WO2021136392A1 - Lag-3 antibody and medical use thereof - Google Patents

Abstract

Disclosed are an LAG-3 antibody and the medical use thereof. The antibody can specifically bind to the extracellular domain of LAG-3 and has excellent affinity and specificity; and the antibody is a functional antibody, and has the activity of blocking the binding of LAG-3 and the ligand MHC II thereof. In addition, the humanized modification of the antibody is further performed, and the present invention provides a method for verifying the function of the antibody. The antibody can treat multiple cancers or immune diseases by regulating human immune functions.

Description

LAG-3 antibody and its medical use Technical field

The present invention relates to the field of immunomedicine, in particular to a LAG-3 antibody and its medical use.

Background technique

For a long time, tumor immunotherapy has been a hot spot in the field of tumor therapy, and T cell immunotherapy has been at the core of tumor immunotherapy. Under normal circumstances, T cells have the function of anti-viral and anti-tumor immune response. Under appropriate antigen stimulation, antigen-specific T cells can be clonally expanded and can effectively exert their cytotoxic function. However, in chronic infection, due to long-term The antigenic stimulation of T cells leads to T cell dysfunction, causing T cells to lose activation, proliferation, secretion of cytokines, and cytotoxicity. This state is called "exhaustion" (Nat Rev Immunol, 2015; 15(1): 45-56). There are dysfunctions in the T cells of chronic infections, autoimmune diseases and tumor patients, and they cannot effectively remove pathogenic bacteria or tumor cells. Recent studies have found that the main reason for the dysfunction of T cells is related to some immunosuppressive receptors such as PD-1, MHC II, CTLA-4, LAG-3 and TIM-3, which participate in the negative regulation of T cell immunity and promote T cell exhaustion. (Cancer Res, 2012; 72(4):887-896), and blocking these immune negative regulatory receptor pathways can restore part or all of T cells to function.

Lymphocyte activation gene-3 (LAG-3, also known as CD223) is a membrane protein that is a member of the immunoglobulin superfamily and is located on chromosome 12 (12P13). Lymphocyte activation gene-3 is composed of three parts: extracellular region, transmembrane region and cytoplasmic region. It is similar in structure to CD4. The ligands are all major histocompatibility complex class II molecules (major histocompatibility complex class II, MHC II). However, the physiological function of lymphocyte activation gene-3 is completely opposite to that of CD4. Lymphocyte activation gene-3 has a higher affinity with MHC-II molecules, thereby interfering with CD4 ⁺ T lymphocyte TCR activation and inhibiting T lymphocyte activation ( J Exp Med, 1990, 171(5): 1393-1405; Eur J Immunol, 2003, 33(4): 970-979), has a significant negative regulatory effect on T lymphocytes.

LAG-3 molecules are mainly expressed on the surface of activated NK cells, T lymphocytes, B lymphocytes, Treg cells, etc., bind with MHC II with high affinity, and participate in the activation of lymphocytes (Eur J Immunol, 2002, 32(8): 2255-2263). LAG-3 inhibits the proliferation of Th1 cells and the secretion of cytokines such as IFN-γ, IL-2 and TNF-α. Anti-LAG-3 antibodies block LAG-3, which can restore the function of Th1 cells. LAG-3 also inhibits CD8 ⁺ T cell activity. In mouse experiments, inhibiting LAG-3 molecules can increase ^{the proliferation of CD8 +} T cells, and can also promote the secretion of IFN-γ and enhance cytotoxicity. LAG-3 has ^{a direct regulatory effect on CD8 +} T cells, and the process of LAG-3 and CD8 ⁺ T cells does not require ^{the participation of CD4 +} T cells (Immunol, 2005, 115(2): 170-178). Studies have found (J Immunol, 2005, 174(2): 688-695; Immunol, 2004, 21(4): 503-513; Blood, 2003, 102(6): 2130-2137; J Immunol, 2008, 180 (9):5916-5926), in LAG-3 ^-/- mice or under the action of anti-LAG-3 antibodies, the number of CD4 ⁺ T cells and CD8 ⁺ T cells expressing CD69 increased significantly. LAG-3 is also a necessary molecule for regulatory T cells (Treg), and anti-LAG-3 antibodies can significantly inhibit the function of Treg (Blood, 2006; 108(7): 2280-2289).

Studies have found that the expression of LAG-3 molecules can promote the occurrence and development of tumors, and inhibiting the expression of this molecule can curb the growth of tumors. JosepH et al. (J Clin Invest, 2007, 117(11): 3 383-3 392) believe that naive CD8 ⁺ T cells under-express LAG-3 molecules, and under the action of antigen stimulation, the expression of LAG-3 is significantly increased. The expression level of activated CD8 ⁺ T cells is higher than that of activated CD4 ⁺ T cells. In the process of tumor development, ^{although the number of tumor-specific CD8 +} T cells increases in tumor tissues, their functions are partially lost. If anti-LAG-3 antibodies are used or the LAG-3 gene is removed, the function of ^{CD8 + T cells can be restored.} Increasing the number of CD8 ⁺ T cells and their cytotoxicity also increases their cytokine secretion. In addition, after blocking the function of LAG-3, the number and function of cytotoxic T lymphocytes in tumor tissues increased significantly, and tumor growth was inhibited. Therefore, it is believed that inhibiting the expression of LAG-3 molecules can restore the immune function of lymphocytes and inhibit the development of tumors. Gandhi et al. (Blood, 2006, 108(7): 2280-9) found that in the tumor tissue and peripheral blood of patients with Hodgkin’s lymphoma, LAG-3 is highly expressed in lymphocytes. The function of specific CD8 ⁺ T cells in tumor tissues is significantly impaired and the number is negatively correlated with the number ^{of CD4 +} CD25 ⁺ T cells that highly express LAG-3 and/or FoxP3. If LAG-3 ⁺ T cells are removed, it is found that ^{the anti-tumor function of specific CD8 +} T cells can be restored and the secretion of cytokines increases. Therefore, the expression of LAG-3 is related to the immune negative regulation function of specific T cells. Inhibiting the function of LAG-3 molecules can enhance ^{the anti-tumor effect of specific CD8 +} T cells. This molecule may be a potential tumor immunotherapy target point.

The study of LAG-3 as an anti-tumor target was first carried out in melanoma. Because LAG-3 has high affinity with MHC-II molecules on APC, and tumor cells also express MHC-II molecules, the study found that LAG-3 Combining with MHC-II molecules on melanoma cells can prevent tumor cells from apoptosis. Therefore, LAG-3 specific monoclonal antibodies may interfere with this pathway to promote tumor cell apoptosis. In experimental studies of human and mouse models, it has been found that the dual blockade of anti-LAG-3 antibody and anti-PD-1 antibody is better than the use of a single blocker in improving the body’s effective anti-tumor immune response (Proc Natl Acad Sci USA, 2010; 107(17): 7875-7880; J Immunol, 2013; 190(9): 4899-4909).

At present, many multinational pharmaceutical companies such as BMS, Novartis, GSK, Merck, etc. have launched the development of monoclonal antibodies against LAG-3. The anti-LAG-3 monoclonal antibody stimulates the antigen-specific T cell response and enhances the anti-tumor effect of T cells, thereby maximizing the patient's own immune system response to tumors and achieving the purpose of killing tumor cells. The current related patents are WO2008132601, WO2010019570, WO2014008218, WO2014140180, WO2015042246, WO2015138920, WO2016028672, WO2017037203 or WO2017062888. There is still a need in the art for anti-human LAG-3 antibodies with improved efficacy (for example, high binding affinity, good specificity, and good stability) to LAG-3.

Summary of the invention

One aspect of the present invention relates to an antibody or an antigen-binding fragment thereof, which can specifically bind to the extracellular region of lymphocyte activation gene 3, and comprises a heavy chain complementarity determining region, the heavy chain complementarity determining region including a heavy chain complementarity determining region 1 , Heavy chain complementarity determining region 2, and heavy chain complementarity determining region 3. The amino acid sequence of the heavy chain complementarity determining region 1 is shown in SEQ ID NO:1, and the amino acid sequence of the heavy chain complementarity determining region 2 is shown in SEQ ID NO: 2, the amino acid sequence of the heavy chain complementarity determining region 3 is shown in SEQ ID NO: 3; and the light chain complementarity determining region, the light chain complementarity determining region includes light chain complementarity determining region 1, light chain Complementarity determining region 2 and light chain complementarity determining region 3, the amino acid sequence of the light chain complementarity determining region 1 is shown in SEQ ID NO: 4, and the amino acid sequence of the light chain complementarity determining region 2 is shown in SEQ ID NO: 5 As shown, the amino acid sequence of the light chain complementarity determining region 3 is shown in SEQ ID NO: 6.

The present invention also relates to nucleic acids, vectors and host cells for expressing and producing the antibody or antigen-binding fragments thereof.

The present invention also relates to a method for producing the antibody or antigen-binding fragment thereof.

The present invention also relates to a pharmaceutical composition, which comprises the antibody or antigen-binding fragment thereof as described above or the immune cell as described above, and one or more of pharmaceutically acceptable excipients, diluents or carriers.

The beneficial effects of the present invention are:

The LAG-3 antibody or its antigen-binding fragment provided by the present invention can specifically bind to the extracellular segment of LAG-3, and has excellent affinity and specificity (it basically does not bind to other antigens on the cell membrane surface); And the antibody is a functional antibody, and the antibody or its antigen-binding fragment has the activity of blocking the binding of LAG-3 to its ligand MHC II.

Description of the drawings

In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following will briefly introduce the drawings that need to be used in the specific embodiments or the description of the prior art. Obviously, the appendix in the following description The drawings are some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative work.

FIG 1 one embodiment of candidate antibody assay results to LAG-3 overexpression of the median effect concentration (EC ₅₀₎ CHO-K1 cells binds embodiment of the invention;

Figure 2 shows the results of an experiment in which a candidate antibody blocks the binding of LAG-3 antigen and Daudi cells in an embodiment of the present invention;

Fig. 3 is the test result of PBMC-T lymphocyte activation by candidate antibody in an embodiment of the present invention;

Figure 4 shows the results of the stimulation experiment of the candidate antibody to the antigen-specific T cell response in an embodiment of the present invention.

Fig. 5 shows the tumor volume measured after intraperitoneal injection of tumor-bearing mice with the antibody drug in the experiment of inhibiting the subcutaneous transplantation tumor of MC38-hPD-L1 by the LAG-3 antibody according to an embodiment of the present invention.

Detailed ways

A reference to the embodiments of the present invention will now be provided in detail, one or more examples of which are described below. Each example is provided as an explanation rather than a limitation of the invention. In fact, it is obvious to those skilled in the art that various modifications and changes can be made to the present invention without departing from the scope or spirit of the present invention. For example, features illustrated or described as part of one embodiment can be used in another embodiment to produce a still further embodiment.

