH2 domain, CH2 and CH3 domain, light chain, or heavy chain sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99, 1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7% , at least 99.8%, or at least 99.9% sequence identity with respect to the sequence of the structural loop, CH3 domain, CH2 domain, CH2 and CH3 domain, light chain or heavy chain described in this document, provided that the sequences of the VL and VH domains of the light and heavy chains, respectively, remain unchanged.

In a preferred embodiment, an antibody molecule of the present invention comprises a CH3 domain sequence having at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least at least 99.9% sequence identity to the CH3 domain sequence shown in SEQ ID NO: 4, 5, or 135.

In a further preferred embodiment, an antibody molecule of the present invention comprises a CH3 and CH2 domain sequence having at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99 .2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8 % or at least 99.9% sequence identity with respect to the sequence of the CH2 and CH3 domains shown in SEQ ID NO: 6 or 7.

Sequence identity is usually determined with reference to the GAP algorithm (Wisconsin GCG package, Accelerys Inc, San Diego, USA). GAP uses the Needleman and Wunsch algorithm to align two complete sequences to maximize matches and minimize gaps. In general, default parameters are used, with gap creation penalty = 12 and gap lengthening penalty = 4. GAP may be preferred, but other algorithms may be used, such as BLAST (which uses the method from Altschul et al. ( 1990) J. MoI. Biol. 215: 405-410), FASTA (which uses the method from Pearson and Lipman (1988) PNAS USA 85: 2444-2448) or the Smith-Waterman algorithm (Smith and Waterman (1981) J. MoI Biol. 147: 195-197) or the TBLASTN program from Altschul et al. (1990) supra, generally using the default parameters. In particular, the psi-Blast algorithm (Nucl. Acids Res. (1997) 25 3389-3402) can be used.

The antibody molecule of the present invention may also contain a structural loop, CH3 domain, CH2 domain, CH2 and CH3 domain, light chain or heavy chain sequence containing one or more amino acid sequence modifications (addition, deletion, substitution and/or insertion amino acid residue), preferably 20 modifications or less, 15 modifications or less, 10 modifications or less, 5 modifications or less, 4 modifications or less, 3 modifications or less, 2 modifications or less, or 1 modification compared to the structural loop sequence, CH3 domain, CH2 domain, CH2 and CH3 domain, light chain or heavy chain described herein, provided that the sequences of the VL and VH domains of the light and heavy chain, respectively, remain unchanged.. In particular, modifications can be made to one or more of the framework regions of the antibody molecule outside of the sequences of the VH and VL domains.

In a preferred embodiment, the antibody molecule may comprise a CH3 domain sequence containing one or more amino acid sequence modifications (addition, deletion, substitution and/or amino acid residue insertion), preferably 20 modifications or less, 15 modifications or less, 10 modifications or less, 5 modifications or less, 4 modifications or less, 3 modifications or less, 2 modifications or less, or 1 modification compared to the CH3 domain sequence shown in SEQ ID NOS: 4, 5, or 135.

In a further preferred embodiment, the antibody molecule of the present invention comprises a CH3 and CH2 domain sequence containing one or more amino acid sequence modifications (addition, deletion, substitution and/or insertion of an amino acid residue), preferably 20 modifications or less, 15 modifications or less , 10 modifications or less, 5 modifications or less, 4 modifications or less, 3 modifications or less, 2 modifications or less, or 1 modification compared to the CH2 and CH3 domain sequence shown in SEQ ID NO: 6 or 7.

In addition, the present invention contemplates an antibody molecule that competes for binding to LAG-3 and/or PD-L1 with an antibody molecule of the present invention, or that binds to the same LAG-3 and/or PD-L1 epitope as an antibody molecule of the present invention. of the present invention, said antibody molecule having both a CDR-based PD-L1 antigen binding site and a LAG-3 antigen binding site located in the CH3 domain of the antibody molecule. Methods are known in the art for determining competition between two antibodies for an antigen. For example, the competition of two antibodies for binding to an antigen can be determined using BIAcore. Similarly, methods for mapping an epitope bound by an antibody are known in the art.

The antibody molecule of the present invention preferably binds to LAG-3 with an affinity (K _D ) of 1 x 10 ^-9 M or higher. For example, an antibody molecule of the present invention can bind to LAG-3 with an affinity (K _D ) of 8 x 10 ^-10 M or higher.

Fcabs have a smaller binding interaction surface than monoclonal antibodies because the Fcab binding sites form a relatively compact antibody fragment with two binding sites located in relative proximity. In contrast, the Fab arms of a typical mAb are separated by a flexible hinge region. The two antigen-binding sites of Fcab are also spatially close to each other compared to the antigen-binding sites of a typical mAb. Given the smaller binding surface area and the reduced flexibility of the two binding sites, the ability of the anti-LAG-3 Fcab to bind to and inhibit LAG-3 with similar affinity and potency to that of a reference monoclonal antibody was unexpected.

The antibody molecule of the present invention preferably binds to PD-L1 with an affinity (KD) of 1 x 10 ^-9 M or higher.

The binding affinity of an antibody molecule to its specific antigen, eg LAG-3 or PD-L1, can be determined using, for example, surface plasmon resonance. The binding affinity of an antibody molecule for its specific antigen, such as LAG-3 or PD-L1, expressed on the cell surface can be determined using flow cytometry.

The antibody molecule of the present invention can preferably bind to LAG-3 and PD-L1 expressed on the cell surface. Said cell is preferably a cancer cell.

The antibody molecule of the present invention can preferably simultaneously bind to LAG-3 and PD-L1. In a preferred embodiment, an antibody molecule of the present invention can simultaneously bind to LAG-3 and PD-L1, with LAG-3 and PD-L1 expressed on the surface of a single cell or on the surface of two separate cells.

An antibody molecule of the present invention can bind to human LAG-3, mouse LAG-3 and/or cynomolgus LAG-3. The antibody molecule of the present invention preferably binds to human LAG-3. More preferably, the antibody molecule of the present invention binds to human LAG-3 and human PD-L1.

The antibody molecule of the present invention comprises (i) a CDR-based PD-L1 antigen binding site and (ii) a LAG-3 antigen binding site located in the constant domain of the antibody molecule. Thus, antibody molecules that do not contain a LAG-3 antigen binding site located in a constant domain, such as the CH3 domain of the antibody molecule, are not relevant to the present invention. Similarly, a molecule lacking the CDR-based PD-L1 antigen-binding site is not relevant to the present invention.

An antibody molecule of the present invention can be conjugated to a therapeutic agent or a detectable label. In this case, the antibody molecule can be called a conjugate. For example, an antibody molecule can be conjugated to an immune system modulator, a cytotoxic molecule, a radioactive isotope, or a detectable label. The immune system modulator or cytotoxic molecule may be a cytokine. The detectable label may be a radioactive isotope, such as a non-therapeutic radioactive isotope.

An antibody molecule can be conjugated to a therapeutic agent or a detectable label via a peptide bond or linker, i.e. as part of a hybrid polypeptide containing the specified therapeutic agent or a detectable label and an antibody molecule or a component of its polypeptide chain. Other means of conjugation include chemical conjugation, such as cross-linking using a bifunctional reagent (eg, using the DOUBLE-REAGENTS™ Cross-linking Reagents Selection Guide, Pierce).

Thus, the antibody molecule and the therapeutic agent or detectable label can be attached directly to each other, for example, through any suitable chemical bond, or through a linker, for example, a peptide linker.

The peptide linker may be short (chain of 2-20, preferably 2-15 amino acid residues). Suitable examples of peptide linker sequences are known in the art. You can use one or more of the various linkers. The linker may be about 5 amino acids long.

The chemical bond may be, for example, a covalent or ionic bond. Examples of covalent bonds include peptide bonds (amide bonds) and disulfide bonds. For example, an antibody molecule and a therapeutic or diagnostic agent may be covalently linked. For example, peptide bonds (amide bonds). Thus, the antibody molecule and the therapeutic or diagnostic agent can be produced (secreted) as a single chain polypeptide.

The present invention also provides isolated nucleic acids encoding the antibody molecules of the present invention. Obtaining such nucleic acids using methods well known in the art will not be difficult for a person skilled in the art. An isolated nucleic acid can be used to express an antibody molecule of the present invention, for example, by expression in a bacterial, yeast, insect, or mammalian host cell. A preferred host cell is a mammalian cell, such as a CHO, HEK or NS0 cell. The nucleic acid can generally be provided in the form of a recombinant expression vector.

The isolated nucleic acid may, for example, contain the sequence shown in SEQ ID NO: 142, 4, 9, 14, 19, 24, 29, 34, 39, 44 or 49 encoding the CH3 domains of FS18-7-9 (codon- optimized CHO nucleotide sequence), FS18-7-9 (HEK293-expressed nucleotide sequence), FS18-7-32, FS18-7-33, FS18-7-36, FS18-7-58, FS18-7-62, FS18 -7-65, FS18-7-78, FS18-7-88 and FS18-7-95, respectively.

The present invention includes in vitro host cells containing such nucleic acids and vectors, as well as their use for the expression of antibody molecules of the present invention, which can subsequently be isolated from cell culture and optionally formulated as a pharmaceutical composition. Thus, the present invention further provides a method for producing an antibody molecule of the present invention, comprising culturing a recombinant host cell of the present invention under conditions for producing an antibody molecule. Methods for culturing the appropriate host cells mentioned above are known in the art. Said method may further comprise isolating and/or purifying the antibody molecule. Said method may also include formulating the antibody molecule as a pharmaceutical composition, optionally with a pharmaceutically acceptable excipient or other substance, as described below.

It is known that PD-L1 is expressed on many cancer cells, while LAG-3 expression on cancer cells is more limited. Both of these antigens are expressed on cells of the immune system. In particular, LAG-3 is known to be expressed on depleted T cells in the tumor environment. In addition, the present inventors have demonstrated that the use of an antibody molecule that binds to both LAG-3 and PD-L1 effectively inhibits tumor growth in syngeneic mouse models, and that such antibody molecules are more effective than administration of two binding molecules that bind to LAG-3 and PD-L1, respectively.

Thus, an antibody molecule of the present invention can be used in a method for treating cancer in a patient. The patient is preferably a human patient.

Cancer cells to be treated with an antibody molecule of the present invention may express LAG-3, for example, on their surface. In one embodiment, the cancer cells to be treated can be found to express LAG-3, for example, on their surface. For example, B-cell lymphomas have been shown to express LAG-3 on the surface of their cells. Methods for determining antigen expression on a cell surface are known in the art and include, for example, flow cytometry.

Example 4 below shows that the antibody molecules of the present invention can be used to treat tumors with high levels of LAG-3-expressing immune cells, such as LAG-3-expressing TIL in mice. Thus, in addition or alternatively, cancers to be treated using the antibody molecule of the present invention may contain LAG-3-expressing immune cells. LAG-3 expressing immune cells, such as LAG-3 expressing TIL, are present between tumor cells in many cancers. In one embodiment, cancers to be treated with an antibody molecule of the present invention are found to contain LAG-3 expressing immune cells. Methods for determining the presence of LAG-3 expressing immune cells in a tumor or in the periphery of a tumor are known in the art.

Example 4 below also shows that the antibody molecules of the present invention can be used to treat tumors whose cells express PD-L1 on their surface. Thus, in addition or alternatively, cancer cells to be treated with an antibody molecule of the present invention may express PD-L1, for example, on their surface. In addition or alternatively, the cancers to be treated may contain immune cells, such as TIL, that express PD-L1. The cells of the cancer to be treated can be found to express PD-L1, for example, on their surface. In addition or alternatively, the cancers to be treated may be found to contain immune cells, eg TIL, that express PD-L1.

Expression of LAG-3 and PD-L1 on the cell surface is expected to allow the antibody molecule to bind to LAG-3 and PD-L1 expressed on the surface of the immune cell and/or cancer cell. This is believed to provide targeted therapy, bridging, and localization of cancer and immune cells.

The cancer to be treated using the antibody molecule of the present invention can be selected from the group consisting of Hodgkin's lymphoma, non-Hodgkin's lymphoma (e.g., diffuse large B-cell lymphoma, slow-growing non-Hodgkin's lymphoma, mantle cell lymphoma), ovarian cancer, prostate cancer cancer of the colon and rectum, fibrosarcoma, renal cell carcinoma, melanoma, pancreatic cancer, breast cancer, glioblastoma multiforme, lung cancer (for example, non-small cell lung cancer), head and neck cancer (for example, squamous cell carcinoma of the head and neck), stomach cancer, bladder cancer, cervical cancer, uterine cancer, vulvar cancer, testicular cancer, penile cancer, leukemia (eg, chronic lymphocytic leukemia, myeloid leukemia, acute lymphoblastic leukemia, or chronic lymphoblastic leukemia), multiple myeloma, squamous cell carcinoma, cancer testicular cancer of the esophagus (eg, adenocarcinoma of the gastroesophageal junction mentia), Kaposi's sarcoma and central nervous system (CNS) lymphoma, hepatocellular carcinoma, nasopharyngeal cancer, Merkel cell carcinoma and mesothelioma. Tumors of these cancers are known or suspected to express PD-L1 on their cell surface and/or contain immune cells such as TIL expressing PD-L1 and/or LAG-3.

Treatment of renal cell carcinoma, lung cancer (eg, non-small cell lung cancer), nasopharyngeal cancer, colon and rectal cancer, melanoma, gastric cancer, esophageal cancer (eg, adenocarcinoma of the gastroesophageal junction), ovarian cancer, cervical cancer, bladder cancer, cancer head and neck (eg, head and neck squamous cell carcinoma), leukemia (eg, chronic lymphocytic leukemia, Hodgkin's lymphoma, non-Hodgkin's lymphoma (eg, diffuse large B-cell lymphoma, slow-growing non-Hodgkin's lymphoma, mantle cell lymphoma), and multiple myeloma with The use of antibodies against LAG-3 is being studied in clinical trials and shows promising results.Thus, the cancer to be treated using the antibody molecules of the present invention may be renal cell carcinoma, lung cancer (e.g., non-small cell lung cancer), nasopharyngeal cancer, cancer colon and rectum, melanoma, stomach cancer, esophageal cancer (eg, adenocarcinoma of the gastroesophageal junction), ovarian cancer, cervical cancer, bladder cancer, head and neck cancer (eg, head and neck squamous cell carcinoma), leukemia (eg, chronic lymphocytic leukemia, Hodgkin's lymphoma, non-Hodgkin's lymphoma (eg , diffuse large B-cell lymphoma, slow-growing non-Hodgkin's lymphoma, mantle cell lymphoma), or multiple myeloma.

Treatment of melanoma, colorectal cancer, breast cancer, bladder cancer, renal cell carcinoma, stomach cancer, head and neck cancer (eg, head and neck squamous cell carcinoma), mesothelioma, lung cancer (eg, non-small cell lung cancer), cancer ovarian, Merkel cell carcinomas, pancreatic cancers, melanomas and hepatocellular carcinomas using anti-PD-L1 antibodies are also being investigated in clinical trials with promising results. Thus, the cancer to be treated using the antibody molecules of the present invention may be melanoma, colorectal cancer, breast cancer, bladder cancer, renal cell carcinoma, gastric cancer, head and neck cancer (e.g. head and neck squamous cell carcinoma and neck), mesothelioma, lung cancer (eg, non-small cell lung cancer), ovarian cancer, Merkel cell carcinoma, pancreatic cancer, melanoma, and hepatocellular carcinoma.

Preferred cancers for treatment with antibody molecules of the present invention are lung cancer (e.g., non-small cell lung cancer), bladder cancer, head and neck cancer (head and neck squamous cell carcinoma), diffuse large B-cell lymphoma, gastric cancer, cancer pancreas and hepatocellular carcinoma. Tumors in these cancers are known to contain LAG-3 expressing immune cells and express PD-L1 on their cell surface or contain immune cells expressing PD-L1.

Where an application relates to a specific type of cancer, eg breast cancer, it relates to the malignant transformation of the relevant tissue, in this case breast tissue. Cancer derived from malignant transformation of another tissue, such as ovarian tissue, may result in metastatic lesions elsewhere in the body, such as the breast, but is therefore not breast cancer as described herein, but ovarian cancer .

The cancer may be primary or secondary cancer. Thus, an antibody molecule of the present invention may be for use in a method of treating cancer in a patient, wherein the cancer is a primary tumor and//or a tumor metastasis.

The antibody molecules of the present invention are intended for use in methods of treating patients, preferably human patients. Antibody molecules will generally be administered in the form of a pharmaceutical composition, which may contain at least one component in addition to the antibody molecule, such as a pharmaceutically acceptable excipient. For example, the pharmaceutical composition of the present invention may contain, in addition to the active ingredient, a pharmaceutically acceptable excipient, carrier, buffer, stabilizer, or other materials well known to those skilled in the art. Such materials should be non-toxic and should not interfere with the effectiveness of the active ingredient. The exact nature of the carrier or other material depends on the route of administration, which can be carried out, for example, by injection, such as intravenous or subcutaneous injection. The antibody molecule can be administered intravenously or subcutaneously.

Liquid pharmaceutical compositions generally contain a liquid carrier such as water, hydrocarbon, animal or vegetable oils, mineral oil or synthetic oil. Saline, dextrose or other saccharide solutions, or glycols such as ethylene glycol, propylene glycol or polyethylene glycol may be included.

For intravenous administration or injection into the affected area, the antibody molecule or pharmaceutical composition containing the antibody molecule is preferably in the form of a parenterally acceptable pyrogen-free aqueous solution with suitable pH, isotonicity and stability. Suitable solutions can be easily prepared by those skilled in the art using, for example, isotonic vehicles, eg sodium chloride injection, Ringer's solution for injection, Ringer's solution with lactate for injection. If necessary, preservatives, stabilizers, buffers, antioxidants and/or other additives can be used. Many methods of making pharmaceutical formulations are known to those skilled in the art. See, for example, Robinson ed., Sustained and Controlled Release Drug Delivery Systems, Marcel Dekker, Inc., New York, 1978.

The composition containing the antibody molecules of the present invention may be administered alone or in combination with other therapeutic agents, simultaneously or sequentially, or as a combination preparation with another therapeutic agent or agents, depending on the condition being treated. For example, an antibody molecule of the present invention may be administered in combination with an existing therapeutic agent for the disease being treated, such as cancer, as described above. For example, an antibody molecule of the present invention can be administered to a patient in combination with a second cancer therapeutic agent, e.g. a chimeric antigen receptor (CAR) or a hormone therapeutic agent.

It is expected that the antibody molecule according to the present invention can act as an adjuvant in anticancer therapy, such as chemotherapy, antitumor vaccination or radiation therapy. Regardless of theory, it is believed that administration of an antibody molecule to a patient as part of chemotherapy, cancer vaccination, or radiation therapy will elicit a stronger immune response against cancer-associated LAG-3 and PD-L1 antigens than that achieved with chemotherapy alone, cancer therapy alone. vaccination or radiation therapy. For example, anti-LAG-3 therapeutics have been shown to be effective in treating viral conditions in mice (Blackburn SD, et al., 2009).

Thus, a method of treating cancer in a patient may include administering to the patient a therapeutically effective amount of an antibody molecule of the present invention in combination with a chemotherapeutic agent, a tumor vaccine, a radionuclide, an immunotherapeutic agent, oncolytic viruses, CAR-T cells, or a hormone therapy agent. The chemotherapeutic agent, tumor vaccine, radionuclide, immunotherapeutic agent, oncolytic viruses, CAR-T cells, or hormone therapy agent is preferably a chemotherapeutic agent, tumor vaccine, radionuclide, immunotherapeutic agent, oncolytic viruses, CAR-T cells, or hormone therapy agent. therapy of the cancer in question, i.e. a chemotherapeutic agent, a tumor vaccine, a radionuclide, an immunotherapeutic agent, oncolytic viruses, CAR-T cells, or a hormone therapy agent that has been shown to be effective in treating the cancer in question. The practitioner is able to select an appropriate chemotherapeutic agent, tumor vaccine, radionuclide, immunotherapeutic agent, oncolytic viruses, CAR-T cells, or hormone therapy agent that has been shown to be effective in treating the cancer in question.

For example, if the method comprises administering an antibody molecule of the present invention to a patient in a therapeutically effective amount in combination with a chemotherapeutic agent, said chemotherapeutic agent is selected from the group consisting of taxanes, cytotoxic antibiotics, tyrosine kinase inhibitors, PARP inhibitors, B_RAF enzyme inhibitors, alkylating agents, analogs platinum, nucleoside analogs, thalidomide derivatives, antitumor chemotherapeutic agents, and the like. Taxanes include docetaxel, paclitaxel and paclitaxel bound to albumin nanoparticles; cytotoxic antibiotics include actinomycin, bleomycin, anthracyclines, doxorubicin and valrubicin; tyrosine kinase inhibitors include erlotinib, gefitinib, axitinib, PLX3397, imatinib, cobimetinib, and trametinib; PARP inhibitors include niraparib; B-Raf enzyme inhibitors include vemurafenib and dabrafenib; alkylating agents include dacarbazine, cyclophosphamide, temozolomide; platinum analogs include carboplatin, cisplatin and oxaplatin; nucleoside analogs include gemcitabine and azacitidine; anticancer agents include fludarabine. Other chemotherapeutic agents suitable for use in the present invention include methotrexate, defactinib, entinostat, pemetrexed, capecitabine, eribulin, irinotecan, fluorouracil, and vinblastine.

Vaccination strategies for cancer treatment have been introduced into clinical practice and are discussed in the scientific literature (eg, Rosenberg, S. 2000 Development of Cancer Vaccines). The main strategies used are to stimulate the immune system to respond to various cellular markers expressed by autologous or allogeneic cancer cells using these cells in the vaccination method with and without granulocyte-macrophage colony stimulating factor (GM-CSF). GMCSF stimulates a strong response to antigen presentation and is particularly effective when used within these strategies.

The introduction can be carried out in a "therapeutically effective amount", which is sufficient to provide a beneficial effect on the patient. Such a beneficial effect may be at least an improvement in at least one symptom. Such "treatment" of said disease refers to the improvement of at least one symptom. The actual amount administered, the rate and period of effect upon administration will depend upon the nature and severity of the disease being treated, the particular patient being treated, the clinical condition of the individual patient, the cause of the disorder, the site of delivery of the composition, the type of antibody molecule, the mode of administration, the schedule of administration, and other factors. known to practitioners. Prescribing treatment, such as dosage decisions, etc., is the responsibility of general practitioners and other physicians; it may depend on the severity of the symptoms and/or the progression of the disease being treated. Suitable doses of antibody molecules are well known in the art (Ledermann et al. (1991) Int. J. Cancer 47: 659-664; and Bagshawe et al. (1991) Antibody, Immunoconjugates and Radiopharmaceuticals 4: 915-922). Specific dosages indicated herein or in the Physician's Desk Reference (2003) may be used as appropriate for the antibody molecule administered. A therapeutically effective amount or appropriate dose of an antibody molecule can be determined by comparing its in vitro and in vivo activity in an animal model. Methods are known for extrapolating effective dosages in mice and other experimental animals to humans. The exact dose depends on a number of factors, including the size and location of the area to be treated and the exact nature of the antibody molecule. Treatment may be repeated at daily, twice weekly, weekly or monthly intervals at the discretion of the physician. Therapeutic agents can be given before and/or after surgery, and can be administered or applied directly to the anatomical area of the surgical treatment.

Additional aspects and embodiments of the present invention are obvious to those skilled in the art in light of the present description, including their experimental implementation.

All documents mentioned in the present description are incorporated into this application by reference in their entirety.

The phrase "and/or", as used herein, should be considered as a specific description of each of the two specified features or components with each other or separately. For example, "A and/or B" should be considered as a specific description of each of (i) A, (ii) B, and (iii) A and B, as if each of them were separately described in this document.

Unless otherwise follows from the context, the descriptions and definitions of the features set forth above are not limited to any particular aspect or embodiment of the present invention and are equally applicable to all aspects and embodiments described.

Certain aspects and embodiments of the present invention will be illustrated by way of examples with reference to the figures described above.

Examples

Example 1 Selection and Characterization of Fcab Molecules

1.1 Naive selection and affinity maturation of Fcab against LAG-3

1.1.1 Naive selection

Naive phage display libraries containing the CH3 domain of human IgG1 (IMGT numbering 1.4-130) randomized within loops AB (residues 14-18) and EF (residues 92-101) were used for selection using recombinant Fc-labeled antigen human LAG-3 (LAG-3 Fc) (R&D systems, 2319-L3-050). Library selection was performed in three rounds using antigen immobilized on protein A (Life Technologies, 10002D) or protein G (Life Technologies, 10004D) beads. The results were screened by ELISA; molecules showing positive binding results were subcloned and expressed as soluble Fcabs (containing a truncated hinge region) in Pichia pastoris using the EasySelect Pichia Expression Kit (Life Technologies, K1740-01). The Fcab was then screened for binding to recombinant human LAG-3 Fc on a Biacore 3000 (GE Healthcare). Briefly, LAG-3 Fc (R&D systems, 2319-L3-050) was attached to a CM5 chip (GE Healthcare, BR-100012) at 7200 RU using amine coupling reagents (GE Healthcare, BR-1000-50). Fcab was diluted in HBS-P buffer (GE Healthcare, BR100368) and injected at 250 nM, 500 nM and 1000 nM for 3 minutes and then allowed to dissociate in buffer for 5 minutes. The data after subtracting the reference control (LAG-3 Fc 2 flow cell - blank flow cell) was analyzed using BIAevaluation 3.2 software to detect binding. Fcab was then tested for binding to human LAG-3 expressed on HEK cells (LAG-3 was cloned in the pcDNA5FRT vector [Life Technologies, V6010-20] [see section 1.4.5 for methodology]). Briefly, HEK 293 cells overexpressing human LAG-3 grown in DMEM (Life Technologies, 61965-026) containing 10% FBS (Life Technologies, 10270-1-6), 100 mg/mL hygromycin B (Melford Laboratories Ltd , Z2475), 15 mg/mL blasticidin (Melford Laboratories Ltd, B1105) and 1 µg/mL doxycycline (Sigma, D9891) were separated from tissue culture flasks using cell dissociation buffer (Life Technologies, 13151-014) and plated in 96-well V-bottom plates based on 2x10 ⁵ cells/well. Fcab was incubated with cells at a concentration of 5 μM in a volume of 100 μl for 1 h at 4°C. The plates were washed, the secondary antibody (anti-human Fc-488 antibody, Jackson ImmunoResearch, 109-546-098) was diluted 1:1000 with PBS, added 100 μl to the cells and incubated for 30 min at 4°C. The plates were washed and the cells were resuspended in 100 μl PBS containing 1 μg/ml DAPI (Biotium, 40043). The plate was read on a BD FACSCanto II cytometer (BD Biosciences) and analyzed using a FlowJoX. Fcabs were then expressed in mammalian cells by transformation using Lipofectamine (Life Technologies, 11668-019) into Flp-In T-Rex 293 cells (Life Technologies, R780-07). LAG-3 binding Fcabs were tested for inhibition of human MHC class II binding on A375 cells (ATCC, CRL-1619) to recombinant LAG-3 Fc (using the methodology described in Example 1.6). Three rounds of phage selection revealed 54 unique Fcab sequences; found that 12 of these Fcabs bind to LAG-3 Fc when analyzed by BIAcore and/or bind to HEK cells expressing LAG-3. Three of the selected Fcabs could also inhibit the interaction of LAG-3 with MHC class II and were selected for affinity maturation. These three Fcabs were named FS18-3, FS18-7 and FS18-21.

1.1.2 Affinity maturation

The first maturation of affinity

Six affinity maturation phage display libraries were constructed by randomizing five residues in the AB loop (residues 14-18) and another (residues 92-94 and 97-98) or eight (residues 92-94 and 97-101) residues in the EF loop of each of three Fcabs identified using the naive selection described above.

Library selection for affinity maturation was performed using recombinant human LAG-3 Fc (R&D systems, 2319-L3-050) and HEK cells expressing human LAG-3 (as described above). The results were screened by ELISA; molecules showing positive binding results were subcloned and expressed as soluble Fcabs (containing a shortened hinge region) in HEK Expi293 cells (Fcabs were cloned into the pTT5 vector [National Research Council of Canada] transfected using the ExpiFectamine 293 Transfection kit [Life Technologies, A14524] into Expi293F cells [Life technologies, A14527]). HEK-expressed soluble Fcabs were then screened for binding to cells expressing human LAG-3, binding to cells expressing cynomolgus monkey LAG-3 (methodology according to Example 1.4. LAG-3 Fc (methodology according to example 1.6). Blocking Fcabs were further tested to determine their ability to reverse LAG-3-induced inhibition of IL-2 secretion in a T cell activation assay (methodology according to Example 2.1). In six affinity maturation libraries, 61 unique anti-LAG-3 Fcabs were identified using these screening methods. It was shown that Fcabs from the FS18-7 line, after affinity maturation, had the maximum level of cross-reactivity with LAG-3 of the cynomolgus monkey. Three Fcabs from this line with the strongest binding to LAG-3 cynomolgus monkey Fc and maximum activity in the T-cell activation assay (named FS18-7-7, FS18-7-9 and FS18-7-11) were selected for further maturation. affinity. It was shown that these three Fcabs also blocked the interaction of LAG-3 Fc with MHC class II expressed by cells.

Second affinity maturation

A pool of three Fcabs (FS18-7-7, FS18-7-9 and FS18-7-11) generated from first affinity maturation were used to further generate libraries for affinity maturation. The CD loop was extensively randomized using randomized primers manufactured by ELLA Biotech. A portion of the amino acid positions in the CD loop (residues 45.1-78) were randomized using an equimolar distribution of amino acids except for cysteine. In addition, a reduced precision PCR was performed over the entire CH3 domain sequence in order to introduce additional mutations that could enhance binding.

Phage affinity maturation libraries were generated, selected using biotinylated recombinant LAG-3 avi-Fc (BPS Bioscience, 71147) and HEK hLAG-3 cells, and screened for binding to recombinant LAG-3 Fc (R&D systems, 2319-L3 -050) by phage solid-phase ELISA. 86 unique Fcabs (containing a shortened hinge region) were expressed in HEK293F cells. Selected Fcabs were also screened for activity in a T cell activation assay as described above. Characteristics of the nine Fcabs with maximum activity in the T cell activation assay identified at second affinity maturation (FS18-7-32; FS18-7-33; FS18-7-36; FS18-7-58; FS18-7-62; FS18 -7-65; FS18-7-78; FS18-7-88; and FS18-7-95), as well as the original Fcab clone, FS18-7-9, were further investigated as described below. The alignment of these nine Fcabs with respect to the sequence of the original Fcab clone, FS18-7-9, is shown in Figure 1A. Figure 1B details the percent sequence identity of each of the nine Fcab clones with respect to the original Fcab clone, FS18-7-9. The Fcabs generated by affinity maturation of the other two original Fcab clones, FS18-7-7 and FS18-7-11, were not as promising candidates as those generated by affinity maturation of FS18-7-9 and therefore were not further investigated.

1.2 Selection of a surrogate Fcab specific for mouse LAG-3

Fcab FS18-7 selected using the naive selection protocol described above was used to generate phage libraries for selection using mouse LAG-3. Two rounds of affinity maturation were performed, and after affinity maturation, Fcab clones FS18-7-108-29 and FS18-7-108-35 were selected, showing high affinity specific binding to mouse LAG-3. The ability of FS18-7-108-29 and FS18-7-108-35 to inhibit mouse LAG-3 in a T cell activation assay was confirmed. Epitope mapping using Octet (Forteo Bio) showed that mouse anti-LAG-3 Fcabs competed with human anti-LAG-3 Fcabs (selected after second affinity maturation as described above) for binding to human LAG-3. There were 4 to 8 differences between anti-human LAG-3 and anti-mouse LAG-3 Fcab residues. Thus, anti-LAG-3 mouse Fcabs are expected to be suitable surrogates for studying the binding and function of anti-LAG-3 human Fcabs in mice.

1.3 Construction and expression of mock mAb ²

“Sham” mAbs ² containing the basic anti-human LAG-3 and anti-LAG-3 mouse Fcabs identified in Sections 1.1 and 1.2 above were generated in mAb ² format to characterize these Fcabs. These mock mAbs ² were derived from anti-LAG-3 Fcab and variable regions of anti-FITC antibody 4420 (see SEQ ID NO: 83, SEQ ID NO: 84 and SEQ ID NO: 85 for details) (Bedzyk, WD, et al. 1989 and Bedzyk, WD, et al. 1989). A mock mAb ² was generated both with and without a LALA mutation in the heavy chain CH2 domain (SEQ ID NOs: 63, 65, 67, 69, 71, 73, 75, 77, 79 and 81) (SEQ ID NOs: 64). , 66, 68, 70, 72, 74, 76, 78, 80 and 82) (see section 1.5 below for details); they additionally contained the anti-FITC mAb 4420 light chain (SEQ ID NO: 85). Mimic mAbs ^{2 were} produced by transient expression in HEK293-6E cells and purified using mAb Select SuRe protein A mAb selection columns.

1.4 Fcab binding affinity for LAG-3

1.4.1 Fcab binding affinity for human LAG-3 as determined by surface plasmon resonance (SPR)

BIAcore T200 (GE Healthcare) was used to measure the affinity of anti-human LAG-3 Fcab in a mock mAb ² format for human LAG-3. Human LAG-3-Fc (R&D Systems, 2319-L3-050) was immobilized in flow cell 4 on a CM5 sensor chip (GE Healthcare, BR1005-30) and flow cell 3 was buffered for reference comparison using an amine coupling kit (GE Healthcare, BR-1000-50). LAG-3-Fc was diluted to a concentration of 5 μg/ml with sodium acetate, pH 5 (ForteoBio, 18-1069) and injected at a flow rate of 10 μl/min for 12 seconds, followed by deactivation of the surface by injection of ethanolamine for 420 seconds. The level of immobilization was 158 RE. Fake mAb ² (or control mAb 25F7 against human LAG-3) was diluted with HBS-P buffer (GE Healthcare, BR-1003-68) in a series of 2-fold dilutions at a concentration of 4 μg/ml. The control mAb/dummy mAb ² was injected with an association time of 240 seconds at a rate of 30 μl/min and a dissociation time of 300 seconds at a rate of 30 μl/min. The surface was regenerated using 25 mM NaOH for 30 seconds at a rate of 100 μl/min. After double subtraction of the control standard readings, the data was analyzed using the BIAevaluation 3.2 software, calculating the kinetic constants. Fcabs in mock mAb ² format had an affinity for human LAG-3 in the range of 0.8-1.1 nM (Table 1), which is similar to that of the reference mAb 25F7 against human LAG-3. This was unexpected because Fcabs have a smaller binding interaction surface than monoclonal antibodies, since the Fcab binding sites form a relatively compact antibody fragment with two binding sites located in relative proximity. In contrast, the Fab arms of a typical mAb are separated by a flexible hinge region. Given the smaller binding surface area and the associated reduced flexibility of the two binding sites in the Fc region, the ability of the anti-LAG-3 Fcab to bind and inhibit LAG-3 with similar affinity and potency to the 25F7 reference antibody was unexpected.

1.4.2 Binding affinity of mouse LAG-3 specific surrogate Fcab to mouse LAG-3 as determined by SPR

BIAcore 3000 (GE Healthcare) was used to measure the affinity of mouse LAG-3 specific surrogate Fcabs for mouse LAG-3. Amine coupling (amine coupling kit, GE Healthcare, BR-1000-50) was used to immobilize mLAG-3 Fc (R&D Systems, 3328-L3-050) diluted with 10 mM sodium acetate, pH 5.0 (ForteBio, 18- 1069), directly on the CM5 chip (GE Healthcare, BR-1000-12). Mouse Fc (SinoBiological, 51094-MNAH) was immobilized in flow cell 1 and 950 RU of mLAG-3 Fc was immobilized in flow cell 2. Fcab was diluted with HBS-P buffer (GE Healthcare, BR-1003-68) and injected at various concentrations (four-fold dilutions from a concentration of 100 mM) for 3 min at a rate of 20 μl/min, and then left to dissociate in buffer for 12 min . The chip was regenerated by injecting 10 mM glycine, pH 2.5, for 30 seconds at a rate of 30 μl/min. After double subtraction of the control standard readings, the data was analyzed using the BIAevaluation 3.2 software, calculating the kinetic constants. The surrogate Fcabs tested bind to mouse LAG-3 with an affinity in the nanomol region as shown in Table 2.

1.4.3 Fcab binding affinity for cell-expressed human LAG-3 as determined by flow cytometry

Production of cell lines overexpressing LAG-3

Lentiviral transduction methodology using the Lenti-X HTX Packaging System (Clontech, cat# 631249) was used to generate DO11.10 cells (National Jewish Health) overexpressing human, cynomolgus or mouse LAG-3. Lenti-X expression vector (pLVX) (Clontech, cat# 631253) containing mouse LAG-3 cDNA (SEQ ID NO: 96), human LAG-3 cDNA (SEQ ID NO: 95), or cynomolgus macaque LAG-3 cDNA (SEQ ID NO: 97) were co-transfected with Lenti-X HTX Packaging Mix into Lenti-X 293T cell line (Clontech, Cat # 632180) to generate virus. DO11.10 cells were transduced using lentiviral vectors obtained using the Lenti-X HTX Packaging System.

