Antibodies that bind human LAG-3, methods of making, and uses thereof
阅读说明：本技术 结合人lag-3的抗体、其制备方法和用途 (Antibodies that bind human LAG-3, methods of making, and uses thereof ) 是由 赵杰 黄浩旻 朱祯平 于 2019-02-28 设计创作，主要内容包括：本发明公开了能够特异性结合人LAG-3的抗体或其抗原结合片段,其具有全新的与人LAG-3结合的表位以及良好的增强混合淋巴细胞反应的生物学活性,可应用于制备用于癌症免疫疗法的药物,具有良好的临床应用前景。(The invention discloses an antibody or an antigen binding fragment thereof capable of specifically binding human LAG-3, which has a brand-new epitope bound with human LAG-3 and good biological activity for enhancing mixed lymphocyte reaction, can be applied to preparation of a medicament for cancer immunotherapy, and has good clinical application prospect.)
1. An antibody or antigen-binding fragment thereof that binds human LAG-3, characterized in that it binds to an epitope of human LAG-3 comprising the amino acid sequence: AAAPGHPLA (SEQ ID NO: 50).
2. The antibody or antigen-binding fragment thereof that binds human LAG-3 of claim 1, which binds an epitope of human LAG-3 comprising the amino acid sequence: GPPAAAPGHPLA (SEQ ID NO: 48) or AAAPGHPLAPGPHPAAPSS (SEQ ID NO: 49).
3. An antibody or antigen-binding fragment thereof that binds human LAG-3, comprising:
(a) heavy chain complementarity determining regions HCDR1, HCDR2, HCDR3, the amino acid sequence of HCDR1 is shown in SEQ ID NO: 9, the amino acid sequence of the HCDR2 is shown as SEQ ID NO: 10, the amino acid sequence of the HCDR3 is shown as SEQ ID NO: 11 and light chain complementarity determining regions LCDR1, LCDR2 and LCDR3, wherein the amino acid sequence of the LCDR1 is shown as SEQ ID NO: 12, the amino acid sequence of the LCDR2 is shown as SEQ ID NO: 13, the amino acid sequence of the LCDR3 is shown as SEQ ID NO: 14 or SEQ ID NO: 43 is shown; or
(b) Heavy chain complementarity determining regions HCDR1, HCDR2, HCDR3, the amino acid sequence of HCDR1 is shown in SEQ ID NO: 15, the amino acid sequence of the HCDR2 is shown as SEQ ID NO: 16, the amino acid sequence of the HCDR3 is shown as SEQ ID NO: 17 and light chain complementarity determining regions LCDR1, LCDR2 and LCDR3, wherein the amino acid sequence of the LCDR1 is shown as SEQ ID NO: 18, and the amino acid sequence of the L-CDR2 is shown as SEQ ID NO: 19, the amino acid sequence of the LCDR3 is shown as SEQ ID NO: shown at 20.
4. The antibody or antigen-binding fragment thereof that binds human LAG-3 of any one of claims 1-3, wherein the antibody is a monoclonal antibody or a polyclonal antibody.
5. The antibody or antigen-binding fragment thereof that binds human LAG-3 of claim 4, wherein the antibody is a monoclonal antibody.
6. The antibody or antigen-binding fragment thereof that binds human LAG-3 of any one of claims 1-3, wherein the antibody is a murine, chimeric, or humanized antibody.
7. The antibody or antigen-binding fragment thereof that binds human LAG-3 of claim 6, wherein the antibody is a humanized antibody.
8. The antibody or antigen-binding fragment thereof that binds human LAG-3 of any one of claims 1-3, wherein the antigen-binding fragment comprises a Fab fragment, a F (ab') 2 fragment, a Fv fragment.
9. The antibody or antigen-binding fragment thereof that binds human LAG-3 of any one of claims 1-3, wherein the antibody or antigen-binding fragment thereof that binds human LAG-3 comprises a heavy chain variable region and a light chain variable region selected from the group consisting of:
(a) the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO: 2, the amino acid sequence of the light chain variable region is shown as SEQ ID NO: 4 is shown in the specification;
(b) the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO: 6, the amino acid sequence of the light chain variable region is shown as SEQ ID NO: 8 is shown in the specification;
(c) the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO: 22, the amino acid sequence of the light chain variable region is shown as SEQ ID NO: shown at 24; and
(d) the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO: 26, the amino acid sequence of the light chain variable region is shown as SEQ ID NO: shown at 28.