Therefore, it is intended that the present invention covers such modifications and changes that fall within the scope of the appended claims and their equivalents. Other objects, features, and aspects of the present invention are disclosed in or are obvious from the following detailed description. Those of ordinary skill in the art should understand that this discussion is only a description of exemplary embodiments and is not intended to limit the broader aspects of the present invention.

The present invention relates to antibodies or antigen-binding fragments thereof, which can specifically bind to the extracellular region of lymphocyte activation gene 3 and comprise a heavy chain complementarity determining region, which includes heavy chain complementarity determining region 1 (H- CDR1), heavy chain complementarity determining region 2 (H-CDR2), and heavy chain complementarity determining region 3 (H-CDR3). The amino acid sequence of the heavy chain complementarity determining region 1 is shown in SEQ ID NO:1. The amino acid sequence of the heavy chain complementarity determining region 2 is shown in SEQ ID NO: 2, the amino acid sequence of the heavy chain complementarity determining region 3 is shown in SEQ ID NO: 3; and the light chain complementarity determining region, the light chain is complementary The determining regions include light chain complementarity determining region 1 (L-CDR1), light chain complementarity determining region 2 (L-CDR2), and light chain complementarity determining region 3 (L-CDR3). The amino acids of the light chain complementarity determining region 1 The sequence is shown in SEQ ID NO: 4, the amino acid sequence of the light chain complementarity determining region 2 is shown in SEQ ID NO: 5, and the amino acid sequence of the light chain complementarity determining region 3 is shown in SEQ ID NO: 6.

As used herein, the technical term "antibody" refers to a protein that binds to a specific antigen, which generally refers to all proteins and protein fragments containing complementarity determining regions (CDR regions), especially full-length antibodies or antibody functional fragments. The term "full-length antibody" includes polyclonal antibodies and monoclonal antibodies. The term "antibody functional fragment" is a substance that contains part or all of the CDR of an antibody, which lacks at least some of the amino acids present in the full-length chain but still has specificity. Binding to antigen. Such fragments are biologically active because they bind to the target antigen and can compete with other antigen-binding molecules (including intact antibodies) for binding to a given epitope. In some embodiments, the antibody functional fragment has the effect of specifically recognizing and binding LAG-3. In some embodiments, the antibody functional fragment is a fragment that has the function of blocking the binding of LAG-3 to its ligand MHC-II molecule. In some embodiments, the antibody functional fragment can block or reduce the activity of LAG-3. In some embodiments, such fragments will comprise a single heavy chain and a single light chain, or portions thereof. The fragments can be produced by recombinant nucleic acid technology, or can be produced by enzymatic or chemical cleavage of antigen binding molecules (including intact antibodies).

The term "complementarity determining region" or "CDR" refers to the hypervariable regions of the heavy and light chains of immunoglobulins, as defined by Kabat et al. (Kabat et al. Sequences of proteins of immunological interest, 5th Ed" US Department of Health and Human Services, NIH, 1991, and later versions). There are three heavy chain CDRs (H-CDR1, H-CDR2, and H-CDR3) and three light chain CDRs (L-CDR1, L-CDR) 2. And L-CDR 3). Here, depending on the situation, the terms "CDR" and "CDRs" are used to refer to one or more or even all of the binding affinity for the antibody and the antigen or epitope it recognizes. The region of the main amino acid residues. In another specific embodiment, the CDR region or CDR refers to the highly variable regions of the heavy and light chains of an immunoglobulin defined by IMGT.

The terms "specifically binds", "selectively binds", "selectively binds" and "specifically binds" refer to the binding of an antibody to an epitope on a predetermined antigen. Typically, the antibody is less than about 10 ^-6 M, e.g. less than about ^{10 -7 M, 10 -8 M,} 10 - 9 M or 10 ^-10 M or smaller affinity (KD) binding.

Variants of antibodies are also within the scope of the present invention, for example, each CDR or framework region (FR), or variable region (heavy chain variable region VL and/or light chain variable region VH) described in the present invention, Or the full-length amino acid or nucleotide sequence of the antibody has at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% % Or greater than 99% sequence identity. In some cases, the variant of the antibody includes at least the above 6 CDRs; in some cases, the variant of the antibody includes at least one heavy chain and one light chain, while in other cases, the variant form contains two identical light chains. Chain and two identical heavy chains (or sub-portions thereof). In some cases, antibody variants are obtained by conservative modifications or conservative substitutions or substitutions on the antibody sequence provided by the present invention. "Conservative modification" or "conservative substitution or substitution" means that other amino acids with similar characteristics (such as charge, side chain size, hydrophobicity/hydrophilicity, main chain conformation and rigidity, etc.) replace amino acids in a protein so that they can be frequently Make changes without changing the biological activity of the protein. Those skilled in the art know that, generally speaking, a single amino acid substitution in a non-essential region of a polypeptide does not substantially change the biological activity (see, for example, Watson et al. (1987) Molecular Biology of the Gene, The Benjamin/Cummings Pub. Co., Page 224, (4th edition)). In addition, the substitution of amino acids with similar structure or function is unlikely to disrupt biological activity. In some cases, the variant retains the ability to block the binding of LAG-3 to its ligand MHC class II molecule. Those skilled in the art will be able to use well-known techniques to determine suitable variants of the antigen binding molecules as set forth herein. In certain embodiments, those skilled in the art can identify suitable regions of the molecule that can be altered by targeting regions that are believed to be unimportant for activity without destroying activity. For nucleotide and amino acid sequences, the term "identity" indicates the degree of identity between two nucleic acid or two amino acid sequences when optimally aligned and compared with appropriate insertions or deletions.

The antibody provided by the present invention can specifically bind to the extracellular segment of LAG-3, and has excellent specificity (it basically does not bind to other antigens on the cell membrane surface). In particular, an important advantage of the antibody is that it has the activity of blocking the binding of LAG-3 to its ligand MHC-II, so it is preferably used as an antibody drug.

In some embodiments, the antibody is a mouse-derived antibody, a human-mouse chimeric antibody, or a humanized antibody.

The term "mouse-derived antibody" in the present invention is an anti-human LAG-3 monoclonal antibody prepared according to the knowledge and skills in the art. During preparation, the test subject is injected with LAG-3 antigen, and then hybridomas expressing antibodies with the desired sequence or functional properties are isolated. In a preferred embodiment of the present invention, the murine LAG-3 antibody or antigen-binding fragment thereof may further comprise the light chain constant region of murine kappa, lambda chains or variants thereof, or further comprise murine IgG1 , IgG2, IgG3 or its variant heavy chain constant region.

The term "chimeric antibody" is an antibody formed by fusing the variable region of a murine antibody with the constant region of a human antibody, which can alleviate the immune response induced by the murine antibody. To establish a chimeric antibody, it is necessary to first establish a hybridoma secreting murine-derived specific monoclonal antibodies, then clone the variable region genes from the murine hybridoma cells, and then clone the constant region genes of the human antibody as needed, and replace the murine variable region genes. It is connected with the human constant region gene to form a chimeric gene and inserted into an expression vector, and finally the chimeric antibody molecule is expressed in a eukaryotic system or a prokaryotic system. In a preferred embodiment of the present invention, the antibody light chain of the LAG-3 chimeric antibody further comprises a light chain constant region of a human kappa, lambda chain or a variant thereof. The antibody heavy chain of the LAG-3 chimeric antibody further comprises the heavy chain constant region of human IgG1, IgG2, IgG3, IgG4 or a variant thereof, preferably comprises a human IgG1, IgG2 or IgG4 heavy chain constant region, or has IgG1, IgG2 or IgG4 variants with amino acid mutations (such as YTE mutations).

The term "humanized antibody", also known as CDR-grafted antibody, refers to the transplantation of mouse CDR sequences into the variable region framework of human antibodies, that is, different types of human germline antibodies The antibody produced in the framework sequence. It can overcome the heterogeneous reaction induced by the chimeric antibody because it carries a large amount of murine protein components. Such framework sequences can be obtained from public DNA databases or published references that include germline antibody gene sequences. For example, the germline DNA sequences of human heavy chain and light chain variable region genes can be obtained from the "VBase" human germline sequence database (www.mrccpe.com.ac.uk/vbase), and in Kabat, EA et al., 1991 , Sequences of Proteins of Immunological Interest, found in the 5th edition. In order to avoid a decrease in immunogenicity and at the same time cause a decrease in functional activity, such as a decrease in LAG-3 binding activity, the human antibody variable region framework sequence may be subjected to minimal reverse mutations or back mutations to maintain activity. The humanized antibody of the present invention also includes a humanized antibody that has been further subjected to affinity maturation for CDR by phage display. In a preferred embodiment of the present invention, the murine CDR sequence of the LAG-3 humanized antibody is selected from SEQ ID NO: 9-20; the human antibody variable region framework is selected by design, wherein the antibody The heavy chain FR region sequence on the heavy chain variable region is derived from the combination of human germline heavy chain IGHV1-18*01 and hjh6.1, or the combination of human germline heavy chain IGHV1-3*01 and hjh6.1 Sequence; wherein the light chain FR region sequence on the light chain variable region of the antibody is derived from the combined sequence of human germline heavy chain IGKV1-39*01 and hjk4.1. In order to avoid the decrease of immunogenicity and the resulting decrease in binding activity, the variable region of the human antibody can be subjected to minimal reverse mutations to maintain the activity.

The grafting of the CDR may cause the LAG-3 antibody or its antigen-binding fragment to weaken the affinity for the antigen due to the framework residues in contact with the antigen. Such interactions can be the result of hypermutation of somatic cells. Therefore, it may still be necessary to transplant such donor framework amino acids into the framework of the humanized antibody. The amino acid residues from the non-human LAG-3 antibody or its antigen-binding fragment involved in antigen binding can be identified by examining the sequence and structure of the murine monoclonal antibody variable region. Residues in the CDR donor framework that differ from the germline can be considered related. If the closest germline cannot be determined, the sequence can be compared with the consensus sequence of a subtype consensus sequence or a murine sequence with a high percentage of similarity. Rare framework residues are thought to be the result of somatic hypermutation and thus play an important role in binding.

In some embodiments, the antibody further comprises a heavy chain framework region, which includes heavy chain framework region 1 (H-FR1), heavy chain framework region 2 (H-FR2), and heavy chain framework region 3. (H-FR3) and heavy chain framework region 4 (H-FR4), the amino acid sequence of the heavy chain framework region 1 is shown in SEQ ID NO: 7, and the amino acid sequence of the heavy chain framework region 2 is shown in SEQ ID NO: 8, the amino acid sequence of the heavy chain framework region 3 is shown in SEQ ID NO: 9, and the amino acid sequence of the heavy chain framework region 4 is shown in SEQ ID NO: 10; and/or light chain Framework region, the light chain framework region includes light chain framework region 1 (L-FR1), light chain framework region 2 (L-FR2), light chain framework region 3 (L-FR3), and light chain framework region 4 ( L-FR4), the amino acid sequence of the light chain framework region 1 is shown in SEQ ID NO: 11, the amino acid sequence of the light chain framework region 2 is shown in SEQ ID NO: 12, and the light chain framework region 3 The amino acid sequence of is shown in SEQ ID NO: 13, and the amino acid sequence of the light chain framework region 4 is shown in SEQ ID NO: 14.

