CN118221816A

CN118221816A - LAG3 monoclonal antibody and application thereof

Info

Publication number: CN118221816A
Application number: CN202410287305.9A
Authority: CN
Inventors: 丰明乾; 左殿宝; 朱元奎; 徐金国
Original assignee: Suzhou Tongqian Medical Technology Co ltd
Current assignee: Suzhou Tongqian Medical Technology Co ltd
Filing date: 2024-03-13
Publication date: 2024-06-21

Abstract

The present invention provides a plurality of monoclonal antibodies capable of recognizing lymphocyte activating gene 3 (LAG 3) with high affinity. The antibodies have high affinity and are capable of blocking the binding of LAG3 protein to its ligand MHC-II or FGL 1. The neutralizing monoclonal antibody provided by the invention not only has good in-vitro blocking activity, but also has very strong thermal stability, and can promote proliferation of PBMC cells and release of cytokines in vitro, and enhance tumor killing activity of bispecific antibodies and CAR-T.

Description

LAG3 monoclonal antibody and application thereof

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a high-affinity monoclonal antibody for recognizing LAG3 and application thereof.

Background

Lymphocyte activating gene 3 (LAG 3, CD 223) is a member of the immunoglobulin superfamily and is an important negative regulator of T cell surface. LAG3 was found in 1990 on activated NK cells and T cells, and it contains 4 extracellular IgSF domains and 1 transmembrane domain and 1 intracellular domain, which can be cleaved into secreted sLAG3 by proteases ADAM10 and ADAM17 between the D4 domain and transmembrane domain. In addition, LAG3 was detected on DC cells, B cells, tumor infiltrating lymphocytes, NK cells, regulatory T cells, depleted cd8+ T cells, and the like.

LAG3 has about 20% homology with MHC-II in the gene structure, and LAG3 has higher affinity with MHC-II than CD4 protein. However, unlike CD4, CD4 is mainly expressed on the cell surface, whereas LAG3 is largely intracellular and LAG3 rapidly migrates to the cell surface to function upon antigen stimulation. Upregulation of expression of LAG3 molecules on the T-cell surface has led to downregulation of T-cell proliferation, and since LAG3 has a higher affinity for MHC-II molecules than CD4, while blocking LAG3 interaction with MHC-II by antibodies can significantly enhance T-cell activity, LAG3 has in the past been widely recognized as downregulating T-cell activity by interaction with MHC-II molecules. However, in recent years, it has been found that antibodies against LAG3 which do not block binding to MHC-II still have some antitumor activity, and thus LAG3 may have other ligands to function, including Galectin-3, LSECtin, FGL1, etc. One article on Cell 2019 shows that the primary inhibitory ligand (Fibrinogen-like Protein 1Is a Major Immune Inhibitory Ligand of LAG3,Wang J,et al,Cell,2019;176:334-347). of LAG3, which FGL1 may also be, is a new strategy in tumor immunotherapy by preparing antibodies to block LAG3-MHC-II or LAG3-FGL1 interactions.

Disclosure of Invention

The present invention aims to provide monoclonal antibodies that recognize LAG3 with high affinity. Antibodies provided include immunoconjugates of antibody fragments (e.g., single chain variable region fragments (scFv)) and effector molecules (e.g., toxin proteins). Also provided are compositions comprising antibodies that specifically bind LAG3, nucleic acid molecules encoding these antibodies, expression vectors comprising the nucleic acid molecules, and isolated host cells expressing the nucleic acid molecules.

The specific technical scheme of the invention is as follows:

A monoclonal antibody or antigen-binding fragment thereof of lymphocyte activation gene 3 comprising a heavy chain variable region and a light chain variable region, said monoclonal antibody or antigen-binding fragment thereof comprising the amino acid sequence of SEQ ID NO:1, a heavy chain variable region CDR1, SEQ ID NO:2, a heavy chain variable region CDR2, SEQ ID NO:3, a heavy chain variable region CDR3 as set forth in SEQ ID NO:4, the light chain variable region CDR1, SEQ ID NO:5, the light chain variable region CDR2, SEQ ID NO:6, and a light chain variable region CDR3.