10. The antibody or antigen-binding fragment thereof that binds human LAG-3 of claim 9, wherein the antibody or antigen-binding fragment thereof that binds human LAG-3 comprises a heavy chain constant region selected from the group consisting of IgG1, IgG2, IgG3, and IgG4 heavy chain constant regions and a light chain constant region selected from the group consisting of kappa or lambda light chain constant regions.
11. The antibody or antigen-binding fragment thereof that binds human LAG-3 of claim 10, wherein the heavy chain constant region is an IgG4 heavy chain constant region and the light chain constant region is a kappa light chain constant region.
12. The antibody or antigen-binding fragment thereof that binds human LAG-3 of claim 11, wherein the amino acid sequence of the heavy chain constant region is as set forth in SEQ ID NO: 30, and the amino acid sequence of the light chain constant region is shown as SEQ ID NO: shown at 34.
13. The antibody or antigen-binding fragment thereof that binds human LAG-3 of any one of claims 9-12, wherein the antibody or antigen-binding fragment thereof that binds human LAG-3 comprises a heavy chain and a light chain selected from the group consisting of:
(a) the amino acid sequence of the heavy chain is shown as SEQ ID NO: 32, and the amino acid sequence of the light chain is shown as SEQ ID NO: 36 is shown; and
(b) the amino acid sequence of the heavy chain is shown as SEQ ID NO: 38, and the amino acid sequence of the light chain is shown as SEQ ID NO: shown at 40.
14. A nucleotide sequence encoding the antibody or antigen-binding fragment thereof that binds human LAG-3 according to any one of claims 1-13.
15. The nucleotide sequence of claim 14, wherein the nucleotide sequence comprises:
(a) as shown in SEQ ID NO: 1, as shown in SEQ ID NO: 3, a nucleotide sequence encoding a light chain variable region;
(b) as shown in SEQ ID NO: 5, as shown in SEQ ID NO: 7, a nucleotide sequence encoding a light chain variable region;
(c) as shown in SEQ ID NO: 21, as shown in SEQ ID NO: 23, a nucleotide sequence encoding a light chain variable region; or
(d) As shown in SEQ ID NO: 25, as shown in SEQ ID NO: 27, and a nucleotide sequence encoding a light chain variable region.
16. The nucleotide sequence of claim 15, comprising the nucleotide sequence set forth as SEQ ID NO: 29, and the nucleotide sequence encoding the heavy chain constant region as set forth in SEQ ID NO: 33, and a nucleotide sequence encoding a light chain constant region.
17. The nucleotide sequence of any one of claims 14-16, comprising:
(a) as shown in SEQ ID NO: 31, as shown in SEQ ID NO: 35, a nucleotide sequence encoding a light chain; or
(b) As shown in SEQ ID NO: 37, as shown in SEQ ID NO: 39 encoding a light chain.
18. An expression vector comprising the nucleotide sequence of any one of claims 14 to 17.
19. A host cell comprising the expression vector of claim 18.
20. A method of producing an antibody or antigen-binding fragment thereof that binds human LAG-3 according to any one of claims 1-13, comprising the steps of:
(a) culturing the host cell of claim 19 under expression conditions such that the antibody or antigen-binding fragment thereof that binds human LAG-3 is expressed;
(b) isolating and purifying the antibody or antigen-binding fragment thereof that binds human LAG-3 of (a).
21. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof that binds human LAG-3 of any one of claims 1-13 and a pharmaceutically acceptable carrier.
22. The pharmaceutical composition of claim 21, wherein the pharmaceutical composition further comprises a PD-1 inhibitor.
23. The pharmaceutical composition of claim 22, wherein the PD-1 inhibitor is an antibody or antigen-binding fragment thereof that binds PD-1.
24. Use of an antibody or antigen-binding fragment thereof that binds human LAG-3 according to any one of claims 1-13 or a pharmaceutical composition according to any one of claims 21-23 in the manufacture of a medicament for the treatment of cancer.
25. The use of claim 24, wherein the cancer comprises melanoma, renal cell carcinoma, non-small cell lung cancer, classical hodgkin's lymphoma, urothelial cancer, colorectal cancer, and liver cancer.