In a further aspect, the present invention further provides humanized antibodies. In some embodiments, the antibody further comprises a heavy chain framework region, which includes heavy chain framework region 1 (H-FR1), heavy chain framework region 2 (H-FR2), and heavy chain framework region 3. (H-FR3) and heavy chain framework region 4 (H-FR4), the amino acid sequence of the heavy chain framework region 1 is shown in SEQ ID NO: 15, and the amino acid sequence of the heavy chain framework region 2 is shown in SEQ ID As shown in NO: 16, the amino acid sequence of the heavy chain framework region 3 is shown in SEQ ID NO: 17, and the amino acid sequence of the heavy chain framework region 4 is shown in SEQ ID NO: 18; and/or light chain Framework region, the light chain framework region includes light chain framework region 1 (L-FR1), light chain framework region 2 (L-FR2), light chain framework region 3 (L-FR3), and light chain framework region 4 ( L-FR4), the amino acid sequence of the light chain framework region 1 is shown in SEQ ID NO: 19, the amino acid sequence of the light chain framework region 2 is shown in SEQ ID NO: 20, and the light chain framework region 3 The amino acid sequence of is shown in SEQ ID NO: 21, and the amino acid sequence of the light chain framework region 4 is shown in SEQ ID NO: 22.

It should be understood that the present invention also claims a variant of the aforementioned framework region (FR). In addition to the homology limitation as described above, the variant is compared with the amino acid sequence shown in any one of SEQ ID NO: 15-22 , Can have 1, 2, 3 or 4 amino acid mutations. In some specific embodiments, the antibody comprises at least one selected from the group consisting of the following mutations:

a. The 15th amino acid of H-FR2 is mutated to I;

b. The amino acid at position 10 of H-FR3 is mutated to A;

c. The amino acid at position 12 of H-FR3 is mutated to L;

d. The 14th amino acid of H-FR3 is mutated to V;

e. The 37th amino acid of H-FR3 is mutated to F;

f. The 16th amino acid of L-FR2 is mutated to M;

g. The 21st amino acid of L-FR3 is mutated to F; and

h. The 35th amino acid of L-FR3 was mutated to F.

In some specific embodiments, the antibody comprises any one of a combination of mutations selected from the following:

Combination 1: a, b, c;

Combination 2: a, b, c, d;

Combination 3: a, b, c, d, e;

Combination 4: f, g;

Combination 5: a, b, c, f, g;

Combination 6: a, b, c, d, f, g;

Combination 7: a, b, c, d, e, f, g;

Combination 8: f, g, h;

Combination 9: a, b, c, f, g, h;

Combination 10: a, b, c, d, f, g, h; or

Combination 11: a, b, c, d, e, f, g, h.

Those skilled in the art can assemble various CDRs and FRs provided by the present invention to obtain the light chain and heavy chain variable regions of the antibody, and the assembly sequence is: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

In some embodiments, the antibody is one of F(ab') ₂ , Fab, scFv, and bispecific antibody.

The term "scFv" means a molecule comprising an antibody heavy chain variable domain (or region; VH) and an antibody light chain variable domain (or region; VL) connected by a linker. Such scFv molecules may have the general structure: NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH. Suitable prior art linkers can, for example, consist of a repetitive GGGGS amino acid sequence or variants thereof, for example using 1-4 repetitive variants (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90: 6444 -6448). Other linkers that can be used in the present invention are described by Alfthan et al. (1995), Protein Eng. 8: 725-731, Choi et al. (2001), Eur. J. Immunol. 31: 94-106, Hu et al. (1996), Cancer Res. 56: 3055-3061, Kipriyanov et al. (1999), J. Mol. Biol. 293: 41-56 and Roovers et al. (2001), Cancer Immunol.

In some embodiments, the antibody has a heavy chain constant region and a light chain constant region, and the sequence of the heavy chain constant region is selected from a constant region sequence of any one of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, and IgD ; The light chain constant region is κ or λ chain.

In some embodiments, the species from which the constant region is derived is selected from cattle, horses, dairy cows, pigs, sheep, goats, rats, mice, dogs, cats, rabbits, camels, donkeys, deer, minks, chickens, ducks , Goose, turkey, cockfighting or people.

In some embodiments, the antibody has a constant region, and the heavy chain constant region sequence of the antibody is shown in SEQ ID NO: 23, and the light chain constant region sequence is shown in SEQ ID NO: 24.

The present invention also relates to nucleic acids, which encode antibodies as described above.

The nucleic acid is usually RNA or DNA, and the nucleic acid molecule may be single-stranded or double-stranded, but is preferably double-stranded DNA. When a nucleic acid is placed in a functional relationship with another nucleic acid sequence, the nucleic acid is "operably linked." For example, if a promoter or enhancer affects the transcription of a coding sequence, then the promoter or enhancer is effectively linked to the coding sequence. Preferably, DNA nucleic acid is used when it is ligated into a vector.

In addition, since antibodies are membrane proteins, nucleic acids usually carry signal peptide sequences.

The present invention also relates to a vector, which contains the nucleic acid as described above.

The term "vector" refers to a nucleic acid delivery vehicle into which polynucleotides can be inserted. When the vector can express the protein encoded by the inserted polynucleotide, the vector is called an expression vector. The vector can be introduced into the host cell through transformation, transduction or transfection, so that the genetic material elements it carries can be expressed in the host cell. Vectors are well known to those skilled in the art, including but not limited to: plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or P1 derived artificial chromosomes (PAC) ; Phages such as lambda phage or M13 phage and animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillary viruses. Polyoma vacuole virus (such as SV40). In some embodiments, the vector of the present invention contains regulatory elements commonly used in genetic engineering, such as enhancers, promoters, internal ribosome entry sites (IRES) and other expression control elements (such as transcription termination signals, or multiple Adenylation signal and poly U sequence etc.).

In some embodiments, the vector also contains a reporter gene, and the reporter gene can be selected from metabolic markers, catalytic reporter genes, antibiotic markers, antibiotic resistance genes, herbicide resistance genes, and nutritional markers that are well known to those skilled in the art. Defective reporter genes, compound detoxification enzyme genes, and carbohydrate metabolizing enzyme selectable marker genes; in some preferred embodiments, for the convenience of observation and detection, the expression product of the reporter gene is self-luminous or capable of catalyzing a substrate reaction. A substance that produces a color change, or can cause a substrate to emit light or produce a color change by catalyzing the reaction of the substrate, or emit light or produce a color change through excitation light irradiation. Representative examples of such substances include fluorescent protein, luciferase, and LacZ. Both fluorescent protein and luciferase are luminescent proteins, and a camera or similar device can be used to detect fluorescent expression. Fluorescent proteins work by absorbing light of one color (excitation) and then emitting lower energy light of a different color (emission). In contrast, luciferase (and other bioluminescent enzymes) emit light by catalyzing a chemical reaction with a substrate (i.e., luciferin). Unlike the above two markers, LacZ does not emit light. The product of LacZ gene β-galactosidase can catalyze the conversion of X-gal into an opaque blue compound similar to indigo.

Further, the fluorescent protein can be selected from green fluorescent protein, blue fluorescent protein, yellow fluorescent protein, orange fluorescent protein or red fluorescent protein. The green fluorescent protein can be common GFP or modified GFP gene, such as enhanced GFP gene EGFP, etc.; blue fluorescent protein can be selected from EBFP, Azuritc, TagBFP, etc.; yellow fluorescent protein can be selected from EYFP, Ypct , PhiYFP, etc.; orange fluorescent protein can be selected from mKO, mOrange, mBanana, etc.; red fluorescent protein can be selected from TagRFP, mRuby, mCherry, mKate, and the like.

The present invention also provides a cell comprising the nucleic acid as described above or the vector as described above.

As used herein, the expressions "cell", "cell line" and "cell culture" are used interchangeably, and all such names include their progeny. Therefore, the words "transformant" and "transformed cell" include primary test cells and cultures derived therefrom, regardless of the number of transfers. It should also be understood that due to deliberate or unintentional mutations, all offspring cannot be exactly the same in terms of DNA content. Including mutant progeny with the same function or biological activity as screened in the original transformed cell. Where a different name is meant, it is clearly visible from the context.

Suitable host cells or cell lines for expressing the antigen binding protein of the present invention include mammalian cells such as NS0, Sp2/0, CHO, COS, HEK, fibroblasts and myeloma cells. Human cells can be used, thus allowing the molecule to be modified with human glycosylation patterns. Alternatively, other eukaryotic cell lines can be used. The selection of suitable mammalian host cells, as well as methods for transformation, culture, amplification, screening, and product production and purification, are known in the art.

It can be shown that bacterial cells can be used as host cells, which are suitable for expressing the recombinant Fab of the present invention or other embodiments. However, since proteins expressed in bacterial cells tend to be in an unfolded form or incorrectly folded form or non-glycosylated form, any recombinant Fab produced in bacterial cells must be screened to retain antigen binding ability. If the molecule expressed by the bacterial cell is produced in a properly folded form, the bacterial cell will be the desired host, or, in an alternative embodiment, the molecule can be expressed in the bacterial host and then refolded subsequently. For example, various strains of E. coli used for expression are well-known host cells in the field of biotechnology. Various strains of Bacillus subtilis, Streptomyces, and other Bacillus genera can also be used in this method.

If necessary, yeast cell strains and insect cells known to those skilled in the art, such as Drosophila and Lepidopteran insects and virus expression systems, can also be used as host cells.

In some embodiments, the nucleic acid is inserted into the cell genome and can be stably expressed.

The method of insertion can be the vector as described above, or the nucleic acid can be directly transferred into the cell without being connected to the vector (for example, liposome-mediated transfection technology).

The present invention also provides a method for producing the antibody as described above, including:

Culturing the host cell as described above under suitable culture conditions; and

The antibody thus produced is recovered from the culture medium or from the cultured host cells.

The general method of constructing the vector, the transfection method required to produce the cell of the present invention, and the culture method necessary to produce the antibody of the present invention from the cell may all be conventional techniques. The culture method of the present invention is usually a serum-free culture method, usually by culturing cells in a serum-free suspension. Similarly, once the antibody of the present invention is produced, it can be purified from cell culture contents according to standard procedures in the art, including ammonium sulfate precipitation, affinity column, column chromatography, gel electrophoresis, and the like. These techniques are well known within the technical scope of the art. Another method of expressing antibodies can utilize expression in animals (especially transgenic animals or nude mice). This involves an expression system that utilizes the animal casein promoter, which when it is transgenicly incorporated into a mammal, allows female animals to produce the desired recombinant protein in their milk. The culture medium from which the antibody is secreted can be purified by conventional techniques. For example, use A or G Sepharose FF column with adjusted buffer for purification. Wash away non-specifically bound components. Then the bound antibody was eluted by the pH gradient method, and the antibody fragment was detected by SDS-PAGE and collected. The antibody can be filtered and concentrated by conventional methods. Soluble mixtures and polymers can also be removed by conventional methods, such as molecular sieves and ion exchange. The resulting product needs to be frozen immediately, such as -70°C, or lyophilized.

