CN113004407B

CN113004407B - LAG3 antibodies and uses thereof

Info

Publication number: CN113004407B
Application number: CN201911344025.2A
Authority: CN
Inventors: 霍永庭; 路力生; 张婵; 涂晶晶; 芦迪; 张先鹏; 张嘉美
Original assignee: Guangdong Fapon Biopharma Inc
Current assignee: Guangdong Fapon Biopharma Inc
Priority date: 2019-12-20
Filing date: 2019-12-20
Publication date: 2022-11-11
Anticipated expiration: 2039-12-20
Also published as: CN113004407A

Abstract

The present invention relates to anti-human LAG3 antibodies and uses thereof. The antibody can block the interaction of LAG-3 protein expressed on the surface of cells and histocompatibility complex II, and shows biological functional activity of stimulating cytokine production in vitro cell function experiment.

Description

LAG3 antibodies and uses thereof

Technical Field

The present invention relates to the field of immunology. In particular, it relates to anti-human LAG3 antibodies and uses thereof.

Background

Lymphocyte activation gene-3 or LAG-3 (also known as CD 223) is a negative costimulatory receptor that regulates T cell homeostasis, proliferation, and activation (Sierro S et al (2010), expert opin. The LAG-3 gene was successfully cloned in 1990 by Triebel (Triebel et al (1990) J.exp.Med.171: 1393-1405) and after two years it was demonstrated that the LAG-3 protein is a ligand protein for the histocompatibility complex MHC II (Baixeras et al (1992) J.exp.Med.176: 327-337) with twice the binding affinity of LAG3 to MHC II compared to CD4 protein and a different binding site (Huard B et al (1997), proc Nat Acad Sci USA, 94. The gene encoding LAG-3 protein is located on chromosome 12 and is structurally and genetically related to the CD4 molecule. LAG-3 proteins typically remember CD4 in activated T cells (Huard et al (1994) immunology 39 213) ⁺ And CD8 ⁺ T cells, regulatory T cells (Huang et al (2004) Immunity 21. LAG-3, as a negative regulatory protein of immune checkpoints, negatively regulates the proliferation and activity of CD4+ and CD8+ T cells. In lentivirusTogether with PD-1, immune tolerance of CD8+ T cells is mediated during infection. LAG-3 molecules on activated CD4 ⁺ CD25 ⁺ Selective up-regulation on regulatory T cells inhibits DC cell maturation by binding to MHC class II molecules on the DC cell surface, resulting in immune tolerance.

Given the importance of LAG-3 in down-regulating immune responses, it was possible to use LAG-3 antibodies for tumor immunotherapy by developing anti-LAG-3 antibodies that block the interaction of LAG-3 proteins with MHCII-type molecules.

Disclosed herein are anti-human LAG-3 antibodies that bind LAG-3 protein with high affinity and specificity, which series of antibodies block the interaction of cell surface-expressed LAG-3 protein with histocompatibility complex II and exhibit biological functional activity that stimulates cytokine production in vitro cell function assays.

Disclosure of Invention

The present invention relates to the following aspects:

1. an anti-human LAG3 antibody or antigen-binding fragment thereof, characterized by comprising heavy chain CDRs 1-3 and light chain CDRs 1-3 selected from the group consisting of:

(1) HCDR1: consisting of SEQ ID NO:86 in seq id no; HCDR2: consisting of SEQ ID NO:87 by sequence number; HCDR3: consisting of SEQ ID NO: 88;

LCDR1: consisting of SEQ ID NO:89 sequence representation; LCDR2: consisting of SEQ ID NO:90, sequence representation; LCDR3: consisting of SEQ ID NO:91 sequence representation;

(2) HCDR1: consisting of SEQ ID NO: 92; HCDR2: consisting of SEQ ID NO:93 with a sequence representation; HCDR3: consisting of SEQ ID NO:94, sequence representation;

LCDR1: consisting of SEQ ID NO:95 in seq id no; LCDR2: consisting of SEQ ID NO:96 is represented by the sequence; LCDR3: consisting of SEQ ID NO:97, sequence representation;

(3) HCDR1: consisting of SEQ ID NO:98 in sequence; HCDR2: consisting of SEQ ID NO:99, sequence representation; HCDR3: consisting of SEQ ID NO:100 in sequence;

LCDR1: consisting of SEQ ID NO:101, sequence representation; LCDR2: consisting of SEQ ID NO:102, sequence representation; LCDR3: consisting of SEQ ID NO: 103;