Technical Field
The invention relates to the field of antibodies, and more particularly discloses an antibody binding to human LAG-3, a preparation method and a use thereof.
Background
Various types of malignancies have now become the major killers of humans and the incidence is rising year by year. Although the conventional operations, chemotherapy and radiotherapy have certain curative effect, the traditional Chinese medicine composition usually brings great damage and toxic and side effects to the body of a patient. Although the existing targeted monoclonal antibody medicines (such as trastuzumab targeting HER 2) and targeted small molecule kinase inhibitors (such as imatinib targeting certain tyrosine kinases) have favorable clinical effects in tumor treatment, the medicines are only effective on tumors expressing specific targets, the types of cancers which can be treated are limited, the objective response rate is low, and the tumors applying the antibodies are easy to generate drug resistance so as to relapse and progress again. The tumor immunotherapy applies immunological principle and method to activate immune system, break immune tolerance state of immune system to tumor, enhance the recognition of immune cell to tumor antigen, and stimulate and enhance anti-tumor immune response of organism, so that the immune system of organism can inhibit and kill tumor cell, and finally the goal of treating tumor can be achieved. Tumor immunotherapy has recently received much attention and is the focus of the tumor therapy field. In recent years, significant information on tumor immunotherapy has emerged. At present, programmed death receptor 1 (PD-1) is undoubtedly the most dazzling target in cancer immunotherapy, and monoclonal anti-tumor drugs such as nivolumab and pembrolizumab, which target it, have demonstrated strong anti-tumor activity in clinical treatment of some tumor types such as melanoma, non-small cell lung cancer, etc., and have been approved for clinical application by FDA in the united states. Because of its excellent curative effect and innovativeness, immunotherapy for tumors is judged by the journal of science in 2013 as the most important scientific breakthrough every year. The tumor immunotherapy is expected to become a revolution in the field of tumor therapy after operations, chemotherapy, radiotherapy and targeted therapy.
T cells play an important role in the immune system, but activation of T cells relies on the antigen presenting cells to digest and re-present foreign harmful antigens to an antigen form that the T cells can recognize. T cells and antigen presenting cells have a population of proteins involved in regulation, which function to help regulate T cell receptor signaling, and these helper receptors are divided into 2 major classes, one class being costimulatory receptors responsible for transmitting activating signals, and the other class being co-inhibitory receptors transmitting inhibitory signals, and these inhibitory molecules are now referred to as immune checkpoint receptors and ligands. The star molecule PD-1 mentioned above is a typical receptor of immune checkpoints and belongs to the CD28 superfamily members, and its important ligand is programmed death ligand-1 (PD-L1). The binding of PD-1 to PD-L1 mediates a co-inhibitory signal of T cell activation, negatively regulating T cell activation and proliferation. In addition, chinese scientists have demonstrated that PD-L1 is highly expressed in tumor tissue and modulates the function of tumor-infiltrating CD8+ T cells. Currently, immunomodulatory therapies targeting PD-1/PD-L1 have met with great success in clinical antitumor therapy.
Lymphocyte activation gene-3 (LAG-3) is mainly expressed in activated T lymphocytes and is an immune negative regulation molecule similar to PD-1, and the main ligand of LAG-3 is a MHC II molecule family, which can compete with CD4 molecule to bind MHC II ligand with higher affinity and transduce inhibitory signals to the inside of the cell, thereby negatively regulating the activation, proliferation and dynamic balance of the cell, and the molecular mechanism is very similar to PD-1. LAG-3 can help T cells to maintain an immune tolerance state or enable continuously activated T cells to be in a depletion state, has the functions of maintaining homeostasis and participating in immune negative regulation, and is closely related to the occurrence and development of tumors. At present, some preliminary studies show that the antibody for blocking LAG-3 can enhance the immune response, and can play a remarkable synergistic activation role when being combined with the PD-1 antibody, so that the potential in the field of cancer immunotherapy is huge.