The present invention also provides a pharmaceutical composition, which includes the antibody as described above, and one or more of pharmaceutically acceptable excipients, diluents or carriers. The term "pharmaceutically acceptable excipient, diluent or carrier" refers to an excipient, diluent or carrier that is pharmacologically and/or physiologically compatible with the subject and the active ingredient, which is well known in the art , Including but not limited to: pH adjusters, surfactants, adjuvants, ionic strength enhancers. For example, pH adjusters include, but are not limited to, phosphate buffer; surfactants include, but are not limited to, cationic, anionic or nonionic surfactants, such as Tween-80; and ionic strength enhancers include, but are not limited to, sodium chloride.

In some embodiments, the pharmaceutical composition is used to treat or prevent immune diseases or tumors.

In some embodiments, the immune disease is selected from: systemic lupus erythematosus, multiple sclerosis, type I diabetes, psoriasis, ulcerative colitis, Sjogren syndrome, scleroderma, polymyositis, rheumatoid Arthritis, mixed connective tissue disease, primary biliary cirrhosis, autoimmune hemolytic anemia, Hashimoto's thyroiditis, Addisons disease, leukoplakia, Graves disease, myasthenia gravis, ankylosing spondylitis, allergies Osteoarthritis, allergic vasculitis, autoimmune neutropenia, idiopathic thrombocytopenic purpura, lupus nephritis, chronic atrophic gastritis, autoimmune infertility, endometriosis , Pasture disease, pemphigus, discoid lupus and dense deposits disease.

In some embodiments, the tumor is selected from: bone, bone connection, muscle, lung, trachea, heart, spleen, artery, vein, blood, capillary, lymph node, lymphatic vessel, lymphatic fluid, oral cavity, pharynx, esophagus, Stomach, duodenum, small intestine, colon, rectum, anus, appendix, liver, gallbladder, pancreas, parotid gland, sublingual gland, urinary kidney, ureter, bladder, urethra, ovary, fallopian tube, uterus, vagina, vulva, scrotum , Testicles, vas deferens, penis, eyes, ears, nose, tongue, skin, brain, brainstem, medulla oblongata, barren medulla, brain barren fluid, nerves, thyroid, parathyroid gland, adrenal gland, pituitary, pineal gland, pancreatic islets, thymus , Gonads, sublingual glands, and parotid glands.

In some embodiments, the pharmaceutical composition is used in combination with one or two or more selected from the group consisting of: antifolate, calcineurin inhibitor, corticosteroid, antithymocyte globulin, nucleic acid Antimetabolites, nucleic acid synthesis inhibitors, biological agents that target cell surface antigens, biological agents that target cytokines or cytokine receptors, and intravenous immunoglobulins.

In addition, the pharmaceutical composition may also contain at least one of an anticancer agent, a cytotoxic agent, and a chemotherapeutic agent.

The term "anti-cancer agent" means any agent that can be used to treat cell proliferative disorders such as cancer, and includes, but is not limited to, cytotoxic agents, cytostatic agents, anti-angiogenic agents, radiotherapy and radiotherapeutics, targeted anti-cancer agents Agents, BRM, therapeutic antibodies (which do not include LAG-3 as defined in the present invention), cancer vaccines, cytokines, hormone therapy, radiotherapy and anti-metastatic agents and immunotherapeutics. It should be understood that in selected embodiments as described above, such anti-cancer agents may comprise conjugates and may bind to the disclosed site-specific antibodies prior to administration. More specifically, in certain embodiments, the selected anticancer agent is linked to the unpaired cysteines of the engineered antibody to provide the engineered conjugate as described herein. Therefore, such engineered conjugates are clearly considered within the scope of the present invention. In other embodiments, the disclosed anticancer agents will be administered in combination with site-specific conjugates comprising different therapeutic agents as described above.

As used herein, the term "cytotoxic agent" refers to a substance that is toxic to cells and reduces or inhibits cell function and/or causes cell destruction. In certain embodiments, the substance is a naturally occurring molecule derived from a living organism. Examples of cytotoxic agents include, but are not limited to: bacteria (e.g., diphtheria toxin, Pseudomonas endotoxin and exotoxin, staphylococcal enterotoxin A), fungi (e.g. α-sarcin, limited toxin), Small molecular toxins or enzymatically active toxins of plants (acacia, ricin, capsula root toxin, quercetin, pokeweed antiviral protein, saponin, gelonin, momoridin, trichosanthin, barley Toxins, Aleurites fordii protein, caryophyllin protein, Phytolacca mericana protein (PAPI, PAPII and PAP-S), bitter melon inhibitor, jatropha toxin protein, croton toxin, sulphur inhibitor, gelonin, mitigellin , Limited to aspergillin, phenomycin, neomycin and trichothecenes), or animal small molecule toxins or enzymatically active toxins (such as cytotoxic RNase, such as extracellular pancreatic RNase; DNase I, including fragments and/or variants thereof).

For the purposes of the present invention, "chemotherapeutic agents" include chemical compounds that non-specifically reduce or inhibit the growth, proliferation, and/or survival of cancer cells (e.g., cytotoxic agents or cytostatic agents). These chemical reagents generally target intracellular processes required for cell growth or division, and therefore are particularly effective for cancer cells that usually grow and divide rapidly. For example, vincristine depolymerizes microtubules, thereby inhibiting cells from entering mitosis. Generally, chemotherapeutic agents can include any chemical agent that inhibits or is designed to inhibit cancer cells or cells that may become or produce tumorigenic progeny (e.g., TIC). These agents can usually be used in combination and are usually the most effective, for example, in regimens such as CHOP or FOLFIRI.

Examples of anticancer agents that can be used in combination with the antibodies of the present invention, antigen-binding fragments thereof, or variants thereof include, but are not limited to, alkylating agents, alkyl sulfonates, aziridines, ethyleneimine and methylmelamine, poly Acetogenins, camptothecin, bryostatin, callistatin, CC-1065, cryptophycins, dolastatin, docarmycin, eleutherobin , Saccharine, sarcodictyin, spongistatin, nitrogen mustard, antibiotics, enediyne antibiotics, dynemicin, bisphosphonates, espotomycin, pigment protein enediyne antibiotics Chromophore, aclacinomysins, actinomycin, antoxomycin, azoserine, bleomycin, actinomycin C, carabicin, carabicin, carcinoma Chromomycin, chromomycin (chromomycinis), dactinomycin, daunorubicin, ditorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin Picin, esorubicin, idarubicin, methiromycin, mitomycin, mycophenolic acid, nogamycin, olivemycin, pelomycin, potfiromycin (potfiromycin), Puromycin, triiron doxorubicin, rhodoubicin, streptozotocin, streptozotocin, tuberculin, ubiquitin, gentatin, zorubicin; anti-metabolite, Erlotinib, verofenib, crizotinib, sorafenib, ibrutinib, enzalutamide, folate analogs, purine analogs, androgens, anti-adrenaline, folic acid supplements such as Folinic acid, acetoglucurolactone, aldophosphamide glycoside, aminolevulinic acid, eniluracil, amsacrine, bestrabucil, bisantrene, edatrexate, Diff method Defofamine, colchicine, diacridone, elfornithine, elfornithine, elfornithine, Ebosirone, Etoglu, gallium nitrate, hydroxyurea, lentinan, clonidamine, maytan Maytansinoids, mitoxantrone, mitoxantrone, mopidanmol, nitraerine, pentostatin, chlorambucil, pirarubicin, losoxantrone, Podophyllic acid, 2-Ethylhydrazine, Procarbazine, Polysaccharide Complex (JHS Natural Products, Eugene, OR), Razosan; Rhizomycin; Cizonan; Gespiramine; Tinozoric acid; Triimine quinone ; 2,2',2”-Trichlorotriethylamine; Trichothecenes (especially T-2 toxin, verracurin A (verracurin A), baculosporin A and serpentine) ; Uratan; vindesine; dacarbazine; mannmostine; dibromomannitol; dibromodulcol; pipepobromide; cassitosin (gacytosi ne); arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxanes; chloranbucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum Analogues; Vinblastine; Platinum; Etoposide (VP-16); Ifosfamide; Mitoxantrone; Vincristine, Vinorelbine; Nuoxiaoling; Teniposide; Edaroxa; Erythromycin; Aminopterin; Xeloda; Ibandronate; Irinotecan (Camptosar, CPT-11); Topoisomerase inhibitor RFS 2000; Difluoromethylornithine; Retinoids; Capecitabine; combstatin; leucovorin; oxaliplatin; inhibitors of PKC-α, Raf, H-Ras, EGFR and VEGF-A (which reduce cell proliferation), and any of the above The pharmaceutically acceptable salt, acid or derivative of. Anti-cancer agents that can be used in combination with the antibody, antigen-binding fragment thereof, or variants of the present invention also include: anti-hormonal agents used to modulate or inhibit the hormonal effect on tumors, such as anti-estrogens and selective estrogen receptors Modulators, aromatase inhibitors that inhibit aromatase that regulates estrogen production in the adrenal glands, and anti-androgens; and troxatabine (1,3-dioxolane nucleoside cytosine analogue); Antisense oligonucleotides, ribozymes such as VEGF expression inhibitors and HER2 expression inhibitors; vaccines, rIL-2; topoisomerase 1 inhibitors; rmRH; vinorelbine and espermycin, and any of the above The pharmaceutically acceptable salt, acid or derivative of.

The present invention also provides a kit, which contains at least one of the following components:

i) The antibody or its antigen-binding fragment as described above, and optionally a container for holding the antibody or its antigen-binding fragment;

ii) The pharmaceutical composition as described above, and optionally a container for holding the pharmaceutical composition.

In some embodiments, the components of i) or ii) in the kit are separately packaged in the form of a unit dose, wherein the unit dose contains a predetermined amount of the composition or antibody or antigen-binding fragment thereof. For other embodiments, this unit dose is supplied in a single-use pre-filled injection syringe. In other embodiments, the composition contained in the unit dose may include saline, sucrose, or the like; buffers, such as phosphate, etc.; and/or formulated in a stable and effective pH range. Alternatively, in certain embodiments, the antibody can be provided as a lyophilized powder, which can be reconstituted after adding a suitable liquid (e.g., sterile water or saline solution). In certain preferred embodiments, the composition includes one or more substances that inhibit protein aggregation/degradation, including but not limited to sucrose and arginine. Any label on or associated with the container indicates that the encapsulated conjugate composition is used to treat the selected tumor or immune disease condition.

The pharmaceutical composition can be used for LAG-3 related diseases, especially immune diseases or tumors. Therefore, in particular, the present invention also relates to a method for treating/preventing an immune disease or tumor in a subject in need thereof, the method comprising:

a) providing the pharmaceutical composition; and

b) Administration of a therapeutically effective amount of the pharmaceutical composition to the subject.