(4) HCDR1: consisting of SEQ ID NO: 104; HCDR2: consisting of SEQ ID NO:105 in sequence; HCDR3: consisting of SEQ ID NO: 106;

LCDR1: consisting of SEQ ID NO: 107; LCDR2: consisting of SEQ ID NO: 108; LCDR3: consisting of SEQ ID NO:109, or a sequence representation of;

(5) HCDR1: consisting of SEQ ID NO: 110; HCDR2: consisting of SEQ ID NO: 111; HCDR3: consisting of SEQ ID NO: 112;

LCDR1: consisting of SEQ ID NO:113, sequence representation; LCDR2: consisting of SEQ ID NO: 114; LCDR3: consisting of SEQ ID NO:115, sequence representation;

(6) HCDR1: consisting of SEQ ID NO:116 in sequence; HCDR2: consisting of SEQ ID NO: 117; HCDR3: consisting of SEQ ID NO: 118;

LCDR1: consisting of SEQ ID NO:119 with sequence representation; LCDR2: consisting of SEQ ID NO:120, sequence representation; LCDR3: consisting of SEQ ID NO:121 in sequence representation;

(7) HCDR1: consisting of SEQ ID NO: 122; HCDR2: consisting of SEQ ID NO:123 is represented by the sequence; HCDR3: consisting of SEQ ID NO: 124;

LCDR1: consisting of SEQ ID NO: 125; LCDR2: consisting of SEQ ID NO:126 with a sequence representation; LCDR3: consisting of SEQ ID NO: 127;

(8) HCDR1: consisting of SEQ ID NO:128 in sequence; HCDR2: consisting of SEQ ID NO:129, sequence representation; HCDR3: consisting of SEQ ID NO: 130;

LCDR1: consisting of SEQ ID NO:131, or a sequence representation thereof; LCDR2: consisting of SEQ ID NO: 132; LCDR3: consisting of SEQ ID NO: 133;

(9) HCDR1: consisting of SEQ ID NO: 134; HCDR2: consisting of SEQ ID NO:135, sequence representation; HCDR3: consisting of SEQ ID NO: 136;

LCDR1: consisting of SEQ ID NO: 137; LCDR2: consisting of SEQ ID NO: 138; LCDR3: consisting of SEQ ID NO:139 sequence representation;

(10) HCDR1: consisting of SEQ ID NO: 140; HCDR2: consisting of SEQ ID NO: 141; HCDR3: consisting of SEQ ID NO: 142;

LCDR1: consisting of SEQ ID NO: 143; LCDR2: consisting of SEQ ID NO:144, sequence representation; LCDR3: consisting of SEQ ID NO: 145;

(11) HCDR1: consisting of SEQ ID NO: 146; HCDR2: consisting of SEQ ID NO: 147; HCDR3: consisting of SEQ ID NO: 148;

LCDR1: consisting of SEQ ID NO:149 in seq id no; LCDR2: consisting of SEQ ID NO: 150; LCDR3: consisting of SEQ ID NO:151, sequence representation;

(12) HCDR1: consisting of SEQ ID NO:152 sequence representation; HCDR2: consisting of SEQ ID NO:153, sequence representation; HCDR3: consisting of SEQ ID NO: 154;

LCDR1: consisting of SEQ ID NO: 155; LCDR2: consisting of SEQ ID NO: 156; LCDR3: consisting of SEQ ID NO:157, sequence representation;

(13) HCDR1: consisting of SEQ ID NO: 158; HCDR2: consisting of SEQ ID NO:159 in seq id no; HCDR3: consisting of SEQ ID NO: 160;

LCDR1: consisting of SEQ ID NO:161 is shown in sequence; LCDR2: consisting of SEQ ID NO: 162; LCDR3: consisting of SEQ ID NO: 163;

(14) HCDR1: consisting of SEQ ID NO:164 in sequence representation; HCDR2: consisting of SEQ ID NO:165, sequence representation; HCDR3: consisting of SEQ ID NO:166, sequence representation;

LCDR1: consisting of SEQ ID NO:167, sequence representation; LCDR2: consisting of SEQ ID NO:168 sequence representation; LCDR3: consisting of SEQ ID NO:169, sequence representation;

(15) HCDR1: consisting of SEQ ID NO: 170; HCDR2: consisting of SEQ ID NO:171 sequence representation; HCDR3: consisting of SEQ ID NO: 172;