At present, most of the research and development of monoclonal antibody medicines taking LAG-3 as a target point internationally are still in an early stage, main research and development companies are international medicine headaches such as Bristol-Myers Squibb (BMS) and Novartis (Novartis), wherein the anti-LAG-3 monoclonal antibody BMS-986016 of the Baishimei Guibao is in a first clinical stage, the main research targets are the safety and effectiveness of combined use with PD-1 antibody nivolumab, and research results show that BMS-986016 and nivolumab show good curative effect in clinical treatment of melanoma. In addition, anti-LAG-3 antibodies can also be used to treat autoimmune diseases, the basic mechanism is to kill activated T cells through antibody-dependent cell-mediated cytotoxicity (ADCC), thereby achieving the purpose of suppressing immune response and treating autoimmune diseases. However, there is still a need to develop more novel, specific and efficient LAG-3 targeted drugs for clinical application in immunotherapy.
Disclosure of Invention
In order to solve the technical problems, the inventor of the invention carries out a large number of experiments, from antigen immunization, hybridoma screening, antibody expression purification to biological activity identification, obtains two antibodies of
Accordingly, it is a first object of the present invention to provide an antibody or antigen-binding fragment thereof that binds human LAG-3.
It is a second object of the invention to provide another antibody or antigen-binding fragment thereof that binds human LAG-3.
It is a third object of the present invention to provide a nucleotide sequence encoding said antibody or antigen-binding fragment thereof that binds human LAG-3.
The fourth purpose of the invention is to provide an expression vector containing the nucleotide sequence.
The fifth object of the present invention is to provide a host cell containing the expression vector.
The sixth object of the present invention is to provide a method for producing the antibody or antigen-binding fragment thereof that binds human LAG-3.
The seventh object of the present invention is to provide a pharmaceutical composition comprising the antibody or antigen-binding fragment thereof that binds human LAG-3.
An eighth object of the present invention is to provide the use of the antibody or antigen-binding fragment thereof that binds human LAG-3 or the pharmaceutical composition.
In order to achieve the purpose, the invention adopts the following technical scheme:
a first aspect of the invention provides an antibody or antigen-binding fragment thereof that binds human LAG-3, which binds to an epitope of human LAG-3 comprising the amino acid sequence: AAAPGHPLA (SEQ ID NO: 50).
According to a preferred embodiment of the invention, the human LAG-3 epitope to which it binds comprises the amino acid sequence: GPPAAAPGHPLA (SEQ ID NO: 48) or AAAPGHPLAPGPHPAAPSS (SEQ ID NO: 49).
A second aspect of the invention provides an antibody or antigen-binding fragment thereof that binds human LAG-3, comprising:
(a) heavy chain complementarity determining regions HCDR1, HCDR2, HCDR3, the amino acid sequence of HCDR1 is shown in SEQ ID NO: 9, the amino acid sequence of the HCDR2 is shown as SEQ ID NO: 10, the amino acid sequence of the HCDR3 is shown as SEQ ID NO: 11 and light chain complementarity determining regions LCDR1, LCDR2 and LCDR3, wherein the amino acid sequence of the LCDR1 is shown as SEQ ID NO: 12, the amino acid sequence of the LCDR2 is shown as SEQ ID NO: 13, the amino acid sequence of the LCDR3 is shown as SEQ ID NO: 14 or SEQ ID NO: 43 is shown; or
(b) Heavy chain complementarity determining regions HCDR1, HCDR2, HCDR3, the amino acid sequence of HCDR1 is shown in SEQ ID NO: 15, the amino acid sequence of the HCDR2 is shown as SEQ ID NO: 16, the amino acid sequence of the HCDR3 is shown as SEQ ID NO: 17 and light chain complementarity determining regions LCDR1, LCDR2 and LCDR3, wherein the amino acid sequence of the LCDR1 is shown as SEQ ID NO: 18, the amino acid sequence of the LCDR2 is shown as SEQ ID NO: 19, the amino acid sequence of the LCDR3 is shown as SEQ ID NO: shown at 20.
According to the invention, the antibody is a monoclonal antibody or a polyclonal antibody. Preferably, the antibody is a monoclonal antibody.
According to the present invention, the antibody is a murine antibody, a chimeric antibody, a humanized antibody or the like. Preferably, the antibody is a humanized antibody.
According to the present invention, the antigen binding fragment includes a Fab fragment, a F (ab') 2 fragment, a Fv fragment, and the like.