The term "effective amount" refers to an amount sufficient to obtain or at least partially obtain the desired effect. For the desired effect, for example, prevention or treatment of immune diseases or tumors, an effective amount is usually an amount sufficient to prevent, prevent, or delay the occurrence of the disease. It is completely within the abilities of those skilled in the art to determine such an effective amount. For example, the effective amount for therapeutic use will depend on the severity of the disease to be treated, the overall state of the subject’s own immune system, the subject’s general conditions such as age, weight, and sex, the way the drug is administered, and other treatments that are administered at the same time and many more. Through any clinical testing methods commonly used by doctors or other professional health care professionals to evaluate the severity or progression of the symptoms, it can be evaluated whether the symptoms of the disease have been alleviated.

In some embodiments, the method of administration may be injection administration or the like, for example.

The method can also be combined with radiotherapy (ie, any mechanism used to locally induce DNA damage in tumor cells, such as γ-irradiation, X-ray, UV-irradiation, microwave, electron emission, etc.) to treat/prevent the above disease. Combination therapy using targeted delivery of radioisotopes to tumor cells is also considered, and the disclosed conjugates can be used in combination with targeted anticancer agents or other targeted means. Generally, radiation therapy is administered in pulses over a period of about 1 week to about 2 weeks. Radiation therapy can be administered to subjects with head and neck cancer for about 6 to 7 weeks. Optionally, radiation therapy can be administered as a single dose or as multiple sequential doses.

In the present invention, vocabulary such as "subject", "subject", "patient", etc. can be used universally as needed. The subject may be a mammal, preferably a human.

The embodiments of the present invention will be described in detail below in conjunction with examples. The experimental methods that do not specify specific conditions in the examples of the present invention usually follow conventional conditions, such as Cold Spring Harbor's antibody technology experiment manual, molecular cloning manual; or according to the conditions recommended by the raw material or commodity manufacturer. The reagents without specific sources are the conventional reagents purchased on the market.

Example 1 Preparation of LAG-3 Antigen and Antibody

1. Protein design and expression

Using UniProt LympHocyte activation gene 3 protein (human LAG-3, Uniprot number: P18627) as the template of LAG-3 of the present invention, design the amino acid sequence of the antigen and detection protein involved in the present invention, optionally on the basis of LAG-3 protein Fusion of different tags, respectively cloned into pHL vector (Shanghai Hailu Biotechnology Co., Ltd.) or pTT5 vector (Biovector, Cat#: 102762) or pTargeT vector (promega, A1410), transient expression in 293 cells or CHO- S is stably expressed and purified to obtain the antigen encoding the present invention and the protein for detection.

The following LAG-3 antigens without special instructions refer to human LAG-3

LAG-3-Flag-his: LAG-3 extracellular region with Flag-his label, used to immunize mice.

Note: The underlined part is the signal peptide, and the italicized part is the flag-his-tag tag.

Full length LAG-3: used to construct LAG-3 overexpression cell line, immunize mice and detect

Note: The underlined part is the signal peptide, and the double-lined part is the transmembrane region.

LAG-3-hFc: A fusion protein of the extracellular region of LAG-3 and the Fc segment of human IgG1 (hIgG1Fc) for detection or immunization

Note: The underlined part is the signal peptide, the double-lined part is the linker, and the italicized part is the hIgG1 part.

LAG-3-mFc: A fusion protein of LAG-3 extracellular domain and mouse IgG2a Fc segment (mIgG2a Fc), used for detection or immunization

Note: The underlined part is the signal peptide, and the italicized part is the Fc segment of mouse IgG2a.

2. Purification of LAG-3 related recombinant proteins, and purification of hybridoma antibodies and recombinant antibodies

1) Purification method of Flag-his tagged protein:

Centrifuge the cell expression solution at high speed, collect the supernatant, and discard the precipitate. The HisTrap FF prepacked column is equilibrated with phosphate buffered saline (PBS) at 5-10 column volumes. The cell expression supernatant was loaded at a rate of 2ml/min. Rinse the pre-packed column with PBS until the mAu reading reaches the baseline, and then eluate the target protein with 20mM, 50mM, 250mM pH7.4 imidazole and collect it, and finally transfer the target protein solution eluted with 250mM imidazole to the concentration tube and centrifuge , Change the medium, and replace the target protein in the PBS solution for storage for subsequent experiments.

2) Purification of hybridomas, recombinant antibodies, and Fc fusion proteins

The cell expression supernatant sample was centrifuged at high speed to remove impurities, the hybridoma expression supernatant was purified with a Protein G column, and the recombinant antibody and Fc fusion protein expression supernatant was purified with a Protein A column. Rinse the column with PBS until the A280 reading drops to baseline. The target protein was eluted with 100mM acetic acid, pH3.0, and neutralized with 1M Tris-HCl, pH8.0.

After the eluted sample is appropriately concentrated, it is further purified by PBS-balanced gel chromatography Superdex200 (GE) to remove the components corresponding to the polymer peaks, and collect the components corresponding to the monomer peaks for use in equipment.

Example 2 Preparation of anti-human LAG-3 hybridoma monoclonal antibody

1. Immunity

Anti-human LAG-3 monoclonal antibody is produced by immunizing mice. Experimental Balb/C mice, female, 6 weeks old (Beijing Weitong Lihua Experimental Animal Technology Co., Ltd., animal production license number: SCXK (Beijing) 2012-0001). Feeding environment: SPF level. After the mice are purchased, they are reared in a laboratory environment for 1 week, 12/12 hours light/dark cycle adjustment, temperature 20℃～25℃; humidity 40%～60%. Mice that have adapted to the environment were immunized according to the following protocol. The immune antigen is the extracellular region of human LAG-3 with Fc and Flag-his tags (SEQ ID NO: 25, 27, 28).

Immunization program: CFA (sigma&F5506-10ML) and IFA (sigma&F5881-10ML) are used for routine immunization. For the first immunization, use a mixture of antigen and adjuvant CFA (sigma&F5506-10ML) with a volume ratio of 1:1, 50μg/head/time, and a total volume of 200ul/head (first immunization); booster immunization (from the second immunization, a total of 2 Second booster immunization) Use antigen and adjuvant IFA (sigma&F5881-10ML) with a volume ratio of 1:1, 25 μg/head/time, total volume 200ul/head (boost immunization). After antigen emulsification, vaccination is performed on the 0th, 14th, and 28th days. On day 0, the mixture of emulsified antigen and adjuvant CFA was injected intraperitoneally (IP) with 50 μg/head for the first immunization. On the 14th day, 25 μg/mouse of emulsified antigen and adjuvant IFA were injected intraperitoneally (IP) for the first booster immunization. On the 28th day, 25 μg/mouse of emulsified antigen and adjuvant IFA were injected intraperitoneally (IP) for the second booster immunization. Blood was taken on the 21st and 35th day, and the antibody titer in the mouse serum was determined by ELISA. After 3 immunizations, mice with high antibody titer in the serum and the titer tending to the plateau were selected for splenocyte fusion. Three days before the fusion of spleen cells, the booster immunization was an antigen solution prepared by intraperitoneal (IP) injection of 50 μg/head of physiological saline.

2. Spleen cell fusion

The optimized PEG (polyethylene glycol)-mediated fusion step was used to combine splenic lymphocytes with myeloma cells Sp2/0 cells (

CRL-8287 ^™ ) was fused to obtain hybridoma cells. The fused hybridoma cells are resuspended in complete medium (DMEM medium containing 20% FBS, 1×HAT, 1×OPI) at a density of ^{0.5-1×10 6 /ml, and seeded in a 96-well plate with 100 μl/well} After incubating at 37°C and 5% CO ₂ for 3 to 4 days, supplement with 100 μl/well of HAT complete medium, and continue to culture for 3 to 4 days until a needle-like clone is formed. Remove the supernatant, add 200μl/well of HT complete medium (RPMI-1640 medium containing 20% FBS, 1×HT and 1×OPI), culture at 37°C, 5% CO _{2 for} 3 days, and then perform ELISA detection.

3. Hybridoma cell screening

According to the growth density of hybridoma cells, the hybridoma culture supernatant was detected by the combined ELISA method (see Test Example 1). The cell supernatant of positive wells detected by ELISA was used for cell blocking experiment (see Test Example 3). The well cells that are positive for binding and blocking are expanded in time, cryopreserved for seed preservation, and subcloned two to three times until a single cell clone is obtained.

Each batch of subcloned cells needs to be tested by LAG-3 binding ELISA and cell blocking experiment (see Test Example 1 and Test Example 3). The hybridoma clones were obtained through the above experiment screening, and the antibody was further prepared by the serum-free cell culture method. The antibody was purified according to the purification example for use in the test example.

4. Sequence determination of hybridoma positive clones

The process of obtaining the clone sequence from the positive hybridoma is as follows. The logarithmic growth phase hybridoma cells were collected, and RNA was extracted with Trizol (Invitrogen, Cat No. 15596-018) according to the instructions of the ^{kit, and reverse transcription with PrimeScript TM} Reverse Transcriptase kit (Takara, Cat No. 2680A). The cDNA obtained by reverse transcription was amplified by PCR using mouse Ig-Primer Set (Novagen, TB326 Rev. B 0503) and sent to a sequencing company for sequencing. The amino acid sequences corresponding to the variable region DNA sequences of the heavy chain and light chain of the obtained hybridoma clone 209B1 are as follows:

209B1-VL

209B1-VH

Note: The italic in the sequence is the FR sequence, and the underline is the CDR sequence.

Table 1 The sequence of each heavy chain and light chain CDR region

Example 3 Humanization of anti-human LAG-3 murine hybridoma monoclonal antibody mAb209B1

By comparing the IMGT human antibody heavy and light chain variable region germline gene database and MOE software, the heavy chain and light chain variable region germline genes with high homology to mAb209B1 were selected as templates, and the CDRs of the murine antibody were respectively selected. Transplanted into the corresponding human template to form the variable region sequence of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. The amino acid residues are identified and annotated by the Kabat numbering system.

1. Humanized framework selection of hybridoma clone mAb209B1

The humanized light chain templates of the murine antibody mAb209B1 are IGKV1-39*01 and hjk4.1, and the humanized heavy chain templates are IGHV1-18*01 and hjh6.1. The humanized variable region sequences are as follows:

Hu209B1VL1 CDR graft

Hu209B1VH1 CDR graft

Note: The italic in the sequence is the FR sequence, and the underline is the CDR sequence.

2. The template selection and back mutation design of hybridoma clone mAb209B1 are shown in Table 2 below:

Table 2 209B1 humanized design

Note: For example, I48M means that the 48-bit I is mutated back to M according to the Kabat numbering system. Grafted represents the sequence of the mouse antibody CDR implanted into the human germline FR region.

Table 3 Mouse anti-mAb209B1 humanized sequence combination

Note: This table shows the sequence of various combinations of mutations. As indicated by Hu209B1L1H1, the humanized mouse antibody Hu209B1L1H1 has two mutations in the light chain Hu209B1VL1 and the heavy chain Hu209B1VH1. Other analogy.