LCDR1: consisting of SEQ ID NO:173, sequence number; LCDR2: consisting of SEQ ID NO: 174; LCDR3: consisting of SEQ ID NO: 175;

(16) HCDR1: consisting of SEQ ID NO: 176; HCDR2: consisting of SEQ ID NO:177 of the sequence representation; HCDR3: consisting of SEQ ID NO: 178;

LCDR1: consisting of SEQ ID NO: 179; LCDR2: consisting of SEQ ID NO:180, sequence representation; LCDR3: consisting of SEQ ID NO:181 in seq id no;

(17) HCDR1: consisting of SEQ ID NO:182, a sequence representation; HCDR2: consisting of SEQ ID NO: 183; HCDR3: consisting of SEQ ID NO: 184;

LCDR1: consisting of SEQ ID NO:185 of the sequence; LCDR2: consisting of SEQ ID NO: 186; LCDR3: consisting of SEQ ID NO:187, sequence representation;

(18) HCDR1: consisting of SEQ ID NO: 188; HCDR2: consisting of SEQ ID NO:189 a sequence representation; HCDR3: consisting of SEQ ID NO: 190;

LCDR1: consisting of SEQ ID NO:191, sequence representation; LCDR2: consisting of SEQ ID NO:192 of seq id no; LCDR3: consisting of SEQ ID NO:193, sequence representation;

(19) HCDR1: consisting of SEQ ID NO:194, sequence representation; HCDR2: consisting of SEQ ID NO: 195; HCDR3: consisting of SEQ ID NO:196, sequence representation;

LCDR1: consisting of SEQ ID NO:197 sequence representation; LCDR2: consisting of SEQ ID NO:198 by sequence representation; LCDR3: consisting of SEQ ID NO:199 for a sequence representation; or

(20) HCDR1: consisting of SEQ ID NO:200 in sequence; HCDR2: consisting of SEQ ID NO:201 in sequence; HCDR3: consisting of SEQ ID NO: 202;

LCDR1: consisting of SEQ ID NO:203 of the sequence; LCDR2: consisting of SEQ ID NO: 204; LCDR3: consisting of SEQ ID NO: 205.

2. The antibody or antigen-binding fragment thereof of 1 above, wherein the antibody comprises:

(1) A heavy chain variable region comprising or consisting of the sequence:

SEQ ID NO:6, or

And SEQ ID NO:6, and a sequence having at least 60%,70%,80%,85%, preferably at least 86%,87%,88%,89%,90%,91%,92%,93%,94%,95%,96%,97%,98% or 99% sequence identity to the sequence set forth in seq id no, and

a light chain variable region comprising or consisting of the sequence:

SEQ ID NO:7, or

And SEQ ID NO:7, preferably at least 86%,87%,88%,89%,90%,91%,92%,93%,94%,95%,96%,97%,98% or 99% sequence identity;

(2) A heavy chain variable region comprising or consisting of the sequence:

SEQ ID NO:10, or

And SEQ ID NO:10, and a sequence having at least 60%,70%,80%,85%, preferably at least 86%,87%,88%,89%,90%,91%,92%,93%,94%,95%,96%,97%,98% or 99% sequence identity to the sequence depicted in seq id No. 10, and

a light chain variable region comprising or consisting of the sequence:

SEQ ID NO:11, or

And SEQ ID NO:11, preferably at least 86%,87%,88%,89%,90%,91%,92%,93%,94%,95%,96%,97%,98% or 99% sequence identity;

(3) A heavy chain variable region comprising or consisting of the sequence:

SEQ ID NO:14, or

And SEQ ID NO:14, and a sequence having at least 60%,70%,80%,85%, preferably at least 86%,87%,88%,89%,90%,91%,92%,93%,94%,95%,96%,97%,98% or 99% sequence identity to the sequence set forth in seq id no, and

a light chain variable region comprising or consisting of the sequence:

SEQ ID NO:15, or

And SEQ ID NO:15, preferably at least 86%,87%,88%,89%,90%,91%,92%,93%,94%,95%,96%,97%,98% or 99% sequence identity;

(4) A heavy chain variable region comprising or consisting of the sequence:

SEQ ID NO:18, or

And SEQ ID NO:18, preferably at least 86%,87%,88%,89%,90%,91%,92%,93%,94%,95%,96%,97%,98% or 99% sequence identity, and

a light chain variable region comprising or consisting of the sequence:

SEQ ID NO:19, or

And SEQ ID NO:19, preferably at least 86%,87%,88%,89%,90%,91%,92%,93%,94%,95%,96%,97%,98% or 99% sequence identity;

(5) A heavy chain variable region comprising or consisting of the sequence:

the amino acid sequence of SEQ ID NO:22, or

And SEQ ID NO:22, and a sequence having at least 60%,70%,80%,85%, preferably at least 86%,87%,88%,89%,90%,91%,92%,93%,94%,95%,96%,97%,98% or 99% sequence identity, and

a light chain variable region comprising or consisting of the sequence:

SEQ ID NO:23, or

And SEQ ID NO:23, preferably at least 86%,87%,88%,89%,90%,91%,92%,93%,94%,95%,96%,97%,98% or 99% sequence identity;

(6) A heavy chain variable region comprising or consisting of the sequence:

SEQ ID NO:26, or

And SEQ ID NO:26, has at least 60%,70%,80%,85%, preferably at least 86%,87%,88%,89%,90%,91%,92%,93%,94%,95%,96%,97%,98% or 99% sequence identity, and

a light chain variable region comprising or consisting of the sequence:

SEQ ID NO:27, or

And SEQ ID NO:27, or a sequence having at least 60%,70%,80%,85%, preferably at least 86%,87%,88%,89%,90%,91%,92%,93%,94%,95%,96%,97%,98% or 99% sequence identity;

(7) A heavy chain variable region comprising or consisting of the sequence:

the amino acid sequence of SEQ ID NO:30, or

And SEQ ID NO:30, and a sequence having at least 60%,70%,80%,85%, preferably at least 86%,87%,88%,89%,90%,91%,92%,93%,94%,95%,96%,97%,98% or 99% sequence identity to the sequence set forth in seq id no, and

a light chain variable region comprising or consisting of the sequence:

SEQ ID NO:31, or

And SEQ ID NO:31, having at least 60%,70%,80%,85%, preferably at least 86%,87%,88%,89%,90%,91%,92%,93%,94%,95%,96%,97%,98% or 99% sequence identity;

(8) A heavy chain variable region comprising or consisting of the sequence:

the amino acid sequence of SEQ ID NO:34, or

And SEQ ID NO:34, and a sequence having at least 60%,70%,80%,85%, preferably at least 86%,87%,88%,89%,90%,91%,92%,93%,94%,95%,96%,97%,98% or 99% sequence identity to the sequence depicted in seq id no, and

a light chain variable region comprising or consisting of the sequence:

SEQ ID NO:35, or

And SEQ ID NO:35, preferably at least 86%,87%,88%,89%,90%,91%,92%,93%,94%,95%,96%,97%,98% or 99% sequence identity;

(9) A heavy chain variable region comprising or consisting of the sequence:

the amino acid sequence of SEQ ID NO:38, or

And SEQ ID NO:38, and a sequence having at least 60%,70%,80%,85%, preferably at least 86%,87%,88%,89%,90%,91%,92%,93%,94%,95%,96%,97%,98% or 99% sequence identity to the sequence depicted in seq id no, and

a light chain variable region comprising or consisting of the sequence:

SEQ ID NO:39, or

And SEQ ID NO:39, has at least 60%,70%,80%,85%, preferably at least 86%,87%,88%,89%,90%,91%,92%,93%,94%,95%,96%,97%,98% or 99% sequence identity;

(10) A heavy chain variable region comprising or consisting of the sequence:

the amino acid sequence of SEQ ID NO:42, or

And SEQ ID NO:42, has at least 60%,70%,80%,85%, preferably at least 86%,87%,88%,89%,90%,91%,92%,93%,94%,95%,96%,97%,98% or 99% sequence identity, and

a light chain variable region comprising or consisting of the sequence:

SEQ ID NO:43, or

And SEQ ID NO:43, preferably at least 86%,87%,88%,89%,90%,91%,92%,93%,94%,95%,96%,97%,98% or 99% sequence identity;

(11) A heavy chain variable region comprising or consisting of the sequence:

SEQ ID NO:46, or

And SEQ ID NO:46, and a sequence having at least 60%,70%,80%,85%, preferably at least 86%,87%,88%,89%,90%,91%,92%,93%,94%,95%,96%,97%,98% or 99% sequence identity to the sequence depicted in seq id no