There are three known subtypes of human LAG3 (subtypes 1-3). The nucleic acid and amino acid sequences of three subtypes of LAG3 are known, including GenBank accession numbers: NM_002286.6 and NP_002277.4 (subtype 1); NM_001414176.1 and NP_001401105.1 (subtype 2); NM-001414177.14 and NP-001401106.1 (subtype 3). The antibodies of the invention may bind to one or more of the three human LAG3 subtypes, or conservative variants thereof.

Specifically, the LAG3 antibody or antigen-binding fragment thereof of the present invention is:

(1) The amino acid sequence of the heavy chain variable region of the antibody or antigen binding fragment thereof is shown as SEQ ID NO. 7, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 8;

Or (2) the heavy chain variable region amino acid sequence of the antibody or antigen binding fragment thereof is shown as SEQ ID NO. 9, and the light chain variable region amino acid sequence is shown as SEQ ID NO. 10.

Or (3) the amino acid sequence of the heavy chain variable region of the antibody or antigen binding fragment thereof is shown as SEQ ID NO. 11, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 12.

Or (4) the heavy chain variable region amino acid sequence of the antibody or antigen binding fragment thereof is shown as SEQ ID NO. 13, and the light chain variable region amino acid sequence is shown as SEQ ID NO. 14.

Or (5) the heavy chain variable region amino acid sequence of the antibody or antigen binding fragment thereof is shown as SEQ ID NO. 15, and the light chain variable region amino acid sequence is shown as SEQ ID NO. 16.

The antibodies of the invention are single chain antibodies, diabodies, monoclonal antibodies or chimeric antibodies.

The monoclonal antibodies of the invention may be of any isotype. May be, for example, igM or IgG antibodies, such as IgG1 or IgG2. The types of antibodies that can specifically bind LAG3 can be converted to each other according to known methods (e.g., igG can be converted to IgM). Class conversion may also be used to convert one subclass of IgG to another, e.g., from IgG1 to IgG2.

The antibodies of the invention may be:

(1) Fab, fragments comprising monovalent antigen binding fragments of an antibody molecule, which can be produced by digesting an intact antibody with papain to produce an intact light chain and a portion of one heavy chain;

(2) Fab', antibody molecule fragments obtainable by treating an intact antibody with pepsin followed by reduction to yield a portion of intact light and heavy chains; two Fab' fragments are obtained per antibody molecule;

(3) (Fab') 2, an antibody fragment obtainable by treating an intact antibody with pepsin, but without subsequent reduction; f (ab ') 2 is a dimer of two Fab' fragments linked together by two disulfide bonds;

(4) Fv, gene Cheng Pianduan containing the light chain variable region and heavy chain variable region expressed as 2 chains;

(5) A single chain antibody (e.g., scFv), a genetically engineered molecule comprising a light chain variable region and a heavy chain variable region linked by a suitable polypeptide linker to form a genetically fused single chain molecule;

(6) A dimer of single chain antibodies (scFv 2), defined as a dimer of scFv (also known as a "minibody");

(7) VH single domain antibodies, antibody fragments consisting of heavy chain variable regions.

Those skilled in the art will appreciate that conservative variants of an antibody may be made. Amino acid substitutions (e.g., 1,2, 3, 4, or 5 amino acid substitutions) may be made in the VH and/or VL regions, with the substituted VH and VL still retaining the ability to bind LAG3, or having greater binding ability to LAG 3. Conservative substitutions of functionally similar amino acids are well known to those of ordinary skill in the art. The following six groups are examples of amino acids that are considered to be conservative substitutions for one another:

1) Alanine (a), serine (S), threonine (T);

2) Aspartic acid (D), glutamic acid (E);

3) Asparagine (N), glutamine (Q);

4) Arginine (R), lysine (K);

5) Isoleucine (I), leucine (L), methionine (M), valine (V);

6) Phenylalanine (F), tyrosine (Y), tryptophan (W).