The specific sequence of mAb209B1 humanization is as follows:

>Hu209B1VL1 (same as Hu209B1VL1 CDR graft)

>Hu209B1VL2

>Hu209B1VL3

>Hu209B1VH1 (same as Hu209B1VH1 CDR graft)

>Hu209B1VH2

>Hu209B1VH3

>Hu209B1VH4

Example 4. Preparation of recombinant and humanized antibodies

For the antibody, the constant region of human heavy chain IgG4/light chain kappa is combined with each variable region, and the S228P mutation is made in the Fc segment to increase the stability of the IgG4 antibody. Other mutations known in the art can also be used to increase its performance.

Heavy chain constant region:

Light chain constant region:

1. Molecular cloning of recombinant antibodies

After the positive antibody molecules obtained from hybridoma screening are sequenced, the variable region coding gene sequence is obtained. Design the head and tail primers according to the sequence obtained by sequencing, use the sequenced gene as a template to amplify each antibody VH/VK gene fragment by PCR, and then combine it with the expression vector pHL (with signal peptide and hIgG4/hkappa constant region gene (CH1-FC/CL) fragment ) Carry out homologous recombination to construct the recombinant antibody full-length expression plasmid VH-CH1-FC-pHL/VL-CL-pHL.

2. Molecular cloning of humanized antibodies

The antibody sequence after human design, after codon optimization, produces the coding gene sequence of human codon preference. The primer PCR is designed to amplify the VH/VK gene fragments of each antibody, and then it is combined with the expression vector pHL (with signal peptide and hIgG4/hkappa). The constant region gene (CH1-FC/CL) fragment) undergoes homologous recombination to construct the humanized antibody full-length expression plasmid VH-CH1-FC-pHL/VL-CL-pHL.

3. Expression and purification of recombinant and humanized antibodies

The plasmids expressing the antibody light and heavy chains were transfected into HEK293 cells at a ratio of 1:1.2, and the expression supernatant was collected 6 days later, centrifuged at high speed to remove impurities, and purified with a Protein A column. Rinse the column with PBS until the A280 reading drops to baseline. The target protein was eluted with pH3.0 acidic eluent, and neutralized with 1M Tris-HCl, pH8.0. After the eluted sample is properly concentrated, it is further purified by PBS-balanced gel chromatography Superdex200 (GE) to remove aggregates, collect monomer peaks, and divide them for use.

The following biochemical test methods are used to verify the performance and beneficial effects of the antibody of the present invention.

Test Example 1: ELISA experiment of LAG-3 antibody binding to human LAG-3 protein

The binding force of anti-LAG-3 antibody was tested by ELISA test of antibody and human LAG-3 protein.

The LAG-3 fusion protein with hFc or mFc tag is fixed to 96-well microtiter plate by binding to the anti-hFc or mFc antibody coated in the microtiter plate. The strength of the signal after the antibody is added is used to determine the antibody and LAG-3 binding activity. The specific experimental methods are as follows:

Dilute the goat anti-mouse Fc antibody (Sigma-Aldrich, Cat No.M4280-1ML) to a concentration of 1μg/ml with PBS (Hyclone, Cat No. SH30256.01) buffer of pH 7.4, and add in a volume of 100μl/well Block the 96-well microtiter plate in a refrigerator at 4°C overnight. The next day, the plate was taken out and returned to room temperature, the coating solution was discarded, and the plate was washed once with PBST buffer (pH7.4PBS containing 0.05% tween-20), and then 2% bovine serum albumin (BSA) diluted with PBS was added ( Sangon Biotech, Cat No. A602449) blocking solution 200μl/well, room temperature or 37°C incubator for 1 hour for blocking. After blocking, discard the blocking solution and wash the plate 3 times with PBST buffer, then add 100μl/well of LAG-3-mFc (IgG2a) fusion protein (ACRO) diluted to 1ug/ml with sample diluent (2%BSA) , Cat No. LA3-H52Aa or self-produced protein: SEQ ID NO 4), incubate in an incubator at room temperature or 37°C for 1 hour. After the incubation, discard the reaction solution in the ELISA plate, wash the plate 3 times with PBST, add 100μl/well of different concentrations of the antibody to be tested diluted with the sample diluent, and incubate at room temperature or 37°C in an incubator for 1 hour. After incubation, wash the plate with PBST 3 times, add 100μl/well HRP-labeled goat anti-human secondary antibody (Jackson Immuno Research, Cat No.109-035-033) diluted with sample diluent, and incubate at room temperature or 37°C for 1 hour . After washing the plate 6 times with PBST, add 50μl/well TMB chromogenic substrate (KPL, Cat No.52-00-03), incubate at room temperature for 5-15min, add 50μl/well 1M H ₂ SO _{4 to} stop the reaction, and use The Thermo MultisKan Fc microplate reader reads the absorbance value at the wavelength of 450nm, and calculates the EC50 value of LAG-3 antibody binding to human LAG-3. The results are shown in Table 4. The data shows that the chimeric antibodies screened by the present invention have high binding activity to human LAG-3 protein.

Table 4 Determination of EC50 of candidate antibodies in binding experiments

Test Example 2: Binding experiment between LAG-3 antibody and human LAG-3 overexpressing CHO-K1 cells

The binding capacity of anti-LAG-3 antibody was tested by binding the antibody to CHO-K1 cells overexpressing LAG-3 protein. The LAG-3 full-length plasmid (internally produced, SEQ ID NO: 2) was transfected into CHO-K1 cells by liposome transfection method, and after two weeks of pressurized screening, the expression of LAG-3 was detected. Then subcloned to obtain the appropriate expression amount of monoclonal cell line. The overexpression cell line is incubated with the antibody, and the signal strength after the antibody is added is used to determine the binding activity of the antibody and LAG-3 overexpressing CHO-K1 cells. The specific experimental method is as follows.

The cells were plated in a 96-well U bottom plate at a density of ^{1×10 5 /ml and 100 μl/well.} Centrifuge the plate at 1500 rpm for 5 min, and discard the supernatant. Add 100μl/well of the antibody to be tested in different concentrations diluted with sample diluent (pH7.4PBS containing 0.1% BSA), gently pipette to mix, and incubate in a refrigerator at 4°C for 1 hour. Take out the plate and centrifuge at 1500 rpm for 5 min, and discard the supernatant. Add the sample diluent to the plate at 200μl/well, and gently pipette the cells to mix. The mixed solution was centrifuged at 1500rpm for 5min. Repeat the wash 3 times. Add 100 μl/well of PE-labeled goat anti-human secondary antibody (Jackson Immuno Research, Cat No. 109-115-098) diluted with sample diluent, and incubate in a refrigerator at 4° C. for 30 min. Take out the plate at 1500rpm, centrifuge for 5min, and discard the supernatant. Add the sample diluent to the plate at 200μl/well, and gently pipette the cells to mix. The mixed solution was centrifuged at 1500rpm for 5min. Repeat the wash 3 times. 150μl/well sample diluent was resuspended and tested with BD FACScelesta flow cytometer. Calculate the binding EC50 of LAG-3 antibody to LAG-3 overexpressing CHO-K1 cells, as shown in Figure 1 and Table 5.

Table 5 Determination of EC50 of candidate antibodies in binding experiments

Candidate Antibody	EC50(ng/ml)
Hu209B1L1H1	92.14
Hu209B1L1H2	84.68
Hu209B1L1H3	94.08
Hu209B1L1H4	81.57
Hu209B1L2H1	98.94
Hu209B1L2H2	93.15
Hu209B1L2H3	77.39
Hu209B1L2H4	86.04
Hu209B1L3H1	78.77
Hu209B1L3H2	80.43
Hu209B1L3H3	132.8
Hu209B1L3H4	126.1

Test Example 3: Anti-LAG-3 antibody blocking LAG-3 antigen and Daudi cell binding experiment

Daudi cells (human leukemia cells, purchased from the cell bank of the Chinese Academy of Sciences) or Raji cells (human Burkitts lymphoma cells, purchased from the cell bank of the Chinese Academy of Sciences) were plated in a 96-well U bottom plate at an amount of ^{1×10 5 /well.} After centrifuging the plate at 1500 rpm for 5 minutes, add 100 μl/well of sample mixture (the mixture is a mixture of gradient concentration of the test antibody and LAG3-mFc fusion protein with a final concentration of 1 ug/ml, incubated for 1 hour), and set at 4°C Incubate in the refrigerator for 1 hour. After the incubation, the plate was taken out at 1500 rpm, centrifuged for 5 min, and the supernatant was discarded. Add the sample diluent to the plate at 200 μl/well, gently pipette the cells to mix, and centrifuge at 1500 rpm for 5 min. Repeat the wash 3 times. Add 100 μl/well of PE-labeled goat anti-mouse secondary antibody (Jackson Immuno Reasearch, Cat No. 115-116-071) diluted with sample diluent, and incubate in a refrigerator at 4° C. for 30 min. Take out the plate at 1500rpm, centrifuge for 5min, and discard the supernatant. Add the sample diluent to the plate at 200μl/well, gently pipette the cells to mix, and centrifuge at 1500rpm for 5min. Repeat the wash 3 times. 150μl/well sample diluent was resuspended and tested with BD FACScelesta flow cytometer. Calculate the blocking effect of LAG-3 antibody on antigen binding to Daudi cells or Raji cells. The results are shown in Figure 2 and Table 6. The data shows that the humanized antibodies screened in the present invention can significantly block the binding of human LAG-3 antigen to Daudi cells or Raji cells.

Table 6 Determination of IC50 of candidate antibodies in binding and blocking experiments

Candidate Antibody	IC50(ng/ml)
Hu209B1L1H1	7.32
Hu209B1L1H2	10.57
Hu209B1L1H3	8.31
Hu209B1L1H4	7.08
Hu209B1L2H1	5.86
Hu209B1L2H2	6.46
Hu209B1L2H3	7.01
Hu209B1L2H4	7.12
Hu209B1L3H1	6.6
Hu209B1L3H2	6.73
Hu209B1L3H3	10.37
Hu209B1L3H4	10.21

Test Example 4: Biacore detects LAG-3 antibody affinity test

According to the method described in the instructions of the mouse anti-capture kit (GE, Cat.#BR-1008-38), the mouse anti-capture antibody was covalently coupled to the CM5 biosensor chip (Cat.#BR-1000-12, GE ), so as to affinity capture the antibody to be tested, and then flow through the LAG3-his (Acro, Cat No. LA3-H5222) antigen on the chip surface, and use the Biacore instrument to detect the reaction signal in real time to obtain the binding and dissociation curves, which are obtained by fitting For the affinity value, see Table 7 below. After each cycle of dissociation in the experiment is completed, the biochip is washed and regenerated with the regeneration solution configured in the mouse anti-capture kit. The results show that LAG-3 antibody has strong binding activity and affinity to human LAG-3 protein.