The affinity of the anti-human LAG-3 Fcab in mock mAb ² format to cells expressing human LAG-3 (human LAG-3 transfected DO11.10 cells) was measured by flow cytometry. Dilutions of mAb ² and control mAb (2 x final concentration) were made in triplicate in 1 x DPBS (Gibco, 14190-094). DO11.10:LAG-3 cell suspensions were prepared in PBS + 2% BSA (Sigma, A7906) and plated at 4 x 10 ^-6 cells/mL in a volume of 50 μL/well in 96-well V-bottom plates ( Costar, 3897). 50 μl dilutions of mAb ² or control mAb (mAb 25F7 against human LAG-3) were added to wells containing cells (final volume 100 μl) and incubated at 4°C for 1 hour. The plates were washed and then a secondary antibody (anti-human Fc-488 antibody, Jackson ImmunoResearch, 109-546-098) diluted 1:1000 with PBS + 2% BSA at 100 µl/well was added and incubated for 30 min at 4°C in the dark. The plates were washed and resuspended in 100 µl PBS containing DAPI (Biotium, 40043) at a concentration of 1 µg/ml. Plates were read using a Canto II flow cytometer (BD Bioscience). Dead cells were excluded and fluorescence was measured on the FITC channel (488 nm/530/30). The data was fitted with a logarithmic function (for agonistic interaction) versus response in the GraphPad Prism software. All Fcabs tested in dummy mAb ² format and reference mAb 25F7 against human LAG-3 bound human LAG-3 with a similar affinity (EC50) in the 1.2-2.1 nm range as shown in Table 3.

1.4.4 Binding affinity of Fcab to cell-expressed cynomolgus macaque LAG-3 as determined by flow cytometry

The affinity of the anti-human LAG-3 Fcab in mock mAb ² format to cells expressing cynomolgus macaque LAG-3 (DO11.10 cells transfected with cynomolgus macaque LAG-3) was measured by flow cytometry. Dilutions of mAb ² and control mAb (2 x final concentration) were made in triplicate in 1 x DPBS (Gibco, 14190-094). DO11.10:LAG-3 cell suspensions were prepared in PBS + 2% BSA (Sigma, A7906) and plated at 4 x 10 ^-6 cells/mL in a volume of 50 μL/well in 96-well V-bottom plates ( Costar, 3897). 50 μl dilutions of mAb ² or control mAb (mAb 25F7 against human LAG-3) were added to wells containing cells (final volume 100 μl) and incubated at 4°C for 1 hour. The plates were washed and then a secondary antibody (anti-human Fc-488 antibody, Jackson ImmunoResearch, 109-546-098) diluted 1:1000 with PBS + 2% BSA at 100 µl/well was added and incubated for 30 min at 4°C in the dark. The plates were washed and resuspended in 100 µl PBS containing DAPI (Biotium, 40043) at a concentration of 1 µg/ml. Plates were read using a Canto II flow cytometer (BD Bioscience). Dead cells were excluded and fluorescence was measured on the FITC channel (488 nm/530/30). The data was fitted with a logarithmic function (for agonistic interaction) versus response in the GraphPad Prism software. The tested Fcabs in mock mAb ² format bound cynomolgus macaque LAG-3 with an affinity of 0.5-0.6 nM, suggesting the possibility that toxicology studies in cynomolgus monkeys would predict effects seen in humans (see table). four). The reference mAb 25F7 against human LAG-3 bound cynomolgus monkey LAG-3 with a 15-fold lower affinity (EC50) (Table 4).

1.4.5 Binding Affinity of Surrogate Anti-LAG-3 Mouse Fcabs and Fcabs against Human LAG-3 to Mouse LAG-3 Expressed on Cells as Determined by Flow Cytometry

Production of HEK cells overexpressing mLAG-3

The mouse LAG-3 sequence (SEQ ID NO: 96) was subcloned into the pcDNA5FRT vector (Life Technologies, V6010-20) using restriction enzyme digestion with KpnI (NEB, R0142) and NotI (NEB, R0146). The vector was then transformed into Flp-In T-REx 293 HEK cell line (Life Technologies, R780-07) using Lipofectamine 2000 (Life Technologies, 11668-019). Transformed Flp-In T-REx 293 cells were grown in DMEM (Life Technologies, 61965-026) containing 10% FBS (Life Technologies, 10270-1-6), 100 μg/ml hygromycin B (Melford Laboratories Ltd, Z2475) , 15 µg/ml of blasticidin (Melford Laboratories Ltd, B1105) for 3-4 weeks until visible colonies of stably transformed cells appear. These colonies were amplified in the presence of 1 μg/ml doxycycline (Sigma, D9891) and tested for mouse LAG-3 expression using a PE-conjugated anti-mouse LAG-3 antibody (clone C9B7W, BD Biosciences, 552380).

The affinity of mouse anti-LAG-3 surrogate Fcabs (containing a shortened hinge region; SEQ ID NO: 58) for mouse LAG-3 expressed on cells was determined by flow cytometry. mLAG-3 expressing HEK cells grown in DMEM (Life Technologies, 61965-026) containing 10% FBS (Life Technologies, 10270-1-6), 100 mg/ml hygromycin B (Melford Laboratories Ltd, Z2475), 15 mg/mL blasticidin (Melford Laboratories Ltd, B1105) and 1 μg/mL doxycycline (Sigma, D9891) were separated from tissue culture flasks using cell dissociation buffer (Life Technologies, 13151-014) and seeded into 96-well V-bottom plates (Costar, 3897) based on 2x10 ⁵ cells/well. The plates were centrifuged at 1500 rpm for 3 min at 4°C to pellet the cells. A series of dilutions of Fcab (or control mAb) were incubated with cells in a volume of 100 μl for 1 h at 4°C. Plates were washed, secondary antibody (anti-human Fc-488, Jackson ImmunoResearch, 109-546-098 for Fcab or anti-rat IgG (H+L), Alexa Fluor 488 conjugate, ThermoFisher, A-11006 for C9B7W) was diluted with PBS in a ratio of 1:1000 and added in an amount of 100 μl to the cells for 30 min at 4°C (the plates were kept in the dark). After the plates were washed, the cells were resuspended in 100 μl PBS containing 1 μg/ml DAPI (Biotium, 40043). Plates were read using a Canto II flow cytometer (BD Bioscience). Dead cells were excluded and fluorescence was measured on the FITC channel (488 nm/530/30). The data was fitted with a logarithmic function (for agonistic interaction) versus response in the GraphPad Prism software. The tested Fcabs bound to mouse LAG-3 with similar affinity (see Table 5). The reference anti-LAG-3 mAb C9B7W (2B Scientific, BE0174-50MG) bound mouse LAG-3 with 17-fold lower affinity (EC50) compared to Fcab (Table 5).

The affinity of anti-human LAG-3 Fcab FS18-7-9 in mock mAb ² format for mouse LAG-3 expressed by the cells was determined by flow cytometry. mLAG-3 expressing HEK cells grown in DMEM (Life Technologies, 61965-026) containing 10% FBS (Life Technologies, 10270-1-6), 100 mg/ml hygromycin B (Melford Laboratories Ltd, Z2475), 15 mg/mL blasticidin (Melford Laboratories Ltd, B1105) and 1 μg/mL doxycycline (Sigma, D9891) were separated from tissue culture flasks using cell dissociation buffer (Life Technologies, 13151-014) Cells were harvested by centrifugation at 1500 rpm /min for 3 min at 4°C, pelleting the cells and then resuspending them in 1 x DPBS followed by seeding in 96-well V-bottom plates (Costar, 3897) at a rate of 1.2x10 ⁵ cells/well in volume of 30 µl. Added a series of dilutions mAb ² (or control mAb) in a volume ratio of 1:1 and incubated them with cells for 1 h at 4°C. The plates were washed, the secondary antibody (anti-human Fc-488 antibody, Jackson ImmunoResearch, 109-546-098) was diluted 1:1000 with PBS and added 60 µl to the cells for 30 min at 4°C (plates were kept in the dark) . After the plates were washed, the cells were resuspended in 60 μl PBS containing 1 μg/ml DAPI (Biotium, 40043). Plates were read using a Canto II flow cytometer (BD Bioscience). Dead cells were excluded and fluorescence was measured on the FITC channel (488 nm/530/30). The data was fitted with a logarithmic function (for agonistic interaction) versus response in the GraphPad Prism software. Fcab FS18-7-9 against human LAG-3 in mock mAb ² format bound mouse LAG-3 with an EC50 of 19 nM compared to an EC50 of 2.6 nM for surrogate Fcab FS18-7-9-108 against LAG- 3 mice (Table 6). The human 25F7 mAb showed no detectable binding to mouse LAG-3, indicating a different binding epitope for anti-human LAG-3 Fcab FS18-7-9 on LAG-3 compared to the binding epitope for 25F7.

1.5 Fcab binding affinity for Fc receptors

Introduction of a LALA mutation into the CH2 domain of human IgG1 is known to impair binding to the Fcγ receptor (Bruhns, P., et al. (2009); and Xu, D. et al. (2000)). BIAcore was used to confirm that the LALA mutation reduced the binding affinity of Fcab (in mock mAb ² format) to Fcγ receptors. Human FcγR binding assay was performed on a Biacore T200 instrument (GE Healthcare) using Fcab in mock mAb ² format. Human FcγR (R&D Systems, 1257-FC, 1330-CD, 1875-CD, 4325-FC) was immobilized using amine coupling (amine coupling kit, GE Healthcare, BR-1000-50) on a Series S CM5 chip (GE Healthcare , BR-1005-30) at an areal density of 370 RU for FcγRI, 264 RU for FcγRIII (high affinity human FcγR) and 500 RU for FcγRIIa and FcγRIIb (low affinity human FcγR). For each immobilized chip, a blank flow cell was left to subtract background values. FcγR was immobilized at a concentration of 5 μg/ml in sodium acetate, pH 5 (ForteBio, 18-1069) and injected at a flow rate of 10 μl/min in 15 second cycles until the desired level of immobilization was achieved.

For high affinity FcγRI and FcγRIII, 200 μg/mL mAbs or spoof mAbs ² were flowed through the chip for 3 min at a flow rate of 30 μL/min followed by dissociation for 5 min. The running buffer was HBS-P (0.01 M HEPES, pH 7.4, 0.15 M NaCl, 0.005% v/v surfactant P20, GE Healthcare, BR-1003-68). For low affinity FcγRIIa and FcγRIIb, the mock mAb ² concentration was increased to 500 μg/ml.

The positive control was a wild-type isotype IgG1 mAb that was compared to control IgG1 LALA mAbs and monoclonal non-specific isotypic IgG2 and IgG4 mAbs. The flow cells were regenerated by injecting 10 mM sodium hydroxide (VWR, 28244.262) at a flow rate of 100 μl/min for 30 seconds. Data analysis was performed using BiaEvaluation version 3.2 RC1 software, double-corrected for the blank flow cell value (no immobilized FcγR) and subtracting the buffer cycle from the tested mAb ² . The results are shown in Table 7.

All mock mAbs ² tested (containing the LALA mutation above) showed significantly reduced binding to the tested Fcγ receptors compared to the control antibody (mock IgG1 mAb) without the LALA mutation, indicating that these mock mAbs ² were attenuated binding to Fcγ receptors. due to LALA mutation and, consequently, the expected decrease in ADCC activity of these mAb ² .

1.6 Blocking the binding of MHC class II to LAG-3

The ability of Fcabs (containing a truncated hinge region; SEQ ID NO: 58) to block the interaction between recombinant human or mouse LAG-3 Fc and human class II MHC was investigated by measuring the binding of LAG-3 Fc to A375 cells, a melanoma cell line expressing class II MHC person. A375 cells (ATCC, CRL-1619) grown in DMEM (Life Technologies, 61965-026) containing 10% FBS (Life Technologies, 10270-106-6) were separated from cell culture flasks using cell dissociation buffer (Life Technologies, 13151-014) and plated in 96-well V-bottom plates (Costar, 3897) based on 2x10 ⁵ cells/well. The plates were centrifuged at 1500 rpm for 3 min at 4°C to pellet the cells. Appropriate concentrations of Fcab or control mAb were incubated with 1 µn/ml LAG-3 Fc (human LAG-3-Fc, R&D Systems, 2319-L3-050 or mouse LAG-3 Fc, R&D Systems, 3328-L3-050) at 100 µl of DMEM containing 10% FBS for 1 h at 4°C. A mixture of LAG-3/Fcab was added to A375 cells and incubated for 1 h at 4°C. The cells were washed. Secondary antibody (F(ab') ₂ goat anti-human Fc conjugated to Alexa Fluor 488, Jackson Immunoresearch, 109-546-098, or goat anti-mouse IgG (H+L) 488 conjugate, Life Technologies, A-1101) diluted with PBS at a ratio of 1:1000, and in the amount of 100 μl was added to the cells for 30 min at 4°C (the plates were kept in the dark). Cells were washed once and resuspended in 100 μl PBS + 1 μg/ml DAPI (Biotium, 40043). Plates were read on a BD FACSCanto II cytometer (BD Biosciences) and analyzed using FlowJo software.

Both anti-LAG-3 mouse Fcabs could inhibit the interaction of human MHC II with mouse LAG-3, while the control anti-LAG-3 mouse mAb (C9B7W, 2B Scientific, BE0174-50MG) did not (see Table 8) .

The tested anti-human LAG-3 Fcabs could also inhibit the interaction of human MHC class II with human LAG-3 with similar potency to the control anti-human LAG-3 mAb (25F7).

Example 2 Preparation and Characterization of Mab and Mab ² Molecules

2.1 Obtaining mAb 84G09

2.1.1 Obtaining a DNA construct

DNA inserts encoding the heavy and light chain variable regions of 84G09 were codon optimized for expression in animal cells and synthesized using DNA2.0 (Menlo Park, CA, USA). Inserts in the pJ-Amp-high host vector were subcloned into the expression vectors pFS-hHC2.1-G1m17(z) LALA (IgG heavy chain containing the LALA mutation) or pFS-hHC2.1-G1m17(z) (IgG heavy chain without mutation LALA) and pFShK1.0 (IgG kappa light chain) using restriction enzyme digestion with EcoRI and NheI.

The correctness of cloning was verified by colony PCR and subsequent nucleotide sequencing by a third party (GATC Biotech).

2.1.2 Cell maintenance

HEK293-6E cells (NRCC) were subcultured in prewarmed F17 medium (Invitrogen, A13835-01) supplemented with 4 mM GlutaMAX-1 (Invitrogen, 35050-038), 0.1% Pluronic F-68 (Invitrogen A13835-01), and 25 µg/ml geneticin (Invitrogen, 10131-027). Cells were incubated at 37°C, 140 rpm, 5% CO ₂ and subcultured at 0.3x10 ⁶ cells/ml for three and four days.

2.1.3 Temporary transfection

HEK293-6E cells were transiently transfected with 1 mg/ml PEIpro (Polyplus, PPLU115). 24 hours before transfection, the cells were seeded at the rate of 0.8x10 ⁶ cells/ml into the culture medium. For every 200 ml of cell culture, a DNA mixture was prepared by mixing 10 ml of warmed Opti-MEMI (Invitrogen, 11058-021), 100 μg of endotoxin-free DNA encoding the heavy chain, and 100 μg of endotoxin-free DNA encoding the light chain. The PEI mixture was prepared by mixing 10 ml of warmed Opti-MEMI medium and 200 µl of PEIpro and vortexing them. The DNA mixture was quickly added to the vortexed PEI mixture, vortexed in the mode of 3 pulses per 1 s, incubated for 3 min at room temperature, and added dropwise to the cells. 48 hours after transfection, 20 μl of F17 medium supplemented with 0.5% N1 tryptone (TekniScience Inc., 19553) was added to each vial.

Cells were harvested 6 days after transfection by centrifugation at 4500 rpm for 40 minutes. The supernatant was filtered through a 0.22 μm polyethersulfone filter block (Millipore, SCGPU01RE, SCGPU02RE, SCGPU05RE, SCGPU11RE) and stored at +4°C until purified.

2.1.4 Protein A chromatography

The clarified supernatant was purified using prepackaged 5 ml HiTrap MabSelect SuRe columns (GE Healthcare, 11-0034-95) on ÄKTAexplorer or ÄKTAxpress. Briefly, the columns were equilibrated with 50 mM Tris-HCl, 250 mM NaCl at pH 7.0, unbound material was washed with the same buffer at a flow rate of 5 ml/min. The products were eluted with 10 mM sodium formate, pH 3.0 at a flow rate of 5 ml/min. The eluted samples were immediately buffer exchanged with PBS, pH 7.4 using PD-10 columns (GE Healthcare, 17-0851-01) pre-equilibrated with PBS, pH 7.4 according to manufacturer's recommendations.

2.1.5 Measurement of product concentration by spectrometry

The absorbance of each purified product at 280 nm was measured using a LabChip DS (PerkinElmer, 133089) with a DropPlate 96 D+ (PerkinElmer, CLS135136). Product concentration was calculated using the extinction coefficient (A280 for 1 mg/mL) calculated using VectorNTI Advance v11.5.4 software (Thermofisher Scientific, A13784)

2.1.6 Product concentration

Purified fractions were concentrated, if necessary, using an Amicon Ultra-4 Centrifugal Filter Unit 30K (Millipore, UFC803024). After equilibration of the regenerated cellulose membrane Ultracel PBS, pH 7.4 by centrifugation for 10 min at 3000 rpm, the samples were loaded into a 4 ml block and centrifuged at 3000 rpm until the desired protein concentration was reached.

2.1.6 Sterilization by filtration

Final samples were filtered using pre-moistened PVDF Millex-GV syringe filters (Millipore, SLGV013SL).

2.2 Obtaining human LAG-3/PD-L1 mAb ²

Heavy chain mAb ² FS18-7-9/84G09 (SEQ ID NOs 94 and 95), FS18-7-32/84G09 (SEQ ID NOs 96 and 97), FS18-7-33/84G09 (SEQ ID NOs 98 and 99), FS18-7-36/84G09 (SEQ ID NOs 100 and 101), FS18-7-58/84G09 (SEQ ID NOs 102 and 103), FS18-7-62/84G09 (SEQ ID NOs 104 and 105) , FS18-7-65/84G09 (SEQ ID NOs 106 and 107), FS18-7-78/84G09 (SEQ ID NOs 108 and 109), FS18-7-88/84G09 (SEQ ID NOs 110 and 111) and FS18 -7-95/84G09 (SEQ ID NOs 112 and 113) was obtained by replacing the CH3 domains of monoclonal antibodies 84G09 (with and without LALA mutation) with the CH3 domains of Fcab FS18-7-9, FS18-7-32, FS18-7 -33, FS18-7-36, FS18-7-58, FS18-7-62, FS18-7-65, FS18-7-78, FS18-7-88 and FS18-7-95 specific for LAG -3 human, within the XhoI and BamHI sites present in the sequence of the unmodified human IgG1 CH3 domain. The heavy chain of mAb ² was co-transfected with the light chain of 84G09 (SEQ ID NO: 116) as described for mAb 84G09 above in section 2.1. Mab ² was then expressed and purified as described for mAb 84G09 in section 2.1 above.

2.2 Preparation of mAb ² against human LAG-3/mock antigen

An anti-FITC mAb (with and without LALA mutation) was generated as described for mAb 84G09 in section 2.1 above, using heavy chains (SEQ ID NOS 83 and 84; with and without LALA mutation) and light chain (SEQ ID NOS: 85) MAT 4420.

Heavy chain mAb ² FS18-7-9/4420 (SEQ ID NOs 63 and 64), FS18-7-32/4420 (SEQ ID NOs 65 and 66), FS18-7-33/4420 (SEQ ID NOs 67 and 68, FS18-7-36/4420 (SEQ ID NOs 69 and 70), FS18-7-58/4420 (SEQ ID NOs 71 and 72), FS18-7-62/4420 (SEQ ID NOs 73 and 74), FS18-7-65/4420 (SEQ ID NOs 75 and 76), FS18-7-78/4420 (SEQ ID NOs 77 and 78), FS18-7-88/4420 (SEQ ID NOs 79 and 80) and FS18- 7-95/4420 (SEQ ID NOS 81 and 82) was obtained by replacing the CH3 domains of monoclonal antibodies 4420 (with and without LALA mutation) with the CH3 domains of Fcab FS18-7-9, FS18-7-32, FS18-7- 33, FS18-7-36, FS18-7-58, FS18-7-62, FS18-7-65, FS18-7-78, FS18-7-88 and FS18-7-95 specific for LAG- 3 human, within the XhoI and BamHI sites present in the sequence of the unmodified human IgG1 CH3 domain mAb ² heavy chains were co-transfected with mAb 4420 light chain as described for mAb 84G09 above in section 2.1 The proteins were then expressed and purified as described for MAT 84G09 above in section 2.1.

2.2 Obtaining mAb ² LAG-3/PD-L1 mice

An anti-mouse PD-L1 mAb (with and without a LALA mutation) was generated as described for mAb 84G09 in Section 2.1 above using the heavy chain (SEQ ID NOs 122 and 123) and light chain (SEQ ID NOs: 119) mAb S1 .

The heavy chain of mAb ² FS18-7-108-29/S1 (SEQ ID NOs 117 and 118) and FS18-7-108-35/S1 (SEQ ID NOs 120 and 121) molecules was obtained by replacing the CH3 domains of monoclonal antibodies S1 ( with and without LALA mutation) CH3 domains of mouse LAG-3 specific Fcab FS18-7-108-29 and FS18-7-108-35 within the XhoI and BamHI sites present in the sequence of the unmodified CH3 domain human IgG1. The mAb ² heavy chain was co-transfected with the S1 light chain as described for mAb 84G09 in section 2.1 above. The proteins were then expressed and purified as described for mAb 84G09 in section 2.1 above.

2.5 Binding affinity and kinetics of mAb ² for human LAG-3 and human PD-L1

Protein L (Thermo, 21189) was immobilized on flow cells 1 and 2 of the S series CM5 chips (GE Healthcare, BR-1005-30) by amine addition (GE Healthcare, BR-1000-50) to an areal density of 2000 PE following the manufacturer's instructions for BIAcore T200 instrument. For LAG-3 binding, mAb ² samples (all of which contained the LALA mutation) were immobilized on flow cell 2 only, and human LAG-3Fc (R&D Systems, 2319-L3) was passed through at 4 concentrations in a 2-fold dilution series starting at 0.5 nM , through both flow cells 1 and 2 at a flow rate of 30 µl/min. The association time was 3 minutes and the dissociation time was 6 minutes. The running buffer was HBS-P (GE Healthcare, BR-1003-68). Both flow cells were regenerated by injection of 10 mM sodium hydroxide (NaOH) at a flow rate of 100 μl/min for 20 seconds. The data were analyzed with a double correction for a blank flow cell.

For PD-L1 binding, four concentrations of a 2-fold dilution series of PD-L1 Fc (R&D Systems, 156-B7) starting at 40 nM were passed through mAb ² immobilized on the same L protein chip. All other conditions were the same as in the LAG-3 binding experiment (see above).

The binding kinetics were approximated by the Langmuir model at a ratio of 1:1, obtaining the rate constants of association (ka) and dissociation (kd) upon binding. Equilibrium binding constants (KD) were calculated by dividing the dissociation rate by the association rate for each sample. Data analysis was performed using BiaEvaluation software version 3.2. The results are shown in tables 10 and 11.

The binding affinity for human PD-L1 and human LAG-3 was comparable for all mAb ² tested. The binding affinity of mAb ² for human PD-L1 was comparable to that of 84G09, indicating no effect of insertion of the LAG-3 binding site into the CH3 domain on PD-L1 binding.

2.6 Co-binding of mAb ² to human LAG-3 and human PD-L1

Capacity MAT ² 84G09 and FS18-7-78/84G09, all of which contained the LALA mutation) to simultaneously bind to LAG-3 and PD-L1 was tested by PPR. Human PD-L1Fc (R&D Systems, 156-B7) was immobilized on a 2 flow cell CM5 S series chip (GE Healthcare, BR-1005-30) to an areal density of 150 PE following the manufacturer's instructions. Flow cell 1 was activated and inactivated without protein immobilization to subtract the background signal. For each sample, 10 μg/ml mAb ² was passed through flow cells 1 and 2 at a flow rate of 10 μl/min for 3 minutes. 40 nM LAG-3Fc (R&D Systems, 2319-L3) was then passed through both flow cells 1 and 2 at a flow rate of 10 µl/min for 3 minutes. Each binding step was followed by dissociation for 3 min. The sensor chip was regenerated after each cycle with a 15-s injection of 25 mM NaOH at a flow rate of 100 μl/min.

All mAb ² tested could simultaneously bind to LAG-3 and PD-L1. The original anti-PD-L1 mAb 84G09 only binds to PD-L1.

2.7. Simultaneous binding of surrogate mAb ² to mouse LAG-3 and mouse PD-L1

The ability of two mouse surrogate mAbs ² (FS18-7-108-29/S1 and FS18-7-108-35/S1, both of which contained the LALA mutation) to simultaneously bind to mouse LAG-3 and mouse PD-L1 was tested by SPR on BIAcore 3000 (GE Healthcare). Mouse PD-L1Fc (R&D Systems, 1019-B7-100) was immobilized on a 4 flow cell CM5 chip to an areal density of 830 RU following the manufacturer's instructions. 820 RE human Fc (R&D system, 110-HG) was immobilized in flow cell 3 to subtract the background signal. For each sample, 50 μg/ml mAb ² was passed through flow cells 1 and 2 at a flow rate of 20 μl/min for 150 s. 50 nM mouse LAG-3Fc (R&D Systems, 3328-L3-050) was then passed through both flow cells 3 and 4 at a flow rate of 20 μl/min for 150 s. Each binding step was followed by dissociation for 3 min. The sensor chip was regenerated after each cycle using 2 x 10 µl 50 mM NaOH. Both mAb ² tested could simultaneously bind to mouse LAG-3 and mouse PD-L1 and were therefore suitable surrogates of human LAG-3/PD-L1 mAb ² .

2.8 Human Fcγ Receptor Binding of mAbs ² Containing the LALA Mutation

Human Fcγ receptors were immobilized on a CM5 chip to an areal density of approximately 200 RU for Fcγ RI (R&D Systems, 1257-FC) and Fcγ RIIIa (R&D Systems, 4325-FC) and approximately 500 RU for Fcγ RIIa (R&D Systems, 1330-CD) and Fcγ RIIb/c (R&D Systems, 1875-CD) according to the manufacturer's instructions for the BIAcore3000 instrument. For Fcγ RI and Fcγ RIIIa, 100 μg/ml mAb or mAb ² was passed through the chip for 3 min at a flow rate of 10 μl/min followed by dissociation for 5 min. The running buffer was PBS (Lonza, BE17-516F) + 0.05% (v/v) surfactant P20 (GE Healthcare, BR-1000-54). The positive control was wild type IgG1 mAb 4420. IgG2 and IgG4 monoclonal mAbs against foreign targets (20H4 and MOR7490) and mouse IgG1 (Sigma, P5305) were included as references. Regeneration was not required due to the rapid dissociation of the complexes upon binding. For testing Fcγ RIIa and Fcγ RIIb/c mAb ² concentration was increased to 500 μg/ml to compensate for the weaker binding to these two receptors. The results are shown in Table 12.

As expected, the LALA variant introduced into the mAb or mAb ² reduced the ability of these molecules to bind human Fcγ receptors.

2.9 Mab ² Binding to Cells Expressing Human and Cynomolgus LAG-3

A human LAG-3 sequence (SEQ ID NO: 126) or a cynomolgus monkey LAG-3 sequence (SEQ ID NO: 128) was subcloned into the pcDNA5FRT vector (Life Technologies, V6010-20) using KpnI restriction enzyme digestion (NEB, R0142) and NotI (NEB, R0146). The vector was then transformed into Flp-In T-REx 293 HEK cell line (Life Technologies, R780-07) using Lipofectamine 2000 (Life Technologies, 11668-019). Transformed Flp-In T-REx 293 cells were grown in DMEM (Life Technologies, 61965-026) containing 10% FBS (Life Technologies, 10270-1-6), 100 μg/ml hygromycin B (Melford Laboratories Ltd, Z2475) , 15 µg/ml of blasticidin (Melford Laboratories Ltd, B1105) for 3-4 weeks until visible colonies of stably transformed cells appear. These colonies were amplified in the presence of 1 μg/ml doxycycline (Sigma, D9891) and tested for LAG-3 expression, which was confirmed by flow cytometry.

The affinity of mAb ² (all of which contained the LALA mutation) for cells expressing human or cynomolgus LAG-3 was determined by flow cytometry. HEK cells expressing human or cynomolgus LAG-3 grown in DMEM (Life Technologies, 61965-026) containing 10% FBS (Life Technologies, 10270-1-6), 100 μg/ml hygromycin B (Melford Laboratories Ltd , Z2475), 15 μg/ml blasticidin (Melford Laboratories Ltd, B1105) and 1 μg/ml doxycycline (Sigma, D9891) were separated from tissue culture flasks using cell dissociation buffer (Life Technologies, 13151-014) and plated in 96-well V-bottom plates (Costar, 3897) at a rate of 2x10 ⁵ cells/well. The plates were centrifuged at 1500 rpm for 3 min at 4°C to pellet the cells. A series of dilutions of mAb ² (or control mAb) were incubated with cells in a volume of 100 μl for 1 h at 4°C. Plates were washed, secondary antibody (anti-human Fc-488 antibody, Jackson ImmunoResearch, 109-546-098) was diluted 1:1000 with PBS and added at 100 µl to cells for 30 min at 4°C (plates were kept in the dark) . The plates were washed and then the cells were resuspended in 100 μl PBS containing 1 μg/ml DAPI (Biotium, 40043). Plates were read using a Canto II flow cytometer (BD Bioscience). Dead cells were excluded and fluorescence was measured on the FITC channel (488 nm/530/30). The data was fitted with a logarithmic function (for agonistic interaction) versus response in the GraphPad Prism software. Plates were read on a BD FACSCanto II cytometer (BD Biosciences) and analyzed using FlowJo software. The results are shown in Table 13.

The results confirmed the binding of mAb ² to human and cynomolgus macaque LAG-3 expressed on HEK cells. In terms of calculated EC ₅₀ values, the tested mAbs ² showed better or equal binding to human LAG-3 and at least twice as good binding to cynomolgus monkey LAG-3 compared to the control anti-LAG-3 antibody 25F7. Cross-reactivity with other proteins expressed on the surface of the HEK cell line was not observed.

2.10 Binding of mAb ² to cells expressing human and cynomolgus monkey PD-L1

The human PD-L1 sequence (SEQ ID NO: 129) or the cynomolgus monkey PD-L1 sequence (SEQ ID NO: 131) were subcloned into the pcDNA5FRT vector (Life Technologies, V6010-20) using restriction enzyme digestion with KpnI (NEB, R0142) and NotI (NEB, R0146). The vector was then transformed into Flp-In T-REx 293 HEK cell line (Life Technologies, R780-07) using Lipofectamine 2000 (Life Technologies, 11668-019). Transformed Flp-In T-REx 293 cells were grown in DMEM (Life Technologies, 61965-026) containing 10% FBS (Life Technologies, 10270-1-6), 100 μg/ml hygromycin B (Melford Laboratories Ltd, Z2475) , 15 µg/ml of blasticidin (Melford Laboratories Ltd, B1105) for 3-4 weeks until visible colonies of stably transformed cells appear. These colonies were amplified in the presence of 1 μg/ml doxycycline (Sigma, D9891) and LAG-3 expression was confirmed by flow cytometry.

The binding affinity of mAb ² (all of which contained the LALA mutation) to cells expressing human or cynomolgus monkey PD-L1 or to parental (non-transformed cells) was determined by flow cytometry. HEK cells expressing human or cynomolgus PD-L1 grown in DMEM (Life Technologies, 61965-026) containing 10% FBS (Life Technologies, 10270-1-6), 100 μg/ml hygromycin B (Melford Laboratories Ltd , Z2475), 15 μg/ml blasticidin (Melford Laboratories Ltd, B1105) and 1 μg/ml doxycycline (Sigma, D9891) were separated from tissue culture flasks using cell dissociation buffer (Life Technologies, 13151-014) and plated in 96-well V-bottom plates (Costar, 3897) at 2x105 cells/well. The plates were centrifuged at 1500 rpm for 3 min at 4°C to pellet the cells. A series of dilutions of mAb ² (or control mAb) were incubated with cells in a volume of 100 μl for 1 h at 4°C. Plates were washed, secondary antibody (anti-human Fc-488 antibody, Jackson ImmunoResearch, 109-546-098) was diluted 1:1000 with PBS and added at 100 µl to cells for 30 min at 4°C (plates were kept in the dark) . The plates were washed and then the cells were resuspended in 100 μl PBS containing 1 μg/ml DAPI (Biotium, 40043). Plates were read using a Canto II flow cytometer (BD Bioscience). Dead cells were excluded and fluorescence was measured on the FITC channel (488 nm/530/30). The data was fitted with a logarithmic function (for agonistic interaction) versus response in the GraphPad Prism software. Plates were read on a BD FACSCanto II cytometer (BD Biosciences) and analyzed using FlowJo software. The results are shown in Table 14.

All tested mAb ² LAG-3/PD-L1 bound to human PD-L1 and cynomolgus monkey PD-L1 with an EC50 close to that of mAb 84G09, demonstrating no effect of insertion of the LAG-3 binding site into the CH3 domain of mAb ² on binding affinity for PD-L1.

2.11 Binding of mouse surrogate mAb ² to cells expressing mouse LAG-3 or mouse PD-L1

The mouse LAG-3 sequence (SEQ ID NO: 127) or mouse PD-L1 sequence (SEQ ID NO: 130) was subcloned into the pcDNA5FRT vector (Life Technologies, V6010-20) using restriction enzyme digestion with KpnI (NEB, R0142) and NotI (NEB, R0146). The vector was then transformed into Flp-In T-REx 293 HEK cell line (Life Technologies, R780-07) using Lipofectamine 2000 (Life Technologies, 11668-019). Transformed Flp-In T-REx 293 cells were grown in DMEM (Life Technologies, 61965-026) containing 10% FBS (Life Technologies, 10270-1-6), 100 μg/ml hygromycin B (Melford Laboratories Ltd, Z2475) , 15 µg/ml of blasticidin (Melford Laboratories Ltd, B1105) for 3-4 weeks until visible colonies of stably transformed cells appear. These colonies were amplified in the presence of 1 μg/ml doxycycline (Sigma, D9891) and LAG-3 or PD-L1 expression was confirmed by flow cytometry.

The binding affinity of mAbs ² (all of which contained LALA mutations) to cells expressing mouse LAG-3 or mouse PD-L1 was determined by flow cytometry. HEK cells expressing mouse LAG-3 or mouse PD-L1 grown in DMEM (Life Technologies, 61965-026) containing 10% FBS (Life Technologies, 10270-1-6), 100 μg/ml hygromycin B (Melford Laboratories Ltd, Z2475), 15 μg/ml blasticidin (Melford Laboratories Ltd, B1105) and 1 μg/ml doxycycline (Sigma, D9891) were separated from tissue culture flasks using cell dissociation buffer (Life Technologies, 13151-014) and plated in 96-well V-bottom plates (Costar, 3897) at 2x10 ⁵ cells/well. The plates were centrifuged at 1500 rpm for 3 min at 4°C to pellet the cells. A series of dilutions of mAb ² (or control mAb) were incubated with cells in a volume of 60 μl for 1 h at 4°C. Plates were washed, secondary antibody (anti-human Fc-488, Jackson ImmunoResearch, 109-546-098 for Fcab or anti-rat IgG (H+L), conjugate with Alexa Fluor 488, ThermoFisher, A-11006 for control anti-C9B7W antibody LAG-3) was diluted with PBS at a ratio of 1:1000 and added in an amount of 50 μl to the cells for 30 min at 4°C (the plates were kept in the dark). The plates were washed and then the cells were resuspended in 50 µl FACS Cell Fix (BD Bioscience, 340181) for 15 min, then washed and resuspended in 100 µl PBS containing 1 µg/ml DAPI (Biotium, 40043). Plates were read using a Canto II flow cytometer (BD Bioscience). Dead cells were excluded and fluorescence was measured on the FITC channel (488 nm/530/30). The data was fitted with a logarithmic function (for agonistic interaction) versus response in the GraphPad Prism software. Plates were read on a BD FACSCanto II cytometer (BD Biosciences) and analyzed using FlowJo software. The results are shown in Table 15.

Surrogate mAbs ² could bind to mouse LAG-3 expressed by cells and mouse PD-L1 expressed by cells. The binding affinity of the surrogate mAb ² to mouse LAG-3 expressed on cells was approximately the same as that of the anti-human LAG-3/PD-L1 mAb ² to human LAG-3 (2.3-3.8 nM versus 2.5 -4.2 nM), and the binding affinity of the surrogate mAb ² to mouse cell-expressed PD-L1 was up to 3-fold higher than the affinity of mAb ² against human LAG-3/PD-L1 to human PD-L1 (11.9-12 .3 nM versus 3.1-4.0 nM), demonstrating that these mAb ² were suitable surrogates for human anti-LAG-3/PD-L1 mAb ² for use in in vivo studies in mice.