According to a preferred embodiment of the invention, the antibody or antigen-binding fragment thereof that binds human LAG-3 comprises a heavy chain variable region and a light chain variable region selected from the group consisting of:
(a) the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO: 2, the amino acid sequence of the light chain variable region is shown as SEQ ID NO: 4 is shown in the specification;
(b) the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO: 6, the amino acid sequence of the light chain variable region is shown as SEQ ID NO: 8 is shown in the specification;
(c) the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO: 22, the amino acid sequence of the light chain variable region is shown as SEQ ID NO: shown at 24; and
(d) the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO: 26, the amino acid sequence of the light chain variable region is shown as SEQ ID NO: shown at 28.
According to a preferred embodiment of the invention, the antibody or antigen-binding fragment thereof that binds human LAG-3 comprises a heavy chain constant region selected from the group consisting of IgG1, IgG2, IgG3 and IgG4 heavy chain constant regions and a light chain constant region selected from the group consisting of kappa or lambda light chain constant regions. Preferably, the heavy chain constant region is an IgG4 heavy chain constant region and the light chain constant region is a kappa light chain constant region. More preferably, the amino acid sequence of the heavy chain constant region is as set forth in SEQ ID NO: 30, and the amino acid sequence of the light chain constant region is shown as SEQ ID NO: shown at 34.
According to a preferred embodiment of the invention, the antibody or antigen-binding fragment thereof that binds human LAG-3 comprises a heavy chain and a light chain selected from the group consisting of:
(a) the amino acid sequence of the heavy chain is shown as SEQ ID NO: 32, and the amino acid sequence of the light chain is shown as SEQ ID NO: 36 is shown; and
(b) the amino acid sequence of the heavy chain is shown as SEQ ID NO: 38, and the amino acid sequence of the light chain is shown as SEQ ID NO: shown at 40.
A third aspect of the invention provides a nucleotide sequence encoding an antibody or antigen-binding fragment thereof that binds human LAG-3 as described in any one of the above.
According to a preferred embodiment of the invention, the nucleotide sequence comprises:
(a) as shown in SEQ ID NO: 1, as shown in SEQ ID NO: 3, a nucleotide sequence encoding a light chain variable region;
(b) as shown in SEQ ID NO: 5, as shown in SEQ ID NO: 7, a nucleotide sequence encoding a light chain variable region;
(c) as shown in SEQ ID NO: 21, as shown in SEQ ID NO: 23, a nucleotide sequence encoding a light chain variable region; or
(d) As shown in SEQ ID NO: 25, as shown in SEQ ID NO: 27, and a nucleotide sequence encoding a light chain variable region.
According to a preferred embodiment of the invention, the nucleotide sequence comprises the nucleotide sequence as set forth in SEQ ID NO: 29, and the nucleotide sequence encoding the heavy chain constant region as set forth in SEQ ID NO: 33, and a nucleotide sequence encoding a light chain constant region.
According to a preferred embodiment of the invention, the nucleotide sequence comprises:
(a) as shown in SEQ ID NO: 31, as shown in SEQ ID NO: 35, a nucleotide sequence encoding a light chain; or
(b) As shown in SEQ ID NO: 37, as shown in SEQ ID NO: 39 encoding a light chain.
In a fourth aspect, the present invention provides an expression vector comprising a nucleotide sequence as defined in any one of the above.
In a fifth aspect, the present invention provides a host cell comprising an expression vector as described above.
A sixth aspect of the invention provides a method of making an antibody or antigen-binding fragment thereof that binds human LAG-3, as described above, comprising the steps of:
(a) culturing a host cell as described above under expression conditions such that the antibody or antigen-binding fragment thereof that binds human LAG-3 is expressed;
(b) isolating and purifying the antibody or antigen-binding fragment thereof that binds human LAG-3 of (a).
A seventh aspect of the invention provides a pharmaceutical composition comprising an antibody or antigen-binding fragment thereof that binds human LAG-3 as described in any one of the above, and a pharmaceutically acceptable carrier.
According to a preferred embodiment of the invention, the pharmaceutical composition further comprises a PD-1 inhibitor. Preferably, the PD-1 inhibitor is an antibody or antigen-binding fragment thereof that binds to PD-1.
An eighth aspect of the invention provides the use of an antibody or antigen-binding fragment thereof that binds human LAG-3, as defined in any one of the above, or a pharmaceutical composition as defined in any one of the above, in the manufacture of a medicament for the treatment of cancer.