It is another object of the present invention to provide a recombinant protein comprising the antibody or antigen-binding fragment thereof of the present invention and a tag sequence that facilitates expression and/or purification. The tag sequence includes but is not limited to a 6x His tag.

The invention also aims to disclose the application of the monoclonal antibody or antigen binding fragment, the bispecific antibody or fusion protein, the immunoconjugate and the nucleic acid molecule in the preparation of immune checkpoint inhibitors. The immune checkpoint inhibitor is an agent for diagnosing or treating autoimmune diseases, chronic reinfection or tumors. The tumor is non-Hodgkin's lymphoma, chronic lymphocytic leukemia, hodgkin's disease, solid tumor or metastasis.

The detectable label of the present invention is a substance that can be detected by an isotope analyzer, an enzyme-labeled instrument, a bioluminescence detector, a chemiluminescent detector, an electrochemiluminescence detector, a fluorescence analyzer, or a naked eye visualization, including, but not limited to, radioisotopes (e.g., ³H、¹⁴C、¹⁵N、³⁵S、⁹⁰Y、、⁹⁹Tc、¹¹¹In、¹²⁵I、¹³¹I)、 enzymes useful for detection (e.g., horseradish peroxidase, beta-galactosidase, alkaline phosphatase, glucose oxidase, etc.), fluorescent proteins (e.g., green Fluorescent Protein (GFP), yellow Fluorescent Protein (YFP), allophycocyanin APC, phycoerythrin PE), bioluminescent labels (e.g., luciferase), fluorescent compounds (e.g., fluorescein isothiocyanate, rhodamine, 5-dimethylamino-1-naphthalenesulfonyl chloride, phycoerythrin, fluorescent dye Cy3, cy5, rare earth phosphors, quantum dots, etc.), biotin, magnetic agents (e.g., gadolinium), electrochemiluminescent agents (e.g., ruthenium terpyridyl), colloidal gold.

The effector molecule may be linked to the antibodies of the invention using any means known to those skilled in the art. For example, the antibody may be functionally linked (by chemical coupling, gene fusion, non-covalent association, or otherwise) to one or more other molecular entities. The method of linking the effector molecule to the antibody varies depending on the chemical structure of the effector molecule. Polypeptides generally contain multiple functional groups; such as carboxylic acid (COOH), free amino (-NH ₂) or thiol (-SH), which can be used to react with suitable functional groups on antibodies to bind to the effector molecule. Alternatively, the antibody may be derivatized to expose or attach additional reactive functional groups. The derivatization may include attachment of any of a variety of known linker molecules. The linker may be any molecule for binding the antibody to an effector molecule. The linker is capable of forming a covalent bond with the antibody and effector molecule. Suitable linkers are well known to those skilled in the art and include, but are not limited to, straight or branched chain carbon linkers, heterocyclic carbon linkers, or peptide linkers. Where the antibodies and effector molecules are polypeptides, the linker may be attached to the constituent amino acids or to the alpha carbon amino and carboxyl groups of the terminal amino acids through its side groups (e.g., through disulfide bonds of cysteines). Typically, the antibody or portion thereof is derivatized such that binding to the target antigen is not adversely affected by the derivatization or labeling.

In some cases, it is desirable to release the effector molecule from the antibody when the immunoconjugate has reached its target site. Thus, in these cases, the immunoconjugate will comprise a cleavable bond near the target site. Cleavage of the linker to release the effector molecule from the antibody may be caused by enzymatic activity or by conditions under which the immunoconjugate is located within the target cell or near the target site.

It is another object of the present invention to provide a polynucleotide encoding an antibody, recombinant protein or immunoconjugate according to the invention.

Another object of the present invention is to provide a vector comprising the polynucleotide of the present invention. The carrier comprises: bacterial plasmids, phages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses or other vectors.