Example 3 Activity of Mab ² Molecules in T Cell Activation Assay and SEB Assay

3.1 T cell activation assay

An IL-2 release assay based on hybridoma lines of T-lymphocytes DO11.10 OVA and B-lymphocytes LK35.2 was used for mAb ² functional screening. The release of IL-2 is a marker of T-cell activation. T cells expressing endogenous mouse PD-1 were transfected with an empty vector (pLVX) or a human LAG-3 construct. B cells were transfected with an empty vector (pLVX) or a human PD-L1 construct.

Three combinations of these four cell lines in parallel were used to test T cell activation by mAb ² :

• DO11.10 pLVX + LK35.2 hPD-L1 for anti-PD-L1 activity;

• DO11.10 hLAG-3 + LK35.2 pLVX for anti-LAG-3 activity;

• DO11.10 hLAG-3 + LK35.2 hPD-L1 for simultaneous activity against LAG-3/PD-L1.

All mAbs ² (all of which contained the LALA mutation) were tested in duplicate in this T cell activation assay. Cross-reactivity with cynomolgus monkey LAG-3 and PD-L1 was tested in a functional T cell activation assay using cells overexpressing cynomolgus targets (cPD-L1 and cLAG-3).

Generation of T cell lines overexpressing LAG-3

Lentiviral transduction methodology using the Lenti-X HTX Packaging System (catalog # 631249) was used to generate DO11.10 cells (National Jewish Health) overexpressing human, cynomolgus or mouse LAG-3. Lenti-X expression vector (pLVX) (catalog # 631253) containing mouse LAG-3 cDNA (SEQ ID NO: 127), human LAG-3 cDNA (SEQ ID NO: 126), or cynomolgus macaque LAG-3 cDNA (SEQ ID NO: 128) were co-transfected with Lenti-X HTX Packaging Mix into Lenti-X 293T cell line (catalog # 632180) to generate virus. DO11.10 cells were transduced using lentiviral vectors obtained using the Lenti-X HTX Packaging System.

Production of antigen-presenting cells overexpressing PD-L1

To generate LK35.2 B cell lymphoma (ATCC, HB-98) overexpressing human, cynomolgus or mouse PD-L1, a lentiviral transduction methodology was used using the Lenti-X HTX Packaging System (catalog # 631249). Lenti-X expression vector (pLVX) (catalog # 631253) containing mouse PD-L1 cDNA (SEQ ID NO: 130), human PD-L1 cDNA (SEQ ID NO: 129), or cynomolgus monkey PD-L1 cDNA (SEQ ID NO: 131) were co-transfected with Lenti-X HTX Packaging Mix into Lenti-X 293T cell line (catalog # 632180) to generate virus. LK35.2 cells were transduced using lentiviral vectors obtained using the Lenti-X HTX Packaging System.

Medium and peptide

Cell culture medium: DMEM (Gibco, 61965-026) 10% FBS (Gibco, 10270-106), 1 mM sodium pyruvate (Gibco, 11360-070), 1 μg/ml puromycin (Gibco, A11138-03)

Experimental medium: complete culture medium for DO11.10 without puromycin. Peptide OVA (molecular weight = 1773.9 Da): H-ISQAVHAAHAEINEAGR-OH (Pepscan)

Cells:

• DO11.10 hLAG-3: DO11.10 T-cell hybridoma transduced with a lentiviral vector and overexpressing human LAG-3;

• DO11.10 pLVX: DO11.10 T-cell hybridoma transduced with an empty lentiviral vector;

• DO11.10 cLAG-3: DO11.10 T-cell hybridoma transduced with a lentiviral vector and overexpressing cynomolgus monkey LAG-3.

• LK 35.2 hPD-L1: B cell hybridoma transduced with a lentiviral vector containing hPD-L1 and overexpressing human PD-L1;

• LK 35.2 PLVX: B-cell hybridoma transduced with an empty lentiviral (pLVX) vector;

• LK 35.2 cPD-L1: B-cell hybridoma transduced with a lentiviral vector and overexpressing cynomolgus monkey PD-L1.

DO11.10 cells (DO11.10 pLVX cells or DO11.10 hLAG-3 cells) at a concentration of 0.3 x 10 ⁶ cells/ml were mixed 1:1 with antibodies at 3 times the final concentration. Antibodies and DO11.10 cells were incubated at 37°C with 5% CO ₂ for 1 hour. LK 35.2 cells (both pLVX and PD-L1-pLVX) were incubated at a concentration of 3x10 ⁵ cells/ml of experimental medium with OVA peptide at a concentration of 1.5 μM for 30 min. LK 35.2 + OVA cells were added to DO11.10 cells/treatment mix in a 1:2 ratio in the following combinations:

Functional screening with human antigens

DO11.10 pLVX + LK35.2 hPD-L1,

DO11.10 hLAG-3 + LK35.2 pLVX,

DO11.10 hLAG-3 + LK35.2 hPD-L1;

Cross-reactivity screening with cynomolgus monkey antigens

DO11.10 pLVX + LK35.2 cPD-L1,

DO11.10 cLAG-3 + LK35.2 pLVX,

DO11.10 cLAG-3 + LK35.2 cPD-L1;

Cells were incubated at 37°C, 5% CO ₂ for 24 hours. The supernatant was collected and analyzed using a mouse IL-2 solid phase ELISA kit (eBioscience, 88-7024-88 or R&D systems, SM2000) according to the manufacturer's instructions. Plates were read at 450 nm using a plate reader and software Gen5, BioTek. The absorbance values at 570 nm were subtracted from the values at 450 nm (correction). The standard curve for cytokine concentration calculation was based on a four-parameter logistic model fit (Gen5 software, BioTek). The concentration of mIL-2 was plotted against the logarithm of the Fcab or mAb concentration, the resulting curves were fitted with a logarithmic function equation (for agonistic interaction) depending on the response in the GraphPad Prism software. Table 16 shows EC50 values and maximum IL-2 release for mAb ² and control mAbs calculated as percentage of control (84G09+25F7). Figure 2A-F shows representative IL-2 release schedules for a panel of cell lines treated with FS18-7-9/84G09, FS18-7-62/84G09 or FS18-7-78/84G09 and control antibodies. In the analysis of T cell activation in the presence of both targets (DO11.10 hLAG-3 + LK35.2 hPD-L1), IL-2 release was induced only upon inhibition of both LAG-3 and PD-L1, for example, when using mAb ² or combinations of antibodies against LAG-3 and PD-L1. Thus, mAb ² had an advantage over single agents alone. In assays in the presence of LAG-3 (DO11.10 hLAG-3 + LK35.2 pvlx) or PD-L1 (DO11.10 pvlx + LK35.2 PD-L1) alone, mAbs ² showed similar activity in inhibiting these targets compared to single agents, demonstrating that mAb ² was the only single molecule capable of leading to T cell activation in the presence of LAG-3, PD-L1, or LAG-3+PD-L1.

One of the mAbs ² (FS18-7-9/84G09 containing the LALA mutation) was tested for functional cross-reactivity with cynomolgus monkey antigens in the DO11.1-/LK35.2 T cell activation assay. Figure 3 A-C shows the results for a panel of three cell-based assays. FS18-7-9/84G09 could induce T-cell activation in the presence of either cLAG-3 + cPD-L1 or cLAG-3 or cPD-L1 alone, indicating functional cross-reactivity of this mAb ² with antigens cynomolgus monkey and therefore suitability for use in primate safety studies.

3.2 Staphylococcal enterotoxin B assay

Three mAbs ² (all of which contained the LALA mutation) were tested in the human MIC-based staphylococcal enterotoxin B assay (SEB assay). Staphylococcal enterotoxin B is a superantigen and binds to MHC class II molecules on antigen presenting cells (APCs) and the T cell receptor (TCR) vβ chain, causing non-specific T cell activation and cytokine release. There are no requirements for presenting antigen to observe T cell activation. The SEB assay uses stimulated human cells (MPCs) with physiological levels of inhibitors of key components of the immune response; it can be used to confirm the enhancement of T-cell activation by mAb ² in humans. Three mAbs ^{2 were} tested in the SEB system with cells from four different donors.

Obtaining Expanded T Cells

MICs were isolated from the buffy coat by ficoll gradient separation. CD4+ T cells were isolated using the Human CD4+ T Cell Isolation Kit (Miltenyi Biotec Ltd, 130-096-533) according to the manufacturer's instructions. Human T-Activator CD3/CD28 Dynabeads (Life technologies, 11131D) human T-cell activator beads were resuspended by vortexing. The beads were transferred to a sterile 15 ml tube and 10 ml of RPMI medium (Life Technologies, 61870044) with 10% FBS (Life Technologies, 10270106) and 1x penicillin-streptomycin (Life Technologies, 15140122) was added to the washed Dynabeads. The supernatant was discarded. Required number of CD4+ T cells at a concentration of 1.0 x10 ⁶ in RPMI medium with 10% FBS, 1x penicillin-streptomycin solution and 50 IU/ml recombinant human IL-2 (Peprotech, 200-02-50ug ) at a bead to cell ratio 3:1 was transferred to a T75 vial (Greiner Bio-one, 690195) and incubated at 37°C + 5% CO ₂ . After 3 days, the cells were gently resuspended and counted. Cell density was maintained in the range of 0.8-1 x 10 ⁶ cells/ml, adding fresh medium if necessary (RPMI-10% FBS + 1X penicillin-streptomycin solution + 50 IU/ml rhuIL2). On day 7 or 8, CD3/28 beads were removed and CD4+ T cells were left overnight at 1 x 10 ⁶ cells/ml fresh RPMI-10% FBS + penicillin-streptomycin 1X solution with 10 IU/ml rhuIL2. Cells were stored frozen until use.

Getting MoiDC

Intact monocytes were isolated from human PBMC using the Pan Monocyte Isolation Kit (Miltenyi Biotec Ltd, 130-096-537) according to the manufacturer's instructions. Monocytes were differentiated to iDC using human Mo-DC differentiation medium (Miltenyi Biotec Ltd, 130-094-812) according to the manufacturer's instructions.

SEB Analysis

Expanded T cells were thawed the day before the experiment, washed with AIM medium (Gibco, 12055-091) and incubated at 37°C, 5% CO ₂ overnight at a concentration of 1x10 ⁶ cells/ml AIM medium. A 2 μM concentration of each antibody/mixture was prepared in DPBS (Gibco, 14190-169) and diluted 1:10 with medium (30 μl + 270 μl) to a concentration of 200 nM. 1:10 serial dilutions were made in a 96-well plate (30 µl + 270 µl experimental medium; 2x final conc.). MoiDCs were thawed, washed with AIM medium and mixed with T cells from the same donor at a ratio of 1:10 (5 ml of iDC at a concentration of 2x10 ⁵ cells/ml were combined with 5 ml of T cells at a concentration of 2x10 ⁶ cells/ml). 20 μl of SEB (Sigma, S4881) at a concentration of 0.1 μg/ml was added to 10 ml of cells. In a round bottom 96-well plate, 100 µl of cell/SEB mixture was added to 100 µl of antibody dilution, resulting in a ratio of 104 iDC cells to 105 T cells with 0.1 ng/ml SEB in 200 µl of AIM medium per well at a final antibody concentration of 100, 10, 1, 0.1, 0.01, 0.001 nM. Cells were incubated at 37°C, 5% CO ₂ for 3 days. The supernatant was collected and immediately analyzed using a human IFNγ solid phase ELISA kit (R&D Systems, _PDIF50 ) or frozen at -20°C for further analysis. The assay was performed according to the kit manufacturer's instructions using a supernatant diluted with PBA (DPBS, 2% BSA (Sigma, A7906-100G)) at a ratio of 1:30. The concentration of human IFN _γ was plotted against the logarithm of the mAb ² or mAb concentration in 10, the resulting curves were approximated by the equation of the logarithmic function (for agonistic interaction) depending on the response in the GraphPad Prism software. Table 17 shows the EC ₅₀ values and the amount of IFNγ release in the SEB assay with cells from four different cell donors (AD donors). Figure 4 shows a representative plot of the single donor SEB assay. In all six assays, mAb ² LAG-3/PD-L1 and mAb ² LAG-3/FITC + 84G09LALA showed more activation than mAb 84G09LALA alone, while mAb ² LAG-3/FITC or mAb 4420 did not. significant activation during analysis. Thus, the results of the SEB assay confirmed the results of the DO11.10/LK35.2 assay in a more physiological system.

Example 4 In Vivo Activity of Mab ² Molecules in Mouse Tumor Models

3.2 Mab ² activity in the MC38 null tumor model

The syngeneic MC38 tumor model was used in this experiment because MC38 tumors are known to express PD-L1 on their cell surface and are highly immunogenic, resulting in increased expression of LAG-3 on immune cells in the tumor and at the tumor periphery.

A mouse FS18-7-108-29/S1 surrogate mAb ² containing the LALA mutation (SEQ ID NOS: 117 and 119), referred to as FS18-29/S1, was tested for in vivo activity using the MC38 syngeneic tumor growth model in mice. The ability of mAb ² to inhibit tumor growth was compared with the effect of LAG-3/mock mAb ² FS18-7-108-29/4420 containing the LALA mutation (SEQ ID NOS: 132 and 85), called FS18-29/4420, reference mAb C9B7W against LAG-3 (2B scientific; cat. no. BE0174-50MG), a reference mAb S1 against PD-L1 containing the LALA mutation (SEQ ID NOs: 122 and 119), and a combination of mAbs C9B7W and S1.

Female C57BL/6 mice (Charles River) aged 8-10 weeks and weighing 20-25 g were kept for one week before the start of the study. All animals were microchipped and assigned unique identifiers. Each group included 10 mice. The MC38 colon carcinoma cell line (S. Rosenberg, NIH) was initially expanded, stored, and pre-screened for pathogens using IDEXX Bioresearch and the IMPACT I protocol; it was demonstrated that this line did not contain pathogenic organisms. MC38 cells were thawed from storage at -150°C and added to 20 ml DMEM (Gibco, 61965-026) with 10% FCS (Gibco, 10270-106) in T175 tissue culture flasks. Mice were anesthetized with isoflurane (Abbott Laboratories), each animal was injected subcutaneously with 2x10 ⁶ cells in the left iliac region. 7-8 days after cell inoculation, mice that were tumor-free at that time were removed from the study.

All mAb ² molecules and control antibodies were analyzed within 24 hours prior to administration, obtaining a size exclusion HPLC profile and checking them for impurities. Antibodies were prepared at a final concentration of 10 mg/kg in PBS and combined with a second antibody in combination studies. Mab ² molecules or control antibodies were administered to mice by intraperitoneal (ip) injection on days 8, 11 and 14 after tumor inoculation. At the appointed date, accurate measurements of the tumors and administration of formulations were performed; during the remainder of the study, mice were closely monitored. The tumor volume was measured with a caliper, determining the longest and shortest axis of the tumor. The following formula was used to calculate the tumor volume:

L x (S ² ) / 2

where L = longest axis; S = shortest axle

The study was stopped on day 20 when the tumor burden was considered close to the target limit. All mice were humanely sacrificed, tumors were surgically removed and weighed.

The results are shown in Figures 5 and 6. Mice treated with mAb ² LAG-3/PD-L1 (FS18-29/S1) had significantly smaller end tumors compared to mice treated with the combination of reference mAbs C9B7W and S1. More specifically, statistical analysis of final tumor mass was performed using two-tailed Student's t-test in the GraphPad Prism software package, demonstrating a statistically significant difference between FS18-29/S1 administration and administration of the combination of C9B7W and S1 reference antibodies (p = 0.0125), indicating that inhibition of LAG-3 and PD-L1 by the same molecule resulted in a synergistic effect on the final mass of tumors compared with inhibition of LAG-3 and PD-L1 by separate molecules.

The surrogate mAb ² FS18-29/S1 also had a pronounced effect on tumor growth, preventing the development of 6 out of 8 growing MC38 tumors and slowing down the growth in the remaining 2 cases. The introduction of reference antibodies against LAG-3 and PD-L1 in combination slowed tumor growth in 7 animals, however, animals without tumors were absent.

FS18-29/4420 alone had no significant effect on tumor growth, indicating that anti-PD-L1 Fab is required for maximum efficacy of mAb ² . The reference anti-LAG-3 mouse antibody alone had little or no effect on the outcome of tumor growth, while the reference anti-mouse PD-L1 antibody prevented the development of 1 of 7 tumors in this group and had some overall growth inhibiting effect. tumors.

Syngeneic mouse models are considered suitable mouse systems for testing the antitumor effects of inhibition of therapeutic targets and are being extensively used to validate emerging therapeutic agents.

4.2 Activity of mAb2 molecules in the developed tumor model MC38

A mouse FS18-7-108-29/S1 surrogate mAb ² containing the LALA mutation (SEQ ID NOS: 117 and 119), referred to as FS18-29/S1, was tested for in vivo activity in a syngeneic mouse MC38 tumor growth model. The ability of mAb ² to inhibit tumor growth was compared with the effect of LAG-3/mock mAb ² FS18-7-108-29/4420 containing the LALA mutation (SEQ ID NOS: 132 and 85), called FS18-29/4420, reference mAb C9B7W against LAG-3, a reference mAb S1 against PD-L1 containing the LALA mutation (SEQ ID NOs: 122 and 119), and a combination of C9B7W and S1.

Female C57BL/6 mice (Charles River) aged 8-10 weeks and weighing 20-25 g were kept for one week before the start of the study. All animals were microchipped and assigned unique identifiers. Each group included 10 mice. The MC38 colon carcinoma cell line (S. Rosenberg, NIH) was initially expanded, stored, and pre-screened for pathogens using IDEXX Bioresearch and the IMPACT I protocol; it was demonstrated that this line did not contain pathogenic organisms. MC38 cells (approximately 3-5x10 ⁶ ) were thawed from storage at -150°C and added to 20 ml of DMEM (Gibco, 61965-026) with 10% FCS (Gibco, 10270-106) in T175 tissue culture flasks. Mice were anesthetized with isoflurane (Abbott Laboratories), each mouse was injected subcutaneously with 2x106 cells in a volume of 100 μl in the left iliac region. 7-8 days after cell inoculation, the mice were routinely examined for health and tumor growth suitable for starting the study. When the majority of mice showed tumors of 5-10 mm in diameter, they were sorted and randomized into study groups. Mice that did not have tumors of the appropriate size at the indicated time were removed from the study.

All mAb ² molecules and control antibodies were analyzed within 24 hours prior to administration, obtaining a size exclusion HPLC profile and checking them for impurities. Antibodies were prepared at a final concentration of 10 mg/kg in PBS and combined with a second antibody for combination studies. Mab ² molecules or control antibodies were administered to mice by ip injection on days 15, 18 and 21 after tumor inoculation. Animals were screened for health status under anesthesia three times a week in blinded mode, while performing accurate measurement of tumors. The tumor volume was measured with a caliper, determining the longest and shortest axis of the tumor. The formula used to calculate tumor volume is given in section 4.1 above.

The study was stopped on day 24, when the tumor burden was considered close to the target limit. All mice were humanely sacrificed, tumors were surgically removed and weighed. The results are shown in figures 7 and 8.

Effective control or suppression of tumor growth in syngeneic mouse tumor models is best achieved by therapeutic intervention at an early stage (beginning with tumor volumes less than 40 mm3). The later the intervention is performed, the more difficult it is to observe positive effects in relation to tumor growth, although this may be more true in the human clinical situation.

FS18-29/S1 was beneficial in both suppressing tumor growth and preventing the development of MC38 colon carcinoma in immunocompetent mice when administered to C57BL/6 mice at an early stage. When administered at later stages (beginning with tumor volumes of 50-125 mm ³ ), FS18-29/S1 suppressed tumor growth as effectively as the combination of reference antibodies. FS18-29/4420 alone had no significant effect on tumor growth, while S1 and C9B7W slightly affected the overall tumor growth.

3.2 Mab ² activity in the CT26 null tumor model

A syngeneic CT26 tumor model was used in this experiment because CT26 tumors are known to express PD-L1 on their cell surface and are highly immunogenic, resulting in increased LAG-3 expression on immune cells in the tumor and in the periphery.

Mouse surrogate mAb ² FS18-7-108-29/S1 containing the LALA mutation (SEQ ID NOs: 117 and 119) referred to as FS18-29/S1 and FS18-7-108-35/S1 containing the LALA mutation (SEQ ID NOs: 120 and 119), referred to as FS18-35/S1, was tested for in vivo activity in a syngeneic CT26 mouse tumor growth model. The ability of mAb ² to inhibit tumor growth was compared to the effect of LAG-3/mock mAb ² FS18-7-108-29/4420 containing the LALA mutation (SEQ ID NOS: 132 and 85) called FS18-29/4420 and FS18-7 -108-35/4420 containing the LALA mutation (SEQ ID NOs: 133 and 85), referred to as FS18-35/4420, reference mAb C9B7W against LAG-3, reference mAb S1 against PD-L1, containing the LALA mutation (SEQ ID NO : 122 and 119), and combinations of C9B7W and S1.

Female BALB/c mice (Charles River) aged 8-10 weeks and weighing 20-25 g were kept for one week before the start of the study. All animals were microchipped and assigned unique identifiers. Each group included 10 mice. The CT26 colon carcinoma cell line (ATCC, CRL-2638) was initially expanded, stored, and pre-screened for pathogens using IDEXX Bioresearch and the IMPACT I protocol; it was demonstrated that this line did not contain pathogenic organisms. CT26 cells (approximately 3-5x10 ⁶ ) were thawed from storage at -150°C and added to 20 ml of DMEM (Gibco, 61965-026) with 10% FCS (Gibco, 10270-106) in T175 tissue culture flasks. 7-8 days after cell inoculation, the mice were routinely examined for health and tumor growth suitable for starting the study. When the majority of mice showed tumors of 3-5 mm3 in diameter, they were sorted and randomized into study groups. Mice that were tumor-free at that point were removed from the study.

All Mab ² molecules and control antibodies were analyzed within 24 hours prior to administration, obtaining a size exclusion HPLC profile and checking them for impurities. Antibodies were prepared at a final concentration of 10 mg/kg in PBS and combined with a second antibody in combination studies. Mab ² molecules and control antibodies were administered to mice on days 8, 11 and 14 after tumor inoculation. Animals were subjected to health screening while accurately measuring tumors. The tumor volume was measured with a caliper, determining the longest and shortest axis of the tumor. The formula used to calculate tumor volume is given in section 4.1 above.

The study was stopped on day 20 when the tumor burden was considered close to the target limit. All mice were humanely sacrificed, tumors were surgically removed and weighed. The results are shown in figures 9 and 10.

Statistical analysis of the final mass of tumors was performed using a two-tailed Student's t-test in the GraphPad Prism software package. Statistical analysis of tumor growth curves was performed by comparing the Growth Curves function from the Statistical Modeling package, statmod (Elso et al., 2004 and Baldwin et al., 2007), available in the R project for statistical calculations (R Project for Statistical Computing).

Demonstrated a statistically significant difference between mAb ² FS18-35/S1 and control IgG (normal growth) in the suppression of tumor growth. No such statistically significant difference was observed when using the combination of reference antibodies or mAb ² FS18-29/S1 compared to the control IgG group or any other group in this study.

The CT26 tumor model is an aggressive, rapidly growing tumor model that is inherently prone to metastasis in the mouse intestine, resulting in a very limited therapeutic window.

Surprisingly, the combination of reference antibodies against LAG-3 and PD-L1 did not significantly suppress tumor growth compared to the IgG control group. At the same time, the FS18-35/S1 treated group showed significant growth inhibition compared to the IgG control group. Although FS18-29/S1 suppressed tumor growth compared to control IgG, this effect was not statistically significant. This study shows a second tumor model in which mouse anti-LAG-3/PD-L1 mAbs ² showed a positive effect in slowing down tumor growth at least to the same extent as administration of a combination of reference monoclonal antibodies.

4.4 Effect of LALA Mutation on Tumor Growth Inhibition in the MC38 Non-Developed Tumor Model

Two mAbs ² (FS18-7-108-29/S1 LALA and FS18-7-108-29/S1) were tested to evaluate the potential differences in antitumor activity of these mAbs ² with or without a LALA mutation in the Fc region. A mouse FS18-7-108-29/S1 surrogate mAb ² , referred to as FS18-29/S1, containing (SEQ ID NOs: 117 and 119) and not containing the LALA mutation (SEQ ID NOs: 118 and 119) was tested for activity in vivo using a syngeneic mouse MC38 tumor growth model. The ability of mAb ² to inhibit tumor growth was compared with the effect of LAG-3/mock mAb ² FS18-7-108-29/4420 containing (SEQ ID NOS: 132 and 85) and not containing the LALA mutation (SEQ ID NOS: 134 and 85) , called FS18-29/4420LALA and FS18-29/4420, and a combination of LAG-3/sham mAb ² containing and not containing LALA mutations with a reference mAb S1 against PD-L1 containing (SEQ ID NOS: 122 and 119) and not containing the LALA mutation (SEQ ID NOS: 123 and 119).

Female C57BL/6 mice (Charles River) aged 8-10 weeks and weighing 20-25 g were kept for one week before the start of the study. All animals were microchipped and assigned unique identifiers. Each group included 10 mice. The MC38 colon carcinoma cell line (S. Rosenberg, NIH) was initially expanded, stored, and pre-screened for pathogens using IDEXX Bioresearch and the IMPACT I protocol; it was demonstrated that this line did not contain pathogenic organisms. MC38 cells (approximately 3-5x10 ⁶ ) were thawed from storage at -150°C and added to 20 ml of DMEM (Gibco, 61965-026) with 10% FCS (Gibco, 10270-106) in T175 tissue culture flasks. Mice were anesthetized with isoflurane (Abbott Laboratories), each mouse was injected subcutaneously with 2x10 ⁶ cells in a volume of 100 μl in the left iliac region. The mice were allowed to recover under observation; the date of inoculation was noted as day 0. 7-8 days after cell inoculation, the mice were routinely examined for health and tumor growth suitable for starting the study. Mice that were tumor-free at that point were removed from the study.

All mAb ² molecules and control antibodies were analyzed within 24 hours prior to administration, obtaining a size exclusion HPLC profile and checking them for impurities. Antibodies were prepared at a final concentration of 10 mg/kg in PBS and combined with a second antibody in combination studies. Mab ² molecules or control antibodies were administered to mice by ip injection on days 8, 11 and 14 after tumor inoculation. Animals were screened for health status under anesthesia three times a week in blinded mode, while performing accurate measurement of tumors. The tumor volume was measured with a caliper, determining the longest and shortest axis of the tumor. The formula used to calculate tumor volume is given in Section 4.1.

All mice were humanely sacrificed, tumors were surgically removed and weighed. The results are shown in figures 11 and 12.

This study confirmed that the presence or absence of a LALA mutation abolishing ADCC activity had no statistically significant effect on tumor growth in the MC38 colon carcinoma model; at the same time, mAbs ² containing the LALA mutation tended to enhance suppression of tumor growth. In any case, the rationale for including a mutation potentially inhibiting ADCC activity against T cells expressing LAG-3 or PD-L1 is supported, since the LALA mutation did not adversely affect the antitumor activity of mAb ² against LAG-3/PD -L1. There was evidence indicating that the inclusion of the LALA mutation was only critical for the anti-PD-L1 antibody.

The present study also evaluated the possibility that the anti-LAG-3/PD-L1 mAb ² has increased efficacy compared to the administration of single antibodies (LAG-3 LALA + PD-L1 LALA). In this case, there were no significant differences between the two indicated groups. Both groups showed growth suppression in the MC38 colon carcinoma model.

4.5 Conclusion

Taken as a whole, the above results clearly show a synergistic effect with respect to tumor growth suppression when a mAb ² molecule containing both LAG-3 and PD-L1 binding sites is administered to mice in the models tested. Based on these results, it is expected that the antibody molecules of the present invention will show a better effect in the treatment of cancer in human patients, in particular with respect to the suppression of tumor growth, than the introduction of two separate molecules that bind to LAG-3 and PD-L1, respectively.

Example 5: Effect of mAb ² on LAG-3 expression by T cells

The mechanism by which a mouse FS18-7-108-29/S1 surrogate mAb ² containing a LALA mutation (SEQ ID NOs: 117 and 119), called FS18-29/S1, reduced tumor burden was tested in a syngeneic MC38 tumor growth model. expressing ovalbumin (MC38.OVA) in mice. The effect of FS18-29/S1 was compared with the effect of LAG-3/sham mAb ² FS18-7-108-29/4420 containing the LALA mutation (SEQ ID NOS: 132 and 85), referred to as FS18-29/4420, reference mAb S1 against PD-L1 containing the LALA mutation (SEQ ID NOs: 122 and 119) and combinations of FS18-29/4420 and S1.

On the day of implantation, cultured MC38.OVA cells were harvested during the logarithmic growth phase (~75% confluence) and resuspended in PBS at a concentration of 1 x 10 ⁷ cells/ml. Tumors were initiated by first anesthetizing each animal with isoflurane and then subcutaneously implanting 1 x 10 ⁶ MC38.OVA cells (0.1 ml suspension) into the left iliac region of each test animal. Eleven days after tumor cell implantation, animals were randomized using the deterministic randomization method into five groups with individual tumor volumes ranging from 32 to 62.5 mm3. Animals were injected with 10 mg/kg of antibody or mAb ² at 12, 14 and 16 days after tumor inoculation; tumors were harvested from three animals/group on days 19 and 23 after tumor inoculation. A GentleMACS™ Dissociator was used to dissociate the tumors and then the cells were passed through a 70 μm cell sieve to obtain a single cell suspension. 1x10 ⁶ cells/well in a 96-well plate were resuspended in FACS buffer with viability dye 1:3000 and Fc blocking reagent (anti-CD16/32 antibody). Cells for FACS analysis were stained using a standard mixture containing labeled antibodies against CD43, CD8a, CD4, FoxP3 and LAG-3. For intracellular FoxP3 staining, cells were fixed and permeabilized before staining with anti-FoxP3 antibody. Samples were analyzed on a Canto II flow cytometer with compensation matrix and a minimum of 500,000 countable events.

In this experiment, TIL was examined for LAG-3 expression after the third antibody/mAb ² administration, when a difference in tumor growth between control antibody and mAb ² treatment became apparent, but before large differences in tumor size appeared, which could skew the results. It was found that at the indicated time point, the expression of LAG-3 on TIL was significantly reduced in animals treated with mAb ² FS18-29/S1 or a combination of FS18-29/4420 and S1. Specifically, as shown in Figure 13, LAG-3 expression on CD8, CD4, and FoxP3 tumor-infiltrating lymphocytes (TILs) decreased after treatment with FS18-29/S1 on days 19 and 23 post-tumor inoculation, corresponding to days 3 and 7 after the last administration of antibody/mAb ² , respectively. The decrease in LAG-3 expression was more pronounced on day 23, but was evident as early as day 19. Animals treated with the combination of FS18-29/4420 and S1 also showed a reduction in LAG-3 expression on TIL, however this effect was delayed until day 23, while treatment with FS18-29/4420 or S1 alone had little effect. or had no effect on LAG-3 expression on TIL.

These results indicate that double inhibition of LAG-3 and PD-L1 is required to reduce LAG-3 expression in TIL, as this phenomenon was not observed in animals treated with anti-LAG-3 or PD-L1 agents alone. Regardless of theory, it is believed that dual treatment against LAG-3 and against PD-L1 led to a decrease in LAG-3 expression on TIL, which attenuated the inhibitory effect of LAG-3 and allowed TIL to overcome depletion. Once activated, TILs can recognize tumor-expressed neoantigens and elicit a response against it, thereby reducing tumor burden. Thus, this is thought to be the mechanism by which anti-LAG3/PD-L1 mAb ² treatment resulted in a reduction in tumor burden.

Example 6 Antibody-Dependent Cellular Cytotoxicity and Complement-Dependent Cytotoxicity of Mab ²

IgG1 antibodies usually have effector functions due to conserved interaction sites in the constant region of the molecule. These include antibody dependent cellular cytotoxicity (ADCC) mediated by binding to FcγR expressed on monocytes/macrophages, dendritic cells, NK cells, neutrophils and other granulocytes, as well as complement dependent cytotoxicity (CDC) mediated by induction of the complement cascade initiated by binding to the C1q component of the complement. Since LAG-3 is predominantly expressed on activated T cells, and PD-L1 is expressed on them, as well as on tumor cells in a significant amount, the ability of mAb ² FS18-7-9/84G09 (SEQ ID NO 94 and 116) to induce ADCC was investigated. and KZTs.

More specifically, the ability to induce lysis of the respective target cells by treating cells expressing LAG-3 or PD-L1 with FS18-7-9/84G09 antibody followed by incubation with NK cells or complement was tested. In addition, since FS18-7-9/84G09 is a bispecific antibody, the effect of target binding to one of the specific binding sites on effector function was also tested for cells expressing the target for the other specific binding site.

Information about the effector functions of mAb ² FS18-7-9/84G09 is useful for a number of reasons, including the need to determine whether an effector-damaging mutation, such as a LALA mutation, should be included in the molecule in order to protect effector T cells expressing LAG-3 and involved in tumor eradication from FS18-7-9/84G09-mediated ADCC and/or CDC.

6.1 Design of the experiment

All assays used Raji cells recombinantly expressing LAG-3 or PD-L1, using their endogenous CD20 expression as a control to target the generic version of rituximab to demonstrate the functional activity of complement preparations and NK cells, independent of recombinant target protein expression. Target expression was confirmed prior to these experiments.

For the simplest determination of CSC activity against LAG-3 or PD-L1 expressing cells, this activity was measured by LDH release by converting the substrate to a fluorescent dye (CytoTox-ONE™ from Promega). To determine which target cells in a mixture of cells containing both LAG-3 expressing cells and PD-L1 expressing cells were lysed, differential CDC was measured by flow cytometry of target cells with different fluorescent labels after incubation with mAb. ² /antibody, detecting dead cells using a fluorescent dye derived from live cells.

To determine ADCC activity against cells expressing LAG-3 or PD-L1, this activity was measured using NK cells isolated from frozen PBMC, measuring LDH release colorimetrically (CytoTox 96 manufactured by Promega). In all these studies, rituximab in various isotypes and Fc configurations was used as a control. A reliable way to differentially measure ADCC is not known, so differential measurement of ADCC activity was not performed.

In all experiments, PD-L1-specific mAb (84G09) and LAG-3-specific mAb (25F7) were used as controls. In addition, an isotype control IgG (4420) was used as a negative control or to obtain background CSC activity. In addition, in the CDC and ADCC assays, the LALA versions of the respective antibodies and mAbs ² (with the exception of 25F7) were compared with wild-type versions of IgG1 to determine the effect of this mutation on their effector functions.

6.2 Materials and methods

6.2.1 CSC analyzes

All antibodies/mAb ² , including rituximab, were diluted into 10 serial 1 to 2 dilutions. In addition, control wells containing IgG (4420 LALA) at the maximum concentration used were prepared. Suspensions of Raji cells recombinantly expressing LAG-3 or PD-L1, respectively, were prepared in serum free medium for LDH release assay and added in equal volume to the prepared antibodies/mAb ² .

For flow cytometry-based CCC analysis, 5 x 10 ⁷ cell suspensions were prepared and resuspended in 0.5 µM CellTracker deep red (CellTracker™ Deep Red, Thermo Fisher, #C34565) or 5 µM CellTracker Green (CellTracker™ Green CMFDA ( 5-chloromethylfluorescein diacetate, Thermo Fisher, #C7025) with serum-free medium After 30 min incubation at 37°C, cells were washed with serum-free medium and added to wells containing prepared antibody/mAb ² or combined with another differentially stained cell line in equal volumes, and then added to wells containing prepared antibody/mAb ² as described above.For both assays, after 30 min incubation under cell culture conditions, wells were filled to the top with an equal volume of baby rabbit complement, 10% in serum-free medium (Baby Rabbit Complement, TEBU-bio, #CL3441) Plates were incubated for 4 hours under cell culture conditions. 100% lysed samples by adding Triton X 100 to half of the wells treated with 4420 LALA and performing a Cytotox assay according to the manufacturer's instructions (CytotoxOne, Promega, G7891).

After reading the signal of the control samples with 100% lysis was taken as 100%, and the signals of the wells with samples were recalculated as a percentage of this level.

For flow cytometry-based CSC analysis, at the end of the incubation period, dead cell stain (SYTOX® Blue Dead Cell Stain, Thermo Fisher, #S34857) was diluted 1:500 in PBS and the wells filled to the top with an equal volume. Flow cytometry was performed on a Cytoflex flow cytometer tuned to intact cell populations based on FSC and SSC to determine the percentage of Sytox-positive cells (channel PB450) or both CellTracker™ Deep Red-positive and CellTracker™ Green CMFDA-positive cell populations .

6.2.2 Analysis of ADCC

ADCC was measured as previously described (Broussas, Matthieu; Broyer, Lucile; and Goetsch, Liliane. (2013) Evaluation of Antibody-Dependent Cell Cytotoxicity Using Lactate Dehydrogenase (LDH) Measurement in Glycosylation Engineering of Biopharmaceuticals: Methods and Protocols, Methods in Molecular Biology, New York: Springer Science + Business Media, Volume 988, pp 305-317). Briefly, target cells were pre-incubated with antibodies and then primary NK cells isolated from human PBMCs (NK Cell Isolation Kit, Miltenyi Biotec, 130-092-657) were added at a ratio of 20:1 for 4 hours. cytotoxicity was performed according to the manufacturer's instructions (non-radioactive cytotoxicity assay CytoTox 96 Non-Radioactive Cytotoxicity Assay, Promega, G1780). % lysis was calculated based on 100% lysis of target cells, taking into account spontaneous lysis of effector cells and target cells.