According to the invention, the cancer is selected from melanoma, renal cell carcinoma, non-small cell lung cancer, classical Hodgkin lymphoma, urothelial cancer, colorectal cancer, liver cancer and the like.
Has the advantages that:
the invention discloses an antibody or an antigen binding fragment thereof capable of specifically binding human LAG-3, which has a brand-new epitope bound with human LAG-3 and good biological activity for enhancing mixed lymphocyte reaction, can be applied to preparation of a medicament for cancer immunotherapy, and has good clinical application prospect.
Drawings
FIG. 1 shows the results of the effect of murine anti-human LAG-3 antibody on mixed lymphocyte reaction, wherein FIG. 1A shows the first round of screening and FIG. 1B shows the second round of screening.
FIG. 2 is a result of comparison of relative affinities between a humanized antibody and a chimeric antibody against human LAG-3 antibody.
FIG. 3 is a graph showing the effect of the humanized anti-human LAG-3 antibody on the enhancement of IL-2 secretion by PBMC from different donors in FIGS. 3A and 3B, respectively.
FIG. 4 shows the results of the binding effect of humanized anti-human LAG-3 antibody to cell surface LAG-3.
FIG. 5 is a schematic representation of epitopes of anti-human LAG-3 antibody.
FIG. 6 shows the blocking effect of humanized anti-human LAG-3 antibody on the binding of LAG-3 to Raji cells.
Detailed Description
In the present invention, the term "LAG-3" refers to
In the present invention, the terms "antibody (Ab)" and "immunoglobulin G (IgG)", which are heterotetrameric glycan proteins of about 150000 daltons having the same structural features, are composed of two identical light chains (L) and two identical heavy chains (H). Each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide bonds varies between heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bonds. Each heavy chain has at one end a variable region (VH) followed by a constant region consisting of three domains, CH1, CH2, and
The antibody of the present invention includes a monoclonal antibody, a polyclonal antibody, a multispecific antibody (e.g., bispecific antibody) formed from at least two antibodies, and the like.
In the present invention, the term "monoclonal antibody (mab)" refers to an antibody obtained from a substantially homogeneous population, i.e., the individual antibodies comprised in the population are identical, except for a few naturally occurring mutations that may be present. Monoclonal antibodies are directed against a single antigenic site with high specificity. Moreover, unlike conventional polyclonal antibody preparations (typically having different antibodies directed against different determinants), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, monoclonal antibodies are also advantageous in that they are synthesized by hybridoma culture and are not contaminated with other immunoglobulins. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method.
In the present invention, the term "antigen-binding fragment" refers to a fragment of an antibody that is capable of specifically binding to an antigen (e.g., human LAG-3). Examples of the antigen-binding fragment of the present invention include Fab fragment, F (ab') 2 fragment, Fv fragment, and the like. Fab fragments are fragments generated by papain digestion of antibodies. The F (ab') 2 fragment is a fragment produced by digestion of an antibody with pepsin. Fv fragments are composed of dimers in which the heavy and light chain variable regions of an antibody are in close, non-covalent association.
In the present invention, the term "Fc fragment" means that papain can cleave an antibody into two identical Fab fragments and one Fc fragment, i.e., a crystallizable fragment (Fc), which is composed of the CH2 and CH3 domains of the antibody. The Fc region has no antigen binding activity and is the site of antibody interaction with effector molecules or cells.
In the present invention, the term "variable" means that certain portions of the variable regions of an antibody differ in sequence, which results in the binding and specificity of each particular antibody for its particular antigen. However, the variability is not evenly distributed throughout the antibody variable region. It is concentrated in three segments called complementarity-determining regions (CDRs) or hypervariable regions in the heavy chain variable region and the light chain variable region. The more conserved portions of the variable regions are called Framework Regions (FR). The variable regions of native heavy and light chains each comprise four FR regions, which are in a substantially β -sheet configuration, connected by three CDRs that form a connecting loop, and in some cases may form part of a β -sheet structure. The CDRs in each chain are held close together by the FR region and form the antigen binding site of the antibody with the CDRs of the other chain (see Kabat et al, NIH Publ. No.91-3242, Vol I, 647-669 (1991)).
The antibody of the present invention includes a murine antibody, a chimeric antibody, a humanized antibody and the like.