It is another object of the present invention to provide a pharmaceutical composition comprising one or more of the antibodies or antigen binding fragments thereof, recombinant proteins, bispecific antibodies, immunoconjugates, polynucleotides, vectors, or genetically engineered host cells of the invention. The pharmaceutical composition further comprises a pharmaceutically acceptable carrier. The antibody, recombinant protein, bispecific antibody, immunoconjugate, polynucleotide, vector or genetically engineered host cell is soluble in an aqueous carrier, such as buffered saline or the like. Pharmaceutically acceptable excipients required to approximate physiological conditions, such as pH adjusting and buffering agents, sodium acetate, sodium chloride, potassium chloride, calcium chloride or sodium lactate, and the like, may also be included.

The medicament or the pharmaceutical composition can form a pharmaceutical composition with one or more of GITR, CD137, PD-1, PD-Ll, OX40, CTLA4, TIGIT, LAG3, CD47, SIRPa immune checkpoint inhibitor. The medicament or the pharmaceutical composition further comprises a pharmaceutically acceptable carrier, excipient and/or diluent.

It is a further object of the present invention to provide the use of an antibody or antigen binding fragment thereof, recombinant protein, immunoconjugate, polynucleotide, vector or genetically engineered host cell according to the invention for the preparation of a therapeutic or diagnostic agent for autoimmune diseases, viral infections or cancer.

The cancer is liver cancer, gastric cancer, colorectal cancer, lung cancer, ovarian cancer, or any other type of cancer that expresses LAG 3.

The monoclonal antibodies or antigen binding fragments thereof disclosed herein may also be used to prepare chimeric antigen receptors (CARs; also known as chimeric T cell receptors, artificial T cell receptors or chimeric immune receptors) or bispecific antibodies.

The pharmaceutical or neutraceutical compositions of the invention may also include activated or non-activated T cells, genetically modified T cells, human lymphocytes induced, activated and cultured in vitro.

The invention also provides a detection reagent, a diagnostic reagent or a diagnostic kit which comprises one or more of the antibody or antigen binding fragment, the bispecific antibody or fusion protein, the immunoconjugate, the nucleic acid molecule or the carrier, and is used for detecting whether LAG3 protein exists in a biological sample and/or detecting the content of the LAG3 protein.

In specific embodiments of the invention, high affinity LAG3 monoclonal antibodies are disclosed. The invention also discloses the in vitro activity of the antibody and the effect of treating liver cancer by combining hYP7-OKT3 bispecific antibody (anti-tumor activity research of T cell type bispecific antibody in liver cancer and small cell lung cancer, chen Xin, 2020, doctor's laboratory paper of Huazhong university). The antibodies and compositions can be used to treat tumors that are positive for LAG3 expression.

The antibodies and compositions provided herein can be used for a variety of purposes, such as for molecular diagnosis of tumors. The sample may be any sample including, but not limited to, tissue from biopsies, autopsies and pathological specimens. Biological samples also include sections of tissue, such as frozen sections taken for histological purposes. Biological samples also include body fluids such as blood, serum, plasma, sputum, spinal fluid or urine. Biological samples are typically obtained from mammals, including humans, non-human primates, mice, and the like.

The invention has the advantages that:

the monoclonal antibody provided by the invention has high affinity (Kd values are in nM and pM levels and even higher), and has good thermal stability. The monoclonal antibody provided by the invention can block the interaction of LAG3 and ligand MHC-II or FGL1 thereof with high activity. The monoclonal antibody provided by the invention can obviously enhance the anti-tumor activity of the single drug by being combined with the bispecific antibody or the PD1 antibody.

Drawings

FIG. 1 shows the affinity assay of the LAG3 monoclonal antibody M234 scFv-HF and LAG3ECD-hFc according to the present invention.

FIG. 2 shows the affinity assay of the LAG3 monoclonal antibody M234 scFv-HF and LAG3D1D2-hFc according to the present invention.

FIG. 3 shows the affinity assay of M234 IgG for Bio-LAG3D1D 2-hFc.