6.3 Results and conclusions

6.3.1 CSC analyzes

Raji cells expressing PD-L1 were exposed to the anti-CD20 antibody rituximab, which resulted in a maximum <60% lysis as measured by CSC by characteristic LDH release. Anti-PD-L1 antibody 84G09 (containing the F(ab) ₂ fragment of mAb ² FS18-7-9/84G09) and FS18-7-9/84G09 in IgG1 format showed higher maximum lysis values and, in addition, increased lysis efficiency , and the calculated half-maximal doses were approximately two times less than the dose of rituximab in the IgG1 format. This allowed us to demonstrate that the introduction of the LAG-3 binding site into the 84G09 antibody did not affect its specific activity for PD-L1 in terms of power or maximum response, since both of these indicators were very close when comparing 84G09 and FS18-7-9/84G09. The introduction of the LALA mutation led to a decrease in the maximum response for rituximab, 84G09 and FS18-7-9/84G09, but the efficiency decreased only for 84G09 and FS18-7-9/84G09. As expected, the 25F7 anti-LAG-3 antibody did not affect the viability of Raji cells expressing PD-L1, since these cells did not express human LAG-3. These results are shown in Figure 14A.

Raji cells expressing LAG-3 were exposed to the anti-CD20 antibody rituximab to evaluate CSC, but the anti-LAG-3 antibody 25F7 showed even better efficacy, with its calculated half-maximal dose being approximately half that of rituximab. None of the other antibodies (including FS18-7-9/84G09) showed CSC activity against Raji cells expressing LAG-3. The introduction of the LALA mutation had a very limited effect on the CSC activity of rituximab (Figure 14B).

6.3.2 Differential analysis of CSC

The present inventors developed a differential analysis of CSC using flow cytometry to recognize lysed cells expressing targets when treated with FS18-7-9/84G09 or control antibodies. This assay was used to confirm the results of the simplest LDH release CPC assay described above. Unlike the IgG isotype control antibody (4420), which did not affect the percentage of live cells, rituximab mediated a decrease in the number of live cells and an increase in the number of dead cells, both among cells expressing PD-L1 and among cells expressing LAG -3. At the same time, FS18-7-9/84G09 did not affect cells expressing LAG-3, but lysed cells expressing PD-L1 very efficiently. Similarly, a mixture of LAG-3-specific antibody 25F7 and PD-L1-specific antibody 84G09 also showed a dose-dependent decrease in live cells and an inverse increase in dead cells; however, the maximum lysis of cells expressing LAG-3 was only 50% of the total number of cells, but was already achieved at a concentration of approximately 1 nM, which was the minimum dose to achieve maximum lysis among all tested antibodies. This confirms previous results showing that the LAG-3 binding site in the CH3 domain of FS18-7-9/84G09 does not induce CSC-mediated lysis of target cells expressing LAG-3. In addition, this experiment showed that the presence of cells expressing LAG-3 did not affect the CSC activity of FS18-7-9/84G09 towards cells expressing PD-L1. The results are shown in figure 15.

6.3.3 Analysis of ADCC

For ADCC analysis, Raji cells expressing PD-L1 were exposed to the anti-CD20 antibodies rituximab, FS18-7-9/84G09 and 84G09, which showed very similar efficacy and resulted in a maximum lysis of approximately 40% of the total cells. Rituximab and 84G09 containing the LALA mutation did not show ADCC-mediated lysis, and FS18-7-9/84G09 containing the LALA mutation showed very little or no ADCC-mediated lysis of target cells expressing PD-L1. The LAG-3 specific antibody 25F7 and isotype control 4420 with and without the LALA mutation showed no activity in this assay.

These results indicate that the insertion of the LAG-3 binding site into the 84G09 antibody did not affect its PD-L1 specific activity in terms of potency or maximal response, both of which were very close to those of the PD-L1-specific 84G09 antibody. The introduction of the LALA mutation resulted in the elimination of ADCC activity (Figure 16A).

Raji cells expressing LAG-3 were exposed to rituximab and 25F7 to assess ADCC-mediated lysis, resulting in a maximum lysis of approximately 40%. FS18-7-9/84G09 also mediated the lysis of cells expressing LAG-3 by ADCC, albeit with a significantly reduced efficiency, allowing only 20% lysis to be achieved at a concentration of 2.5 nM. The introduction of the LALA mutation completely eliminated the ADCC activity of rituximab and FS18-7-9/84G09. As expected, 84G09 and isotype control 4420 with and without the LALA mutation did not show ADCC activity in this assay because these antibodies do not bind to LAG-3 (Figure 16B).

Sequence list

Amino acid sequences of Fcab loop regions FS18-7-9

Hinge AB FS18-7-9 - WDEPWGED (SEQ ID NO: 1)

CD hinge FS18-7-9 - SNGQPENNY (SEQ ID NO: 2)

Hinge EF FS18-7-9 - PYDRWVWPDE (SEQ ID NO: 3)

Nucleotide sequence of the CH3 domain of Fcab FS18-7-9 (SEQ ID NO: 4)

CHO-codon-optimized nucleotide sequence of the CH3 domain of Fcab FS18-7-9 (SEQ ID NO: 142)

Amino acid sequence of the CH3 domain of Fcab FS18-7-9 (SEQ ID NO: 5)

Amino acid sequence of the CH3 domain of Fcab FS18-7-9 containing a C-terminal lysine residue (SEQ ID NO: 135)

Amino acid sequence of the CH2 and CH3 domains of Fcab FS18-7-9 containing the LALA mutation (underlined) (SEQ ID NO: 6)

Amino acid sequence of the CH2 and CH3 domains of Fcab FS18-7-9 without LALA mutation (SEQ ID NO: 7)

Amino acid sequences of Fcab loop regions FS18-7-32

Hinge AB FS18-7-32 - WDEPWGED (SEQ ID NO: 1)

CD hinge FS18-7-32 - SNGQPENNY (SEQ ID NO: 8)

Hinge EF FS18-7-32 - PYDRWVWPDE (SEQ ID NO: 3)

Nucleotide sequence of the CH3 domain of Fcab FS18-7-32 (SEQ ID NO: 9)

Amino acid sequence of the CH3 domain of Fcab FS18-7-32 (SEQ ID NO: 10)

Amino acid sequence of the CH2 and CH3 domains of Fcab FS18-7-32 containing the LALA mutation (underlined) (SEQ ID NO: 11)

Amino acid sequence of CH2 and CH3 domains of Fcab FS18-7-32 without LALA mutation (SEQ ID NO: 12)

Amino acid sequences of Fcab loop regions FS18-7-33

Hinge AB FS18-7-33 - WDEPWGED (SEQ ID NO: 1)

CD hinge FS18-7-33 - SNGQPEDNY (SEQ ID NO: 13)

Hinge EF FS18-7-33 - PYDRWVWPDE (SEQ ID NO: 3)

Nucleotide sequence of the CH3 domain of Fcab FS18-7-33 (SEQ ID NO: 14)

Amino acid sequence of the CH3 domain of Fcab FS18-7-33 (SEQ ID NO: 15).

Amino acid sequence of the CH2 and CH3 domains of Fcab FS18-7-33 containing the LALA mutation (underlined) (SEQ ID NO: 16)

Amino acid sequence of CH2 and CH3 domains of Fcab FS18-7-33 without LALA mutation (SEQ ID NO: 17)

Amino acid sequences of Fcab loop regions FS18-7-36

Hinge AB FS18-7-36 - WDEPWGED (SEQ ID NO: 1)

CD hinge FS18-7-36 - SNGQPENNY (SEQ ID NO: 18)

Hinge EF FS18-7-36 - PYDRWVWPDE (SEQ ID NO: 3)

Nucleotide sequence of the CH3 domain of Fcab FS18-7-36 (SEQ ID NO: 19)

Amino acid sequence of the CH3 domain of Fcab FS18-7-36 (SEQ ID NO: 20)

Amino acid sequence of the CH2+CH3 CH2 and CH3 domains of Fcab FS18-7-36 containing the LALA mutation (underlined) (SEQ ID NO: 21)

Amino acid sequence of CH2 and CH3 domains of Fcab FS18-7-36 without LALA mutation (SEQ ID NO: 22)

Amino acid sequences of Fcab loop regions FS18-7-58

Hinge AB FS18-7-58 - WDEPWGED (SEQ ID NO: 1)

CD hinge FS18-7-58 - SNGYPEIEF (SEQ ID NO: 23)

Hinge EF FS18-7-58 - PYDRWVWPDE (SEQ ID NO: 3)

Nucleotide sequence of the CH3 domain of Fcab FS18-7-58 (SEQ ID NO: 24)

Amino acid sequence of the CH3 domain of Fcab FS18-7-58 (SEQ ID NO: 25)

Amino acid sequence of the CH2 and CH3 domains of Fcab FS18-7-58 containing the LALA mutation (underlined) (SEQ ID NO: 26)

Amino acid sequence of CH2 and CH3 domains of Fcab FS18-7-58 without LALA mutation (SEQ ID NO: 27)

Amino acid sequences of Fcab loop regions FS18-7-62

Hinge AB FS18-7-62 - WDEPWGED (SEQ ID NO: 1)

CD hinge FS18-7-62 - SNGIPEWNY (SEQ ID NO: 28)

Hinge EF FS18-7-62 - PYDRWVWPDE (SEQ ID NO: 3)

Nucleotide sequence of the CH3 domain of Fcab FS18-7-62 (SEQ ID NO: 29)

Amino acid sequence of the CH3 domain of Fcab FS18-7-62 (SEQ ID NO: 30)

Amino acid sequence of the CH2 and CH3 domains of Fcab FS18-7-62 containing the LALA mutation (underlined) (SEQ ID NO: 31)

Amino acid sequence of CH2 and CH3 domains of Fcab FS18-7-62 without LALA mutation (SEQ ID NO: 32)

Amino acid sequences of Fcab loop regions FS18-7-65

Hinge AB FS18-7-65 - WDEPWGED (SEQ ID NO: 1)

CD hinge FS18-7-65 - SNGYAEYNY (SEQ ID NO: 33)

Hinge EF FS18-7-65 - PYDRWVWPDE (SEQ ID NO: 3)

Nucleotide sequence of the CH3 domain of Fcab FS18-7-65 (SEQ ID NO: 34)

Amino acid sequence of the CH3 domain of Fcab FS18-7-65 (SEQ ID NO: 35)

Amino acid sequence of the CH2 and CH3 domains of Fcab FS18-7-65 containing the LALA mutation (underlined) (SEQ ID NO: 36)

Amino acid sequence of CH2 and CH3 domains of Fcab FS18-7-65 without LALA mutation (SEQ ID NO: 37)

Amino acid sequences of Fcab loop regions FS18-7-78

Hinge AB FS18-7-78 - WDEPWGED (SEQ ID NO: 1)

CD hinge FS18-7-78 - SNGYKEENY (SEQ ID NO: 38)

Hinge EF FS18-7-78 - PYDRWVWPDE (SEQ ID NO: 3)

Nucleotide sequence of the CH3 domain of Fcab FS18-7-78 (SEQ ID NO: 39)

Amino acid sequence of the CH3 domain of Fcab FS18-7-78 (SEQ ID NO: 40)

Amino acid sequence of the CH2 and CH3 domains of Fcab FS18-7-78 containing the LALA mutation (underlined) (SEQ ID NO: 41)

Amino acid sequence of CH2 and CH3 domains of Fcab FS18-7-78 without LALA mutation (SEQ ID NO: 42)

Amino acid sequences of Fcab loop regions FS18-7-88

Hinge AB FS18-7-88 - WDEPWGED (SEQ ID NO: 1)

CD hinge FS18-7-88 - SNGVPELNV (SEQ ID NO: 43)

Hinge EF FS18-7-88 - PYDRWVWPDE (SEQ ID NO: 3)

Nucleotide sequence of the CH3 domain of Fcab FS18-7-88 (SEQ ID NO: 44)

Amino acid sequence of the CH3 domain of Fcab FS18-7-88 (SEQ ID NO: 45)

Amino acid sequence of the CH2 and CH3 domains of Fcab FS18-7-88 containing the LALA mutation (underlined) (SEQ ID NO: 46)

Amino acid sequence of CH2 and CH3 domains of Fcab FS18-7-88 without LALA mutation (SEQ ID NO: 47)

Amino acid sequences of Fcab loop regions FS18-7-95

Hinge AB FS18-7-95 - WDEPWGED (SEQ ID NO: 1)

CD hinge FS18-7-95 - SNGYQEDNY (SEQ ID NO: 48)

Hinge EF FS18-7-95 - PYDRWVWPDE (SEQ ID NO: 3)

Nucleotide sequence of the CH3 domain of Fcab FS18-7-95 (SEQ ID NO: 49)

Amino acid sequence of the CH3 domain of Fcab FS18-7-95 (SEQ ID NO: 50)

Amino acid sequence of the CH2 and CH3 domains of Fcab FS18-7-95 containing the LALA mutation (underlined) (SEQ ID NO: 51)

Amino acid sequence of CH2 and CH3 domains of Fcab FS18-7-95 without LALA mutation (SEQ ID NO: 52)

Amino acid sequence of wild-type human IgG1 CH2 domain (SEQ ID NO: 53)

Amino acid sequence of the CH2 domain of human IgG1 containing "LALA mutation" (highlighted in underlined font) (SEQ ID NO: 54)

Amino acid sequence of wild-type Fcab CH2 and CH3 domains without LALA mutation (SEQ ID NO: 55).

Amino acid sequence of wild-type Fcab CH2 and CH3 domains containing the LALA mutation (highlighted in underlined font) (SEQ ID NO: 56)

Human IgG1 Hinge Amino Acid Sequence (SEQ ID NO: 57)

Human IgG1 truncated hinge amino acid sequence (SEQ ID NO: 58)

Amino acid sequence of anti-mouse LAG-3 Fcab FS18-7-108-29 containing the LALA mutation (underlined) (SEQ ID NO: 59)

The CH3 domain is shown in italics. The AB, CD, and EF loops of the CH3 domain are shown in bold underlined font.

Amino acid sequence of Fcab FS18-7-108-29 against mouse LAG-3 without LALA mutation (SEQ ID NO: 60)

The CH3 domain is shown in italics. The AB, CD, and EF loops of the CH3 domain are shown in bold underlined font.

Amino acid sequence of anti-mouse LAG-3 Fcab FS18-7-108-35 containing the LALA mutation (underlined) (SEQ ID NO: 61)

The CH3 domain is shown in italics. Loop regions AB, CD, and EF are shown in bold underlined font.

Amino acid sequence of Fcab FS18-7-108-35 against mouse LAG-3 without LALA mutation (SEQ ID NO: 62)

The CH3 domain is shown in italics. Loop regions AB, CD, and EF are shown in bold underlined font.

Heavy chain amino acid sequence of mAb ² FS18-7-9/4420 against human LAG-3/FITC containing the LALA mutation (SEQ ID NO: 63)

The CDR positions are underlined, and the AB, CD, and EF loop sequences are shown in bold underlined font. The position of the LALA mutation is shown in bold.

Heavy chain amino acid sequence of mAb ² FS18-7-9/4420 against human LAG-3/FITC without LALA mutation (SEQ ID NO: 64)

The CDR positions are underlined, and the AB, CD, and EF loop sequences are shown in bold underlined font.

Heavy chain amino acid sequence of mAb ² FS18-7-32/4420 against human LAG-3/FITC containing the LALA mutation (SEQ ID NO: 65)

The CDR positions are underlined, and the AB, CD, and EF loop sequences are shown in bold underlined font. The position of the LALA mutation is shown in bold.

Heavy chain amino acid sequence of mAb ² FS18-7-32 against human LAG-3/FITC without LALA mutation (SEQ ID NO: 66)

The CDR positions are underlined, and the AB, CD, and EF loop sequences are shown in bold underlined font.

Heavy chain amino acid sequence of mAb ² FS18-7-33/4420 against human LAG-3/FITC containing the LALA mutation (SEQ ID NO: 67)

The CDR positions are underlined, and the AB, CD, and EF loop sequences are shown in bold underlined font. The position of the LALA mutation is shown in bold.

Heavy chain amino acid sequence of mAb ² FS18-7-33/4420 against human LAG-3/FITC without LALA mutation (SEQ ID NO: 68)

The CDR positions are underlined, and the AB, CD, and EF loop sequences are shown in bold underlined font.

Heavy chain amino acid sequence of mAb ² FS18-7-36/4420 against human LAG-3/FITC containing the LALA mutation (SEQ ID NO: 69)

The CDR positions are underlined, and the AB, CD, and EF loop sequences are shown in bold underlined font. The position of the LALA mutation is shown in bold.

Heavy chain amino acid sequence of mAb ² FS18-7-36/4420 against human LAG-3/FITC without LALA mutation (SEQ ID NO: 70)

The CDR positions are underlined, and the AB, CD, and EF loop sequences are shown in bold underlined font.

Heavy chain amino acid sequence of mAb ² FS18-7-58/4420 against human LAG-3/FITC containing the LALA mutation (SEQ ID NO: 71)

The CDR positions are underlined, and the AB, CD, and EF loop sequences are shown in bold underlined font. The position of the LALA mutation is shown in bold.

Heavy chain amino acid sequence of mAb ² FS18-7-58/4420 against human LAG-3/FITC without LALA mutation (SEQ ID NO: 72)

The CDR positions are underlined, and the AB, CD, and EF loop sequences are shown in bold underlined font.

Heavy chain amino acid sequence of mAb ² FS18-7-62/4420 against human LAG-3/FITC containing the LALA mutation (SEQ ID NO: 73)

The CDR positions are underlined, and the AB, CD, and EF loop sequences are shown in bold underlined font. The position of the LALA mutation is shown in bold.

Heavy chain amino acid sequence of mAb ² FS18-7-62/4420 against human LAG-3/FITC without LALA mutation (SEQ ID NO: 74)

The CDR positions are underlined, and the AB, CD, and EF loop sequences are shown in bold underlined font.

Heavy chain amino acid sequence of mAb ² FS18-7-65/4420 against human LAG-3/FITC containing LALA mutation (SEQ ID NO: 75)

The CDR positions are underlined, and the AB, CD, and EF loop sequences are shown in bold underlined font. The position of the LALA mutation is shown in bold.

Heavy chain amino acid sequence of mAb ² FS18-7-65/4420 against human LAG-3/FITC without LALA mutation (SEQ ID NO: 76)

The CDR positions are underlined, and the AB, CD, and EF loop sequences are shown in bold underlined font.

Heavy chain amino acid sequence of mAb ² FS18-7-78/4420 against human LAG-3/FITC containing LALA mutation (SEQ ID NO: 77)

The CDR positions are underlined, and the AB, CD, and EF loop sequences are shown in bold underlined font. The position of the LALA mutation is shown in bold.

Amino acid sequence of mAb ² heavy chain FS18-7-78/4420 against human LAG-3/FITC without LALA mutation (SEQ ID NO: 78)

The CDR positions are underlined, and the AB, CD, and EF loop sequences are shown in bold underlined font.

Heavy chain amino acid sequence of mAb ² FS18-7-88/4420 against human LAG-3/FITC containing LALA mutation (SEQ ID NO: 79)

The CDR positions are underlined, and the AB, CD, and EF loop sequences are shown in bold underlined font. The position of the LALA mutation is shown in bold.

Heavy chain amino acid sequence of mAb ² FS18-7-88/4420 against human LAG-3/FITC without LALA mutation (SEQ ID NO: 80)

The CDR positions are underlined, and the AB, CD, and EF loop sequences are shown in bold underlined font.

Heavy chain amino acid sequence of mAb ² FS18-7-95/4420 against human LAG-3/FITC containing LALA mutation (SEQ ID NO: 81)

The CDR positions are underlined, and the AB, CD, and EF loop sequences are shown in bold underlined font. The position of the LALA mutation is shown in bold.

Heavy chain amino acid sequence of mAb ² FS18-7-95/4420 against human LAG-3/FITC without LALA mutation (SEQ ID NO: 82)

The CDR positions are underlined, and the AB, CD, and EF loop sequences are shown in bold underlined font.

Heavy chain amino acid sequence of anti-FITC mAb 4420 containing LALA mutation (SEQ ID NO: 83)

CDR positions are underlined. The position of the LALA mutation is shown in bold.

Anti-FITC mAb 4420 heavy chain amino acid sequence without LALA mutation (SEQ ID NO: 84)

CDR positions are underlined.

Anti-FITC mAb 4420 light chain amino acid sequence (SEQ ID NO: 85)

CDR positions are underlined.

Amino acid sequences of the CDRs of anti-PD-L1 antibody 84G09 (according to IMGT)

Amino acid sequences of the CDRs of anti-PD-L1 antibody 84G09 (according to Kabat)

Amino acid sequence of the VH domain of anti-PD-L1 antibody 84G09 (SEQ ID NO: 92)

Amino acid sequence of the VL domain of anti-PD-L1 antibody 84G09 (SEQ ID NO: 93)

Heavy chain amino acid sequence of mAb ² FS18-7-9/84G09 against human LAG-3/PD-L1 with LALA mutation (SEQ ID NO: 94)

The CDR positions are underlined, and the AB, CD, and EF loop sequences are shown in bold underlined font. The position of the LALA mutation is shown in bold.

Heavy chain amino acid sequence of mAb ² FS18-7-9/84G09 against human LAG-3/PD-L1 (SEQ ID NO: 95)

The CDR positions are underlined, and the AB, CD, and EF loop sequences are shown in bold underlined font.

Heavy chain amino acid sequence of mAb ² FS18-7-32/84G09 against human LAG-3/PD-L1 with LALA mutation (SEQ ID NO: 96)

The CDR positions are underlined, and the AB, CD, and EF loop sequences are shown in bold underlined font. The position of the LALA mutation is shown in bold.

Heavy chain amino acid sequence of mAb ² FS18-7-32/84G09 against human LAG-3/PD-L1 (SEQ ID NO: 97)

The CDR positions are underlined, and the AB, CD, and EF loop sequences are shown in bold underlined font.

Heavy chain amino acid sequence of mAb ² FS18-7-33/84G09 against human LAG-3/PD-L1 with LALA mutation (SEQ ID NO: 98)

The CDR positions are underlined, and the AB, CD, and EF loop sequences are shown in bold underlined font. The position of the LALA mutation is shown in bold.

Heavy chain amino acid sequence of mAb ² FS18-7-33/84G09 against human LAG-3/PD-L1 (SEQ ID NO: 99)

The CDR positions are underlined, and the AB, CD, and EF loop sequences are shown in bold underlined font.

Heavy chain amino acid sequence of mAb ² FS18-7-36/84G09 against human LAG-3/PD-L1 with LALA mutation (SEQ ID NO: 100)

The CDR positions are underlined, and the AB, CD, and EF loop sequences are shown in bold underlined font. The position of the LALA mutation is shown in bold.

Heavy chain amino acid sequence of mAb ² FS18-7-36/84G09 against human LAG-3/PD-L1 (SEQ ID NO: 101)

The CDR positions are underlined, and the AB, CD, and EF loop sequences are shown in bold underlined font.

Heavy chain amino acid sequence of mAb ² FS18-7-58/84G09 against human LAG-3/PD-L1 with LALA mutation (SEQ ID NO: 102)

The CDR positions are underlined, and the AB, CD, and EF loop sequences are shown in bold underlined font. The position of the LALA mutation is shown in bold.

Heavy chain amino acid sequence of mAb ² FS18-7-58/84G09 against human LAG-3/PD-L1 (SEQ ID NO: 103)

The CDR positions are underlined, and the AB, CD, and EF loop sequences are shown in bold underlined font.

Heavy chain amino acid sequence of mAb ² FS18-7-62/84G09 against human LAG-3/PD-L1 with LALA mutation (SEQ ID NO: 104)

The CDR positions are underlined, and the AB, CD, and EF loop sequences are shown in bold underlined font. The position of the LALA mutation is shown in bold.

Heavy chain amino acid sequence of mAb ² FS18-7-62/84G09 against human LAG-3/PD-L1 (SEQ ID NO: 105)

The CDR positions are underlined, and the AB, CD, and EF loop sequences are shown in bold underlined font.

Heavy chain amino acid sequence of mAb ² FS18-7-65/84G09 against human LAG-3/PD-L1 with LALA mutation (SEQ ID NO: 106)

The CDR positions are underlined, and the AB, CD, and EF loop sequences are shown in bold underlined font. The position of the LALA mutation is shown in bold.

Heavy chain amino acid sequence of mAb ² FS18-7-65/84G09 against human LAG-3/PD-L1 (SEQ ID NO: 107)

The CDR positions are underlined, and the AB, CD, and EF loop sequences are shown in bold underlined font.

Heavy chain amino acid sequence of mAb ² FS18-7-78/84G09 against human LAG-3/PD-L1 with LALA mutation (SEQ ID NO: 108)

The CDR positions are underlined, and the AB, CD, and EF loop sequences are shown in bold underlined font. The position of the LALA mutation is shown in bold.

Heavy chain amino acid sequence of mAb ² FS18-7-78/84G09 against human LAG-3/PD-L1 (SEQ ID NO: 109)

The CDR positions are underlined, and the AB, CD, and EF loop sequences are shown in bold underlined font.

Heavy chain amino acid sequence of mAb ² FS18-7-88/84G09 against human LAG-3/PD-L1 with LALA mutation (SEQ ID NO: 110)

The CDR positions are underlined, and the AB, CD, and EF loop sequences are shown in bold underlined font. The position of the LALA mutation is shown in bold.

Heavy chain amino acid sequence of mAb ² FS18-7-88/84G09 against human LAG-3/PD-L1 (SEQ ID NO: 111)

The CDR positions are underlined, and the AB, CD, and EF loop sequences are shown in bold underlined font.

Heavy chain amino acid sequence of mAb ² FS18-7-95/84G09 against human LAG-3/PD-L1 with LALA mutation (SEQ ID NO: 112)

The CDR positions are underlined, and the AB, CD, and EF loop sequences are shown in bold underlined font. The position of the LALA mutation is shown in bold.

Heavy chain amino acid sequence of mAb ² FS18-7-95/84G09 against human LAG-3/PD-L1 (SEQ ID NO: 113)

The CDR positions are underlined, and the AB, CD, and EF loop sequences are shown in bold underlined font. The position of the LALA mutation is shown in bold.

Heavy chain amino acid sequence of mAb 84G09 against human PD-L1 with LALA mutation (SEQ ID NO: 114)

CDR positions are underlined. The position of the LALA mutation is shown in bold.

Anti-human PD-L1 mAb 84G09 heavy chain amino acid sequence (SEQ ID NO: 115)

CDR positions are underlined.

Anti-human PD-L1 mAb 84G09 light chain amino acid sequence (SEQ ID NO: 116)

CDR positions are underlined.

Heavy chain amino acid sequence of mAb ² FS18-7-108-29/S1 against LAG-3/PD-L1 mouse with LALA mutation (SEQ ID NO: 117)

The CDR positions are underlined, and the AB, CD, and EF loop sequences are shown in bold underlined font. The position of the LALA mutation is shown in bold.

Heavy chain amino acid sequence of mAb ² FS18-7-108-29/S1 against mouse LAG-3/PD-L1 (SEQ ID NO: 118)

The CDR positions are underlined, and the AB, CD, and EF loop sequences are shown in bold underlined font.

Anti-PD-L1 mouse mAb S1 light chain amino acid sequence (SEQ ID NO: 119) CDR positions are underlined.

Heavy chain amino acid sequence of mAb ² FS18-7-108-35/S1 against LAG-3/PD-L1 mouse with LALA mutation (SEQ ID NO: 120)

The CDR positions are underlined, and the AB, CD, and EF loop sequences are shown in bold underlined font. The position of the LALA mutation is shown in bold.

Heavy chain amino acid sequence of mAb ² FS18-7-108-35/S1 against mouse LAG-3/PD-L1 (SEQ ID NO: 121)

The CDR positions are underlined, and the AB, CD, and EF loop sequences are shown in bold underlined font.

Heavy chain amino acid sequence of control anti-PD-L1 mAb S1 with LALA mutation (SEQ ID NO: 122)

CDR positions are underlined. The position of the LALA mutation is shown in bold.

Heavy chain amino acid sequence of control mAb S1 against PD-L1 (SEQ ID NO: 123)

CDR positions are underlined. The position of the LALA mutation is shown in bold.

Heavy chain amino acid sequence of control anti-LAG-3 mAb 25F7 (SEQ ID NO: 124)

CDR positions are underlined.

Light chain amino acid sequence of control anti-LAG-3 mAb 25F7 (SEQ ID NO: 125)

CDR positions are underlined.

Amino acid sequence of human LAG-3 (SEQ ID NO: 126)

Mouse LAG-3 amino acid sequence (SEQ ID NO: 127)

Cynomolgus macaque LAG-3 amino acid sequence (SEQ ID NO: 128)

Amino acid sequence of human PD-L1 (SEQ ID NO: 129)

Mouse PD-L1 amino acid sequence (SEQ ID NO: 130)

Amino acid sequence of cynomolgus monkey PD-L1 (SEQ ID NO: 131)

Amino acid sequence of mAb heavy chain ² FS18-7-108-29/4420 vs.

Mouse LAG-3/FITC containing the LALA mutation (SEQ ID NO: 132)

The CDR positions are underlined, and the AB, CD, and EF loop sequences are shown in bold underlined font. The position of the LALA mutation is shown in bold.

Amino acid sequence of mAb heavy chain ² FS18-7-108-35/4420 vs.

Mouse LAG-3/FITC containing the LALA mutation (SEQ ID NO: 133)

The CDR positions are underlined, and the AB, CD, and EF loop sequences are shown in bold underlined font. The position of the LALA mutation is shown in bold.

Amino acid sequence of mAb ² heavy chain FS18-7-108-29/4420 against mouse LAG-3/FITC, (SEQ ID NO: 134)

The CDR positions are underlined, and the AB, CD, and EF loop sequences are shown in bold underlined font.

Bibliography

All documents mentioned in the present description are incorporated into this application by reference in their entirety.

Bae J, Lee SJ, Park CG, Lee YS, Chun T. Trafficking of LAG-3 to the surface on activated T cells via its cytoplasmic domain and protein kinase C signaling. J Immunol. 193(6),3101-12 (2014).

Baecher-Allan C, Wolf E, Hafler DA. MHC class II expression identifies functionally distinct human regulatory T cells. J Immunol. 176(8),4622-31 (2006).

Camisaschi C, Casati C, Rini F, Perego M, De Filippo A, Triebel F, Parmiani G, Belli F, Rivoltini L, Castelli C. LAG-3 expression defines a subset of CD4(+)CD25(high)Foxp3(+ ) regulatory T cells that are expanded at tumor sites. J Immunol. 184(11), 6545-51 (2010).

Curran MA, Montalvo W, Yagita H, Allison JP. PD-1 and CTLA-4 combination blockade expands infiltrating T cells and reduces regulatory T and myeloid cells within B16 melanoma tumors. Proc Natl Acad Sci U S A. 107(9),4275-80 (2010).

Demeure, C. E., Wolfers, J., Martin-Garcia, N., Gaulard, P. & Triebel, F. T Lymphocytes infiltrating various tumor types express the MHC class II ligand lymphocyte activation gene-3 (LAG-3): role of LAG-3/MHC class II interactions in cell-cell contacts. European journal of cancer 37, 1709-1718 (2001).

Durham NM, Nirschl CJ, Jackson CM, Elias J, Kochel CM, Anders RA, Drake CG. Lymphocyte Activation Gene 3 (LAG-3) modulates the ability of CD4 T cells to be suppressed in vivo. PLOS One. 9(11), e109080 (2015).

Engels B, Engelhard VH, Sidney J, Sette A, Binder DC, Liu RB, Kranz DM, Meredith SC, Rowley DA, Schreiber H. Relapse or eradication of cancer is predicted by peptide-major histocompatibility complex affinity. cancer cell. 23(4),516-26 (2013).

Gandhi MK, Lambley E, Duraiswamy J, Dua U, Smith C, Elliott S, Gill D, Marlton P, Seymour J, Khanna R. Expression of LAG-3 by tumor-infiltrating lymphocytes is coincident with the suppression of latent membrane antigen- specific CD8+ T cell function in Hodgkin lymphoma patients. Blood 108(7),2280-9 (2006).

Grosso J, Inzunza D, Wu Q, et al. Programmed death-ligand 1 (PD-L1) expression in various tumor types. Journal for Immunotherapy of Cancer. 1(Suppl 1):P53. (2013).

Herbst RS, Soria JC, Kowanetz M, Fine GD, Hamid O, Gordon MS, Sosman JA, McDermott DF, Powderly JD, Gettinger SN, Kohrt HE, Horn L, Lawrence DP, Rost S, Leabman M, Xiao Y, Mokatrin A , Koeppen H, Hegde PS, Mellman I, Chen DS, Hodi FS. Predictive correlates of response to the anti-PD-L1 antibody MPDL3280A in cancer patients. Nature 515(7528),563-7 (2014).

Huard B, Mastrangeli R, Prigent P, et al. Characterization of the major histocompatibility complex class II binding site on LAG-3 protein. Proc Natl Acad Sci USA 94.5744-9 (1997).

Iwai Y, Ishida M, Tanaka Y, Okazaki T, Honjo T, Minato N. Involvement of PD-L1 on tumor cells in the escape from host immune system and tumor immunotherapy by PD-L1 blockade. Proc Natl Acad Sci U S A. 99(19),12293-7.

Jing W, Gershan JA, Weber J, Tlomak D, McOlash L, Sabatos-Peyton C, Johnson BD1. Combined immune checkpoint protein blockade and low dose whole body irradiation as immunotherapy for myeloma. J Immunother Cancer. 3(1):2 (2015).

Kabat, E.A. et al., In: Sequences of Proteins of Immunological Interest. NIH Publication, 91-3242 (1991).

Larkin J, Hodi FS, Wolchok JD. Combined Nivolumab and Ipilimumab or Monotherapy in Untreated Melanoma. N Engl J Med. 373(13),1270-1 (2015).

Huard B, Mastrangeli R, Prigent P, et al. Characterization of the major histocompatibility complex class II binding site on LAG-3 protein. Proc Natl Acad Sci USA 94.5744-9 (1997).

Powles T, Eder JP, Fine GD, Braiteh FS, Loriot Y, Cruz C, Bellmunt J, Burris HA, Petrylak DP, Teng SL, Shen X, Boyd Z, Hegde PS, Chen DS, Vogelzang NJ. MPDL3280A (anti-PD-L1) treatment leads to clinical activity in metastatic bladder cancer. Nature. 515(7528),558-62 (2014).

Sega EI, Leveson-Gower DB, Florek M, Schneidawind D, Luong RH, Negrin RS. Role of lymphocyte activation gene-3 (Lag-3) in conventional and regulatory T cell function in allogeneic transplantation.PLoS One. 9(1), e86551 (2014).

Wolchok J et al; Nivolumab plus ipilimumab in advanced melanoma. N Engl J Med. 369(2),122-33 (2013).

Woo SR, Turnis ME, Goldberg MV, Bankoti J, Selby M, Nirschl CJ, Bettini ML, Gravano DM, Vogel P, Liu CL, Tangsombatvisit S, Grosso JF, Netto G, Smeltzer MP, Chaux A, Utz PJ, Workman CJ , Pardoll DM, Korman AJ, Drake CG, Vignali DA. Immune inhibitory molecules LAG-3 and PD-1 synergistically regulate T cell function to promote tumoral immune escape. Cancer Res. 72(4),917-2 (2012).

Workman CJ, Vignali DA. Negative regulation of T cell homeostasis by lymphocyte activation gene-3 (CD223). J Immunol. 2005 Jan 15;174(2):688-95.