Table 7 Affinity of anti-LAG-3 antibodies

Candidate Antibody	Ka(1/MS)	Kd(1/s)	KD(M)
mAb209B1	1.37E+07	7.17E-05	5.22E-12

According to the method described in the instructions of the human anti-capture kit (GE, Cat No. BR-1008-39), the human anti-capture antibody was covalently coupled to the CM5 biosensor chip (GE, Cat No. BR-1000-12). ), to affinity capture the antibody to be tested, and then flow through the LAG3-his (Acro, Cat No. LA3-H5222) antigen on the chip surface, and use the Biacore T200 instrument to monitor the reaction signal in real time to obtain the binding and dissociation curves. Obtain the affinity constant, see Table 8 below. After each cycle of dissociation in the experiment is completed, the CM5 chip is regenerated with the regeneration solution configured in the human anti-capture kit. The results show that the antibodies screened by the present invention have strong binding activity and affinity to human LAG-3 protein.

Table 8: Affinity of anti-LAG-3 antibodies

Candidate Antibody	Ka(1/MS)	Kd(1/s)	KD(M)
Hu209B1L1H1	2.23E+07	2.28E-04	1.02E-11
Hu209B1L1H2	4.22E+06	1.25E-04	2.96E-11
Hu209B1L1H3	1.14E+07	1.58E-04	1.39E-11
Hu209B1L1H4	1.12E+07	1.49E-04	1.33E-11
Hu209B1L3H1	1.39E+07	1.07E-04	7.70E-12
Hu209B1L3H2	1.27E+07	1.03E-04	8.11E-12
Hu209B1L3H3	1.17E+07	1.19E-04	1.02E-11
Hu209B1L3H4	1.16E+07	1.25E-04	1.08E-11
Hu209B1L2H1	1.43E+07	1.23E-04	8.60E-12
Hu209B1L2H2	1.23E+07	1.12E-04	9.11E-12
Hu209B1L2H3	1.05E+07	1.23E-04	1.17E-11
Hu209B1L2H4	7.02E+06	1.44E-04	2.05E-11

Test Example 5: PBMC-T lymphocyte activation experiment

In order to study the activation of T lymphocytes by LAG-3 antibody, human peripheral blood mononuclear cells (PBMC) were collected and purified, and the superantigen Staphylococcus aureus enterotoxin B (SEB) was used to stimulate in vitro for 72 hours to detect the IL-2 cytokine Secretion level. The experiment process is briefly described as follows:

Freshly isolated and purified PBMCs were inoculated into a 96-well cell culture plate with a cell density of about 1×10 ⁵ /well, 100ng/mL SEB super antigen was added for stimulation, and a serially diluted antibody sample (diluted with medium) or medium was added at the same time As a blank control. After culturing in a 37°C, 5% CO ₂ incubator for 72 hours, the cell culture supernatant was collected. ELISA (BD, CAT#550611) method was used to detect IL-2 secretion level in cell culture supernatant. Refer to the reagent instructions for specific operations. The results are shown in Figure 3, the LAG-3 humanized candidate antibody can enhance the secretion of the cytokine IL-2 by activated T lymphocytes to varying degrees, and has a drug concentration and dose effect. The corresponding EC _{50 is} shown in Table 9.

Table 9 PBMC-T lymphocyte activation test results

Candidate Antibody	EC 50
Hu209B1L1H1	404
Hu209B1L1H2	0.008878
Hu209B1L1H3	2.40E-06
Hu209B1L1H4	2.63E-13
Hu209B1L2H1	450.2
Hu209B1L2H2	246.7
Hu209B1L2H3	482.8
Hu209B1L2H4	527.3
Hu209B1L3H1	706.6
Hu209B1L3H2	945.2
Hu209B1L3H3	2340
Hu209B1L3H4	1011

Test Example 6: Antigen-specific T cell response stimulation experiment

LAG-3 interacts with its ligand MHCII expressed on target cells (including APC or cancer cells), and inhibits T cell activation by turning off the positive signal initiated by the TCR signal.

In order to identify the ability of anti-LAG-3 antibodies to antagonize LAG-3 mediated signal transduction in T cells, we used the LAG-3 Blockade bioassay system (purchased from Promega, Cat No. CS194811) for functional activity detection experiments. MHCII APC cells (MHCII APC cells: TCR activating antigen = 1:1) were plated according to the method described in the instructions the day before the experiment, and cultured overnight in a carbon dioxide incubator at 37°C for 18-22 hours. The next day, the dilution of control antibody and test antibody and preparation of LAG-3 effector cells were performed. Finally, add antibody and LAG-3 effector cells to the APC cell plate according to the instructions and template. Incubate the APC cell plate in a carbon dioxide incubator at 37°C for 6 hours. According to the requirements of the Bio-GloTM Luciferase Assay System (Promega, Cat No. G7940) kit, add the substrate for color development, and incubate for 10 minutes at room temperature in the dark. Use Perkin Elmer Envision microplate reader for Luminescence reading. The EC50 of LAG-3 antibody antagonizing LAG-3 signal transduction in Jurkat cells was calculated. The results shown in Figure 4 and Table 10 show that the antibodies screened in the present invention can significantly block the binding of human LAG-3 antigen to MHC II on APC cells.

Table 10 Stimulation results of anti-LAG-3 monoclonal antibodies to antigen-specific T cell responses

Antibody number	IC50(ng/ml)
IgG4	NA
BMS986016	1200
209B1	314

Test Example 7: Inhibition experiment of LAG-3 antibody on MC38-hPD-L1 subcutaneous xenograft tumor

B-hPD1/hPD-L1/hLAG(V3) humanized mice were used to test the inhibitory effect of antibody drugs on the growth of MC38-hPD-L1 colon cancer subcutaneously transplanted tumors. MC38-hPD-L1 cells were inoculated subcutaneously on the right side of B-hPD1/hPD-L1/hLAG(V3) humanized mice at a concentration of 5×105 cells/0.1mL. When the tumor grew to about 84mm ³ , press the tumor Eighteen mice were selected by volume and randomly grouped, each with 6 mice, 4 female mice and 2 male mice, for a total of 3 groups. They are: G1Human IgG (10mg/kg), G2PD-1 monoclonal antibody (Keytruda) (0.3mg/kg), G3 209B1H1L1 (10mg/kg). The route of administration in all groups was intraperitoneal injection, twice a week for 6 consecutive administrations, and the experiment ended one day after the last administration. During the administration and observation period, the mouse body weight and tumor volume were measured twice a week, and the measured values were recorded. At the end of the experiment, the animals were euthanized, the tumor was stripped and the lymphocytes infiltrated by the tumor were detected by flow cytometry.

Table 11 Experimental administration of humanized anti-LAG-3 antibodies to MC38-hPD-L1 mice subcutaneously transplanted tumors

Tumor volume:

After grouping, the tumor volume was measured twice a week with a vernier caliper. The tumor volume was measured before euthanasia, and the long diameter and short diameter of the tumor were measured. The volume calculation formula is: tumor volume = 0.5 × long diameter × short diameter2.

Weight detection:

Inoculation, grouping (before the first administration), twice a week during the administration period, and weigh the animals before euthanasia.

General clinical observation:

Observe once a day during the adaptive feeding period and the experimental period. Observations include but are not limited to the rupture of tumor nodules, animal mental state, diet, etc.

The calculation formula of tumor volume inhibition rate (TGITV) is as follows:

TGITV(%)=[1-(Ti-T0)/(Vi-V0)]×100%

(Ti: mean tumor volume of the treatment group on day i of administration, T0: mean tumor volume of the treatment group on day 0 of administration; Vi: mean tumor volume of the solvent control group on day i of administration, V0: solvent (The mean tumor volume of the control group on day 0 of administration)

The calculation formula of tumor weight inhibition rate (TGITW) is as follows:

Tumor weight inhibition rate TGITW%=(W solvent control group-W treatment group)/W solvent control group×100%, W refers to tumor weight.

During the experiment, all animals were in good activity and eating state during the administration period, and their body weight increased to a certain extent, indicating that the animals tolerated each test product well. At the end of the experiment (the 17th day of group administration), the average tumor volume of the Human IgG group was 1482±156mm3, and the average tumor volume of the G2Keytruda (0.3mg/kg) and G3209B1H1L1 (10mg/kg) dose groups were 816±176mm3, respectively. 865±194mm3, the tumor volume growth inhibition rate TGITV was 47.7% and 44.1%, respectively. The experimental results were also confirmed in the tumor weight.

Table 12: The inhibitory effect of humanized anti-LAG-3 antibodies on MC38-hPD-L1 mice subcutaneous xenografts

Test Example 8: Physical stability of antibody

This test case is used to detect the stability of the anti-LAG-3 humanized antibody Hu209B1L1H1.

DSC (Differential Scanning Calorimetry, Differential Scanning Calorimetry) was used to detect the thermal stability of different antibodies and compared with the commercially available drug Herceptin in PBS (Hyclone, Cat No. SH30256.01, pH 7.4).

Control the sample concentration at about 1mg/ml and use MicroCal*VP-Capillary DSC (Malvern) for detection. Before testing, degas each sample and blank buffer with a vacuum degasser for 1 min to 2 min. Add 400μl of sample or blank buffer to each well of the sample plate (the loading volume of the instrument is 300μl). Add 14% Decon 90 and ddH ₂ O to the last two pairs of well plates for cleaning. After the sample plate is loaded, put on the plastic soft cover. The scanning temperature starts at 25°C and ends at 100°C, and the scanning rate is 60°C/h. The specific results are shown in Table 11. Compared with the marketed drug Herceptin, Hu209B1L1H1 and Hu209B1L3H1 both showed better thermal stability.

Table 13: Tm values of antibodies

The purity of the sample was monitored by SEC-HPLC, and the periodic stability under certain concentration conditions was investigated. The concentration of the sample was controlled at about 5mg/ml. The stability of the antibody stored at 40°C for one month was detected in the PBS system. Use Xbridge protein BEH SEC 200A (Waters) HPLC column detection. The test results are shown in Table 12. Compared with the marketed drug Herceptin, the antibody Hu209B1L1H1 showed better stability in PBS.

Table 14 Antibody purity test

Remarks: △% refers to the decrease rate of HPLC purity

Test Example 9. Chemical stability of antibodies

Deamidation modification is a common chemical modification in antibodies that may affect the stability of the later stage. In particular, the highly deamidated modification of some amino acids in the CDR region is generally avoided or minimized. Take out 100μg samples at different time points (5mg/mL), add to 30μL denaturation buffer (8M guanidine hydrochloride, 60mM Tris-HCl, 6mM EDTA), then add 1μl 1M DTT, water bath at 56℃ for 30min, add 2.5 μl 1M IAM, incubate at room temperature and dark for 30min. Add 200μl of enzyme digestion buffer (50mM Tris-HCl, 5mM EDTA), mix well, take out 125μl, add 5μl 0.5mg/mL trypsin, and incubate at 37°C for 4h. Use Waters Xevo G2-XS to perform LC-MS to detect deamidation modification. The results of mass spectrometry showed that the antibody did not have a significant high proportion of deamidation modification sites, suggesting that its later chemical stability was better. The results are shown in Table 13. Compared with the commercially available drug Herceptin, the antibody Hu209B1L1H1 showed better stability. .