FIG. 4 shows M234 IgG binding activated PBMC cells according to the invention.

FIG. 5 affinity of Bio-LAG3D1D2-hFc with FGL 1-hFc.

FIG. 6 shows the ability of M234 IgG to block BioLAG D1D2-hFc binding to FGL 1-hFc.

FIG. 7M 234 IgG blocks BioLAG interaction of 3D1D2-hFc with Raji cell surface MHC-II.

FIG. 8M 234 IgG inhibits growth of Huh-7 tumor cells in NOD-SCID mice.

Figure 9 weight change after treatment of Huh7 tumor bearing mice with m234 IgG.

FIG. 10 affinity detection of humanized M234 monoclonal antibodies.

Detailed Description

The present disclosure describes the preparation and identification of monoclonal antibodies that bind LAG 3. Particular embodiments disclose the isolation and characterization of LAG 3-targeting monoclonal antibodies.

The invention will now be described in further detail with reference to the following specific examples, which are to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever. The terms used in the present invention generally have meanings commonly understood by those of ordinary skill in the art unless otherwise indicated. In the following examples, various processes and methods, which are not described in detail, are conventional methods well known in the art.

Abbreviations (abbreviations)

CDR complementarity determining region

CTL cytotoxic T lymphocytes

ELISA enzyme-linked immunosorbent assay

FACS fluorescence activated cell sorting

GPC3 glypican 3

BLI biological film interference technique

HCC hepatocellular carcinoma

HFc human Fc

Ig immunoglobulin

MAb monoclonal antibodies

PE phycoerythrin

EXAMPLE 1 preparation of monoclonal antibodies to LAG3 according to the invention

This example describes the preparation of high affinity monoclonal antibodies against tumor-associated LAG 3.

The truncated protein of LAG3ECD and LAG3D1D2 fusion hffc was expressed and purified using 293F cells (see np_002277.4 for truncation information). Mice were immunized with LAG3ECD-hFc and LAG3D1D2-hFc proteins at 6 weeks of age, 50 μg each time, 3 weeks apart, the spleens of the mice were harvested 2 times after immunization, total RNA in the spleens was extracted, reverse transcribed into cDNA as a template for the establishment of phage antibody libraries, and mouse antibody phage libraries (inventory capacity 4.6x10 ⁹) were constructed. After 3 times of panning on phage library, single clones were randomly picked for sequencing using LAG3ECD-hFc protein as antigen, obtaining high affinity LAG3 monoclonal antibody M234.

The amino acid sequences of the complementarity determining regions (CDR regions) and the variable regions of antibody M234 are shown in tables 1 and 2:

TABLE 1 CDR amino acid sequences of LAG 3M 234 monoclonal antibodies (according to Kabat and IMGT)

TABLE 2 amino acid sequence of variable region of LAG3M234 monoclonal antibody

EXAMPLE 2 affinity of monoclonal antibody M234 scFv-HF of LAG3 to LAG3 protein according to the present invention

The monoclonal antibody scFv-HF of example 1 was prokaryotic expressed using HB2151, and the expression product was purified using Ni-NTA chromatography column (GE HEALTHCARE). The antibody affinity was detected by ELISA.

First, LAG3ECD-hFc protein or LAG3D1D2-hFc protein is respectively coated on the bottom of ELISA plate, then M234 scFv-HF prepared above is respectively added, the first hole is 200nM, gradient dilution is carried out at 1:3, incubation is carried out, and goat anti-FLAG antibody marked by HRP is used as secondary antibody for detection. And detecting absorbance by using a multifunctional enzyme-labeled instrument after color development by using TMB color development liquid, and finally analyzing the result by using GRAPHPAD PRISM. As shown in FIGS. 1 and 2, M234 had affinities of 0.2nM and 0.14nM for LAG3ECD-hFc and LAG3D1D2-hFc, respectively.