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SEQUENCE LIST

<110> F-STAR DELTA LIMITED

<120> Bondage partners (2)

<130> TEK/CP7282171

<140> PCT/EP2017/065073

<141> 20.06.2017

<150> US 62/352482

<151> 06/20/2016

<160> 142

<170> PatentIn version 3.5

<210> 1

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Hinge AB FS18-7-9

<400> 1

Trp Asp Glu Pro Trp Gly Glu Asp

fifteen

<210> 2

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CD hinge FS18-7-9

<400> 2

Ser Asn Gly Gln Pro Glu Asn Asn Tyr

fifteen

<210> 3

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Hinge EF FS18-7-9

<400> 3

Pro Tyr Asp Arg Trp Val Trp Pro Asp Glu

1 5 10

<210> 4

<211> 318

<212> DNA

<213> Artificial sequence

<220>

<223> Nucleotide sequence of the CH3 domain of Fcab FS18-7-9

<400> 4

ggccagcctc gagaaccaca ggtgtacacc ctgcccccat cctgggatga gccgtggggt 60

gaagacgtca gcctgacctg cctggtcaaa ggcttctatc ccagcgaaat cgccgtggag 120

tgggagagca atgggcagcc ggagaacaac tacaagacca cgcctcccgt gctggactcc 180

gacggctcct tcttcctcta cagcaagctc accgtgccgt atgataggtg ggtttggccg 240

gatgagttct catgctccgt gatgcatgag gctctgcaca accactacac acagaagagc 300

ctctccctgt ctccggggt 318

<210> 5

<211> 106

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Amino acid sequence of the CH3 domain of Fcab FS18-7-9

<400> 5

Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Trp Asp

1 5 10 15

Glu Pro Trp Gly Glu Asp Val Ser Leu Thr Cys Leu Val Lys Gly Phe

20 25 30

Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu

35 40 45

Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe

50 55 60

Phe Leu Tyr Ser Lys Leu Thr Val Pro Tyr Asp Arg Trp Val Trp Pro

65 70 75 80

Asp Glu Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr

85 90 95

Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

100 105

<210> 6

<211> 216

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Amino acid sequence of CH2 and CH3 domains of Fcab FS18-7-9,

containing the LALA mutation

<400> 6

Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys

1 5 10 15

Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val

20 25 30

Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr

35 40 45

Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu

50 55 60

Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His

65 70 75 80

Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys

85 90 95

Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln

100 105 110

Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Ser Trp Asp Glu Pro

115 120 125

Trp Gly Glu Asp Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro

130 135 140

Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn

145 150 155 160

Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu

165 170 175

Tyr Ser Lys Leu Thr Val Pro Tyr Asp Arg Trp Val Trp Pro Asp Glu

180 185 190

Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln

195 200 205

Lys Ser Leu Ser Leu Ser Pro Gly

210 215

<210> 7

<211> 216

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Amino acid sequence of the CH2 and CH3 domains of Fcab

FS18-7-9 without LALA mutation

<400> 7

Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys

1 5 10 15

Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val

20 25 30

Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr

35 40 45

Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu

50 55 60

Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His

65 70 75 80

Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys

85 90 95

Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln

100 105 110

Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Ser Trp Asp Glu Pro

115 120 125

Trp Gly Glu Asp Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro

130 135 140

Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn

145 150 155 160

Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu

165 170 175

Tyr Ser Lys Leu Thr Val Pro Tyr Asp Arg Trp Val Trp Pro Asp Glu

180 185 190

Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln

195 200 205

Lys Ser Leu Ser Leu Ser Pro Gly

210 215

<210> 8

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CD hinge FS18-7-32

<400> 8

Ser Asn Gly Gln Pro Glu Asn Asn Tyr

fifteen

<210> 9

<211> 318

<212> DNA

<213> Artificial sequence

<220>

<223> Nucleotide sequence of the CH3 domain of Fcab FS18-7-32

<400> 9

ggccagcctc gagaaccaca ggtgtacacc ctgcccccat cctgggatga gccgtggggt 60

gaagacgtca gcctgacctg cctggtcaaa ggcttctatc ccagcgaaat cgccgtggag 120

tgggagagca atgggcagcc ggagaacaac tacaagacca cgcctcccgt gctggactcc 180

gacggctcct tcttcctcta cagcaagctc accgtgccgt atgataggtg ggtttggccg 240

gatgagttct catgctccgt gatgcatgag gctctgcaca accactacac acagaagagc 300

ctctccctgt ctccggggt 318

<210> 10

<211> 106

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Amino acid sequence of the CH3 domain of Fcab FS18-7-32

<400> 10

Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Trp Asp

1 5 10 15

Glu Pro Trp Gly Glu Asp Val Ser Leu Thr Cys Leu Val Lys Gly Phe

20 25 30

Tyr Pro Ser Glu Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu

35 40 45

Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe

50 55 60

Phe Leu Tyr Ser Lys Leu Thr Val Pro Tyr Asp Arg Trp Val Trp Pro

65 70 75 80

Asp Glu Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr

85 90 95

Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

100 105

<210> 11

<211> 216

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Amino acid sequence of CH2 and CH3 domains of Fcab FS18-7-32,

containing the LALA mutation

<400> 11

Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys

1 5 10 15

Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val

20 25 30

Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr

35 40 45

Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu

50 55 60

Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His

65 70 75 80

Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys

85 90 95

Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln

100 105 110

Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Ser Trp Asp Glu Pro

115 120 125

Trp Gly Glu Asp Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro

130 135 140

Ser Glu Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn

145 150 155 160

Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu

165 170 175

Tyr Ser Lys Leu Thr Val Pro Tyr Asp Arg Trp Val Trp Pro Asp Glu

180 185 190

Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln

195 200 205

Lys Ser Leu Ser Leu Ser Pro Gly

210 215

<210> 12

<211> 216

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Amino acid sequence of the CH2 and CH3 domains of Fcab

FS18-7-32 without LALA mutation

<400> 12

Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys

1 5 10 15

Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val

20 25 30

Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr

35 40 45

Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu

50 55 60

Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His

65 70 75 80

Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys

85 90 95

Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln

100 105 110

Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Ser Trp Asp Glu Pro

115 120 125

Trp Gly Glu Asp Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro

130 135 140

Ser Glu Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn

145 150 155 160

Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu

165 170 175

Tyr Ser Lys Leu Thr Val Pro Tyr Asp Arg Trp Val Trp Pro Asp Glu

180 185 190

Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln

195 200 205

Lys Ser Leu Ser Leu Ser Pro Gly

210 215

<210> 13

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CD hinge FS18-7-33

<400> 13

Ser Asn Gly Gln Pro Glu Asp Asn Tyr

fifteen

<210> 14

<211> 318

<212> DNA

<213> Artificial sequence

<220>

<223> Nucleotide sequence of the CH3 domain of Fcab FS18-7-33

<400> 14

ggccagcctc gagaaccaca ggtgtacacc ctgcccccat cctgggatga gccgtggggt 60

gaagacgtca gcctgacctg cctggtcaaa ggcttctatc ccagcgacat cgccgtggag 120

tgggagagca atgggcagcc ggaggacaac tacaagacca cgcctcccgt gctggactcc 180

gacggctcct tcttcctcta cagcaagctc accgtgccgt atgataggtg ggtttggccg 240

gatgagttct catgctccgt gatgcatgag gctctgcaca accactacac acagaagagc 300

ctctccctgt ctccggggt 318

<210> 15

<211> 106

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Amino acid sequence of the CH3 domain of Fcab FS18-7-33

<400> 15

Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Trp Asp

1 5 10 15

Glu Pro Trp Gly Glu Asp Val Ser Leu Thr Cys Leu Val Lys Gly Phe

20 25 30

Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu

35 40 45

Asp Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe

50 55 60

Phe Leu Tyr Ser Lys Leu Thr Val Pro Tyr Asp Arg Trp Val Trp Pro

65 70 75 80

Asp Glu Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr

85 90 95

Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

100 105

<210> 16

<211> 216

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Amino acid sequence of CH2 and CH3 domains of Fcab FS18-7-33,

containing the LALA mutation

<400> 16

Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys

1 5 10 15

Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val

20 25 30

Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr

35 40 45

Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu

50 55 60

Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His

65 70 75 80

Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys

85 90 95

Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln

100 105 110

Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Ser Trp Asp Glu Pro

115 120 125

Trp Gly Glu Asp Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro

130 135 140

Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asp Asn

145 150 155 160

Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu

165 170 175

Tyr Ser Lys Leu Thr Val Pro Tyr Asp Arg Trp Val Trp Pro Asp Glu

180 185 190

Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln

195 200 205

Lys Ser Leu Ser Leu Ser Pro Gly

210 215

<210> 17

<211> 216

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Amino acid sequence of the CH2 and CH3 domains of Fcab

FS18-7-33 without LALA mutation

<400> 17

Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys

1 5 10 15

Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val

20 25 30

Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr

35 40 45

Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu

50 55 60

Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His

65 70 75 80

Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys

85 90 95

Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln

100 105 110

Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Ser Trp Asp Glu Pro

115 120 125

Trp Gly Glu Asp Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro

130 135 140

Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asp Asn

145 150 155 160

Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu

165 170 175

Tyr Ser Lys Leu Thr Val Pro Tyr Asp Arg Trp Val Trp Pro Asp Glu

180 185 190

Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln

195 200 205

Lys Ser Leu Ser Leu Ser Pro Gly

210 215

<210> 18

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CD hinge FS18-7-36

<400> 18

Ser Asn Gly Gln Pro Glu Asn Asn Tyr

fifteen

<210> 19

<211> 318

<212> DNA

<213> Artificial sequence

<220>

<223> Nucleotide sequence of the CH3 domain of Fcab FS18-7-36

<400> 19

ggccagcctc gagaaccaca ggtgtacacc ctgcccccat cctgggatga gccgtggggt 60

gaagacgtca gcctgacctg cctggtcaaa ggcttctatc ccagcgacat cgccgtggag 120

tgggagagca atgggcagcc ggagaacaac tacaagacca cgcctcccgt gctggactcc 180

gacggctcct acttcctcta cagcaagctc accgtgccgt atgataggtg ggtttggccg 240

gatgagttct catgctccgt gatgcatgag gctctgcaca accactacac acagaagagc 300

ctctccctgt ctccggggt 318

<210> 20

<211> 106

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Amino acid sequence of the CH3 domain of Fcab FS18-7-36

<400> 20

Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Trp Asp

1 5 10 15

Glu Pro Trp Gly Glu Asp Val Ser Leu Thr Cys Leu Val Lys Gly Phe

20 25 30

Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu

35 40 45

Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Tyr

50 55 60

Phe Leu Tyr Ser Lys Leu Thr Val Pro Tyr Asp Arg Trp Val Trp Pro

65 70 75 80

Asp Glu Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr

85 90 95

Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

100 105

<210> 21

<211> 216

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Amino acid sequence of CH2+CH3 CH2 and CH3 domains of Fcab

FS18-7-36 containing the LALA mutation

<400> 21

Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys

1 5 10 15

Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val

20 25 30

Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr

35 40 45

Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu

50 55 60

Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His

65 70 75 80

Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys

85 90 95

Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln

100 105 110

Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Ser Trp Asp Glu Pro

115 120 125

Trp Gly Glu Asp Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro

130 135 140

Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn

145 150 155 160

Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Tyr Phe Leu

165 170 175

Tyr Ser Lys Leu Thr Val Pro Tyr Asp Arg Trp Val Trp Pro Asp Glu

180 185 190

Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln

195 200 205

Lys Ser Leu Ser Leu Ser Pro Gly

210 215

<210> 22

<211> 216

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Amino acid sequence of the CH2 and CH3 domains of Fcab

FS18-7-36 without LALA mutation

<400> 22

Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys

1 5 10 15

Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val

20 25 30

Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr

35 40 45

Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu

50 55 60

Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His

65 70 75 80

Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys

85 90 95

Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln

100 105 110

Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Ser Trp Asp Glu Pro

115 120 125

Trp Gly Glu Asp Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro

130 135 140

Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn

145 150 155 160

Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Tyr Phe Leu

165 170 175

Tyr Ser Lys Leu Thr Val Pro Tyr Asp Arg Trp Val Trp Pro Asp Glu

180 185 190

Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln

195 200 205

Lys Ser Leu Ser Leu Ser Pro Gly

210 215

<210> 23

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CD hinge FS18-7-58

<400> 23

Ser Asn Gly Tyr Pro Glu Ile Glu Phe

fifteen

<210> 24

<211> 318

<212> DNA

<213> Artificial sequence

<220>

<223> Nucleotide sequence of the CH3 domain of Fcab FS18-7-58

<400> 24

ggccagcctc gagaaccaca ggtgtacacc ctgcccccat cctgggatga gccgtggggt 60

gaagacgtca gcctgacctg cctggtcaaa ggcttctatc ccagcgacat cgccgtggag 120

tgggagagca atgggtatcc agaaatcgaa ttcaagacca cgcctcccgt gctggactcc 180

gacggctcct tcttcctcta cagcaagctc accgtgcctt atgataggtg ggtttggccg 240

gatgagttct catgctccgt gatgcatgag gctctgcaca accactacac acagaagagc 300

ctctccctgt ctccggggt 318

<210> 25

<211> 106

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Amino acid sequence of the CH3 domain of Fcab FS18-7-58

<400> 25

Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Trp Asp

1 5 10 15

Glu Pro Trp Gly Glu Asp Val Ser Leu Thr Cys Leu Val Lys Gly Phe

20 25 30

Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Tyr Pro Glu

35 40 45

Ile Glu Phe Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe

50 55 60

Phe Leu Tyr Ser Lys Leu Thr Val Pro Tyr Asp Arg Trp Val Trp Pro

65 70 75 80

Asp Glu Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr

85 90 95

Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

100 105

<210> 26

<211> 216

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Amino acid sequence of the CH2 and CH3 domains of Fcab

FS18-7-58 containing the LALA mutation

<400> 26

Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys

1 5 10 15

Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val

20 25 30

Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr

35 40 45

Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu

50 55 60

Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His

65 70 75 80

Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys

85 90 95

Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln

100 105 110

Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Ser Trp Asp Glu Pro

115 120 125

Trp Gly Glu Asp Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro

130 135 140

Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Tyr Pro Glu Ile Glu

145 150 155 160

Phe Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu

165 170 175

Tyr Ser Lys Leu Thr Val Pro Tyr Asp Arg Trp Val Trp Pro Asp Glu

180 185 190

Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln

195 200 205

Lys Ser Leu Ser Leu Ser Pro Gly

210 215

<210> 27

<211> 216

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Amino acid sequence of the CH2 and CH3 domains of Fcab

FS18-7-58 without LALA mutation

<400> 27

Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys

1 5 10 15

Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val

20 25 30

Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr

35 40 45

Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu

50 55 60

Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His

65 70 75 80

Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys

85 90 95

Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln

100 105 110

Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Ser Trp Asp Glu Pro

115 120 125

Trp Gly Glu Asp Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro

130 135 140

Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Tyr Pro Glu Ile Glu

145 150 155 160

Phe Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu

165 170 175

Tyr Ser Lys Leu Thr Val Pro Tyr Asp Arg Trp Val Trp Pro Asp Glu

180 185 190

Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln

195 200 205

Lys Ser Leu Ser Leu Ser Pro Gly

210 215

<210> 28

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CD hinge FS18-7-62

<400> 28

Ser Asn Gly Ile Pro Glu Trp Asn Tyr

fifteen

<210> 29

<211> 318

<212> DNA

<213> Artificial sequence

<220>

<223> Nucleotide sequence of the CH3 domain of Fcab FS18-7-62

<400> 29

ggccagcctc gagaaccaca ggtgtacacc ctgcccccat cctgggatga gccgtggggt 60

gaagacgtca gcctgacctg cctggtcaaa ggcttctatc ccagcgacat cgccgtggag 120

tgggagagca atgggatccc agaatggaac tataagacca cgcctcccgt gctggactcc 180

gacggctcct tcttcctcta cagcaagctc accgtgccgt atgataggtg ggtttggccg 240

gatgagttct catgctccgt gatgcatgag gctctgcaca accactacac acagaagagc 300

ctctccctgt ctccggggt 318

<210> 30

<211> 106

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Amino acid sequence of the CH3 domain of Fcab FS18-7-62

<400> 30

Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Trp Asp

1 5 10 15

Glu Pro Trp Gly Glu Asp Val Ser Leu Thr Cys Leu Val Lys Gly Phe

20 25 30

Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Ile Pro Glu

35 40 45

Trp Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe

50 55 60

Phe Leu Tyr Ser Lys Leu Thr Val Pro Tyr Asp Arg Trp Val Trp Pro

65 70 75 80

Asp Glu Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr

85 90 95

Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

100 105

<210> 31

<211> 216

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Amino acid sequence of the CH2 and CH3 domains of Fcab

FS18-7-62 containing the LALA mutation

<400> 31

Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys

1 5 10 15

Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val

20 25 30

Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr

35 40 45

Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu

50 55 60

Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His

65 70 75 80

Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys

85 90 95

Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln

100 105 110

Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Ser Trp Asp Glu Pro

115 120 125

Trp Gly Glu Asp Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro

130 135 140

Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Ile Pro Glu Trp Asn

145 150 155 160

Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu

165 170 175

Tyr Ser Lys Leu Thr Val Pro Tyr Asp Arg Trp Val Trp Pro Asp Glu

180 185 190

Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln

195 200 205

Lys Ser Leu Ser Leu Ser Pro Gly

210 215

<210> 32

<211> 216

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Amino acid sequence of the CH2 and CH3 domains of Fcab

FS18-7-62 without LALA mutation

<400> 32

Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys

1 5 10 15

Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val

20 25 30

Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr

35 40 45

Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu

50 55 60

Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His

65 70 75 80

Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys

85 90 95

Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln

100 105 110

Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Ser Trp Asp Glu Pro

115 120 125

Trp Gly Glu Asp Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro

130 135 140

Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Ile Pro Glu Trp Asn

145 150 155 160

Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu

165 170 175

Tyr Ser Lys Leu Thr Val Pro Tyr Asp Arg Trp Val Trp Pro Asp Glu

180 185 190

Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln

195 200 205

Lys Ser Leu Ser Leu Ser Pro Gly

210 215

<210> 33

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CD hinge FS18-7-65

<400> 33

Ser Asn Gly Tyr Ala Glu Tyr Asn Tyr

fifteen

<210> 34

<211> 318

<212> DNA

<213> Artificial sequence

<220>

<223> Nucleotide sequence of the CH3 domain of Fcab FS18-7-65

<400> 34

ggccagcctc gagaaccaca ggtgtacacc ctgcccccat cctgggatga gccgtggggt 60

gaagacgtca gcctgacctg cctggtcaaa ggcttctatc ccagcgacat cgccgtggag 120

tgggagagca atgggtatgc agaatataac tataagacca cgcctcccgt gctggactcc 180

gacggctcct tcttcctcta cagcaagctc accgtgccgt atgataggtg ggtttggccg 240

gatgagttct catgctccgt gatgcatgag gctctgcaca accactacac acagaagagc 300

ctctccctgt ctccggggt 318

<210> 35

<211> 106

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Amino acid sequence of the CH3 domain of Fcab FS18-7-65

<400> 35

Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Trp Asp

1 5 10 15

Glu Pro Trp Gly Glu Asp Val Ser Leu Thr Cys Leu Val Lys Gly Phe

20 25 30

Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Tyr Ala Glu

35 40 45

Tyr Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe

50 55 60

Phe Leu Tyr Ser Lys Leu Thr Val Pro Tyr Asp Arg Trp Val Trp Pro

65 70 75 80

Asp Glu Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr

85 90 95

Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

100 105

<210> 36

<211> 216

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Amino acid sequence of the CH2 and CH3 domains of Fcab

FS18-7-65 containing the LALA mutation

<400> 36

Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys

1 5 10 15

Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val

20 25 30

Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr

35 40 45

Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu

50 55 60

Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His

65 70 75 80

Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys

85 90 95

Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln

100 105 110

Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Ser Trp Asp Glu Pro

115 120 125

Trp Gly Glu Asp Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro

130 135 140

Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Tyr Ala Glu Tyr Asn

145 150 155 160

Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu

165 170 175

Tyr Ser Lys Leu Thr Val Pro Tyr Asp Arg Trp Val Trp Pro Asp Glu

180 185 190

Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln

195 200 205

Lys Ser Leu Ser Leu Ser Pro Gly

210 215

<210> 37

<211> 216

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Amino acid sequence of the CH2 and CH3 domains of Fcab

FS18-7-65 without LALA mutation

<400> 37

Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys

1 5 10 15

Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val

20 25 30

Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr

35 40 45

Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu

50 55 60

Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His

65 70 75 80

Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys

85 90 95

Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln

100 105 110

Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Ser Trp Asp Glu Pro

115 120 125

Trp Gly Glu Asp Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro

130 135 140

Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Tyr Ala Glu Tyr Asn

145 150 155 160

Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu

165 170 175

Tyr Ser Lys Leu Thr Val Pro Tyr Asp Arg Trp Val Trp Pro Asp Glu

180 185 190

Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln

195 200 205

Lys Ser Leu Ser Leu Ser Pro Gly

210 215

<210> 38

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CD hinge FS18-7-78

<400> 38

Ser Asn Gly Tyr Lys Glu Glu Asn Tyr

fifteen

<210> 39

<211> 318

<212> DNA

<213> Artificial sequence

<220>

<223> Nucleotide sequence of the CH3 domain of Fcab FS18-7-78

<400> 39

ggccagcctc gagaaccaca ggtgtacacc ctgcccccat cctgggatga gccgtggggt 60

gaagacgtca gcctgacctg cctggtcaaa ggcttctatc ccagcgacat cgccgtggag 120

tgggagagca atgggtataa agaagaaaac tataagacca cgcctcccgt gctggactcc 180

gacggctcct tcttcctcta cagcaagctc accgtgccgt atgataggtg ggtttggccg 240

gatgagttct catgctccgt gatgcatgag gctctgcaca accactacac acagaagagc 300

ctctccctgt ctccggggt 318

<210> 40

<211> 106

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Amino acid sequence of the CH3 domain of Fcab FS18-7-78

<400> 40

Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Trp Asp

1 5 10 15

Glu Pro Trp Gly Glu Asp Val Ser Leu Thr Cys Leu Val Lys Gly Phe

20 25 30

Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Tyr Lys Glu

35 40 45

Glu Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe

50 55 60

Phe Leu Tyr Ser Lys Leu Thr Val Pro Tyr Asp Arg Trp Val Trp Pro

65 70 75 80

Asp Glu Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr

85 90 95

Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

100 105

<210> 41

<211> 216

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Amino acid sequence of the CH2 and CH3 domains of Fcab

FS18-7-78 containing the LALA mutation

<400> 41

Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys

1 5 10 15

Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val

20 25 30

Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr

35 40 45

Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu

50 55 60

Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His

65 70 75 80

Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys

85 90 95

Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln

100 105 110

Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Ser Trp Asp Glu Pro

115 120 125

Trp Gly Glu Asp Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro

130 135 140

Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Tyr Lys Glu Glu Asn

145 150 155 160

Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu

165 170 175

Tyr Ser Lys Leu Thr Val Pro Tyr Asp Arg Trp Val Trp Pro Asp Glu

180 185 190

Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln

195 200 205

Lys Ser Leu Ser Leu Ser Pro Gly

210 215

<210> 42

<211> 216

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Amino acid sequence of the CH2 and CH3 domains of Fcab

FS18-7-78 without LALA mutation

<400> 42

Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys

1 5 10 15

Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val

20 25 30

Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr

35 40 45

Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu

50 55 60

Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His

65 70 75 80

Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys

85 90 95

Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln

100 105 110

Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Ser Trp Asp Glu Pro

115 120 125

Trp Gly Glu Asp Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro

130 135 140

Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Tyr Lys Glu Glu Asn

145 150 155 160

Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu

165 170 175

Tyr Ser Lys Leu Thr Val Pro Tyr Asp Arg Trp Val Trp Pro Asp Glu

180 185 190

Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln

195 200 205

Lys Ser Leu Ser Leu Ser Pro Gly

210 215

<210> 43

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CD hinge FS18-7-88

<400> 43

Ser Asn Gly Val Pro Glu Leu Asn Val

fifteen

<210> 44

<211> 318

<212> DNA

<213> Artificial sequence

<220>

<223> Nucleotide sequence of the CH3 domain of Fcab FS18-7-88

<400> 44

ggccagcctc gagaaccaca ggtgtacacc ctgcccccat cctgggatga gccgtggggt 60

gaagacgtca gcctgacctg cctggtcaaa ggcttctatc ccagcgacat cgccgtggag 120

tgggagagca atggggttcc agaactgaac gttaagacca cgcctcccgt gctggactcc 180

gacggctcct tcttcctcta cagcaagctc accgtgccgt atgataggtg ggtttggccg 240

gatgagttct catgctccgt gatgcatgag gctctgcaca accactacac acagaagagc 300

ctctccctgt ctccggggt 318

<210> 45

<211> 106

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Amino acid sequence of the CH3 domain of Fcab FS18-7-88

<400> 45

Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Trp Asp

1 5 10 15

Glu Pro Trp Gly Glu Asp Val Ser Leu Thr Cys Leu Val Lys Gly Phe

20 25 30

Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Val Pro Glu

35 40 45

Leu Asn Val Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe

50 55 60

Phe Leu Tyr Ser Lys Leu Thr Val Pro Tyr Asp Arg Trp Val Trp Pro

65 70 75 80

Asp Glu Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr

85 90 95

Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

100 105

<210> 46

<211> 216

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Amino acid sequence of the CH2 and CH3 domains of Fcab

FS18-7-88 containing the LALA mutation

<400> 46

Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys

1 5 10 15

Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val

20 25 30

Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr

35 40 45

Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu

50 55 60

Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His

65 70 75 80

Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys

85 90 95

Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln

100 105 110

Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Ser Trp Asp Glu Pro

115 120 125

Trp Gly Glu Asp Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro

130 135 140

Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Val Pro Glu Leu Asn

145 150 155 160

Val Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu

165 170 175

Tyr Ser Lys Leu Thr Val Pro Tyr Asp Arg Trp Val Trp Pro Asp Glu

180 185 190

Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln

195 200 205

Lys Ser Leu Ser Leu Ser Pro Gly

210 215

<210> 47

<211> 216

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Amino acid sequence of the CH2 and CH3 domains of Fcab

FS18-7-88 without LALA mutation

<400> 47

Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys

1 5 10 15

Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val

20 25 30

Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr

35 40 45

Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu

50 55 60

Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His

65 70 75 80

Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys

85 90 95

Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln

100 105 110

Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Ser Trp Asp Glu Pro

115 120 125

Trp Gly Glu Asp Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro

130 135 140

Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Val Pro Glu Leu Asn

145 150 155 160

Val Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu

165 170 175

Tyr Ser Lys Leu Thr Val Pro Tyr Asp Arg Trp Val Trp Pro Asp Glu

180 185 190

Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln

195 200 205

Lys Ser Leu Ser Leu Ser Pro Gly

210 215

<210> 48

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CD hinge FS18-7-95

<400> 48

Ser Asn Gly Tyr Gln Glu Asp Asn Tyr

fifteen

<210> 49

<211> 318

<212> DNA

<213> Artificial sequence

<220>

<223> Nucleotide sequence of the CH3 domain of Fcab FS18-7-95

<400> 49

ggccagcctc gagaaccaca ggtgtacacc ctgcccccat cctgggatga gccgtggggt 60

gaagacgtca gcctgacctg cctggtcaaa ggcttctatc ccagcgacat cgccgtggag 120

tgggagagca atgggtatca ggaagataac tataagacca cgcctcccgt gctggactcc 180

gacggctcct tcttcctcta cagcaagctc accgtgccgt atgataggtg ggtttggccg 240

gatgagttct catgctccgt gatgcatgag gctctgcaca accactacac acagaagagc 300

ctctccctgt ctccggggt 318

<210> 50

<211> 106

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Amino acid sequence of the CH3 domain of Fcab FS18-7-95

<400> 50

Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Trp Asp

1 5 10 15

Glu Pro Trp Gly Glu Asp Val Ser Leu Thr Cys Leu Val Lys Gly Phe

20 25 30

Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Tyr Gln Glu

35 40 45

Asp Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe

50 55 60

Phe Leu Tyr Ser Lys Leu Thr Val Pro Tyr Asp Arg Trp Val Trp Pro

65 70 75 80

Asp Glu Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr

85 90 95

Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

100 105

<210> 51

<211> 216

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Amino acid sequence of the CH2 and CH3 domains of Fcab

FS18-7-95 containing the LALA mutation

<400> 51

Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys

1 5 10 15

Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val

20 25 30

Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr

35 40 45

Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu

50 55 60

Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His

65 70 75 80

Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys

85 90 95

Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln

100 105 110

Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Ser Trp Asp Glu Pro

115 120 125

Trp Gly Glu Asp Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro

130 135 140

Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Tyr Gln Glu Asp Asn

145 150 155 160

Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu

165 170 175

Tyr Ser Lys Leu Thr Val Pro Tyr Asp Arg Trp Val Trp Pro Asp Glu

180 185 190

Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln

195 200 205

Lys Ser Leu Ser Leu Ser Pro Gly

210 215

<210> 52

<211> 216

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Amino acid sequence of the CH2 and CH3 domains of Fcab

FS18-7-95 without LALA mutation

<400> 52

Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys

1 5 10 15

Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val

20 25 30

Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr

35 40 45

Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu

50 55 60

Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His

65 70 75 80

Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys

85 90 95

Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln

100 105 110

Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Ser Trp Asp Glu Pro

115 120 125

Trp Gly Glu Asp Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro

130 135 140

Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Tyr Gln Glu Asp Asn

145 150 155 160

Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu

165 170 175

Tyr Ser Lys Leu Thr Val Pro Tyr Asp Arg Trp Val Trp Pro Asp Glu

180 185 190

Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln

195 200 205

Lys Ser Leu Ser Leu Ser Pro Gly

210 215

<210> 53

<211> 110

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Amino acid sequence of wild-type human IgG1 CH2 domain

<400> 53

Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys

1 5 10 15

Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val

20 25 30

Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr

35 40 45

Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu

50 55 60

Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His

65 70 75 80

Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys

85 90 95

Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys

100 105 110

<210> 54

<211> 110

<212> PROTEIN

<213> Artificial sequence

<220>

<223> The amino acid sequence of the CH2 domain of human IgG1 containing

"LALA mutation"

<400> 54

Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys

1 5 10 15

Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val

20 25 30

Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr

35 40 45

Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu

50 55 60

Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His

65 70 75 80

Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys

85 90 95

Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys

100 105 110

<210> 55

<211> 216

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Amino acid sequence of wild-type Fcab CH2 and CH3 domains

no LALA mutation

<400> 55

Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys

1 5 10 15

Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val

20 25 30

Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr

35 40 45

Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu

50 55 60

Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His

65 70 75 80

Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys

85 90 95

Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln

100 105 110

Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Ser Arg Asp Glu Leu

115 120 125

Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro

130 135 140

Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn

145 150 155 160

Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu

165 170 175

Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val

180 185 190

Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln

195 200 205

Lys Ser Leu Ser Leu Ser Pro Gly

210 215

<210> 56

<211> 216

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Amino acid sequence of the CH2 and CH3 domains of Fcab

"wild-type" containing the LALA mutation

<400> 56

Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys

1 5 10 15

Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val

20 25 30

Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr

35 40 45

Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu

50 55 60

Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His

65 70 75 80

Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys

85 90 95

Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln

100 105 110

Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Ser Arg Asp Glu Leu

115 120 125

Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro

130 135 140

Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn

145 150 155 160

Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu

165 170 175

Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val

180 185 190

Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln

195 200 205

Lys Ser Leu Ser Leu Ser Pro Gly

210 215

<210> 57

<211> 15

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Human IgG1 hinge amino acid sequence

<400> 57

Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro

1 5 10 15

<210> 58

<211> 6

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Shortened hinge amino acid sequence

human IgG1

<400> 58

Thr Cys Pro Pro Cys Pro

fifteen

<210> 59

<211> 222

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Anti-LAG-3 mouse Fcab FS18-7-108-29 amino acid sequence,

containing the LALA mutation

<400> 59

Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val

1 5 10 15

Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr

20 25 30

Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu

35 40 45

Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys

50 55 60

Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser

65 70 75 80

Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys

85 90 95

Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile

100 105 110

Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro

115 120 125

Pro Ser Trp Asp Glu Pro Trp Gly Glu Asp Val Ser Leu Thr Cys Leu

130 135 140

Val Lys Gly Phe Tyr Pro Ser Asp Ile Val Val Glu Trp Glu Ser Asn

145 150 155 160

Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser

165 170 175

Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Pro Phe Glu Arg

180 185 190

Trp Met Trp Pro Asp Glu Phe Ser Cys Ser Val Met His Glu Ala Leu

195 200 205

His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

210 215 220

<210> 60

<211> 222

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Amino acid sequence of Fcab FS18-7-108-29 against LAG-3

mice without LALA mutation

<400> 60

Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val

1 5 10 15

Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr

20 25 30

Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu

35 40 45

Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys

50 55 60

Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser

65 70 75 80

Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys

85 90 95

Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile

100 105 110

Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro

115 120 125

Pro Ser Trp Asp Glu Pro Trp Gly Glu Asp Val Ser Leu Thr Cys Leu

130 135 140

Val Lys Gly Phe Tyr Pro Ser Asp Ile Val Val Glu Trp Glu Ser Asn

145 150 155 160

Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser

165 170 175

Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Pro Phe Glu Arg

180 185 190

Trp Met Trp Pro Asp Glu Phe Ser Cys Ser Val Met His Glu Ala Leu

195 200 205

His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

210 215 220

<210> 61

<211> 222

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Amino acid sequence of Fcab FS18-7-108-35 against mouse LAG-3,

containing the LALA mutation

<400> 61

Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val

1 5 10 15

Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr

20 25 30

Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu

35 40 45

Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys

50 55 60

Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser

65 70 75 80

Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys

85 90 95

Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile

100 105 110

Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro

115 120 125

Pro Ser Trp Asp Glu Pro Trp Gly Glu Asp Val Ser Leu Thr Cys Leu

130 135 140

Val Lys Gly Phe Tyr Pro Ser Asp Ile Ser Val Glu Trp Glu Ser Asn

145 150 155 160

Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser

165 170 175

Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Pro Phe Glu Arg

180 185 190

Trp Met Trp Pro Asp Glu Phe Ser Cys Ser Val Met His Glu Ala Leu

195 200 205

His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

210 215 220

<210> 62

<211> 222

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Amino acid sequence of Fcab FS18-7-108-35 against LAG-3

mice without LALA mutation

<400> 62

Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val

1 5 10 15

Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr

20 25 30

Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu

35 40 45

Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys

50 55 60

Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser

65 70 75 80

Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys

85 90 95

Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile

100 105 110

Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro

115 120 125

Pro Ser Trp Asp Glu Pro Trp Gly Glu Asp Val Ser Leu Thr Cys Leu

130 135 140

Val Lys Gly Phe Tyr Pro Ser Asp Ile Ser Val Glu Trp Glu Ser Asn

145 150 155 160

Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser

165 170 175

Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Pro Phe Glu Arg

180 185 190

Trp Met Trp Pro Asp Glu Phe Ser Cys Ser Val Met His Glu Ala Leu

195 200 205

His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

210 215 220

<210> 63

<211> 447

<212> PROTEIN

<213> Artificial sequence

<220>

<223> mAb2 heavy chain amino acid sequence

FS18-7-9/4420 against human LAG-3/FITC containing LALA mutation

<400> 63

Glu Val Lys Leu Asp Glu Thr Gly Gly Gly Leu Val Gln Pro Gly Arg

1 5 10 15

Pro Met Lys Leu Ser Cys Val Ala Ser Gly Phe Thr Phe Ser Asp Tyr

20 25 30

Trp Met Asn Trp Val Arg Gln Ser Pro Glu Lys Gly Leu Glu Trp Val

35 40 45

Ala Gln Ile Arg Asn Lys Pro Tyr Asn Tyr Glu Thr Tyr Tyr Ser Asp

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Ser

65 70 75 80

Val Tyr Leu Gln Met Asn Asn Leu Arg Val Glu Asp Met Gly Ile Tyr

85 90 95

Tyr Cys Thr Gly Ser Tyr Tyr Gly Met Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Ser Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro

115 120 125

Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly

130 135 140

Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn

145 150 155 160

Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln

165 170 175

Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser

180 185 190

Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser

195 200 205

Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr

210 215 220

His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser

225 230 235 240

Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg

245 250 255

Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro

260 265 270

Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala

275 280 285

Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val

290 295 300

Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr

305 310 315 320

Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr

325 330 335

Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu

340 345 350

Pro Pro Ser Trp Asp Glu Pro Trp Gly Glu Asp Val Ser Leu Thr Cys

355 360 365

Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser

370 375 380

Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp

385 390 395 400

Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Pro Tyr Asp

405 410 415

Arg Trp Val Trp Pro Asp Glu Phe Ser Cys Ser Val Met His Glu Ala

420 425 430

Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

435 440 445

<210> 64

<211> 447

<212> PROTEIN

<213> Artificial sequence

<220>

<223> mAb2 heavy chain amino acid sequence

FS18-7-9/4420 vs human LAG-3/FITC without LALA mutation

<400> 64

Glu Val Lys Leu Asp Glu Thr Gly Gly Gly Leu Val Gln Pro Gly Arg

1 5 10 15

Pro Met Lys Leu Ser Cys Val Ala Ser Gly Phe Thr Phe Ser Asp Tyr

20 25 30

Trp Met Asn Trp Val Arg Gln Ser Pro Glu Lys Gly Leu Glu Trp Val

35 40 45

Ala Gln Ile Arg Asn Lys Pro Tyr Asn Tyr Glu Thr Tyr Tyr Ser Asp

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Ser

65 70 75 80

Val Tyr Leu Gln Met Asn Asn Leu Arg Val Glu Asp Met Gly Ile Tyr

85 90 95

Tyr Cys Thr Gly Ser Tyr Tyr Gly Met Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Ser Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro

115 120 125

Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly

130 135 140

Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn

145 150 155 160

Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln

165 170 175

Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser

180 185 190

Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser

195 200 205

Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr

210 215 220

His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser

225 230 235 240

Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg

245 250 255

Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro

260 265 270

Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala

275 280 285

Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val

290 295 300

Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr

305 310 315 320

Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr

325 330 335

Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu

340 345 350

Pro Pro Ser Trp Asp Glu Pro Trp Gly Glu Asp Val Ser Leu Thr Cys

355 360 365

Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser

370 375 380

Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp

385 390 395 400

Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Pro Tyr Asp

405 410 415

Arg Trp Val Trp Pro Asp Glu Phe Ser Cys Ser Val Met His Glu Ala

420 425 430

Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

435 440 445

<210> 65

<211> 447

<212> PROTEIN

<213> Artificial sequence

<220>

<223> mAb2 heavy chain amino acid sequence

FS18-7-32/4420 against human LAG-3/FITC containing LALA mutation

<400> 65

Glu Val Lys Leu Asp Glu Thr Gly Gly Gly Leu Val Gln Pro Gly Arg

1 5 10 15

Pro Met Lys Leu Ser Cys Val Ala Ser Gly Phe Thr Phe Ser Asp Tyr

20 25 30

Trp Met Asn Trp Val Arg Gln Ser Pro Glu Lys Gly Leu Glu Trp Val

35 40 45

Ala Gln Ile Arg Asn Lys Pro Tyr Asn Tyr Glu Thr Tyr Tyr Ser Asp

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Ser

65 70 75 80

Val Tyr Leu Gln Met Asn Asn Leu Arg Val Glu Asp Met Gly Ile Tyr

85 90 95

Tyr Cys Thr Gly Ser Tyr Tyr Gly Met Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Ser Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro

115 120 125

Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly

130 135 140

Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn

145 150 155 160

Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln

165 170 175

Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser

180 185 190

Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser

195 200 205

Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr

210 215 220

His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser

225 230 235 240

Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg

245 250 255

Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro

260 265 270

Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala

275 280 285

Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val

290 295 300

Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr

305 310 315 320

Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr

325 330 335

Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu

340 345 350

Pro Pro Ser Trp Asp Glu Pro Trp Gly Glu Asp Val Ser Leu Thr Cys

355 360 365

Leu Val Lys Gly Phe Tyr Pro Ser Glu Ile Ala Val Glu Trp Glu Ser

370 375 380

Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp

385 390 395 400

Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Pro Tyr Asp

405 410 415

Arg Trp Val Trp Pro Asp Glu Phe Ser Cys Ser Val Met His Glu Ala

420 425 430

Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

435 440 445

<210> 66

<211> 447

<212> PROTEIN

<213> Artificial sequence

<220>

<223> mAb2 heavy chain amino acid sequence

FS18-7-32 vs human LAG-3/FITC without LALA mutation

<400> 66

Glu Val Lys Leu Asp Glu Thr Gly Gly Gly Leu Val Gln Pro Gly Arg

1 5 10 15

Pro Met Lys Leu Ser Cys Val Ala Ser Gly Phe Thr Phe Ser Asp Tyr

20 25 30

Trp Met Asn Trp Val Arg Gln Ser Pro Glu Lys Gly Leu Glu Trp Val

35 40 45

Ala Gln Ile Arg Asn Lys Pro Tyr Asn Tyr Glu Thr Tyr Tyr Ser Asp

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Ser

65 70 75 80

Val Tyr Leu Gln Met Asn Asn Leu Arg Val Glu Asp Met Gly Ile Tyr

85 90 95

Tyr Cys Thr Gly Ser Tyr Tyr Gly Met Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Ser Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro

115 120 125

Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly

130 135 140

Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn

145 150 155 160

Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln

165 170 175

Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser

180 185 190

Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser

195 200 205

210 215 220

His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser

225 230 235 240

Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg

245 250 255

Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro

260 265 270

Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala

275 280 285

Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val

290 295 300

Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr

305 310 315 320

Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr

325 330 335

Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu

340 345 350

Pro Pro Ser Trp Asp Glu Pro Trp Gly Glu Asp Val Ser Leu Thr Cys

355 360 365

Leu Val Lys Gly Phe Tyr Pro Ser Glu Ile Ala Val Glu Trp Glu Ser

370 375 380

Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp

385 390 395 400

Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Pro Tyr Asp

405 410 415

Arg Trp Val Trp Pro Asp Glu Phe Ser Cys Ser Val Met His Glu Ala

420 425 430

Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

435 440 445

<210> 67

<211> 447

<212> PROTEIN

<213> Artificial sequence

<220>

<223> mAb2 heavy chain amino acid sequence

FS18-7-33/4420 against human LAG-3/FITC containing LALA mutation

<400> 67

Glu Val Lys Leu Asp Glu Thr Gly Gly Gly Leu Val Gln Pro Gly Arg

1 5 10 15

Pro Met Lys Leu Ser Cys Val Ala Ser Gly Phe Thr Phe Ser Asp Tyr

20 25 30

Trp Met Asn Trp Val Arg Gln Ser Pro Glu Lys Gly Leu Glu Trp Val

35 40 45

Ala Gln Ile Arg Asn Lys Pro Tyr Asn Tyr Glu Thr Tyr Tyr Ser Asp

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Ser

65 70 75 80

Val Tyr Leu Gln Met Asn Asn Leu Arg Val Glu Asp Met Gly Ile Tyr

85 90 95

Tyr Cys Thr Gly Ser Tyr Tyr Gly Met Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Ser Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro

115 120 125

Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly

130 135 140

Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn

145 150 155 160

Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln

165 170 175

Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser

180 185 190

Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser

195 200 205

Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr

210 215 220

His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser

225 230 235 240

Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg

245 250 255

Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro

260 265 270

Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala

275 280 285

Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val

290 295 300

Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr

305 310 315 320

Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr

325 330 335

Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu

340 345 350

Pro Pro Ser Trp Asp Glu Pro Trp Gly Glu Asp Val Ser Leu Thr Cys

355 360 365

Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser

370 375 380

Asn Gly Gln Pro Glu Asp Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp

385 390 395 400

Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Pro Tyr Asp

405 410 415

Arg Trp Val Trp Pro Asp Glu Phe Ser Cys Ser Val Met His Glu Ala

420 425 430

Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

435 440 445

<210> 68

<211> 447

<212> PROTEIN

<213> Artificial sequence

<220>

<223> mAb2 heavy chain amino acid sequence

FS18-7-33/4420 vs human LAG-3/FITC without LALA mutation

<400> 68

Glu Val Lys Leu Asp Glu Thr Gly Gly Gly Leu Val Gln Pro Gly Arg

1 5 10 15

Pro Met Lys Leu Ser Cys Val Ala Ser Gly Phe Thr Phe Ser Asp Tyr

20 25 30

Trp Met Asn Trp Val Arg Gln Ser Pro Glu Lys Gly Leu Glu Trp Val

35 40 45

Ala Gln Ile Arg Asn Lys Pro Tyr Asn Tyr Glu Thr Tyr Tyr Ser Asp

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Ser

65 70 75 80

Val Tyr Leu Gln Met Asn Asn Leu Arg Val Glu Asp Met Gly Ile Tyr

85 90 95

Tyr Cys Thr Gly Ser Tyr Tyr Gly Met Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Ser Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro

115 120 125

Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly

130 135 140

Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn

145 150 155 160

Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln

165 170 175

Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser

180 185 190

Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser

195 200 205

Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr

210 215 220

His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser

225 230 235 240

Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg

245 250 255

Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro

260 265 270

Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala

275 280 285

Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val

290 295 300

Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr

305 310 315 320

Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr

325 330 335

Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu

340 345 350

Pro Pro Ser Trp Asp Glu Pro Trp Gly Glu Asp Val Ser Leu Thr Cys

355 360 365

Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser

370 375 380

Asn Gly Gln Pro Glu Asp Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp

385 390 395 400

Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Pro Tyr Asp

405 410 415

Arg Trp Val Trp Pro Asp Glu Phe Ser Cys Ser Val Met His Glu Ala

420 425 430

Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

435 440 445

<210> 69

<211> 447

<212> PROTEIN

<213> Artificial sequence

<220>

<223> mAb2 heavy chain amino acid sequence

FS18-7-36/4420 against human LAG-3/FITC containing LALA mutation

<400> 69

Glu Val Lys Leu Asp Glu Thr Gly Gly Gly Leu Val Gln Pro Gly Arg

1 5 10 15

Pro Met Lys Leu Ser Cys Val Ala Ser Gly Phe Thr Phe Ser Asp Tyr

20 25 30

Trp Met Asn Trp Val Arg Gln Ser Pro Glu Lys Gly Leu Glu Trp Val

35 40 45

Ala Gln Ile Arg Asn Lys Pro Tyr Asn Tyr Glu Thr Tyr Tyr Ser Asp

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Ser

65 70 75 80

Val Tyr Leu Gln Met Asn Asn Leu Arg Val Glu Asp Met Gly Ile Tyr

85 90 95

Tyr Cys Thr Gly Ser Tyr Tyr Gly Met Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Ser Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro

115 120 125

Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly

130 135 140

Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn

145 150 155 160

Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln

165 170 175

Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser

180 185 190

Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser

195 200 205

Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr

210 215 220

His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser

225 230 235 240

Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg

245 250 255

Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro

260 265 270

Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala

275 280 285

Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val

290 295 300

Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr

305 310 315 320

Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr

325 330 335

Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu

340 345 350

Pro Pro Ser Trp Asp Glu Pro Trp Gly Glu Asp Val Ser Leu Thr Cys

355 360 365

Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser

370 375 380

Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp

385 390 395 400

Ser Asp Gly Ser Tyr Phe Leu Tyr Ser Lys Leu Thr Val Pro Tyr Asp

405 410 415

Arg Trp Val Trp Pro Asp Glu Phe Ser Cys Ser Val Met His Glu Ala

420 425 430

Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

435 440 445

<210> 70

<211> 447

<212> PROTEIN

<213> Artificial sequence

<220>

<223> mAb2 heavy chain amino acid sequence

FS18-7-36/4420 vs human LAG-3/FITC without LALA mutation

<400> 70

Glu Val Lys Leu Asp Glu Thr Gly Gly Gly Leu Val Gln Pro Gly Arg

1 5 10 15

Pro Met Lys Leu Ser Cys Val Ala Ser Gly Phe Thr Phe Ser Asp Tyr

20 25 30

Trp Met Asn Trp Val Arg Gln Ser Pro Glu Lys Gly Leu Glu Trp Val

35 40 45

Ala Gln Ile Arg Asn Lys Pro Tyr Asn Tyr Glu Thr Tyr Tyr Ser Asp

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Ser

65 70 75 80

Val Tyr Leu Gln Met Asn Asn Leu Arg Val Glu Asp Met Gly Ile Tyr

85 90 95

Tyr Cys Thr Gly Ser Tyr Tyr Gly Met Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Ser Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro

115 120 125

Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly

130 135 140

Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn

145 150 155 160

Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln

165 170 175

Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser

180 185 190

Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser

195 200 205

Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr

210 215 220

His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser

225 230 235 240

Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg

245 250 255

Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro

260 265 270

Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala

275 280 285

Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val

290 295 300

Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr

305 310 315 320

325 330 335

Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu

340 345 350

Pro Pro Ser Trp Asp Glu Pro Trp Gly Glu Asp Val Ser Leu Thr Cys

355 360 365

Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser

370 375 380

Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp

385 390 395 400

Ser Asp Gly Ser Tyr Phe Leu Tyr Ser Lys Leu Thr Val Pro Tyr Asp

405 410 415

Arg Trp Val Trp Pro Asp Glu Phe Ser Cys Ser Val Met His Glu Ala

420 425 430

Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

435 440 445

<210> 71

<211> 447

<212> PROTEIN

<213> Artificial sequence

<220>

<223> mAb2 heavy chain amino acid sequence

FS18-7-58/4420 against human LAG-3/FITC containing LALA mutation

<400> 71

Glu Val Lys Leu Asp Glu Thr Gly Gly Gly Leu Val Gln Pro Gly Arg

1 5 10 15

Pro Met Lys Leu Ser Cys Val Ala Ser Gly Phe Thr Phe Ser Asp Tyr

20 25 30

Trp Met Asn Trp Val Arg Gln Ser Pro Glu Lys Gly Leu Glu Trp Val

Ala Gln Ile Arg Asn Lys Pro Tyr Asn Tyr Glu Thr Tyr Tyr Ser Asp

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Ser

65 70 75 80

Val Tyr Leu Gln Met Asn Asn Leu Arg Val Glu Asp Met Gly Ile Tyr

85 90 95

Tyr Cys Thr Gly Ser Tyr Tyr Gly Met Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Ser Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro

115 120 125

Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly

130 135 140

Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn

145 150 155 160

Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln

165 170 175

Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser

180 185 190

Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser

195 200 205

Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr

210 215 220

His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser

225 230 235 240

Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg

245 250 255

Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro

260 265 270

Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala

275 280 285

Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val

290 295 300

Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr

305 310 315 320

Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr

325 330 335

Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu

340 345 350

Pro Pro Ser Trp Asp Glu Pro Trp Gly Glu Asp Val Ser Leu Thr Cys

355 360 365

Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser

370 375 380

Asn Gly Tyr Pro Glu Ile Glu Phe Lys Thr Thr Pro Pro Val Leu Asp

385 390 395 400

Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Pro Tyr Asp

405 410 415

Arg Trp Val Trp Pro Asp Glu Phe Ser Cys Ser Val Met His Glu Ala

420 425 430

Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

435 440 445

<210> 72

<211> 447

<212> PROTEIN

<213> Artificial sequence

<220>

<223> mAb2 heavy chain amino acid sequence

FS18-7-58/4420 vs human LAG-3/FITC without LALA mutation

<400> 72

Glu Val Lys Leu Asp Glu Thr Gly Gly Gly Leu Val Gln Pro Gly Arg

1 5 10 15

Pro Met Lys Leu Ser Cys Val Ala Ser Gly Phe Thr Phe Ser Asp Tyr

20 25 30

Trp Met Asn Trp Val Arg Gln Ser Pro Glu Lys Gly Leu Glu Trp Val

35 40 45

Ala Gln Ile Arg Asn Lys Pro Tyr Asn Tyr Glu Thr Tyr Tyr Ser Asp

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Ser

65 70 75 80

Val Tyr Leu Gln Met Asn Asn Leu Arg Val Glu Asp Met Gly Ile Tyr

85 90 95

Tyr Cys Thr Gly Ser Tyr Tyr Gly Met Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Ser Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro

115 120 125

Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly

130 135 140

Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn

145 150 155 160

Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln

165 170 175

Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser

180 185 190

Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser

195 200 205

Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr

210 215 220

His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser

225 230 235 240

Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg

245 250 255

Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro

260 265 270

Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala

275 280 285

Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val

290 295 300

Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr

305 310 315 320

Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr

325 330 335

Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu

340 345 350

Pro Pro Ser Trp Asp Glu Pro Trp Gly Glu Asp Val Ser Leu Thr Cys

355 360 365

Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser

370 375 380

Asn Gly Tyr Pro Glu Ile Glu Phe Lys Thr Thr Pro Pro Val Leu Asp

385 390 395 400

Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Pro Tyr Asp

405 410 415

Arg Trp Val Trp Pro Asp Glu Phe Ser Cys Ser Val Met His Glu Ala

420 425 430

Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

435 440 445

<210> 73

<211> 447

<212> PROTEIN

<213> Artificial sequence

<220>

<223> mAb2 heavy chain amino acid sequence

FS18-7-62/4420 against human LAG-3/FITC containing LALA mutation

<400> 73

Glu Val Lys Leu Asp Glu Thr Gly Gly Gly Leu Val Gln Pro Gly Arg

1 5 10 15

Pro Met Lys Leu Ser Cys Val Ala Ser Gly Phe Thr Phe Ser Asp Tyr

20 25 30

Trp Met Asn Trp Val Arg Gln Ser Pro Glu Lys Gly Leu Glu Trp Val

35 40 45

Ala Gln Ile Arg Asn Lys Pro Tyr Asn Tyr Glu Thr Tyr Tyr Ser Asp

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Ser

65 70 75 80

Val Tyr Leu Gln Met Asn Asn Leu Arg Val Glu Asp Met Gly Ile Tyr

85 90 95

Tyr Cys Thr Gly Ser Tyr Tyr Gly Met Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Ser Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro

115 120 125

Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly

130 135 140

Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn

145 150 155 160

Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln

165 170 175

Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser

180 185 190

Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser

195 200 205

Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr

210 215 220

His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser

225 230 235 240

Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg

245 250 255

Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro

260 265 270

Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala

275 280 285

Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val

290 295 300

Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr

305 310 315 320

Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr

325 330 335

Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu

340 345 350

Pro Pro Ser Trp Asp Glu Pro Trp Gly Glu Asp Val Ser Leu Thr Cys

355 360 365

Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser

370 375 380

Asn Gly Ile Pro Glu Trp Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp

385 390 395 400

Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Pro Tyr Asp

405 410 415

Arg Trp Val Trp Pro Asp Glu Phe Ser Cys Ser Val Met His Glu Ala

420 425 430

Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

435 440 445

<210> 74

<211> 447

<212> PROTEIN

<213> Artificial sequence

<220>

<223> mAb2 heavy chain amino acid sequence

FS18-7-62/4420 vs human LAG-3/FITC without LALA mutation

<400> 74

Glu Val Lys Leu Asp Glu Thr Gly Gly Gly Leu Val Gln Pro Gly Arg

1 5 10 15

Pro Met Lys Leu Ser Cys Val Ala Ser Gly Phe Thr Phe Ser Asp Tyr

20 25 30

Trp Met Asn Trp Val Arg Gln Ser Pro Glu Lys Gly Leu Glu Trp Val

35 40 45

Ala Gln Ile Arg Asn Lys Pro Tyr Asn Tyr Glu Thr Tyr Tyr Ser Asp

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Ser

65 70 75 80

Val Tyr Leu Gln Met Asn Asn Leu Arg Val Glu Asp Met Gly Ile Tyr

85 90 95

Tyr Cys Thr Gly Ser Tyr Tyr Gly Met Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Ser Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro

115 120 125

Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly

130 135 140

Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn

145 150 155 160

Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln

165 170 175

Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser

180 185 190

Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser

195 200 205

Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr

210 215 220

225 230 235 240

Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg

245 250 255

Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro

260 265 270

Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala

275 280 285

Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val

290 295 300

Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr

305 310 315 320

Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr

325 330 335

Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu

340 345 350

Pro Pro Ser Trp Asp Glu Pro Trp Gly Glu Asp Val Ser Leu Thr Cys

355 360 365

Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser

370 375 380

Asn Gly Ile Pro Glu Trp Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp

385 390 395 400

Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Pro Tyr Asp

405 410 415

Arg Trp Val Trp Pro Asp Glu Phe Ser Cys Ser Val Met His Glu Ala

420 425 430

435 440 445

<210> 75

<211> 447

<212> PROTEIN

<213> Artificial sequence

<220>

<223> mAb2 heavy chain amino acid sequence

FS18-7-65/4420 against human LAG-3/FITC containing LALA mutation

<400> 75

Glu Val Lys Leu Asp Glu Thr Gly Gly Gly Leu Val Gln Pro Gly Arg

1 5 10 15

Pro Met Lys Leu Ser Cys Val Ala Ser Gly Phe Thr Phe Ser Asp Tyr

20 25 30

Trp Met Asn Trp Val Arg Gln Ser Pro Glu Lys Gly Leu Glu Trp Val

35 40 45

Ala Gln Ile Arg Asn Lys Pro Tyr Asn Tyr Glu Thr Tyr Tyr Ser Asp

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Ser

65 70 75 80

Val Tyr Leu Gln Met Asn Asn Leu Arg Val Glu Asp Met Gly Ile Tyr

85 90 95

Tyr Cys Thr Gly Ser Tyr Tyr Gly Met Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Ser Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro

115 120 125

Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly

130 135 140

Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn

145 150 155 160

Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln

165 170 175

Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser

180 185 190

Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser

195 200 205

Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr

210 215 220

His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser

225 230 235 240

Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg

245 250 255

Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro

260 265 270

Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala

275 280 285

Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val

290 295 300

Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr

305 310 315 320

Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr

325 330 335

Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu

340 345 350

Pro Pro Ser Trp Asp Glu Pro Trp Gly Glu Asp Val Ser Leu Thr Cys

355 360 365

Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser

370 375 380

Asn Gly Tyr Ala Glu Tyr Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp

385 390 395 400

Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Pro Tyr Asp

405 410 415

Arg Trp Val Trp Pro Asp Glu Phe Ser Cys Ser Val Met His Glu Ala

420 425 430

Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

435 440 445

<210> 76

<211> 447

<212> PROTEIN

<213> Artificial sequence

<220>

<223> mAb2 heavy chain amino acid sequence

FS18-7-65/4420 vs human LAG-3/FITC without LALA mutation

<400> 76

Glu Val Lys Leu Asp Glu Thr Gly Gly Gly Leu Val Gln Pro Gly Arg

1 5 10 15

Pro Met Lys Leu Ser Cys Val Ala Ser Gly Phe Thr Phe Ser Asp Tyr

20 25 30

Trp Met Asn Trp Val Arg Gln Ser Pro Glu Lys Gly Leu Glu Trp Val

35 40 45

Ala Gln Ile Arg Asn Lys Pro Tyr Asn Tyr Glu Thr Tyr Tyr Ser Asp

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Ser

65 70 75 80

Val Tyr Leu Gln Met Asn Asn Leu Arg Val Glu Asp Met Gly Ile Tyr

85 90 95

Tyr Cys Thr Gly Ser Tyr Tyr Gly Met Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Ser Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro

115 120 125

Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly

130 135 140

Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn

145 150 155 160

Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln

165 170 175

Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser

180 185 190

Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser

195 200 205

Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr

210 215 220

His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser

225 230 235 240

Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg

245 250 255

Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro

260 265 270

Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala

275 280 285

Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val

290 295 300

Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr

305 310 315 320

Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr

325 330 335

Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu

340 345 350

Pro Pro Ser Trp Asp Glu Pro Trp Gly Glu Asp Val Ser Leu Thr Cys

355 360 365

Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser

370 375 380

Asn Gly Tyr Ala Glu Tyr Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp

385 390 395 400

Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Pro Tyr Asp

405 410 415

Arg Trp Val Trp Pro Asp Glu Phe Ser Cys Ser Val Met His Glu Ala

420 425 430

Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

435 440 445

<210> 77

<211> 447

<212> PROTEIN

<213> Artificial sequence

<220>

<223> mAb2 heavy chain amino acid sequence

FS18-7-78/4420 against human LAG-3/FITC containing LALA mutation

<400> 77

Glu Val Lys Leu Asp Glu Thr Gly Gly Gly Leu Val Gln Pro Gly Arg

1 5 10 15

Pro Met Lys Leu Ser Cys Val Ala Ser Gly Phe Thr Phe Ser Asp Tyr

20 25 30

Trp Met Asn Trp Val Arg Gln Ser Pro Glu Lys Gly Leu Glu Trp Val

35 40 45

Ala Gln Ile Arg Asn Lys Pro Tyr Asn Tyr Glu Thr Tyr Tyr Ser Asp

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Ser

65 70 75 80

Val Tyr Leu Gln Met Asn Asn Leu Arg Val Glu Asp Met Gly Ile Tyr

85 90 95

Tyr Cys Thr Gly Ser Tyr Tyr Gly Met Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Ser Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro

115 120 125

Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly

130 135 140

Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn

145 150 155 160

Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln

165 170 175

Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser

180 185 190

Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser

195 200 205

Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr

210 215 220

His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser

225 230 235 240

Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg

245 250 255

Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro

260 265 270

Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala

275 280 285

Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val

290 295 300

Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr

305 310 315 320

Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr

325 330 335

Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu

340 345 350

Pro Pro Ser Trp Asp Glu Pro Trp Gly Glu Asp Val Ser Leu Thr Cys

355 360 365

Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser

370 375 380

Asn Gly Tyr Lys Glu Glu Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp

385 390 395 400

Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Pro Tyr Asp

405 410 415

Arg Trp Val Trp Pro Asp Glu Phe Ser Cys Ser Val Met His Glu Ala

420 425 430

Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

435 440 445

<210> 78

<211> 447

<212> PROTEIN

<213> Artificial sequence

<220>

<223> mAb2 heavy chain amino acid sequence

FS18-7-78/4420 vs human LAG-3/FITC without LALA mutation

<400> 78

Glu Val Lys Leu Asp Glu Thr Gly Gly Gly Leu Val Gln Pro Gly Arg

1 5 10 15

Pro Met Lys Leu Ser Cys Val Ala Ser Gly Phe Thr Phe Ser Asp Tyr

20 25 30

Trp Met Asn Trp Val Arg Gln Ser Pro Glu Lys Gly Leu Glu Trp Val

35 40 45

Ala Gln Ile Arg Asn Lys Pro Tyr Asn Tyr Glu Thr Tyr Tyr Ser Asp

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Ser

65 70 75 80

Val Tyr Leu Gln Met Asn Asn Leu Arg Val Glu Asp Met Gly Ile Tyr

85 90 95

Tyr Cys Thr Gly Ser Tyr Tyr Gly Met Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Ser Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro

115 120 125

Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly

130 135 140

Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn

145 150 155 160

Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln

165 170 175

Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser

180 185 190

Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser

195 200 205

Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr

210 215 220

His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser

225 230 235 240

Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg

245 250 255

Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro

260 265 270

Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala

275 280 285

Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val

290 295 300

Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr

305 310 315 320

Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr

325 330 335

Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu

340 345 350

Pro Pro Ser Trp Asp Glu Pro Trp Gly Glu Asp Val Ser Leu Thr Cys

355 360 365

Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser

370 375 380

Asn Gly Tyr Lys Glu Glu Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp

385 390 395 400

Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Pro Tyr Asp

405 410 415

Arg Trp Val Trp Pro Asp Glu Phe Ser Cys Ser Val Met His Glu Ala

420 425 430

Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

435 440 445

<210> 79

<211> 447

<212> PROTEIN

<213> Artificial sequence

<220>

<223> mAb2 heavy chain amino acid sequence

FS18-7-88/4420 against human LAG-3/FITC containing LALA mutation

<400> 79

Glu Val Lys Leu Asp Glu Thr Gly Gly Gly Leu Val Gln Pro Gly Arg

1 5 10 15

Pro Met Lys Leu Ser Cys Val Ala Ser Gly Phe Thr Phe Ser Asp Tyr

20 25 30

Trp Met Asn Trp Val Arg Gln Ser Pro Glu Lys Gly Leu Glu Trp Val

35 40 45

Ala Gln Ile Arg Asn Lys Pro Tyr Asn Tyr Glu Thr Tyr Tyr Ser Asp

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Ser

65 70 75 80

Val Tyr Leu Gln Met Asn Asn Leu Arg Val Glu Asp Met Gly Ile Tyr

85 90 95

Tyr Cys Thr Gly Ser Tyr Tyr Gly Met Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Ser Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro

115 120 125

Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly

130 135 140

Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn

145 150 155 160

Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln

165 170 175

Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser

180 185 190

Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser

195 200 205

Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr

210 215 220

His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser

225 230 235 240

Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg

245 250 255

Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro

260 265 270

Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala

275 280 285

Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val

290 295 300

Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr

305 310 315 320

Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr

325 330 335

Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu

340 345 350

Pro Pro Ser Trp Asp Glu Pro Trp Gly Glu Asp Val Ser Leu Thr Cys

355 360 365

Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser

370 375 380

Asn Gly Val Pro Glu Leu Asn Val Lys Thr Pro Pro Val Leu Asp

385 390 395 400

Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Pro Tyr Asp

405 410 415

Arg Trp Val Trp Pro Asp Glu Phe Ser Cys Ser Val Met His Glu Ala

420 425 430

Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

435 440 445

<210> 80

<211> 447

<212> PROTEIN

<213> Artificial sequence

<220>

<223> mAb2 heavy chain amino acid sequence

FS18-7-88/4420 vs human LAG-3/FITC without LALA mutation

<400> 80

Glu Val Lys Leu Asp Glu Thr Gly Gly Gly Leu Val Gln Pro Gly Arg

1 5 10 15

Pro Met Lys Leu Ser Cys Val Ala Ser Gly Phe Thr Phe Ser Asp Tyr

20 25 30

Trp Met Asn Trp Val Arg Gln Ser Pro Glu Lys Gly Leu Glu Trp Val

35 40 45

Ala Gln Ile Arg Asn Lys Pro Tyr Asn Tyr Glu Thr Tyr Tyr Ser Asp

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Ser

65 70 75 80

Val Tyr Leu Gln Met Asn Asn Leu Arg Val Glu Asp Met Gly Ile Tyr

85 90 95

Tyr Cys Thr Gly Ser Tyr Tyr Gly Met Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Ser Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro

115 120 125

Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly

130 135 140

Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn

145 150 155 160

Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln

165 170 175

Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser

180 185 190

Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser

195 200 205

Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr

210 215 220

His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser

225 230 235 240

Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg

245 250 255

Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro

260 265 270

Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala

275 280 285

Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val

290 295 300

Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr

305 310 315 320

Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr

325 330 335

Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu

340 345 350

Pro Pro Ser Trp Asp Glu Pro Trp Gly Glu Asp Val Ser Leu Thr Cys

355 360 365

Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser

370 375 380

Asn Gly Val Pro Glu Leu Asn Val Lys Thr Pro Pro Val Leu Asp

385 390 395 400

Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Pro Tyr Asp

405 410 415

Arg Trp Val Trp Pro Asp Glu Phe Ser Cys Ser Val Met His Glu Ala

420 425 430

Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

435 440 445

<210> 81

<211> 447

<212> PROTEIN

<213> Artificial sequence

<220>

<223> mAb2 heavy chain amino acid sequence

FS18-7-95/4420 against human LAG-3/FITC containing LALA mutation

<400> 81

Glu Val Lys Leu Asp Glu Thr Gly Gly Gly Leu Val Gln Pro Gly Arg

1 5 10 15

Pro Met Lys Leu Ser Cys Val Ala Ser Gly Phe Thr Phe Ser Asp Tyr

20 25 30

Trp Met Asn Trp Val Arg Gln Ser Pro Glu Lys Gly Leu Glu Trp Val

35 40 45

Ala Gln Ile Arg Asn Lys Pro Tyr Asn Tyr Glu Thr Tyr Tyr Ser Asp

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Ser

65 70 75 80

Val Tyr Leu Gln Met Asn Asn Leu Arg Val Glu Asp Met Gly Ile Tyr

85 90 95

Tyr Cys Thr Gly Ser Tyr Tyr Gly Met Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Ser Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro

115 120 125

Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly

130 135 140

Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn

145 150 155 160

Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln

165 170 175

Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser

180 185 190

Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser

195 200 205

Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr

210 215 220

His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser

225 230 235 240

Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg

245 250 255

Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro

260 265 270

Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala

275 280 285

Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val

290 295 300

Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr

305 310 315 320

Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr

325 330 335

Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu

340 345 350

Pro Pro Ser Trp Asp Glu Pro Trp Gly Glu Asp Val Ser Leu Thr Cys

355 360 365

Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser

370 375 380

Asn Gly Tyr Gln Glu Asp Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp

385 390 395 400

Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Pro Tyr Asp

405 410 415

Arg Trp Val Trp Pro Asp Glu Phe Ser Cys Ser Val Met His Glu Ala

420 425 430

Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

435 440 445

<210> 82

<211> 447

<212> PROTEIN

<213> Artificial sequence

<220>

<223> mAb2 heavy chain amino acid sequence

FS18-7-95/4420 vs human LAG-3/FITC without LALA mutation

<400> 82

Glu Val Lys Leu Asp Glu Thr Gly Gly Gly Leu Val Gln Pro Gly Arg

1 5 10 15

Pro Met Lys Leu Ser Cys Val Ala Ser Gly Phe Thr Phe Ser Asp Tyr

20 25 30

Trp Met Asn Trp Val Arg Gln Ser Pro Glu Lys Gly Leu Glu Trp Val

35 40 45

Ala Gln Ile Arg Asn Lys Pro Tyr Asn Tyr Glu Thr Tyr Tyr Ser Asp

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Ser

65 70 75 80

Val Tyr Leu Gln Met Asn Asn Leu Arg Val Glu Asp Met Gly Ile Tyr

85 90 95

Tyr Cys Thr Gly Ser Tyr Tyr Gly Met Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Ser Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro

115 120 125

Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly

130 135 140

Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn

145 150 155 160

Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln

165 170 175

Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser

180 185 190

Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser

195 200 205

Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr

210 215 220

His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser

225 230 235 240

245 250 255

Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro

260 265 270

Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala

275 280 285

Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val

290 295 300

Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr

305 310 315 320

Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr

325 330 335

Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu

340 345 350

Pro Pro Ser Trp Asp Glu Pro Trp Gly Glu Asp Val Ser Leu Thr Cys

355 360 365

Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser

370 375 380

Asn Gly Tyr Gln Glu Asp Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp

385 390 395 400

Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Pro Tyr Asp

405 410 415

Arg Trp Val Trp Pro Asp Glu Phe Ser Cys Ser Val Met His Glu Ala

420 425 430

Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

435 440 445

<210> 83

<211> 447

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Anti-FITC mAb 4420 heavy chain amino acid sequence

containing the LALA mutation

<400> 83

Glu Val Lys Leu Asp Glu Thr Gly Gly Gly Leu Val Gln Pro Gly Arg

1 5 10 15

Pro Met Lys Leu Ser Cys Val Ala Ser Gly Phe Thr Phe Ser Asp Tyr

20 25 30

Trp Met Asn Trp Val Arg Gln Ser Pro Glu Lys Gly Leu Glu Trp Val

35 40 45

Ala Gln Ile Arg Asn Lys Pro Tyr Asn Tyr Glu Thr Tyr Tyr Ser Asp

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Ser

65 70 75 80

Val Tyr Leu Gln Met Asn Asn Leu Arg Val Glu Asp Met Gly Ile Tyr

85 90 95

Tyr Cys Thr Gly Ser Tyr Tyr Gly Met Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Ser Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro

115 120 125

Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly

130 135 140

Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn

145 150 155 160

Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln

165 170 175

Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser

180 185 190

Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser

195 200 205

Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr

210 215 220

His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser

225 230 235 240

Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg

245 250 255

Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro

260 265 270

Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala

275 280 285

Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val

290 295 300

Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr

305 310 315 320

Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr

325 330 335

Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu

340 345 350

Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys

355 360 365

Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser

370 375 380

Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp

385 390 395 400

Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser

405 410 415

Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala

420 425 430

Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

435 440 445

<210> 84

<211> 447

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Anti-FITC mAb 4420 heavy chain amino acid sequence

no LALA mutation

<400> 84

Glu Val Lys Leu Asp Glu Thr Gly Gly Gly Leu Val Gln Pro Gly Arg

1 5 10 15

Pro Met Lys Leu Ser Cys Val Ala Ser Gly Phe Thr Phe Ser Asp Tyr

20 25 30

Trp Met Asn Trp Val Arg Gln Ser Pro Glu Lys Gly Leu Glu Trp Val

35 40 45

Ala Gln Ile Arg Asn Lys Pro Tyr Asn Tyr Glu Thr Tyr Tyr Ser Asp

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Ser

65 70 75 80

Val Tyr Leu Gln Met Asn Asn Leu Arg Val Glu Asp Met Gly Ile Tyr

85 90 95

Tyr Cys Thr Gly Ser Tyr Tyr Gly Met Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Ser Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro

115 120 125

Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly

130 135 140

Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn

145 150 155 160

Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln

165 170 175

Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser

180 185 190

Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser

195 200 205

Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr

210 215 220

His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser

225 230 235 240

Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg

245 250 255

Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro

260 265 270

Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala

275 280 285

Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val

290 295 300

Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr

305 310 315 320

Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr

325 330 335

Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu

340 345 350

Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys

355 360 365

Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser

370 375 380

Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp

385 390 395 400

Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser

405 410 415

Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala

420 425 430

Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

435 440 445

<210> 85

<211> 219

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Anti-FITC mAb 4420 light chain amino acid sequence

<400> 85

Asp Val Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly

1 5 10 15

Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser

20 25 30

Asn Gly Asn Thr Tyr Leu Arg Trp Tyr Leu Gln Lys Pro Gly Gln Ser

35 40 45

Pro Lys Val Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Phe Cys Ser Gln Ser

85 90 95

Thr His Val Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105 110

Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu

115 120 125

Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe

130 135 140

Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln

145 150 155 160

Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser

165 170 175

Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu

180 185 190

Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser

195 200 205

Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 86

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Amino acid sequences of CDRs of anti-PD-L1 antibody 84G09

(according to IMGT) HCDR1

<400> 86

Gly Phe Thr Phe Asp Asp Tyr Ala

fifteen

<210> 87

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Amino acid sequences of CDRs of anti-PD-L1 antibody 84G09

(according to IMGT) HCDR2

<400> 87

Ile Ser Trp Lys Ser Asn Ile Ile

fifteen

<210> 88

<211> 15

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Amino acid sequences of CDRs of anti-PD-L1 antibody 84G09

(according to IMGT) HCDR3

<400> 88

Ala Arg Asp Ile Thr Gly Ser Gly Ser Tyr Gly Trp Phe Asp Pro

1 5 10 15

<210> 89

<211> 6

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Amino acid sequences of CDRs of anti-PD-L1 antibody 84G09

(according to IMGT) LCDR1

<400> 89

Gln Ser Ile Ser Ser Tyr

fifteen

<210> 90

<211> 3

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Amino acid sequences of the CDRs of antibody 84G09

vs. PD-L1 (according to IMGT) LCDR2

<400> 90

Val Ala Ser

one

<210> 91

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Amino acid sequences of CDRs of anti-PD-L1 antibody 84G09

(according to IMGT) LCDR3

<400> 91

Gln Gln Ser Tyr Ser Asn Pro Ile Thr

fifteen

<210> 92

<211> 122

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Amino acid sequence of the VH domain of anti-PD-L1 antibody 84G09

<400> 92

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp Tyr

20 25 30

Ala Met His Trp Val Arg Gln Thr Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Gly Ile Ser Trp Lys Ser Asn Ile Ile Gly Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Leu Tyr Tyr Cys

85 90 95

Ala Arg Asp Ile Thr Gly Ser Gly Ser Tyr Gly Trp Phe Asp Pro Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 93

<211> 107

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Amino acid sequence of the VL domain of anti-PD-L1 antibody 84G09

<400> 93

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Pro Leu Ile

35 40 45

Tyr Val Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Ser Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Asn Pro Ile

85 90 95

Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys

100 105

<210> 94

<211> 451

<212> PROTEIN

<213> Artificial sequence

<220>

<223> mAb2 heavy chain amino acid sequence

against human LAG-3/PD-L1 FS18-7-9/84G09 with LALA mutation

<400> 94

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp Tyr

20 25 30

Ala Met His Trp Val Arg Gln Thr Pro Gly Lys Gly Leu Glu Glu Trp Val

35 40 45

Ser Gly Ile Ser Trp Lys Ser Asn Ile Ile Gly Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Leu Tyr Tyr Cys

85 90 95

Ala Arg Asp Ile Thr Gly Ser Gly Ser Tyr Gly Trp Phe Asp Pro Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro

115 120 125

Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr

130 135 140

Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr

145 150 155 160

Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro

165 170 175

Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr

180 185 190

Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn

195 200 205

His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser

210 215 220

Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala

225 230 235 240

Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu

245 250 255

Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser

260 265 270

His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu

275 280 285

Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr

290 295 300

Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn

305 310 315 320

Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro

325 330 335

Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln

340 345 350

Val Tyr Thr Leu Pro Pro Ser Trp Asp Glu Pro Trp Gly Glu Asp Val

355 360 365

Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val

370 375 380

Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro

385 390 395 400

Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr

405 410 415

Val Pro Tyr Asp Arg Trp Val Trp Pro Asp Glu Phe Ser Cys Ser Val

420 425 430

Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu

435 440 445

Ser Pro Gly

450

<210> 95

<211> 451

<212> PROTEIN

<213> Artificial sequence

<220>

<223> mAb2 heavy chain amino acid sequence

against human LAG-3/PD-L1 FS18-7-9/84G09

<400> 95

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp Tyr

20 25 30

Ala Met His Trp Val Arg Gln Thr Pro Gly Lys Gly Leu Glu Glu Trp Val

35 40 45

Ser Gly Ile Ser Trp Lys Ser Asn Ile Ile Gly Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Leu Tyr Tyr Cys

85 90 95

Ala Arg Asp Ile Thr Gly Ser Gly Ser Tyr Gly Trp Phe Asp Pro Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro

115 120 125

Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr

130 135 140

Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr

145 150 155 160

Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro

165 170 175

Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr

180 185 190

Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn

195 200 205

His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser

210 215 220

Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu

225 230 235 240

Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu

245 250 255

Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser

260 265 270

His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu

275 280 285

Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr

290 295 300

Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn

305 310 315 320

Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro

325 330 335

Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln

340 345 350

Val Tyr Thr Leu Pro Pro Ser Trp Asp Glu Pro Trp Gly Glu Asp Val

355 360 365

Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val

370 375 380

Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro

385 390 395 400

Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr

405 410 415

Val Pro Tyr Asp Arg Trp Val Trp Pro Asp Glu Phe Ser Cys Ser Val

420 425 430

Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu

435 440 445

Ser Pro Gly

450

<210> 96

<211> 451

<212> PROTEIN

<213> Artificial sequence

<220>

<223> mAb2 heavy chain amino acid sequence against

Human LAG-3/PD-L1 FS18-7-32/84G09 with LALA mutation

<400> 96

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp Tyr

20 25 30

Ala Met His Trp Val Arg Gln Thr Pro Gly Lys Gly Leu Glu Glu Trp Val

35 40 45

Ser Gly Ile Ser Trp Lys Ser Asn Ile Ile Gly Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Leu Tyr Tyr Cys

85 90 95

Ala Arg Asp Ile Thr Gly Ser Gly Ser Tyr Gly Trp Phe Asp Pro Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro

115 120 125

Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr

130 135 140

Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr

145 150 155 160

Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro

165 170 175

Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr

180 185 190

Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn

195 200 205

His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser

210 215 220

Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala

225 230 235 240

Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu

245 250 255

Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser

260 265 270

His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu

275 280 285

Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr

290 295 300

Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn

305 310 315 320

Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro

325 330 335

Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln

340 345 350

Val Tyr Thr Leu Pro Pro Ser Trp Asp Glu Pro Trp Gly Glu Asp Val

355 360 365

Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Glu Ile Ala Val

370 375 380

Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro

385 390 395 400

Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr

405 410 415

Val Pro Tyr Asp Arg Trp Val Trp Pro Asp Glu Phe Ser Cys Ser Val

420 425 430

Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu

435 440 445

Ser Pro Gly

450

<210> 97

<211> 451

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Anti-LAG-3/PD-L1 mAb2 heavy chain amino acid sequence

human FS18-7-32/84G09

<400> 97

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp Tyr

20 25 30

Ala Met His Trp Val Arg Gln Thr Pro Gly Lys Gly Leu Glu Glu Trp Val

35 40 45

Ser Gly Ile Ser Trp Lys Ser Asn Ile Ile Gly Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Leu Tyr Tyr Cys

85 90 95

Ala Arg Asp Ile Thr Gly Ser Gly Ser Tyr Gly Trp Phe Asp Pro Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro

115 120 125

Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr

130 135 140

Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr

145 150 155 160

Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro

165 170 175

Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr

180 185 190

Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn

195 200 205

His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser

210 215 220

Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu

225 230 235 240

Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu

245 250 255

Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser

260 265 270

His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu

275 280 285

Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr

290 295 300

Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn

305 310 315 320

Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro

325 330 335

Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln

340 345 350

Val Tyr Thr Leu Pro Pro Ser Trp Asp Glu Pro Trp Gly Glu Asp Val

355 360 365

Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Glu Ile Ala Val

370 375 380

Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro

385 390 395 400

Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr

405 410 415

Val Pro Tyr Asp Arg Trp Val Trp Pro Asp Glu Phe Ser Cys Ser Val

420 425 430

Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu

435 440 445

Ser Pro Gly

450

<210> 98

<211> 451

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Anti-LAG-3/PD-L1 mAb2 heavy chain amino acid sequence

human FS18-7-33/84G09 with LALA mutation

<400> 98

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp Tyr

20 25 30

Ala Met His Trp Val Arg Gln Thr Pro Gly Lys Gly Leu Glu Glu Trp Val

35 40 45

Ser Gly Ile Ser Trp Lys Ser Asn Ile Ile Gly Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Leu Tyr Tyr Cys

85 90 95

Ala Arg Asp Ile Thr Gly Ser Gly Ser Tyr Gly Trp Phe Asp Pro Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro

115 120 125

Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr

130 135 140

Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr

145 150 155 160

Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro

165 170 175

Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr

180 185 190

Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn

195 200 205

His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser

210 215 220

Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala

225 230 235 240

Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu

245 250 255

Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser

260 265 270

His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu

275 280 285

Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr

290 295 300

Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn

305 310 315 320

Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro

325 330 335

Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln

340 345 350

Val Tyr Thr Leu Pro Pro Ser Trp Asp Glu Pro Trp Gly Glu Asp Val

355 360 365

Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val

370 375 380

Glu Trp Glu Ser Asn Gly Gln Pro Glu Asp Asn Tyr Lys Thr Thr Pro

385 390 395 400

Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr

405 410 415

Val Pro Tyr Asp Arg Trp Val Trp Pro Asp Glu Phe Ser Cys Ser Val

420 425 430

Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu

435 440 445

Ser Pro Gly

450

<210> 99

<211> 451

<212> PROTEIN

<213> Artificial sequence

<220>

<223> mAb2 heavy chain amino acid sequence against

LAG-3/PD-L1 human FS18-7-33/84G09

<400> 99

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp Tyr

20 25 30

Ala Met His Trp Val Arg Gln Thr Pro Gly Lys Gly Leu Glu Glu Trp Val

35 40 45

Ser Gly Ile Ser Trp Lys Ser Asn Ile Ile Gly Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Leu Tyr Tyr Cys

85 90 95

Ala Arg Asp Ile Thr Gly Ser Gly Ser Tyr Gly Trp Phe Asp Pro Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro

115 120 125

Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr

130 135 140

Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr

145 150 155 160

Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro

165 170 175

Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr

180 185 190

Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn

195 200 205

His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser

210 215 220

Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu

225 230 235 240

Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu

245 250 255

Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser

260 265 270

His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu

275 280 285

Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr

290 295 300

Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn

305 310 315 320

Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro

325 330 335

Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln

340 345 350

Val Tyr Thr Leu Pro Pro Ser Trp Asp Glu Pro Trp Gly Glu Asp Val

355 360 365

Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val

370 375 380

Glu Trp Glu Ser Asn Gly Gln Pro Glu Asp Asn Tyr Lys Thr Thr Pro

385 390 395 400

Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr

405 410 415

Val Pro Tyr Asp Arg Trp Val Trp Pro Asp Glu Phe Ser Cys Ser Val

420 425 430

Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu

435 440 445

Ser Pro Gly

450

<210> 100

<211> 451

<212> PROTEIN

<213> Artificial sequence

<220>

<223> mAb2 heavy chain amino acid sequence

against human LAG-3/PD-L1 FS18-7-36/84G09 with LALA mutation

<400> 100

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp Tyr

20 25 30

Ala Met His Trp Val Arg Gln Thr Pro Gly Lys Gly Leu Glu Glu Trp Val

35 40 45

Ser Gly Ile Ser Trp Lys Ser Asn Ile Ile Gly Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Leu Tyr Tyr Cys

85 90 95

Ala Arg Asp Ile Thr Gly Ser Gly Ser Tyr Gly Trp Phe Asp Pro Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro

115 120 125

Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr

130 135 140

Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr

145 150 155 160

Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro

165 170 175

Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr

180 185 190

Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn

195 200 205

His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser

210 215 220

Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala

225 230 235 240

Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu

245 250 255

Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser

260 265 270

His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu

275 280 285

Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr

290 295 300

Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn

305 310 315 320

Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro

325 330 335

Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln

340 345 350

Val Tyr Thr Leu Pro Pro Ser Trp Asp Glu Pro Trp Gly Glu Asp Val

355 360 365

Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val

370 375 380

Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro

385 390 395 400

Pro Val Leu Asp Ser Asp Gly Ser Tyr Phe Leu Tyr Ser Lys Leu Thr

405 410 415

Val Pro Tyr Asp Arg Trp Val Trp Pro Asp Glu Phe Ser Cys Ser Val

420 425 430

Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu

435 440 445

Ser Pro Gly

450

<210> 101

<211> 451

<212> PROTEIN

<213> Artificial sequence

<220>

<223> mAb2 heavy chain amino acid sequence

against human LAG-3/PD-L1 FS18-7-36/84G09

<400> 101

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp Tyr

20 25 30

Ala Met His Trp Val Arg Gln Thr Pro Gly Lys Gly Leu Glu Glu Trp Val

35 40 45

Ser Gly Ile Ser Trp Lys Ser Asn Ile Ile Gly Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Leu Tyr Tyr Cys

85 90 95

Ala Arg Asp Ile Thr Gly Ser Gly Ser Tyr Gly Trp Phe Asp Pro Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro

115 120 125

Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr

130 135 140

Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr

145 150 155 160

Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro

165 170 175

Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr

180 185 190

Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn

195 200 205

His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser

210 215 220

Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu

225 230 235 240

Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu

245 250 255

Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser

260 265 270

His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu

275 280 285

Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr

290 295 300

Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn

305 310 315 320

Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro

325 330 335

Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln

340 345 350

Val Tyr Thr Leu Pro Pro Ser Trp Asp Glu Pro Trp Gly Glu Asp Val

355 360 365

Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val

370 375 380

Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro

385 390 395 400

Pro Val Leu Asp Ser Asp Gly Ser Tyr Phe Leu Tyr Ser Lys Leu Thr

405 410 415

Val Pro Tyr Asp Arg Trp Val Trp Pro Asp Glu Phe Ser Cys Ser Val

420 425 430

Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu

435 440 445

Ser Pro Gly

450

<210> 102

<211> 451

<212> PROTEIN

<213> Artificial sequence

<220>

<223> mAb2 heavy chain amino acid sequence

against human LAG-3/PD-L1 FS18-7-58/84G09 with LALA mutation

<400> 102

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp Tyr

20 25 30

Ala Met His Trp Val Arg Gln Thr Pro Gly Lys Gly Leu Glu Glu Trp Val

35 40 45

Ser Gly Ile Ser Trp Lys Ser Asn Ile Ile Gly Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Leu Tyr Tyr Cys

85 90 95

Ala Arg Asp Ile Thr Gly Ser Gly Ser Tyr Gly Trp Phe Asp Pro Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro

115 120 125

Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr

130 135 140

Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr

145 150 155 160

Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro

165 170 175

Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr

180 185 190

Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn

195 200 205

His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser

210 215 220

Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala

225 230 235 240

Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu

245 250 255

Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser

260 265 270

His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu

275 280 285

Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr

290 295 300

Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn

305 310 315 320

Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro

325 330 335

Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln

340 345 350

Val Tyr Thr Leu Pro Pro Ser Trp Asp Glu Pro Trp Gly Glu Asp Val

355 360 365

Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val

370 375 380

Glu Trp Glu Ser Asn Gly Tyr Pro Glu Ile Glu Phe Lys Thr Pro

385 390 395 400

Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr

405 410 415

Val Pro Tyr Asp Arg Trp Val Trp Pro Asp Glu Phe Ser Cys Ser Val

420 425 430

Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu

435 440 445

Ser Pro Gly

450

<210> 103

<211> 451

<212> PROTEIN

<213> Artificial sequence

<220>

<223> mAb2 heavy chain amino acid sequence

against human LAG-3/PD-L1 FS18-7-58/84G09

<400> 103

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp Tyr

20 25 30

Ala Met His Trp Val Arg Gln Thr Pro Gly Lys Gly Leu Glu Glu Trp Val

35 40 45

Ser Gly Ile Ser Trp Lys Ser Asn Ile Ile Gly Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Leu Tyr Tyr Cys

85 90 95

Ala Arg Asp Ile Thr Gly Ser Gly Ser Tyr Gly Trp Phe Asp Pro Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro

115 120 125

Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr

130 135 140

Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr

145 150 155 160

Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro

165 170 175

Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr

180 185 190

Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn

195 200 205

His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser

210 215 220

Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu

225 230 235 240

Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu

245 250 255

Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser

260 265 270

His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu

275 280 285

Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr

290 295 300

Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn

305 310 315 320

Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro

325 330 335

Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln

340 345 350

Val Tyr Thr Leu Pro Pro Ser Trp Asp Glu Pro Trp Gly Glu Asp Val

355 360 365

Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val

370 375 380

Glu Trp Glu Ser Asn Gly Tyr Pro Glu Ile Glu Phe Lys Thr Pro

385 390 395 400

Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr

405 410 415

Val Pro Tyr Asp Arg Trp Val Trp Pro Asp Glu Phe Ser Cys Ser Val

420 425 430

Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu

435 440 445

Ser Pro Gly

450

<210> 104

<211> 451

<212> PROTEIN

<213> Artificial sequence

<220>

<223> mAb2 heavy chain amino acid sequence

against human LAG-3/PD-L1 FS18-7-62/84G09 with LALA mutation

<400> 104

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp Tyr

20 25 30

Ala Met His Trp Val Arg Gln Thr Pro Gly Lys Gly Leu Glu Glu Trp Val

35 40 45

Ser Gly Ile Ser Trp Lys Ser Asn Ile Ile Gly Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Leu Tyr Tyr Cys

85 90 95

Ala Arg Asp Ile Thr Gly Ser Gly Ser Tyr Gly Trp Phe Asp Pro Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro

115 120 125

Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr

130 135 140

Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr

145 150 155 160

Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro

165 170 175

Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr

180 185 190

Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn

195 200 205

His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser

210 215 220

Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala

225 230 235 240

Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu

245 250 255

Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser

260 265 270

His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu

275 280 285

Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr

290 295 300

Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn

305 310 315 320

Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro

325 330 335

Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln

340 345 350

Val Tyr Thr Leu Pro Pro Ser Trp Asp Glu Pro Trp Gly Glu Asp Val

355 360 365

Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val

370 375 380

Glu Trp Glu Ser Asn Gly Ile Pro Glu Trp Asn Tyr Lys Thr Thr Pro

385 390 395 400

Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr

405 410 415

Val Pro Tyr Asp Arg Trp Val Trp Pro Asp Glu Phe Ser Cys Ser Val

420 425 430

Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu

435 440 445

Ser Pro Gly

450

<210> 105

<211> 451

<212> PROTEIN

<213> Artificial sequence

<220>

<223> mAb2 heavy chain amino acid sequence

against human LAG-3/PD-L1 FS18-7-62/84G09

<400> 105

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp Tyr

20 25 30

Ala Met His Trp Val Arg Gln Thr Pro Gly Lys Gly Leu Glu Glu Trp Val

35 40 45

Ser Gly Ile Ser Trp Lys Ser Asn Ile Ile Gly Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Leu Tyr Tyr Cys

85 90 95

Ala Arg Asp Ile Thr Gly Ser Gly Ser Tyr Gly Trp Phe Asp Pro Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro

115 120 125

Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr

130 135 140

Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr

145 150 155 160

Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro

165 170 175

Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr

180 185 190

Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn

195 200 205

His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser

210 215 220

Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu

225 230 235 240

Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu

245 250 255

Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser

260 265 270

His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu

275 280 285

Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr

290 295 300

Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn

305 310 315 320

Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro

325 330 335

Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln

340 345 350

Val Tyr Thr Leu Pro Pro Ser Trp Asp Glu Pro Trp Gly Glu Asp Val

355 360 365

Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val

370 375 380

Glu Trp Glu Ser Asn Gly Ile Pro Glu Trp Asn Tyr Lys Thr Thr Pro

385 390 395 400

Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr

405 410 415

Val Pro Tyr Asp Arg Trp Val Trp Pro Asp Glu Phe Ser Cys Ser Val

420 425 430

Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu

435 440 445

Ser Pro Gly

450

<210> 106

<211> 451

<212> PROTEIN

<213> Artificial sequence

<220>

<223> mAb2 heavy chain amino acid sequence

against human LAG-3/PD-L1 FS18-7-65/84G09 with LALA mutation

<400> 106

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp Tyr

20 25 30

Ala Met His Trp Val Arg Gln Thr Pro Gly Lys Gly Leu Glu Glu Trp Val

35 40 45

Ser Gly Ile Ser Trp Lys Ser Asn Ile Ile Gly Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Leu Tyr Tyr Cys

85 90 95

Ala Arg Asp Ile Thr Gly Ser Gly Ser Tyr Gly Trp Phe Asp Pro Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro

115 120 125

Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr

130 135 140

Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr

145 150 155 160

Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro

165 170 175

Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr

180 185 190

Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn

195 200 205

His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser

210 215 220

Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala

225 230 235 240

Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu

245 250 255

Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser

260 265 270

His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu

275 280 285

Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr

290 295 300

Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn

305 310 315 320

Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro

325 330 335

Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln

340 345 350

Val Tyr Thr Leu Pro Pro Ser Trp Asp Glu Pro Trp Gly Glu Asp Val

355 360 365

Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val

370 375 380

Glu Trp Glu Ser Asn Gly Tyr Ala Glu Tyr Asn Tyr Lys Thr Thr Pro

385 390 395 400

Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr

405 410 415

Val Pro Tyr Asp Arg Trp Val Trp Pro Asp Glu Phe Ser Cys Ser Val

420 425 430

Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu

435 440 445

Ser Pro Gly

450

<210> 107

<211> 451

<212> PROTEIN

<213> Artificial sequence

<220>

<223> mAb2 heavy chain amino acid sequence

against human LAG-3/PD-L1 FS18-7-65/84G09

<400> 107

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp Tyr

20 25 30

Ala Met His Trp Val Arg Gln Thr Pro Gly Lys Gly Leu Glu Glu Trp Val

35 40 45

Ser Gly Ile Ser Trp Lys Ser Asn Ile Ile Gly Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Leu Tyr Tyr Cys

85 90 95

Ala Arg Asp Ile Thr Gly Ser Gly Ser Tyr Gly Trp Phe Asp Pro Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro

115 120 125

Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr

130 135 140

Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr

145 150 155 160

Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro

165 170 175

Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr

180 185 190

Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn

195 200 205

His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser

210 215 220

Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu

225 230 235 240

Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu

245 250 255

Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser

260 265 270

His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu

275 280 285

Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr

290 295 300

Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn

305 310 315 320

Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro

325 330 335

Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln

340 345 350

Val Tyr Thr Leu Pro Pro Ser Trp Asp Glu Pro Trp Gly Glu Asp Val

355 360 365

Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val

370 375 380

Glu Trp Glu Ser Asn Gly Tyr Ala Glu Tyr Asn Tyr Lys Thr Thr Pro

385 390 395 400

Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr

405 410 415

Val Pro Tyr Asp Arg Trp Val Trp Pro Asp Glu Phe Ser Cys Ser Val

420 425 430

Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu

435 440 445

Ser Pro Gly

450

<210> 108

<211> 451

<212> PROTEIN

<213> Artificial sequence

<220>

<223> mAb2 heavy chain amino acid sequence

against human LAG-3/PD-L1 FS18-7-78/84G09 with LALA mutation

<400> 108

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp Tyr

20 25 30

Ala Met His Trp Val Arg Gln Thr Pro Gly Lys Gly Leu Glu Glu Trp Val

35 40 45

Ser Gly Ile Ser Trp Lys Ser Asn Ile Ile Gly Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Leu Tyr Tyr Cys

85 90 95

Ala Arg Asp Ile Thr Gly Ser Gly Ser Tyr Gly Trp Phe Asp Pro Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro

115 120 125

Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr

130 135 140

Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr

145 150 155 160

Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro

165 170 175

Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr

180 185 190

Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn

195 200 205

His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser

210 215 220

Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala

225 230 235 240

Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu

245 250 255

Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser

260 265 270

His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu

275 280 285

Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr

290 295 300

Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn

305 310 315 320

Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro

325 330 335

Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln

340 345 350

Val Tyr Thr Leu Pro Pro Ser Trp Asp Glu Pro Trp Gly Glu Asp Val

355 360 365

Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val

370 375 380

Glu Trp Glu Ser Asn Gly Tyr Lys Glu Glu Asn Tyr Lys Thr Thr Pro

385 390 395 400

Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr

405 410 415

Val Pro Tyr Asp Arg Trp Val Trp Pro Asp Glu Phe Ser Cys Ser Val

420 425 430

Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu

435 440 445

Ser Pro Gly

450

<210> 109

<211> 451

<212> PROTEIN

<213> Artificial sequence

<220>

<223> mAb2 heavy chain amino acid sequence

against human LAG-3/PD-L1 FS18-7-78/84G09

<400> 109

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp Tyr

20 25 30

Ala Met His Trp Val Arg Gln Thr Pro Gly Lys Gly Leu Glu Glu Trp Val

35 40 45

Ser Gly Ile Ser Trp Lys Ser Asn Ile Ile Gly Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Leu Tyr Tyr Cys

85 90 95

Ala Arg Asp Ile Thr Gly Ser Gly Ser Tyr Gly Trp Phe Asp Pro Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro

115 120 125

Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr

130 135 140

Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr

145 150 155 160

Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro

165 170 175

Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr

180 185 190

Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn

195 200 205

His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser

210 215 220

Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu

225 230 235 240

Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu

245 250 255

Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser

260 265 270

His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu

275 280 285

Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr

290 295 300

Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn

305 310 315 320

Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro

325 330 335

Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln

340 345 350

Val Tyr Thr Leu Pro Pro Ser Trp Asp Glu Pro Trp Gly Glu Asp Val

355 360 365

Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val

370 375 380

Glu Trp Glu Ser Asn Gly Tyr Lys Glu Glu Asn Tyr Lys Thr Thr Pro

385 390 395 400

Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr

405 410 415

Val Pro Tyr Asp Arg Trp Val Trp Pro Asp Glu Phe Ser Cys Ser Val

420 425 430

Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu

435 440 445

Ser Pro Gly

450

<210> 110

<211> 451

<212> PROTEIN

<213> Artificial sequence

<220>

<223> mAb2 heavy chain amino acid sequence

against human LAG-3/PD-L1 FS18-7-88/84G09 with LALA mutation

<400> 110

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp Tyr

20 25 30

Ala Met His Trp Val Arg Gln Thr Pro Gly Lys Gly Leu Glu Glu Trp Val

35 40 45

Ser Gly Ile Ser Trp Lys Ser Asn Ile Ile Gly Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Leu Tyr Tyr Cys

85 90 95

Ala Arg Asp Ile Thr Gly Ser Gly Ser Tyr Gly Trp Phe Asp Pro Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro

115 120 125

Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr

130 135 140

Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr

145 150 155 160

Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro

165 170 175

Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr

180 185 190

Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn

195 200 205

His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser

210 215 220

Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala

225 230 235 240

Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu

245 250 255

Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser

260 265 270

His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu

275 280 285

Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr

290 295 300

Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn

305 310 315 320

Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro

325 330 335

Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln

340 345 350

Val Tyr Thr Leu Pro Pro Ser Trp Asp Glu Pro Trp Gly Glu Asp Val

355 360 365

Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val

370 375 380

Glu Trp Glu Ser Asn Gly Val Pro Glu Leu Asn Val Lys Thr Pro

385 390 395 400

Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr

405 410 415

Val Pro Tyr Asp Arg Trp Val Trp Pro Asp Glu Phe Ser Cys Ser Val

420 425 430

Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu

435 440 445

Ser Pro Gly

450

<210> 111

<211> 451

<212> PROTEIN

<213> Artificial sequence

<220>

<223> mAb2 heavy chain amino acid sequence

against human LAG-3/PD-L1 FS18-7-88/84G09

<400> 111

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp Tyr

20 25 30

Ala Met His Trp Val Arg Gln Thr Pro Gly Lys Gly Leu Glu Glu Trp Val

35 40 45

Ser Gly Ile Ser Trp Lys Ser Asn Ile Ile Gly Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Leu Tyr Tyr Cys

85 90 95

Ala Arg Asp Ile Thr Gly Ser Gly Ser Tyr Gly Trp Phe Asp Pro Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro

115 120 125

Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr

130 135 140

Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr

145 150 155 160

Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro

165 170 175

Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr

180 185 190

Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn

195 200 205

His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser

210 215 220

Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu

225 230 235 240

Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu

245 250 255

Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser

260 265 270

His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu

275 280 285

Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr

290 295 300

Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn

305 310 315 320

Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro

325 330 335

Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln

340 345 350

Val Tyr Thr Leu Pro Pro Ser Trp Asp Glu Pro Trp Gly Glu Asp Val

355 360 365

Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val

370 375 380

Glu Trp Glu Ser Asn Gly Val Pro Glu Leu Asn Val Lys Thr Pro

385 390 395 400

Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr

405 410 415

Val Pro Tyr Asp Arg Trp Val Trp Pro Asp Glu Phe Ser Cys Ser Val

420 425 430

Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu

435 440 445

Ser Pro Gly

450

<210> 112

<211> 451

<212> PROTEIN

<213> Artificial sequence

<220>

<223> mAb2 heavy chain amino acid sequence

against human LAG-3/PD-L1 FS18-7-95/84G09 with LALA mutation

<400> 112

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp Tyr

20 25 30

Ala Met His Trp Val Arg Gln Thr Pro Gly Lys Gly Leu Glu Glu Trp Val

35 40 45

Ser Gly Ile Ser Trp Lys Ser Asn Ile Ile Gly Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Leu Tyr Tyr Cys

85 90 95

Ala Arg Asp Ile Thr Gly Ser Gly Ser Tyr Gly Trp Phe Asp Pro Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro

115 120 125

Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr

130 135 140

Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr

145 150 155 160

Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro

165 170 175

Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr

180 185 190

Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn

195 200 205

His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser

210 215 220

Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala

225 230 235 240

Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu

245 250 255

Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser

260 265 270

His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu

275 280 285

Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr

290 295 300

Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn

305 310 315 320

Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro

325 330 335

Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln

340 345 350

Val Tyr Thr Leu Pro Pro Ser Trp Asp Glu Pro Trp Gly Glu Asp Val

355 360 365

Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val

370 375 380

Glu Trp Glu Ser Asn Gly Tyr Gln Glu Asp Asn Tyr Lys Thr Thr Pro

385 390 395 400

Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr

405 410 415

Val Pro Tyr Asp Arg Trp Val Trp Pro Asp Glu Phe Ser Cys Ser Val

420 425 430

Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu

435 440 445

Ser Pro Gly

450

<210> 113

<211> 451

<212> PROTEIN

<213> Artificial sequence

<220>

<223> mAb2 heavy chain amino acid sequence

against human LAG-3/PD-L1 FS18-7-95/84G09

<400> 113

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp Tyr

20 25 30

Ala Met His Trp Val Arg Gln Thr Pro Gly Lys Gly Leu Glu Glu Trp Val

35 40 45

Ser Gly Ile Ser Trp Lys Ser Asn Ile Ile Gly Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Leu Tyr Tyr Cys

85 90 95

Ala Arg Asp Ile Thr Gly Ser Gly Ser Tyr Gly Trp Phe Asp Pro Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro

115 120 125

Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr

130 135 140

Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr

145 150 155 160

Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro

165 170 175

Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr

180 185 190

Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn

195 200 205

His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser

210 215 220

Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu

225 230 235 240

Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu

245 250 255

Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser

260 265 270

His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu

275 280 285

Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr

290 295 300

Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn

305 310 315 320

Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro

325 330 335

Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln

340 345 350

Val Tyr Thr Leu Pro Pro Ser Trp Asp Glu Pro Trp Gly Glu Asp Val

355 360 365

Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val

370 375 380

Glu Trp Glu Ser Asn Gly Tyr Gln Glu Asp Asn Tyr Lys Thr Thr Pro

385 390 395 400

Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr

405 410 415

Val Pro Tyr Asp Arg Trp Val Trp Pro Asp Glu Phe Ser Cys Ser Val

420 425 430

Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu

435 440 445

Ser Pro Gly

450

<210> 114

<211> 451

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Amino acid sequence of mAb 84G09 against human PD-L1

with LALA mutation, heavy chain

<400> 114

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp Tyr

20 25 30

Ala Met His Trp Val Arg Gln Thr Pro Gly Lys Gly Leu Glu Glu Trp Val

35 40 45

Ser Gly Ile Ser Trp Lys Ser Asn Ile Ile Gly Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Leu Tyr Tyr Cys

85 90 95

Ala Arg Asp Ile Thr Gly Ser Gly Ser Tyr Gly Trp Phe Asp Pro Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro

115 120 125

Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr

130 135 140

Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr

145 150 155 160

Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro

165 170 175

Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr

180 185 190

Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn

195 200 205

His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser

210 215 220

Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala

225 230 235 240

Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu

245 250 255

Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser

260 265 270

His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu

275 280 285

Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr

290 295 300

Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn

305 310 315 320

Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro

325 330 335

Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln

340 345 350

Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val

355 360 365

Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val

370 375 380

Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro

385 390 395 400

Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr

405 410 415

Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val

420 425 430

Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu

435 440 445

Ser Pro Gly

450

<210> 115

<211> 451

<212> PROTEIN

<213> Artificial sequence

<220>

<223> mAb heavy chain amino acid sequence 84G09

against human PD-L1

<400> 115

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp Tyr

20 25 30

Ala Met His Trp Val Arg Gln Thr Pro Gly Lys Gly Leu Glu Glu Trp Val

35 40 45

Ser Gly Ile Ser Trp Lys Ser Asn Ile Ile Gly Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Leu Tyr Tyr Cys

85 90 95

Ala Arg Asp Ile Thr Gly Ser Gly Ser Tyr Gly Trp Phe Asp Pro Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro

115 120 125

Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr

130 135 140

Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr

145 150 155 160

Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro

165 170 175

Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr

180 185 190

Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn

195 200 205

His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser

210 215 220

Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu

225 230 235 240

Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu

245 250 255

Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser

260 265 270

His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu

275 280 285

Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr

290 295 300

Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn

305 310 315 320

Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro

325 330 335

Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln

340 345 350

Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val

355 360 365

Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val

370 375 380

Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro

385 390 395 400

Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr

405 410 415

Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val

420 425 430

Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu

435 440 445

Ser Pro Gly

450

<210> 116

<211> 214

<212> PROTEIN

<213> Artificial sequence

<220>

<223> mAb 84G09 light chain amino acid sequence vs.

Human PD-L1

<400> 116

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Pro Leu Ile

35 40 45

Tyr Val Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Ser Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Asn Pro Ile

85 90 95

Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 117

<211> 447

<212> PROTEIN

<213> Artificial sequence

<220>

<223> mAb2 heavy chain amino acid sequence against

LAG-3/PD-L1 mouse FS18-7-108-29/S1 with LALA mutation

<400> 117

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

20 25 30

Trp Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Trp Ile Ser Pro Tyr Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Arg His Trp Pro Gly Gly Phe Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ala Ala Ser Thr Lys Gly Pro Ser Val Phe Pro

115 120 125

Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly

130 135 140

Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn

145 150 155 160

Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln

165 170 175

Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser

180 185 190

Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser

195 200 205

Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr

210 215 220

225 230 235 240

Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg

245 250 255

Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro

260 265 270

Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala

275 280 285

Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val

290 295 300

Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr

305 310 315 320

Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr

325 330 335

Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu

340 345 350

Pro Pro Ser Trp Asp Glu Pro Trp Gly Glu Asp Val Ser Leu Thr Cys

355 360 365

Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Val Val Glu Trp Glu Ser

370 375 380

Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp

385 390 395 400

Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Pro Phe Glu

405 410 415

Arg Trp Met Trp Pro Asp Glu Phe Ser Cys Ser Val Met His Glu Ala

420 425 430

435 440 445

<210> 118

<211> 447

<212> PROTEIN

<213> Artificial sequence

<220>

<223> mAb2 heavy chain amino acid sequence against

LAG-3/PD-L1 mice FS18-7-108-29/S1

<400> 118

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Ser

20 25 30

Trp Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Trp Ile Ser Pro Tyr Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Arg His Trp Pro Gly Gly Phe Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ala Ala Ser Thr Lys Gly Pro Ser Val Phe Pro

115 120 125

Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly

130 135 140

145 150 155 160

Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln

165 170 175

Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser

180 185 190

Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser

195 200 205

Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr

210 215 220

His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser

225 230 235 240

Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg

245 250 255

Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro

260 265 270

Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala

275 280 285

Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val

290 295 300

Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr

305 310 315 320

Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr

325 330 335

Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu

340 345 350

Pro Pro Ser Trp Asp Glu Pro Trp Gly Glu Asp Val Ser Leu Thr Cys

355 360 365

Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Val Val Glu Trp Glu Ser

370 375 380

Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp

385 390 395 400

Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Pro Phe Glu

405 410 415

Arg Trp Met Trp Pro Asp Glu Phe Ser Cys Ser Val Met His Glu Ala

420 425 430

Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

435 440 445

<210> 119

<211> 214

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Light chain amino acid sequence of mAb S1 vs.

PD-L1 mice

<400> 119

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Ser Thr Ala

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Leu Phe Thr Pro Pro

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 120

<211> 447

<212> PROTEIN

<213> Artificial sequence

<220>

<223> mAb2 heavy chain amino acid sequence against

LAG-3/PD-L1 mouse FS18-7-108-35/S1 with LALA mutation

<400> 120

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Ser

20 25 30

Trp Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Trp Ile Ser Pro Tyr Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Arg His Trp Pro Gly Gly Phe Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ala Ala Ser Thr Lys Gly Pro Ser Val Phe Pro

115 120 125

Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly

130 135 140

Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn

145 150 155 160

Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln

165 170 175

Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser

180 185 190

Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser

195 200 205

Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr

210 215 220

His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser

225 230 235 240

Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg

245 250 255

Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro

260 265 270

Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala

275 280 285

Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val

290 295 300

Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr

305 310 315 320

Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr

325 330 335

Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu

340 345 350

Pro Pro Ser Trp Asp Glu Pro Trp Gly Glu Asp Val Ser Leu Thr Cys

355 360 365

Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ser Val Glu Trp Glu Ser

370 375 380

Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp

385 390 395 400

Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Pro Phe Glu

405 410 415

Arg Trp Met Trp Pro Asp Glu Phe Ser Cys Ser Val Met His Glu Ala

420 425 430

Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

435 440 445

<210> 121

<211> 447

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Anti-LAG-3/PD-L1 mAb2 heavy chain amino acid sequence

mice FS18-7-108-35/S1

<400> 121

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Ser

20 25 30

Trp Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Trp Ile Ser Pro Tyr Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Arg His Trp Pro Gly Gly Phe Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ala Ala Ser Thr Lys Gly Pro Ser Val Phe Pro

115 120 125

Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly

130 135 140

Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn

145 150 155 160

Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln

165 170 175

Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser

180 185 190

Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser

195 200 205

Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr

210 215 220

His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser

225 230 235 240

Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg

245 250 255

Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro

260 265 270

Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala

275 280 285

Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val

290 295 300

Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr

305 310 315 320

Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr

325 330 335

Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu

340 345 350

Pro Pro Ser Trp Asp Glu Pro Trp Gly Glu Asp Val Ser Leu Thr Cys

355 360 365

Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ser Val Glu Trp Glu Ser

370 375 380

Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp

385 390 395 400

Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Pro Phe Glu

405 410 415

Arg Trp Met Trp Pro Asp Glu Phe Ser Cys Ser Val Met His Glu Ala

420 425 430

Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

435 440 445

<210> 122

<211> 447

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Heavy chain amino acid sequence of reference mAb S1

against PD-L1 with LALA mutation,

<400> 122

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Ser

20 25 30

Trp Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Trp Ile Ser Pro Tyr Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Arg His Trp Pro Gly Gly Phe Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ala Ala Ser Thr Lys Gly Pro Ser Val Phe Pro

115 120 125

Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly

130 135 140

Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn

145 150 155 160

Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln

165 170 175

Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser

180 185 190

Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser

195 200 205

Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr

210 215 220

His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser

225 230 235 240

Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg

245 250 255

Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro

260 265 270

Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala

275 280 285

Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val

290 295 300

Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr

305 310 315 320

Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr

325 330 335

Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu

340 345 350

Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys

355 360 365

Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser

370 375 380

Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp

385 390 395 400

Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser

405 410 415

Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala

420 425 430

Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

435 440 445

<210> 123

<211> 447

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Heavy chain amino acid sequence of reference mAb S1

against

PD-L1

<400> 123

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Ser

20 25 30

Trp Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Trp Ile Ser Pro Tyr Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Arg His Trp Pro Gly Gly Phe Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ala Ala Ser Thr Lys Gly Pro Ser Val Phe Pro

115 120 125

Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly

130 135 140

Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn

145 150 155 160

Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln

165 170 175

Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser

180 185 190

Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser

195 200 205

Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr

210 215 220

His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser

225 230 235 240

Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg

245 250 255

Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro

260 265 270

Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala

275 280 285

Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val

290 295 300

Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr

305 310 315 320

Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr

325 330 335

Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu

340 345 350

Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys

355 360 365

Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser

370 375 380

Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp

385 390 395 400

Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser

405 410 415

Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala

420 425 430

Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

435 440 445

<210> 124

<211> 449

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Amino acid sequence of control mAb 25F7 vs.