Table 15: Test results of deamidation modification ratio

Remarks: N represents the modified asparagine detected, and the number represents the position where the N-terminus of the light or heavy chain starts to count. The percentage represents the ratio of the deamidation detected by LC-MS to the total peptide signal at the site.

The technical features of the above-mentioned embodiments can be combined arbitrarily. In order to make the description concise, all possible combinations of the various technical features in the above-mentioned embodiments are not described. However, as long as there is no contradiction in the combination of these technical features, All should be considered as the scope of this specification.

The above-mentioned embodiments only express several implementation modes of the present invention, and their description is relatively specific and detailed, but they should not be understood as a limitation on the scope of the invention patent. It should be pointed out that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can be made, and these all fall within the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention should be subject to the appended claims.

The sequences involved in the present invention are shown in the following table

Claims (22)

1. The antibody or antigen-binding fragment thereof that specifically binds to the extracellular region of lymphocyte activation gene 3 is characterized in that the antibody or antigen-binding fragment thereof comprises:

   The heavy chain complementarity determining region includes a heavy chain complementarity determining region 1, a heavy chain complementarity determining region 2, and a heavy chain complementarity determining region 3. The amino acid sequence of the heavy chain complementarity determining region 1 is as SEQ ID NO: 1, the amino acid sequence of the heavy chain complementarity determining region 2 is shown in SEQ ID NO: 2, and the amino acid sequence of the heavy chain complementarity determining region 3 is shown in SEQ ID NO: 3; and

   The light chain complementarity determining region, the light chain complementarity determining region includes the light chain complementarity determining region 1, the light chain complementarity determining region 2, and the light chain complementarity determining region 3. The amino acid sequence of the light chain complementarity determining region 1 is as SEQ ID As shown in NO: 4, the amino acid sequence of the light chain complementarity determining region 2 is shown in SEQ ID NO: 5, and the amino acid sequence of the light chain complementarity determining region 3 is shown in SEQ ID NO: 6.
2. The antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody is selected from a murine antibody, a human-mouse chimeric antibody, a humanized antibody, a fully human antibody, a single chain antibody, or a domain antibody .
3. The antibody or antigen-binding fragment thereof according to claim 1, further comprising:

   A heavy chain framework region, the heavy chain framework region includes a heavy chain framework region 1, a heavy chain framework region 2, a heavy chain framework region 3, and a heavy chain framework region 4. The amino acid sequence of the heavy chain framework region 1 is as SEQ ID As shown in NO: 7, the amino acid sequence of the heavy chain framework region 2 is shown in SEQ ID NO: 8, and the amino acid sequence of the heavy chain framework region 3 is shown in SEQ ID NO: 9, and the heavy chain framework The amino acid sequence of region 4 is shown in SEQ ID NO: 10;

   and / or

   A light chain framework region, the light chain framework region includes a light chain framework region 1, a light chain framework region 2, a light chain framework region 3, and a light chain framework region 4. The amino acid sequence of the light chain framework region 1 is as SEQ ID As shown in NO: 11, the amino acid sequence of the light chain framework region 2 is shown in SEQ ID NO: 12, and the amino acid sequence of the light chain framework region 3 is shown in SEQ ID NO: 13, and the light chain framework region The amino acid sequence of 4 is shown in SEQ ID NO: 14.
4. The antibody or antigen-binding fragment thereof according to claim 1, further comprising:

   A heavy chain framework region, the heavy chain framework region includes a heavy chain framework region 1, a heavy chain framework region 2, a heavy chain framework region 3, and a heavy chain framework region 4. The amino acid sequence of the heavy chain framework region 1 is as SEQ ID As shown in NO: 15, the amino acid sequence of the heavy chain framework region 2 is shown in SEQ ID NO: 16, the amino acid sequence of the heavy chain framework region 3 is shown in SEQ ID NO: 17, and the heavy chain framework The amino acid sequence of region 4 is shown in SEQ ID NO: 18;

   and / or

   A light chain framework region, the light chain framework region includes a light chain framework region 1, a light chain framework region 2, a light chain framework region 3, and a light chain framework region 4. The amino acid sequence of the light chain framework region 1 is as SEQ ID As shown in NO: 19, the amino acid sequence of the light chain framework region 2 is shown in SEQ ID NO: 20, and the amino acid sequence of the light chain framework region 3 is shown in SEQ ID NO: 21, and the light chain framework region is shown in SEQ ID NO: 21. The amino acid sequence of 4 is shown in SEQ ID NO: 22.
5. The antibody or antigen-binding fragment thereof according to claim 4, characterized by comprising at least one selected from the group consisting of the following mutations:

   a. Compared with SEQ ID NO: 16, the 15th amino acid of the heavy chain framework region 2 is mutated to 1;

   b. Compared with SEQ ID NO: 17, the amino acid at position 10 of the heavy chain framework region 3 is mutated to A;

   c. Compared with SEQ ID NO: 17, the amino acid at position 12 of the heavy chain framework region 3 is mutated to L;

   d. Compared with SEQ ID NO: 17, the amino acid at position 14 of the heavy chain framework region 3 is mutated to V;

   e. Compared with SEQ ID NO: 17, the amino acid at position 37 of the heavy chain framework region 3 is mutated to F;

   f. Compared with SEQ ID NO: 20, the amino acid at position 16 of the light chain framework 2 is mutated to M;

   g. Compared with SEQ ID NO: 21, the 21st amino acid of light chain framework region 3 is mutated to F; and

   h. Compared with SEQ ID NO: 21, the 35th amino acid of light chain framework region 3 is mutated to F.
6. The antibody or antigen-binding fragment thereof according to claim 5, comprising a combination of mutations selected from:

   Combination 1: a, b, c;

   Combination 2: a, b, c, d;

   Combination 3: a, b, c, d, e;

   Combination 4: f, g;

   Combination 5: a, b, c, f, g;

   Combination 6: a, b, c, d, f, g;

   Combination 7: a, b, c, d, e, f, g;

   Combination 8: f, g, h;

   Combination 9: a, b, c, f, g, h;

   Combination 10: a, b, c, d, f, g, h; or

   Combination 11: a, b, c, d, e, f, g, h.
7. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, which is selected from one of F(ab') ₂ , Fab, scFv, and bispecific antibodies.
8. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, wherein the antibody has:

   Heavy chain constant region, the sequence of the heavy chain constant region includes a constant region sequence selected from any one of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, IgD or a variant thereof;

   The light chain constant region includes a kappa or lambda chain or a variant thereof.
9. The antibody or antigen-binding fragment thereof according to claim 8, wherein the species from which the constant region is derived is selected from the group consisting of cows, horses, dairy cows, pigs, sheep, goats, rats, mice, dogs, cats, rabbits, camels, and donkeys , Deer, mink, chicken, duck, goose, turkey, cockfighting or human.
10. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, wherein the antibody has a constant region, the sequence of the heavy chain constant region of the antibody is shown in SEQ ID NO: 23, and the sequence of the light chain constant region is shown in SEQ ID NO: 24 is shown.
11. Nucleic acid, which encodes the antibody or antigen-binding fragment thereof according to any one of claims 1 to 10.
12. A vector comprising the nucleic acid of claim 11.
13. A host cell, which comprises:

    The nucleic acid according to claim 11; or

    The vector of claim 12

    Optional,

    The nucleic acid of claim 11 is integrated into the genome of the cell.
14. A method for producing the antibody or antigen-binding fragment thereof according to any one of claims 1 to 10, comprising:

    Culturing the host cell of claim 13 under suitable culture conditions; and

    The antibody thus produced is recovered from the culture medium or from the cultured host cells.
15. A pharmaceutical composition, which includes:

    The antibody or antigen-binding fragment thereof according to any one of claims 1 to 10, and

    One or more of pharmaceutically acceptable excipients, diluents or carriers.
16. The pharmaceutical composition according to claim 15, which is used to treat or prevent immune diseases or tumors.
17. The pharmaceutical composition according to claim 16, wherein the immune disease is selected from: systemic lupus erythematosus, multiple sclerosis, type I diabetes, psoriasis, ulcerative colitis, Sjogren syndrome, scleroderma, multiple Myositis, rheumatoid arthritis, mixed connective tissue disease, primary biliary cirrhosis, autoimmune hemolytic anemia, Hashimoto's thyroiditis, Addisons disease, leukoplakia, Graves disease, myasthenia gravis, ankylosis Spondylitis, allergic osteoarthritis, allergic vasculitis, autoimmune neutropenia, idiopathic thrombocytopenic purpura, lupus nephritis, chronic atrophic gastritis, autoimmune infertility, uterus Endometriosis, Pasture's disease, pemphigus, discoid lupus and dense deposit diseases.
18. The pharmaceutical composition according to claim 16, wherein the tumor is selected from the group consisting of bone, bone connection, muscle, lung, trachea, heart, spleen, artery, vein, blood, capillary, lymph node, lymphatic vessel, lymphatic fluid, oral cavity , Pharynx, esophagus, stomach, duodenum, small intestine, colon, rectum, anus, appendix, liver, gallbladder, pancreas, parotid gland, sublingual gland, urinary kidney, ureter, bladder, urethra, ovary, fallopian tube, uterus, vagina , Vulva, scrotum, testes, vas deferens, penis, eyes, ears, nose, tongue, skin, brain, brainstem, medulla oblongata, barren medulla, brain barren fluid, nerve, thyroid, parathyroid, adrenal gland, pituitary, pineal Tumors generated from any lesions in the body, pancreatic islets, thymus, gonads, sublingual glands and parotid glands
19. The pharmaceutical composition according to claim 15, which is used in combination with one or two or more selected from the group consisting of antifolates, calcineurin inhibitors, corticosteroids, and antithymocytes Globulins, nucleic acid antimetabolites, nucleic acid synthesis inhibitors, biological agents targeting cell surface antigens, biological agents targeting cytokines or cytokine receptors, and intravenous immunoglobulins.
20. The pharmaceutical composition according to claim 15, comprising at least one of an anticancer agent, a cytotoxic agent, and a chemotherapeutic agent.
21. A kit comprising at least one of the following components:

    i) The antibody or antigen-binding fragment thereof according to any one of claims 1 to 10, and optionally a container for holding the antibody;

    ii) The pharmaceutical composition according to any one of claims 15 to 20, and optionally a container for holding the pharmaceutical composition.
22. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 10, used for preparing targeted drugs, antibody-drug conjugates, or multifunctional antibodies that specifically target LAG-3 related lesions;

    Used for preparing reagents for diagnosing immune diseases or tumors, said immune diseases or tumors are related to LAG-3;

    Used to prepare immune cells modified with LAG-3 on the surface; or

    Used for screening other LAG-3 antibodies, which bind different epitopes with the antibody or antigen-binding fragment of any one of claims 1-10.

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