A light chain variable region comprising or consisting of the sequence:

SEQ ID NO:47, or

And SEQ ID NO:47, preferably at least 86%,87%,88%,89%,90%,91%,92%,93%,94%,95%,96%,97%,98% or 99% sequence identity;

(12) A heavy chain variable region comprising or consisting of the sequence:

SEQ ID NO:50, or

And SEQ ID NO:50, has at least 60%,70%,80%,85%, preferably at least 86%,87%,88%,89%,90%,91%,92%,93%,94%,95%,96%,97%,98% or 99% sequence identity, and

a light chain variable region comprising or consisting of the sequence:

SEQ ID NO:51, or

And SEQ ID NO:51, preferably at least 86%,87%,88%,89%,90%,91%,92%,93%,94%,95%,96%,97%,98% or 99% sequence identity;

(13) A heavy chain variable region comprising or consisting of the sequence:

SEQ ID NO:54, or

And SEQ ID NO:54, has at least 60%,70%,80%,85%, preferably at least 86%,87%,88%,89%,90%,91%,92%,93%,94%,95%,96%,97%,98% or 99% sequence identity, and

a light chain variable region comprising or consisting of the sequence:

SEQ ID NO:55, or

And SEQ ID NO:55, preferably at least 86%,87%,88%,89%,90%,91%,92%,93%,94%,95%,96%,97%,98% or 99% sequence identity;

(14) A heavy chain variable region comprising or consisting of the sequence:

SEq ID NO:58, or

And SEQ ID NO:58, and a sequence having at least 60%,70%,80%,85%, preferably at least 86%,87%,88%,89%,90%,91%,92%,93%,94%,95%,96%,97%,98% or 99% sequence identity, and

a light chain variable region comprising or consisting of the sequence:

SEQ ID NO:59, or

And SEQ ID NO:59, has at least 60%,70%,80%,85%, preferably at least 86%,87%,88%,89%,90%,91%,92%,93%,94%,95%,96%,97%,98% or 99% sequence identity;

(15) A heavy chain variable region comprising or consisting of the sequence:

SEQ ID NO:62, or

And SEQ ID NO:62, preferably at least 86%,87%,88%,89%,90%,91%,92%,93%,94%,95%,96%,97%,98% or 99% sequence identity, and

a light chain variable region comprising or consisting of the sequence:

SEQ ID NO:63, or

And SEQ ID NO:63, has at least 60%,70%,80%,85%, preferably at least 86%,87%,88%,89%,90%,91%,92%,93%,94%,95%,96%,97%,98% or 99% sequence identity;

(16) A heavy chain variable region comprising or consisting of the sequence:

SEQ ID NO:66, or

And SEQ ID NO:66, preferably at least 86%,87%,88%,89%,90%,91%,92%,93%,94%,95%,96%,97%,98% or 99% sequence identity, and

a light chain variable region comprising or consisting of the sequence:

SEQ ID NO:67, or

And SEQ ID NO:67, preferably at least 86%,87%,88%,89%,90%,91%,92%,93%,94%,95%,96%,97%,98% or 99% sequence identity;

(17) A heavy chain variable region comprising or consisting of the sequence:

SEQ ID NO:70, or

And SEQ ID NO:70, preferably at least 86%,87%,88%,89%,90%,91%,92%,93%,94%,95%,96%,97%,98% or 99% sequence identity, and

a light chain variable region comprising or consisting of the sequence:

SEQ ID NO:71, or

And SEQ ID NO:71, having at least 60%,70%,80%,85%, preferably at least 86%,87%,88%,89%,90%,91%,92%,93%,94%,95%,96%,97%,98% or 99% sequence identity;

(18) A heavy chain variable region comprising or consisting of the sequence:

SEQ ID NO:74, or

And SEQ ID NO:74, preferably at least 86%,87%,88%,89%,90%,91%,92%,93%,94%,95%,96%,97%,98% or 99% sequence identity, and

a light chain variable region comprising or consisting of the sequence:

the amino acid sequence of SEQ ID NO:75, or

And SEQ ID NO:75, preferably at least 86%,87%,88%,89%,90%,91%,92%,93%,94%,95%,96%,97%,98% or 99% sequence identity;

(19) A heavy chain variable region comprising or consisting of the sequence:

SEQ ID NO:78, or

And SEQ ID NO:78, preferably at least 86%,87%,88%,89%,90%,91%,92%,93%,94%,95%,96%,97%,98% or 99% sequence identity, and

a light chain variable region comprising or consisting of the sequence:

the amino acid sequence of SEQ ID NO:79, or

And SEQ ID NO:79, preferably at least 86%,87%,88%,89%,90%,91%,92%,93%,94%,95%,96%,97%,98% or 99% sequence identity; or

(20) A heavy chain variable region comprising or consisting of the sequence:

SEQ ID NO:82, or

And SEQ ID NO:82, and a sequence having at least 60%,70%,80%,85%, preferably at least 86%,87%,88%,89%,90%,91%,92%,93%,94%,95%,96%,97%,98% or 99% sequence identity, and

a light chain variable region comprising or consisting of the sequence:

SEQ ID NO:83, or

And SEQ ID NO:83, or a sequence having at least 60%,70%,80%,85%, preferably at least 86%,87%,88%,89%,90%,91%,92%,93%,94%,95%,96%,97%,98% or 99% sequence identity.

3. The antibody or antigen-binding fragment thereof according to any one of items 1 to 2 above, wherein the antibody further comprises a heavy chain constant region and a light chain constant region, preferably from human IgG or IgM, more preferably IgG4, preferably the amino acid sequence of the heavy chain constant region is the sequence shown in SEQ ID No.84 and the amino acid sequence of the light chain constant region is the sequence shown in SEQ ID No. 85.

4. The antibody or antigen-binding fragment thereof of any one of above 1-2, wherein the antigen-binding fragment is selected from the group consisting of Fab, svFv, fab ', dAb, F (ab') ₂ Fv or Fab/c.

5. The antibody or antigen-binding fragment thereof of any one of items 1-2 above, wherein the antibody is a humanized antibody, a chimeric antibody, a multispecific antibody (e.g., bispecific antibody).

6. A polynucleotide encoding the antibody or antigen-binding fragment thereof according to any one of the above items 1 to 5.

7. The polynucleotide of 6 above selected from the group consisting of SEQ ID NO:4 and 5; SEQ ID NO:8 and 9; the amino acid sequence of SEQ ID NO:12 and 13; SEQ ID NO:16 and 17; SEQ ID NO:20 and 21; SEQ ID NO:24 and 25; SEQ ID NO:28 and 29; SEQ ID NO:32 and 33; SEQ ID NO:36 and 37; SEQ ID NO:40 and 41; SEQ ID NO:44 and 45; SEQ ID NO:48 and 49; the amino acid sequence of SEQ ID NO:52 and 53; SEQ ID NO:56 and 57; SEQ ID NO:60 and 61; SEQ ID NO:64 and 65; SEQ ID NO:68 and 69; SEQ ID NO:72 and 73; SEQ ID NO:76 and 77; or SEQ ID NO:80 and 81.

8. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof of any one of 1-5 above; optionally, it further comprises a pharmaceutically acceptable carrier and/or excipient.

9. Use of the antibody or antigen-binding fragment thereof of any one of 1 to 5 above for the prophylactic and/or therapeutic and/or adjunctive therapeutic and/or diagnostic treatment of a tumor disease or for the manufacture of a medicament for the prophylactic and/or therapeutic and/or adjunctive therapeutic and/or diagnostic treatment of a tumor disease, preferably a tumor expressing LAG3, preferably cancer, or for the manufacture of a medicament for modulating an immune response, preferably to stimulate IL-2 or IFN gamma secretion.

10. The pharmaceutical composition of claim 8 or the use of claim 9, in a form suitable for injection, preferably for administration by subcutaneous, intradermal, intravenous, intramuscular or intralesional injection.

As used herein, the term "light chain" includes full-length light chains and fragments thereof having variable region sequences to confer binding specificity. The full-length light chain includes a variable region domain V _L And constant region Domain C _L . The variable region domain of the light chain is at the amino terminus of the polypeptide. Light chains include kappa and lambda chains.

As used herein, the term "heavy chain" includes full-length heavy chains and fragments thereof having variable region sequences to confer binding specificity. Full-length heavy chain includes variable region domain V _H And 3 constant region domains C _H1 、C _H2 And C _H3 。V _H The structural domain is at the amino terminal end of the polypeptide, and C _H Domain at the carboxyl terminus, C _H3 Closest to the carboxy terminus of the polypeptide. The heavy chain may be of any isotype, including IgG (including IgG1, igG2, igG3 and IgG4 subtypes), igA (including IgA1 and IgA2 subtypes), igM and IgE.