Example 3 affinity of M234 IgG for LAG3 protein according to the present invention

The heavy chain variable region of the antibody M234 obtained in example 1 was connected to the heavy chain constant region of an IgG1 type antibody, and after the light chain variable region was connected to the light chain constant region, it was constructed on IgG expression vectors each having two promoters, and expression was performed using 293F cells, and the expression product was purified using a ProteinA column (GE HEALTHCARE). The antibody affinity was detected by ELISA.

Firstly, M234 IgG proteins are respectively coated on the bottom of an ELISA plate, and meanwhile, NHS-Biotin (biological organism) ice is used for marking LAG3D1D2-hFc proteins, so that Biotin marked Bio-LAG3D1D2 is obtained. The biotin-labeled BioLAG D1D2-hFc was then diluted in a first well 200nM 1:3 gradient, incubated therewith, and detected using HRP-labeled streptavidin as a secondary antibody. And detecting absorbance by using a multifunctional enzyme-labeled instrument after color development by using TMB color development liquid, and finally analyzing the result by using GRAPHPAD PRISM. The results are shown in FIG. 3, bioLAG D1D2-hFc had an affinity of 0.45nM for the M234 monoclonal antibody.

EXAMPLE 4 detection of M234 IgG stability

The Tm value is a parameter quantitatively describing the thermal stability of a protein, and is one of the most commonly used indexes in the evaluation of patent drug properties. The higher the Tm value means the more stable the protein conformation. Stability of the antibody prepared in example 1 was determined using Prometheus NT.48 from Nano Temper: the antibody concentration was diluted to 500. Mu.g/ml, then loaded and raised from 25℃to 95℃at a rate of 1℃per minute. Finally, the T _m value of the antibody is obtained. As a result, as shown in Table 3, the antibody M234 had T _m of 61.67 ℃and T _m of 87.73 ℃and an aggregation temperature of 89.12℃and M234 had higher thermostability and aggregation than the LAG3 monoclonal antibody Relatlimab which was already on the market.

TABLE 3T _m values for monoclonal antibodies of the invention

EXAMPLE 4FACS detection of binding of M234 IgG to LAG 3-positive cell lines according to the invention

PBMC cells were incubated with CD3Ab/CD28Ab and IL-2 followed by addition of 10% fetal bovine serum (Hyclone, logan, UT), 1% L-glutamine and 1% penicillin-streptomycin (Invitrogen, carlsbad, calif.) using 1640 medium (Invitrogen, carlsbad, calif.). After harvesting the cells, the antibodies prepared in example 3 were combined with activated PBMC cells, PE-labeled goat anti-human Fc secondary antibody was added, and the antibodies bound to the cell surface were detected. The results are shown in FIG. 4, and the M234 prepared in example 3 has an affinity of 0.10nM for PBMC cells.

EXAMPLE 5M 234 IgG blocking binding of LAG3 to FGL1 according to the invention

FGL1 was first coated on ELISA plate bottom, while using NHS-Biotin (Bio) ice to label LAG3D1D2-hFc protein to obtain Biotin-labeled Bio-LAG3D1D2.Bio-LAG3D1D2 at 1:2 was diluted from 1000nM, incubated at 37℃for 30min after addition to the ELISA plate bottom, and detected using HRP-labeled streptavidin as secondary antibody. And detecting absorbance by using a multifunctional enzyme-labeled instrument after color development by using TMB color development liquid, and finally analyzing the result by using GRAPHPAD PRISM. The results show that LAG3-FGL1 affinity is shown in FIG. 5.

Antibody blocking experiments FGL1 was first coated onto the bottom of ELISA plates, then subjected to first well 200nm 1:3 gradient dilution, and M234 IgG prepared in example 3 was incubated with 100nM BioLAG3D1D2 min separately and then added to ELISA plates, and HRP-labeled streptavidin was used as secondary antibody for detection. And detecting absorbance by using a multifunctional enzyme-labeled instrument after color development by using TMB color development liquid, and finally analyzing the result by using GRAPHPAD PRISM. The antibody blocking activity is shown in FIG. 6, which shows that antibody M234 has LAG3-FGL1 blocking activity with IC ₅₀ of 15nM.