LAG-3 man, heavy chain

<400> 124

Gln Val Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Ser Asp Tyr

20 25 30

Tyr Trp Asn Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Glu Ile Asn His Arg Gly Ser Thr Asn Ser Asn Pro Ser Leu Lys

50 55 60

Ser Arg Val Thr Leu Ser Leu Asp Thr Ser Lys Asn Gln Phe Ser Leu

65 70 75 80

Lys Leu Arg Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Phe Gly Tyr Ser Asp Tyr Glu Tyr Asn Trp Phe Asp Pro Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp

210 215 220

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly

225 230 235 240

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

245 250 255

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

260 265 270

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

275 280 285

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

290 295 300

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

305 310 315 320

Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu

325 330 335

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

340 345 350

Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu

355 360 365

Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

370 375 380

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

385 390 395 400

Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp

405 410 415

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

420 425 430

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440 445

gly

<210> 125

<211> 214

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Amino acid sequence of control mAb 25F7 vs.

LAG-3 human light chain

<400> 125

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

35 40 45

Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro

65 70 75 80

Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser Asn Trp Pro Leu

85 90 95

Thr Phe Gly Gln Gly Thr Asn Leu Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 126

<211> 525

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Human LAG-3 amino acid sequence

<400> 126

Met Trp Glu Ala Gln Phe Leu Gly Leu Leu Phe Leu Gln Pro Leu Trp

1 5 10 15

Val Ala Pro Val Lys Pro Leu Gln Pro Gly Ala Glu Val Pro Val Val

20 25 30

Trp Ala Gln Glu Gly Ala Pro Ala Gln Leu Pro Cys Ser Pro Thr Ile

35 40 45

Pro Leu Gln Asp Leu Ser Leu Leu Arg Arg Ala Gly Val Thr Trp Gln

50 55 60

His Gln Pro Asp Ser Gly Pro Pro Ala Ala Ala Pro Gly His Pro Leu

65 70 75 80

Ala Pro Gly Pro His Pro Ala Ala Pro Ser Ser Trp Gly Pro Arg Pro

85 90 95

Arg Arg Tyr Thr Val Leu Ser Val Gly Pro Gly Gly Leu Arg Ser Gly

100 105 110

Arg Leu Pro Leu Gln Pro Arg Val Gln Leu Asp Glu Arg Gly Arg Gln

115 120 125

Arg Gly Asp Phe Ser Leu Trp Leu Arg Pro Ala Arg Arg Ala Asp Ala

130 135 140

Gly Glu Tyr Arg Ala Ala Val His Leu Arg Asp Arg Ala Leu Ser Cys

145 150 155 160

Arg Leu Arg Leu Arg Leu Gly Gln Ala Ser Met Thr Ala Ser Pro Pro

165 170 175

Gly Ser Leu Arg Ala Ser Asp Trp Val Ile Leu Asn Cys Ser Phe Ser

180 185 190

Arg Pro Asp Arg Pro Ala Ser Val His Trp Phe Arg Asn Arg Gly Gln

195 200 205

Gly Arg Val Pro Val Arg Glu Ser Pro His His His Leu Ala Glu Ser

210 215 220

Phe Leu Phe Leu Pro Gln Val Ser Pro Met Asp Ser Gly Pro Trp Gly

225 230 235 240

Cys Ile Leu Thr Tyr Arg Asp Gly Phe Asn Val Ser Ile Met Tyr Asn

245 250 255

Leu Thr Val Leu Gly Leu Glu Pro Pro Thr Pro Leu Thr Val Tyr Ala

260 265 270

Gly Ala Gly Ser Arg Val Gly Leu Pro Cys Arg Leu Pro Ala Gly Val

275 280 285

Gly Thr Arg Ser Phe Leu Thr Ala Lys Trp Thr Pro Pro Gly Gly Gly

290 295 300

Pro Asp Leu Leu Val Thr Gly Asp Asn Gly Asp Phe Thr Leu Arg Leu

305 310 315 320

Glu Asp Val Ser Gln Ala Gln Ala Gly Thr Tyr Thr Cys His Ile His

325 330 335

Leu Gln Glu Gln Gln Leu Asn Ala Thr Val Thr Leu Ala Ile Ile Thr

340 345 350

Val Thr Pro Lys Ser Phe Gly Ser Pro Gly Ser Leu Gly Lys Leu Leu

355 360 365

Cys Glu Val Thr Pro Val Ser Gly Gln Glu Arg Phe Val Trp Ser Ser

370 375 380

Leu Asp Thr Pro Ser Gln Arg Ser Phe Ser Gly Pro Trp Leu Glu Ala

385 390 395 400

Gln Glu Ala Gln Leu Leu Ser Gln Pro Trp Gln Cys Gln Leu Tyr Gln

405 410 415

Gly Glu Arg Leu Leu Gly Ala Ala Val Tyr Phe Thr Glu Leu Ser Ser

420 425 430

Pro Gly Ala Gln Arg Ser Gly Arg Ala Pro Gly Ala Leu Pro Ala Gly

435 440 445

His Leu Leu Leu Phe Leu Ile Leu Gly Val Leu Ser Leu Leu Leu Leu

450 455 460

Val Thr Gly Ala Phe Gly Phe His Leu Trp Arg Arg Gln Trp Arg Pro

465 470 475 480

Arg Arg Phe Ser Ala Leu Glu Gln Gly Ile His Pro Pro Gln Ala Gln

485 490 495

Ser Lys Ile Glu Glu Leu Glu Gln Glu Pro Glu Pro Glu Pro Glu Pro

500 505 510

Glu Pro Glu Pro Glu Pro Glu Pro Glu Pro Glu Gln Leu

515 520 525

<210> 127

<211> 521

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Mouse LAG-3 amino acid sequence

<400> 127

Met Arg Glu Asp Leu Leu Leu Gly Phe Leu Leu Leu Gly Leu Leu Trp

1 5 10 15

Glu Ala Pro Val Val Ser Ser Gly Pro Gly Lys Glu Leu Pro Val Val

20 25 30

Trp Ala Gln Glu Gly Ala Pro Val His Leu Pro Cys Ser Leu Lys Ser

35 40 45

Pro Asn Leu Asp Pro Asn Phe Leu Arg Arg Gly Gly Val Ile Trp Gln

50 55 60

His Gln Pro Asp Ser Gly Gln Pro Thr Pro Ile Pro Ala Leu Asp Leu

65 70 75 80

His Gln Gly Met Pro Ser Pro Arg Gln Pro Ala Pro Gly Arg Tyr Thr

85 90 95

Val Leu Ser Val Ala Pro Gly Gly Leu Arg Ser Gly Arg Gln Pro Leu

100 105 110

His Pro His Val Gln Leu Glu Glu Arg Gly Leu Gln Arg Gly Asp Phe

115 120 125

Ser Leu Trp Leu Arg Pro Ala Leu Arg Thr Asp Ala Gly Glu Tyr His

130 135 140

Ala Thr Val Arg Leu Pro Asn Arg Ala Leu Ser Cys Ser Leu Arg Leu

145 150 155 160

Arg Val Gly Gln Ala Ser Met Ile Ala Ser Pro Ser Gly Val Leu Lys

165 170 175

Leu Ser Asp Trp Val Leu Leu Asn Cys Ser Phe Ser Arg Pro Asp Arg

180 185 190

Pro Val Ser Val His Trp Phe Gln Gly Gln Asn Arg Val Pro Val Tyr

195 200 205

Asn Ser Pro Arg His Phe Leu Ala Glu Thr Phe Leu Leu Leu Pro Gln

210 215 220

Val Ser Pro Leu Asp Ser Gly Thr Trp Gly Cys Val Leu Thr Tyr Arg

225 230 235 240

Asp Gly Phe Asn Val Ser Ile Thr Tyr Asn Leu Lys Val Leu Gly Leu

245 250 255

Glu Pro Val Ala Pro Leu Thr Val Tyr Ala Ala Glu Gly Ser Arg Val

260 265 270

Glu Leu Pro Cys His Leu Pro Pro Gly Val Gly Thr Pro Ser Leu Leu

275 280 285

Ile Ala Lys Trp Thr Pro Pro Gly Gly Gly Pro Glu Leu Pro Val Ala

290 295 300

Gly Lys Ser Gly Asn Phe Thr Leu His Leu Glu Ala Val Gly Leu Ala

305 310 315 320

Gln Ala Gly Thr Tyr Thr Cys Ser Ile His Leu Gln Gly Gln Gln Leu

325 330 335

Asn Ala Thr Val Thr Leu Ala Val Ile Thr Val Thr Pro Lys Ser Phe

340 345 350

Gly Leu Pro Gly Ser Arg Gly Lys Leu Leu Cys Glu Val Thr Pro Ala

355 360 365

Ser Gly Lys Glu Arg Phe Val Trp Arg Pro Leu Asn Asn Leu Ser Arg

370 375 380

Ser Cys Pro Gly Pro Val Leu Glu Ile Gln Glu Ala Arg Leu Leu Ala

385 390 395 400

Glu Arg Trp Gln Cys Gln Leu Tyr Glu Gly Gln Arg Leu Leu Gly Ala

405 410 415

Thr Val Tyr Ala Ala Glu Ser Ser Ser Gly Ala His Ser Ala Arg Arg

420 425 430

Ile Ser Gly Asp Leu Lys Gly Gly His Leu Val Leu Val Leu Ile Leu

435 440 445

Gly Ala Leu Ser Leu Phe Leu Leu Val Ala Gly Ala Phe Gly Phe His

450 455 460

Trp Trp Arg Lys Gln Leu Leu Leu Arg Arg Phe Ser Ala Leu Glu His

465 470 475 480

Gly Ile Gln Pro Phe Pro Ala Gln Arg Lys Ile Glu Glu Leu Glu Arg

485 490 495

Glu Leu Glu Thr Glu Met Gly Gln Glu Pro Glu Pro Glu Pro Glu Pro

500 505 510

Gln Leu Glu Pro Glu Pro Arg Gln Leu

515 520

<210> 128

<211> 533

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Cynomolgus macaque LAG-3 amino acid sequence

<400> 128

Met Trp Glu Ala Gln Phe Leu Gly Leu Leu Phe Leu Gln Pro Leu Trp

1 5 10 15

Val Ala Pro Val Lys Pro Pro Gln Pro Gly Ala Glu Ile Ser Val Val

20 25 30

Trp Ala Gln Glu Gly Ala Pro Ala Gln Leu Pro Cys Ser Pro Thr Ile

35 40 45

Pro Leu Gln Asp Leu Ser Leu Leu Arg Arg Ala Gly Val Thr Trp Gln

50 55 60

His Gln Pro Asp Ser Gly Pro Pro Ala Ala Ala Pro Gly His Pro Pro

65 70 75 80

Val Pro Gly His Arg Pro Ala Ala Pro Tyr Ser Trp Gly Pro Arg Pro

85 90 95

Arg Arg Tyr Thr Val Leu Ser Val Gly Pro Gly Gly Leu Arg Ser Gly

100 105 110

Arg Leu Pro Leu Gln Pro Arg Val Gln Leu Asp Glu Arg Gly Arg Gln

115 120 125

Arg Gly Asp Phe Ser Leu Trp Leu Arg Pro Ala Arg Arg Ala Asp Ala

130 135 140

Gly Glu Tyr Arg Ala Thr Val His Leu Arg Asp Arg Ala Leu Ser Cys

145 150 155 160

Arg Leu Arg Leu Arg Val Gly Gln Ala Ser Met Thr Ala Ser Pro Pro

165 170 175

Gly Ser Leu Arg Thr Ser Asp Trp Val Ile Leu Asn Cys Ser Phe Ser

180 185 190

Arg Pro Asp Arg Pro Ala Ser Val His Trp Phe Arg Ser Arg Gly Gln

195 200 205

Gly Arg Val Pro Val Gln Gly Ser Pro His His His Leu Ala Glu Ser

210 215 220

Phe Leu Phe Leu Pro His Val Gly Pro Met Asp Ser Gly Leu Trp Gly

225 230 235 240

Cys Ile Leu Thr Tyr Arg Asp Gly Phe Asn Val Ser Ile Met Tyr Asn

245 250 255

Leu Thr Val Leu Gly Leu Glu Pro Ala Thr Pro Leu Thr Val Tyr Ala

260 265 270

Gly Ala Gly Ser Arg Val Glu Leu Pro Cys Arg Leu Pro Pro Ala Val

275 280 285

Gly Thr Gln Ser Phe Leu Thr Ala Lys Trp Ala Pro Pro Gly Gly Gly

290 295 300

Pro Asp Leu Leu Val Ala Gly Asp Asn Gly Asp Phe Thr Leu Arg Leu

305 310 315 320

Glu Asp Val Ser Gln Ala Gln Ala Gly Thr Tyr Ile Cys His Ile Arg

325 330 335

Leu Gln Gly Gln Gln Leu Asn Ala Thr Val Thr Leu Ala Ile Ile Thr

340 345 350

Val Thr Pro Lys Ser Phe Gly Ser Pro Gly Ser Leu Gly Lys Leu Leu

355 360 365

Cys Glu Val Thr Pro Ala Ser Gly Gln Glu His Phe Val Trp Ser Pro

370 375 380

Leu Asn Thr Pro Ser Gln Arg Ser Phe Ser Gly Pro Trp Leu Glu Ala

385 390 395 400

Gln Glu Ala Gln Leu Leu Ser Gln Pro Trp Gln Cys Gln Leu His Gln

405 410 415

Gly Glu Arg Leu Leu Gly Ala Ala Val Tyr Phe Thr Glu Leu Ser Ser

420 425 430

Pro Gly Ala Gln Arg Ser Gly Arg Ala Pro Gly Ala Leu Arg Ala Gly

435 440 445

His Leu Pro Leu Phe Leu Ile Leu Gly Val Leu Phe Leu Leu Leu Leu

450 455 460

Val Thr Gly Ala Phe Gly Phe His Leu Trp Arg Arg Gln Trp Arg Pro

465 470 475 480

Arg Arg Phe Ser Ala Leu Glu Gln Gly Ile His Pro Pro Gln Ala Gln

485 490 495

Ser Lys Ile Glu Glu Leu Glu Gln Glu Pro Glu Leu Glu Pro Glu Pro

500 505 510

Glu Leu Glu Arg Glu Leu Gly Pro Glu Pro Glu Pro Gly Pro Glu Pro

515 520 525

Glu Pro Glu Gln Leu

530

<210> 129

<211> 290

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Human PD-L1 amino acid sequence

<400> 129

Met Arg Ile Phe Ala Val Phe Ile Phe Met Thr Tyr Trp His Leu Leu

1 5 10 15

Asn Ala Phe Thr Val Thr Val Pro Lys Asp Leu Tyr Val Val Glu Tyr

20 25 30

Gly Ser Asn Met Thr Ile Glu Cys Lys Phe Pro Val Glu Lys Gln Leu

35 40 45

Asp Leu Ala Ala Leu Ile Val Tyr Trp Glu Met Glu Asp Lys Asn Ile

50 55 60

Ile Gln Phe Val His Gly Glu Glu Asp Leu Lys Val Gln His Ser Ser

65 70 75 80

Tyr Arg Gln Arg Ala Arg Leu Leu Lys Asp Gln Leu Ser Leu Gly Asn

85 90 95

Ala Ala Leu Gln Ile Thr Asp Val Lys Leu Gln Asp Ala Gly Val Tyr

100 105 110

Arg Cys Met Ile Ser Tyr Gly Gly Ala Asp Tyr Lys Arg Ile Thr Val

115 120 125

Lys Val Asn Ala Pro Tyr Asn Lys Ile Asn Gln Arg Ile Leu Val Val

130 135 140

Asp Pro Val Thr Ser Glu His Glu Leu Thr Cys Gln Ala Glu Gly Tyr

145 150 155 160

Pro Lys Ala Glu Val Ile Trp Thr Ser Ser Asp His Gln Val Leu Ser

165 170 175

Gly Lys Thr Thr Thr Thr Asn Ser Lys Arg Glu Glu Lys Leu Phe Asn

180 185 190

Val Thr Ser Thr Leu Arg Ile Asn Thr Thr Thr Asn Glu Ile Phe Tyr

195 200 205

Cys Thr Phe Arg Arg Leu Asp Pro Glu Glu Asn His Thr Ala Glu Leu

210 215 220

Val Ile Pro Glu Leu Pro Leu Ala His Pro Asn Glu Arg Thr His

225 230 235 240

Leu Val Ile Leu Gly Ala Ile Leu Leu Cys Leu Gly Val Ala Leu Thr

245 250 255

Phe Ile Phe Arg Leu Arg Lys Gly Arg Met Met Asp Val Lys Lys Cys

260 265 270

Gly Ile Gln Asp Thr Asn Ser Lys Lys Gln Ser Asp Thr His Leu Glu

275 280 285

Glu Thr

290

<210> 130

<211> 290

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Mouse PD-L1 amino acid sequence

<400> 130

Met Arg Ile Phe Ala Gly Ile Ile Phe Thr Ala Cys Cys His Leu Leu

1 5 10 15

Arg Ala Phe Thr Ile Thr Ala Pro Lys Asp Leu Tyr Val Val Glu Tyr

20 25 30

Gly Ser Asn Val Thr Met Glu Cys Arg Phe Pro Val Glu Arg Glu Leu

35 40 45

Asp Leu Leu Ala Leu Val Val Tyr Trp Glu Lys Glu Asp Glu Gln Val

50 55 60

Ile Gln Phe Val Ala Gly Glu Glu Asp Leu Lys Pro Gln His Ser Asn

65 70 75 80

Phe Arg Gly Arg Ala Ser Leu Pro Lys Asp Gln Leu Leu Lys Gly Asn

85 90 95

Ala Ala Leu Gln Ile Thr Asp Val Lys Leu Gln Asp Ala Gly Val Tyr

100 105 110

Cys Cys Ile Ile Ser Tyr Gly Gly Ala Asp Tyr Lys Arg Ile Thr Leu

115 120 125

Lys Val Asn Ala Pro Tyr Arg Lys Ile Asn Gln Arg Ile Ser Val Asp

130 135 140

Pro Ala Thr Ser Glu His Glu Leu Ile Cys Gln Ala Glu Gly Tyr Pro

145 150 155 160

Glu Ala Glu Val Ile Trp Thr Asn Ser Asp His Gln Pro Val Ser Gly

165 170 175

Lys Arg Ser Val Thr Thr Ser Arg Thr Glu Gly Met Leu Leu Asn Val

180 185 190

Thr Ser Ser Leu Arg Val Asn Ala Thr Ala Asn Asp Val Phe Tyr Cys

195 200 205

Thr Phe Trp Arg Ser Gln Pro Gly Gln Asn His Thr Ala Glu Leu Ile

210 215 220

Ile Pro Glu Leu Pro Ala Thr His Pro Pro Gln Asn Arg Thr His Trp

225 230 235 240

Val Leu Leu Gly Ser Ile Leu Leu Phe Leu Ile Val Val Ser Thr Val

245 250 255

Leu Leu Phe Leu Arg Lys Gln Val Arg Met Leu Asp Val Glu Lys Cys

260 265 270

Gly Val Glu Asp Thr Ser Ser Lys Asn Arg Asn Asp Thr Gln Phe Glu

275 280 285

Glu Thr

290

<210> 131

<211> 290

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Cynomolgus monkey PD-L1 amino acid sequence

<400> 131

Met Arg Ile Phe Ala Val Phe Ile Phe Thr Ile Tyr Trp His Leu Leu

1 5 10 15

Asn Ala Phe Thr Val Thr Val Pro Lys Asp Leu Tyr Val Val Glu Tyr

20 25 30

Gly Ser Asn Met Thr Ile Glu Cys Lys Phe Pro Val Glu Lys Gln Leu

35 40 45

Asp Leu Thr Ser Leu Ile Val Tyr Trp Glu Met Glu Asp Lys Asn Ile

50 55 60

Ile Gln Phe Val His Gly Glu Glu Asp Leu Lys Val Gln His Ser Asn

65 70 75 80

Tyr Arg Gln Arg Ala Gln Leu Leu Lys Asp Gln Leu Ser Leu Gly Asn

85 90 95

Ala Ala Leu Arg Ile Thr Asp Val Lys Leu Gln Asp Ala Gly Val Tyr

100 105 110

Arg Cys Met Ile Ser Tyr Gly Gly Ala Asp Tyr Lys Arg Ile Thr Val

115 120 125

Lys Val Asn Ala Pro Tyr Asn Lys Ile Asn Gln Arg Ile Leu Val Val

130 135 140

Asp Pro Val Thr Ser Glu His Glu Leu Thr Cys Gln Ala Glu Gly Tyr

145 150 155 160

Pro Lys Ala Glu Val Ile Trp Thr Ser Ser Asp His Gln Val Leu Ser

165 170 175

Gly Lys Thr Thr Thr Thr Asn Ser Lys Arg Glu Glu Lys Leu Leu Asn

180 185 190

Val Thr Ser Thr Leu Arg Ile Asn Thr Thr Ala Asn Glu Ile Phe Tyr

195 200 205

Cys Ile Phe Arg Arg Leu Asp Pro Glu Glu Asn His Thr Ala Glu Leu

210 215 220

Val Ile Pro Glu Leu Pro Leu Ala Leu Pro Pro Asn Glu Arg Thr His

225 230 235 240

Leu Val Ile Leu Gly Ala Ile Phe Leu Leu Leu Gly Val Ala Leu Thr

245 250 255

Phe Ile Phe Tyr Leu Arg Lys Gly Arg Met Met Asp Met Lys Lys Cys

260 265 270

Gly Ile Arg Val Thr Asn Ser Lys Lys Gln Arg Asp Thr Gln Leu Glu

275 280 285

Glu Thr

290

<210> 132

<211> 447

<212> PROTEIN

<213> Artificial sequence

<220>

<223> mAb2 heavy chain amino acid sequence against

LAG-3 mouse/FITC FS18-7-108-29/4420 containing the LALA mutation

<400> 132

Glu Val Lys Leu Asp Glu Thr Gly Gly Gly Leu Val Gln Pro Gly Arg

1 5 10 15

Pro Met Lys Leu Ser Cys Val Ala Ser Gly Phe Thr Phe Ser Asp Tyr

20 25 30

Trp Met Asn Trp Val Arg Gln Ser Pro Glu Lys Gly Leu Glu Trp Val

35 40 45

Ala Gln Ile Arg Asn Lys Pro Tyr Asn Tyr Glu Thr Tyr Tyr Ser Asp

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Ser

65 70 75 80

Val Tyr Leu Gln Met Asn Asn Leu Arg Val Glu Asp Met Gly Ile Tyr

85 90 95

Tyr Cys Thr Gly Ser Tyr Tyr Gly Met Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Ser Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro

115 120 125

Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly

130 135 140

Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn

145 150 155 160

Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln

165 170 175

Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser

180 185 190

Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser

195 200 205

Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr

210 215 220

His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser

225 230 235 240

Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg

245 250 255

Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro

260 265 270

Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala

275 280 285

Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val

290 295 300

Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr

305 310 315 320

Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr

325 330 335

Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu

340 345 350

Pro Pro Ser Trp Asp Glu Pro Trp Gly Glu Asp Val Ser Leu Thr Cys

355 360 365

Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Val Val Glu Trp Glu Ser

370 375 380

Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp

385 390 395 400

Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Pro Phe Glu

405 410 415

Arg Trp Met Trp Pro Asp Glu Phe Ser Cys Ser Val Met His Glu Ala

420 425 430

Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

435 440 445

<210> 133

<211> 447

<212> PROTEIN

<213> Artificial sequence

<220>

<223> mAb2 heavy chain amino acid sequence against

LAG-3 mouse/FITC FS18-7-108-35/4420 containing the LALA mutation

<400> 133

Glu Val Lys Leu Asp Glu Thr Gly Gly Gly Leu Val Gln Pro Gly Arg

1 5 10 15

Pro Met Lys Leu Ser Cys Val Ala Ser Gly Phe Thr Phe Ser Asp Tyr

20 25 30

Trp Met Asn Trp Val Arg Gln Ser Pro Glu Lys Gly Leu Glu Trp Val

35 40 45

Ala Gln Ile Arg Asn Lys Pro Tyr Asn Tyr Glu Thr Tyr Tyr Ser Asp

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Ser

65 70 75 80

Val Tyr Leu Gln Met Asn Asn Leu Arg Val Glu Asp Met Gly Ile Tyr

85 90 95

Tyr Cys Thr Gly Ser Tyr Tyr Gly Met Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Ser Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro

115 120 125

Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly

130 135 140

Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn

145 150 155 160

Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln

165 170 175

Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser

180 185 190

Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser

195 200 205

Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr

210 215 220

His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser

225 230 235 240

Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg

245 250 255

Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro

260 265 270

Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala

275 280 285

Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val

290 295 300

Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr

305 310 315 320

Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr

325 330 335

Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu

340 345 350

Pro Pro Ser Trp Asp Glu Pro Trp Gly Glu Asp Val Ser Leu Thr Cys

355 360 365

Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ser Val Glu Trp Glu Ser

370 375 380

Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp

385 390 395 400

Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Pro Phe Glu

405 410 415

Arg Trp Met Trp Pro Asp Glu Phe Ser Cys Ser Val Met His Glu Ala

420 425 430

Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

435 440 445

<210> 134

<211> 447

<212> PROTEIN

<213> Artificial sequence

<220>

<223> mAb2 heavy chain amino acid sequence against

LAG-3 mouse/FITC FS18-7-108-29/4420

<400> 134

Glu Val Lys Leu Asp Glu Thr Gly Gly Gly Leu Val Gln Pro Gly Arg

1 5 10 15

Pro Met Lys Leu Ser Cys Val Ala Ser Gly Phe Thr Phe Ser Asp Tyr

20 25 30

Trp Met Asn Trp Val Arg Gln Ser Pro Glu Lys Gly Leu Glu Trp Val

35 40 45

Ala Gln Ile Arg Asn Lys Pro Tyr Asn Tyr Glu Thr Tyr Tyr Ser Asp

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Ser

65 70 75 80

Val Tyr Leu Gln Met Asn Asn Leu Arg Val Glu Asp Met Gly Ile Tyr

85 90 95

Tyr Cys Thr Gly Ser Tyr Tyr Gly Met Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Ser Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro

115 120 125

Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly

130 135 140

Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn

145 150 155 160

Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln

165 170 175

Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser

180 185 190

Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser

195 200 205

Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr

210 215 220

His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser

225 230 235 240

Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg

245 250 255

Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro

260 265 270

Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala

275 280 285

Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val

290 295 300

Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr

305 310 315 320

Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr

325 330 335

Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu

340 345 350

Pro Pro Ser Trp Asp Glu Pro Trp Gly Glu Asp Val Ser Leu Thr Cys

355 360 365

Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Val Val Glu Trp Glu Ser

370 375 380

Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp

385 390 395 400

Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Pro Phe Glu

405 410 415

Arg Trp Met Trp Pro Asp Glu Phe Ser Cys Ser Val Met His Glu Ala

420 425 430

Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

435 440 445

<210> 135

<211> 107

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Amino acid sequence of the CH3 domain of Fcab FS18-7-9,

containing a C-terminal lysine residue

<400> 135

Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Trp Asp

1 5 10 15

Glu Pro Trp Gly Glu Asp Val Ser Leu Thr Cys Leu Val Lys Gly Phe

20 25 30

Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu

35 40 45

Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe

50 55 60

Phe Leu Tyr Ser Lys Leu Thr Val Pro Tyr Asp Arg Trp Val Trp Pro

65 70 75 80

Asp Glu Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr

85 90 95

Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

100 105

<210> 136

<211> 5

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Amino acid sequences of the CDRs of antibody 84G09

against PD-L1 (according to Kabat) HCDR1

<400> 136

Asp Tyr Ala Met His

fifteen

<210> 137

<211> 17

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Amino acid sequences of CDRs of anti-PD-L1 antibody 84G09

(according to Kabat) HCDR2

<400> 137

Gly Ile Ser Trp Lys Ser Asn Ile Ile Gly Tyr Ala Asp Ser Val Lys

1 5 10 15

gly

<210> 138

<211> 13

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Amino acid sequences of CDRs of antibody 84G09 against

PD-L1 (according to Kabat) HCDR3

<400> 138

Asp Ile Thr Gly Ser Gly Ser Tyr Gly Trp Phe Asp Pro

1 5 10

<210> 139

<211> 11

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Amino acid sequences of CDRs of antibody 84G09 against

PD-L1 (according to Kabat) LCDR1

<400> 139

Arg Ala Ser Gln Ser Ile Ser Ser Tyr Leu Asn

1 5 10

<210> 140

<211> 7

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Amino acid sequences of CDRs of antibody 84G09 against

PD-L1 (according to Kabat) LCDR2

<400> 140

Val Ala Ser Ser Leu Gln Ser

fifteen

<210> 141

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Amino acid sequences of CDRs of antibody 84G09 against

PD-L1 (according to Kabat) LCDR3

<400> 141

Gln Gln Ser Tyr Ser Asn Pro Ile Thr

fifteen

<210> 142

<211> 318

<212> DNA

<213> Artificial sequence

<220>

<223> CHO-codon-optimized nucleotide sequence of the Fcab CH3 domain

FS18-7-9

<400> 142

ggccagcccc gggaacccca ggtgtacaca ctgcctccat cctgggatga gccctggggc 60

gaggatgtgt ctctgacctg tctcgtgaaa ggcttctacc cctccgatat cgccgtggaa 120

tgggagagca acggccagcc cgagaacaac tacaagacca ccccccctgt gctggactcc 180

gacggctcat tcttcctgta cagcaagctg acagtgccct acgacagatg ggtgtggccc 240

gacgagttct cctgctccgt gatgcacgag gccctgcaca accactacac ccagaagtcc 300

ctgtccctga gccccggc 318

<---

Claims (46)

1. An antibody molecule that binds to programmed cell death protein ligand-1 (PD-L1) and lymphocyte activation gene 3 (LAG-3), said antibody molecule containing: (i) an antigen-binding site based on complementarity determining regions (CDRs) for PD-L1 containing HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 anti-PD-L1 antibodies; and (ii) a LAG-3 antigen binding site located in the CH3 domain of the antibody molecule, wherein the LAG-3 binding site contains the amino acid sequences WDEPWGED (SEQ ID NO: 1) and PYDRWVWPDE (SEQ ID NO: 3), and the amino acid sequence WDEPWGED is located in the AB loop of the CH3 domain, and the amino acid sequence PYDRWVWPDE is located in the EF loop of the CH3 domain. 2. An antibody molecule according to claim 1, characterized in that said LAG-3 antigen binding site additionally contains one of the following sequences in the CD loop of the CH3 domain: (i) SNGQPENNY (SEQ ID NOs: 2, 8 and 18); (ii) SNGQPEDNY (SEQ ID NO: 13); (iii) SNGYPEIEF (SEQ ID NO: 23); (iv) SNGIPEWNY (SEQ ID NO: 28); (v) SNGYAEYNY (SEQ ID NO: 33); (vi) SNGYKEENY (SEQ ID NO: 38); (vii) SNGVPELNV (SEQ ID NO: 43); or (viii) SNGYQEDNY (SEQ ID NO: 48). 3. The antibody molecule according to claim 2, characterized in that the binding site of the LAG-3 antigen contains the amino acid sequence shown in SEQ ID NO: 2, 28 or 38, in the CD loop of the CH3 domain. 4. An antibody molecule according to claim 2, characterized in that the binding site of the LAG-3 antigen contains the amino acid sequence shown in SEQ ID NO: 2 in the CD CH3 loop of the domain. 5. An antibody molecule according to any one of paragraphs. 1-4, characterized in that said antibody molecule is an immunoglobulin G molecule, optionally said antibody molecule is an IgG1, IgG2, IgG3 or IgG4 molecule, and further optionally said antibody molecule is an IgG1 molecule. 6. An antibody molecule according to any one of paragraphs. 1-5, characterized in that said antibody molecule contains the CH3 domain shown in SEQ ID NO: 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50. 7. An antibody molecule according to claim 6, characterized in that said antibody molecule contains the CH3 domain shown in SEQ ID NO: 5, 30 or 40. 8. An antibody molecule according to claim 7, characterized in that said antibody molecule contains the CH3 domain shown in SEQ ID NO: 5. 9. An antibody molecule according to any one of paragraphs. 1-8, characterized in that said antibody molecule contains a CH2 domain and said CH2 domain contains the sequence shown in SEQ ID NO: 53 or SEQ ID NO: 54. 10. An antibody molecule according to any one of paragraphs. 1-9, characterized in that the specified antibody molecule contains the sequence shown in SEQ ID NO: 6, 7, 11, 12, 16, 17, 21, 22, 26, 27, 31, 32, 36, 37, 41, 42, 46, 47, 51 or 52. 11. An antibody molecule according to claim 10, characterized in that said antibody molecule contains the sequence shown in SEQ ID NO: 6, 7, 31, 32, 41, or 42. 12. An antibody molecule according to claim 11, characterized in that said antibody molecule contains the sequence shown in SEQ ID NO: 6 or 7. 13. An antibody molecule according to any one of paragraphs. 1-12, characterized in that the CDR-based antigen-binding site for PD-L1 contains the CDRs shown in SEQ ID NOs: 86-91. 14. An antibody molecule according to any one of paragraphs. 1-12, characterized in that the CDR-based antigen-binding site for PD-L1 contains the CDRs shown in SEQ ID NOs: 136-141. 15. An antibody molecule according to claim 13 or 14, characterized in that said antibody molecule contains the VH and/or VL domains shown in SEQ ID NOs: 92 and 93. 16. An antibody molecule according to any one of paragraphs. 13-15, characterized in that said antibody molecule contains the heavy chain sequence shown in any of SEQ ID NOs: 94-113. 17. An antibody molecule according to claim 16, characterized in that said antibody molecule contains the heavy chain sequence shown in SEQ ID NOs: 94, 95, 104, 105, 108, or 109. 18. An antibody molecule according to claim 17, characterized in that said antibody molecule contains the heavy chain sequence shown in SEQ ID NO: 94 or 95. 19. An antibody molecule according to any one of paragraphs. 13-18, characterized in that said antibody molecule contains the light chain sequence shown in SEQ ID NO: 116. 20. An antibody molecule according to claim 19, characterized in that said antibody molecule contains the heavy chain sequence shown in SEQ ID NO: 94 and the light chain sequence shown in SEQ ID NO: 116. 21. An antibody molecule according to any one of paragraphs. 6-20, characterized in that the sequence of the CH3 domain of the antibody molecule additionally contains a lysine residue (K) at the very C-terminus of the sequence shown in SEQ ID NO: 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50. 22. An antibody molecule according to claim 21, characterized in that the antibody molecule contains the CH3 domain shown in the sequence of SEQ ID NO: 135. 23. An antibody molecule according to any one of paragraphs. 1-22, characterized in that said antibody molecule is capable of simultaneously binding to PD-L1 and LAG-3. 24. An antibody molecule according to claim 23, characterized in that PD-L1 and LAG-3 are present on two different cells. 25. An antibody molecule according to any one of paragraphs. 1-24, characterized in that said antibody molecule is conjugated to an immune system modulator, a cytotoxic molecule, a radioactive isotope, or a detectable label. 26. An antibody molecule according to claim 25, characterized in that the immune system modulator or cytotoxic molecule is a cytokine. 27. Nucleic acid encoding an antibody molecule according to any one of paragraphs. 1-26. 28. An expression vector containing the nucleic acid according to claim 27. 29. A recombinant host cell containing a nucleic acid according to paragraph 27 or a vector according to paragraph 28, for the expression of an antibody molecule according to any one of paragraphs. 1-26. 30. The method of obtaining an antibody molecule according to any one of paragraphs. 1-26, which includes culturing the recombinant host cell according to claim 29 under conditions for producing an antibody molecule. 31. The method of claim 30, further comprising isolating and/or purifying the antibody molecule. 32. Pharmaceutical composition for the treatment of cancer in a patient, containing a therapeutically effective amount of an antibody molecule according to any one of paragraphs. 1-26 and a pharmaceutically acceptable excipient. 33. The use of an antibody molecule according to any one of paragraphs. 1-26 for treating cancer in a patient. 34. Use according to claim 33, characterized in that the cancer is selected from the group consisting of: Hodgkin's lymphoma, non-Hodgkin's lymphoma, ovarian cancer, prostate cancer, colon and rectal cancer, fibrosarcoma, renal cell carcinoma, melanoma, pancreatic cancer, breast cancer, glioblastoma multiforme, lung cancer, head and neck cancer, stomach cancer, bladder cancer, cervical cancer, uterine cancer, vulvar cancer, testicular cancer, penile cancer, leukemia, multiple myeloma, squamous cell carcinoma, testicular cancer, esophageal cancer, Kaposi's sarcoma and central nervous system (CNS) lymphoma, hepatocellular carcinoma, Merkel cell carcinoma, nasopharyngeal cancer and mesothelioma. 35. Use according to claim 33 or 34, characterized in that the treatment further comprises administering an antitumor vaccine to the patient. 36. Use according to claim 33 or 34, characterized in that said treatment further comprises administering a chemotherapeutic agent to a patient.

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