In the present invention, the term "murine antibody" refers to an antibody derived from a rat or a mouse, preferably a mouse. The murine antibody of the invention is obtained by immunizing a mouse by using the extracellular domain of human LAG-3 as an antigen and screening hybridoma cells. Preferably, the murine antibodies of the invention are
In the present invention, the term "chimeric antibody" refers to an antibody comprising heavy chain variable region and light chain variable region sequences derived from one species and constant region sequences derived from another species, for example, an antibody having murine heavy chain variable region and light chain variable region linked to human constant regions. Preferably, the chimeric antibody of the present invention is obtained by splicing the heavy chain variable region and light chain variable region sequences of murine antibody Nos. 134 and 2-34 (i.e., 5E7), respectively, to human constant regions. More preferably, the heavy chain of the chimeric antibody of the present invention is obtained by splicing the heavy chain variable region sequences of
In the present invention, the term "humanized antibody" means an antibody in which the CDRs are derived from a non-human species (preferably a mouse), and the remaining part of the antibody molecule (including the framework region and the constant region) is derived from a human antibody. In addition, framework region residues may be altered to maintain binding affinity. Preferably, the humanized antibody of the present invention is obtained by recombining the CDR regions of murine antibody Nos. 134 and 2-34 (i.e., 5E7) and the non-CDR regions derived from a human antibody and back-mutating the buried residues, residues that directly interact with the CDR regions, and residues that significantly affect the conformation of VL and VH of antibody Nos. 134 and 2-34 (i.e., 5E 7). More preferably, the humanized antibodies of the invention include 134-Hu-IgG4-C91S and 5E7-Hu-
In the present invention, the terms "epitope" and "human LAG-3 epitope" refer to a region on human LAG-3 to which an antibody specifically binds. Preferably, the epitope of human LAG-3 of the present invention is located within the extracellular domain of human LAG-3, said extracellular domain of human LAG-3 comprising the amino acid sequence as set forth in SEQ ID NO: 41. More preferably, the human LAG-3 epitope of the invention comprises the amino acid sequence: AAAPGHPLA (SEQ ID NO: 50). Most preferably, the human LAG-3 epitope of the invention comprises the amino acid sequence: GPPAAAPGHPLA (SEQ ID NO: 48) or AAAPGHPLAPGPHPAAPSS (SEQ ID NO: 49).
In the present invention, the term "antibody that binds human LAG-3" or "anti-human LAG-3 antibody" refers to an antibody that specifically binds to an epitope of human LAG-3 the term "specifically binds" refers to an antibody that binds less than 1 × 10-7M or less, preferably 1 × 10-8M or less, more preferably 1 × 10-9M or less affinity (KD) binding.
In the present invention, the term "expression vector" may be pTT5, pSECtag series, pCGS3 series, pCDNA series vectors, etc., and other vectors for use in mammalian expression systems, etc., the expression vector including a fusion DNA sequence to which appropriate transcription and translation regulatory sequences are ligated.
In the present invention, the term "host cell" refers to a cell suitable for expressing the above expression vector, and may be a eukaryotic cell, such as mammalian or insect host cell culture system, which can be used for the expression of the fusion protein of the present invention, and CHO (Chinese Hamster Ovary), HEK293, COS, BHK and derived cells of the above cell can be suitable for the present invention.
In the present invention, the term "pharmaceutical composition" means that the antibody or antigen-binding fragment thereof binding to human LAG-3 of the present invention can be combined with a pharmaceutically acceptable carrier to form a pharmaceutical formulation composition that can ensure the conformational integrity of the amino acid core sequence of the antibody or antigen-binding fragment thereof binding to human LAG-3 disclosed herein, while also protecting the multiple functional groups of the protein from degradation (including but not limited to aggregation, deamination or oxidation) to exert therapeutic effects more stably.
The following examples and experimental examples are intended to further illustrate the present invention and should not be construed as limiting the present invention. The examples do not include detailed descriptions of conventional methods, such as those used to construct vectors and plasmids, methods of inserting genes encoding proteins into such vectors and plasmids, or methods of introducing plasmids into host cells. Such methods are well known to those having ordinary skill in the art and are described in numerous publications, including Sambrook, j., Fritsch, e.f. and maniis, T. (1989) Molecular Cloning: a Laboratory Manual, 2 interpretation, Cold spring harbor Laboratory Press.
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