EXAMPLE 6M 234 IgG blocking binding of LAG3 to MHC-II according to the invention

Raji cells naturally express MHC-II at a high level, and Raji cells are used for blocking experiments. Raji cells were cultured using 1640 medium (Invitrogen, carlsbad, calif.) with 10% fetal bovine serum (Hyclone, logan, UT), 1% L-glutamine, and 1% penicillin-streptomycin (Invitrogen, carlsbad, calif.). 10. Mu.g/ml LAG3ECD-hFc protein was incubated with gradient diluted antibody M234 IgG prepared in example 3 for 30min, then added to the collected Raji cells and incubated on ice for 30min. Finally, PE-labeled goat anti-human secondary antibodies are added, and antibodies bound to the cell surface are detected. As a result, as shown in FIG. 7, antibody M234 had LAG3-MHC-II blocking activity with blocking IC ₅₀ of 6.2nM.

EXAMPLE 7M234 IgG inhibits tumor growth in mice

The liver cancer cell line Huh-7 is selected as a test cell for the tumor-inhibiting activity of the antibody. Huh-7 cells were cultured using DMEM medium (Invitrogen, carlsbad, calif.) with 10% fetal bovine serum (Hyclone, logan, UT), 1% L-glutamine and 1% penicillin-streptomycin (Invitrogen, carlsbad, calif.). After cell digestion, each mouse was injected with 5X 10 ⁶ cells, after tumor formation, each mouse was injected with 5X 10 ⁶ PBMC cells, and immunotherapy was performed with 0.2mg/kg hYP7-OKT3 bispecific antibody (antibody preparation procedure was referred to the doctor's thesis of Chen Xin, anti-tumor activity study of T cell type bispecific antibody in liver cancer and small cell lung cancer, university of Huazhong agriculture, 2020)) in combination with 10mg/kg of LAG3 monoclonal antibody prepared in example 3. The tumor growth curves of the mice are shown in fig. 8 and table 4, and the weight changes are shown in fig. 9. The results show that the M234 IgG monoclonal antibody combined with the bispecific antibody can remarkably enhance the anti-tumor activity.

Table 4 mouse tumor volume

Example 8 humanized M234 and affinity detection of humanized M234 IgG

Humanized transformation is carried out on the monoclonal antibody M234 by using a skeleton replacement method of a human antibody Germline to obtain a humanized Hu1M234 sequence, a heavy chain of the humanized M234 is subjected to back mutation to obtain Hu2M234, a light chain of the humanized M234 is subjected to back mutation to obtain Hu3M234, the light chain and the heavy chain are subjected to back mutation simultaneously to obtain Hu4M234, and the humanized sequence is shown in a table 5.

The heavy chain variable region of humanized M234 was linked to the heavy chain constant region of a human IgG1 type antibody and a Flag tag was added after the Fc region, and the light chain variable region of humanized M234 was linked to the kappa light chain constant region of a human antibody, and then cloned into IgG expression vectors, respectively, to construct IgG expression plasmids, and expression was performed using 293F cells, and the expression product was purified using a ProteinA column (GE HEALTHCARE). The affinity of HuM234 IgG was detected by ELISA.

TABLE 5 VH and VL sequences of humanized M234

Coating LAG3ECD-hFc protein on the bottom of an ELISA plate, adding humanized M234 IgG-Flag antibody, diluting the first well by 200nM, incubating the rest of the wells according to a 1:3 gradient, detecting by using HRP-labeled FLAG antibody, detecting absorbance by using a multifunctional enzyme-labeled instrument after color development by using TMB color development liquid, and finally analyzing the experimental result by using GRAPHPAD PRISM. The results are shown in FIG. 10, where humanized HuM234 IgG affinities were 0.96nM,0.52nM,0.59nM,0.16nM, respectively, and where Hu4M234 affinities were substantially identical to the original M234 antibody affinities.

Claims

1. A monoclonal antibody or antigen binding fragment thereof that targets human lymphocyte activation gene 3, comprising a heavy chain variable region and a light chain variable region, characterized in that:

The monoclonal antibody or antigen binding fragment thereof comprises SEQ ID NO:1, a heavy chain variable region CDR1, SEQ ID NO:2, a heavy chain variable region CDR2, SEQ ID NO:3, a heavy chain variable region CDR3 as set forth in SEQ ID NO:4, the light chain variable region CDR1, SEQ ID NO:5, the light chain variable region CDR2, SEQ ID NO:6, and a light chain variable region CDR3.

2. The monoclonal antibody or antigen-binding fragment thereof according to claim 1, wherein:

3. The monoclonal antibody or antigen-binding fragment thereof according to claim 1, characterized in that the antibody is a single chain antibody, a diabody, a chimeric antibody or a humanized antibody.

4. The monoclonal antibody or antigen-binding fragment thereof according to claim 1, characterized in that the antibody is a VH single domain antibody, a Fab fragment, a Fab 'fragment, a F (ab)' 2 fragment, a single chain variable fragment scFv or a disulfide stabilized variable fragment dsFv.

5. A recombinant protein comprising the monoclonal antibody or antigen-binding fragment thereof of any one of claims 1-4 and a tag sequence that facilitates expression and/or purification.

6. Bispecific antibody or fusion protein characterized in that it comprises a monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1-4 or a recombinant protein according to claim 5.

7. Immunoconjugates characterized in that they contain the monoclonal antibodies or antigen-binding fragments thereof according to any one of claims 1 to 4, the recombinant proteins according to claim 5 or the bispecific antibodies or fusion proteins according to claim 6.

8. The immunoconjugate of claim 7, wherein the effector molecule of the immunoconjugate is selected from one or more of a fluorescent-tagged radiolabel, avidin, biotin, an enzyme, a toxin, or a chemotherapeutic agent.

9. A polynucleotide encoding an antibody or antigen-binding fragment thereof according to any one of claims 1-4, or a recombinant protein according to claim 5, or a bispecific antibody or fusion protein according to claim 6, or an immunoconjugate according to claim 7 or 8.

10. A vector comprising the polynucleotide of claim 9.

11. Use of the monoclonal antibody or antigen binding fragment thereof of any one of claims 1-4, the recombinant protein of claim 5, the bispecific antibody or fusion protein of claim 6, the immunoconjugate of claim 7 or 8, the polynucleotide of claim 9, or the vector of claim 10 for the preparation of an immune checkpoint inhibitor.

12. Use according to claim 11, characterized in that the immune checkpoint inhibitor is an agent or drug for diagnosing or treating an immune disease, chronic viral infection or tumor.

13. A pharmaceutical composition comprising one or more of the monoclonal antibodies or antigen binding fragments thereof of claims 1-4, the recombinant protein of claim 5, the bispecific antibody or fusion protein of claim 6, the immunoconjugate of claim 7 or 8, the polynucleotide of claim 9, or the vector of claim 10.

14. The pharmaceutical composition of claim 13, further comprising one or more of GITR, CD137, PD-1, PD-L1, OX40, CTLA4, TI GIT, LAG3, CD47, SIRPa immune checkpoint inhibitor.

15. The pharmaceutical composition according to claim 13, characterized in that the pharmaceutical composition further comprises one or more of activated or non-activated T cells, genetically modified T cells or human lymphocytes induced, activated and cultured in vitro.

16. A host cell, characterized in that it carries a polynucleotide according to claim 9 or a vector according to claim 10.

Lag-3 protein detection reagent, diagnostic reagent or diagnostic kit, characterized in that it comprises one or several of the monoclonal antibodies or antigen-binding fragments thereof according to claims 1-4, the bispecific antibodies and fusion proteins according to claims 5 or 6, the immunoconjugates according to claims 7 or 8, the nucleic acid molecules according to claim 9 or the vectors according to claim 10.