As used herein, the term "Fab fragment" consists of one light chain and CH1 and the variable region of one heavy chain. The heavy chain of a Fab molecule is unable to form a disulfide bond with another heavy chain molecule.

As used herein, the term "Fc" region contains two antibody-containing Cs _H1 And C _H2 A heavy chain fragment of a domain. Two heavy chain fragments through two or more disulfide bonds and through C _H3 The hydrophobic interactions of the domains remain together.

As used herein, the term "Fab' fragment" contains one light chain and one heavy chain portion (which contains V) _H Domains and C _H1 Domains and also C _H1 And C _H2 Part of the region between the domains) so that an interchain disulfide bond can be formed between the two heavy chains of the two Fab 'fragments to form F (ab') ₂ A molecule.

As used herein, the term "F (ab') ₂ A fragment "contains two light chains and two heavy chains containing a portion of the constant region between the CH1 and CH2 domains, such that an interchain disulfide bond is formed between the two heavy chains. F (ab') ₂ The fragment thus consists of two Fab' fragments held together by a disulfide bond between the two heavy chains.

As used herein, the term "Fv region" comprises the variable regions from the heavy and light chains, but lacks the constant regions.

As used in the present invention, the term "Fd" fragment means a fragment consisting of V _H And C _H1 Antibody fragments consisting of domains (Ward et al, nature 341 544-546 (1989)).

As used in the present invention, the term "dAb" fragment (Ward et al, nature 341 _H Structural domain composition。

As used herein, the term "Fab/c" fragment is an intermediate in the cleavage of an immunoglobulin by pepsin digestion, which combines the advantages of the Fab and Fc regions, i.e., strong diffusibility and slow metabolic clearance in vivo, while maintaining high affinity (Liu Jianjun, J. Cell and molecular immunology, 1989 (4): 29-29).

As used herein, the term "single chain antibody" is an Fv molecule in which the heavy and light chain variable regions are joined by a flexible linker to form a single polypeptide chain (which forms the antigen binding region) (see, e.g., bird et al, science.242:423-426 (1988) and Huston et al, proc. Natl. Acad. Sci. USA.90:5879-5883 (1988)).

As used herein, the term "multispecific antigen-binding protein" or "multispecific antibody" is an antigen-binding protein or antibody that targets more than one antigen or epitope.

As used herein, the term "bispecific", "dual specificity" or "bifunctional" antigen binding proteins or antibodies are hybrid antigen binding proteins or antibodies, respectively, having two different antigen binding sites. A bispecific antibody is a multispecific antigen-binding protein or multispecific antibody, and may be produced by a variety of methods, including, but not limited to, fusion of hybridomas or linkage of Fab' fragments. See, e.g., songsivilai and Lachmann,1990, clin. Exp. Immunol.79:315 to 321; kostelny et al, 1992, J.Immunol.148:1547-1553. The two binding sites of a bispecific antigen binding protein or antibody will bind two different epitopes that are present on the same or different protein targets.

As used herein, the term "humanized antibody" refers to an antibody or antibody fragment obtained by replacing all or a portion of the CDR regions of a human immunoglobulin (recipient antibody) with the CDR regions of a non-human antibody (donor antibody), which may be a non-human (e.g., mouse, rat, or rabbit) antibody of the desired specificity, affinity, or reactivity. In addition, some amino acid residues in the Framework Region (FR) of the acceptor antibody may be substituted with those of the corresponding non-human antibody, or with those of other antibodies, to further refine or optimize the performance of the antibody. For more details on humanized antibodies, see, e.g., jones et al, nature,321:522 525 (1986); reichmann et al, nature,332:323329 (1988); presta, curr, op, struct, biol, 2:593 596 (1992); and Clark, immunol. Today 21:397 402 (2000).

As used herein, the term "epitope" refers to a site on an antigen to which an immunoglobulin or antibody specifically binds. An "epitope" is also referred to in the art as an "antigenic determinant". Epitopes or antigenic determinants usually consist of chemically active surface groups of molecules such as amino acids or carbohydrates or sugar side chains and usually have specific three-dimensional structural characteristics as well as specific charge characteristics. For example, an epitope typically includes at least 3,4,5,6,7,8,9, 10, 11, 12, 13, 14, or 15 contiguous or non-contiguous amino acids in a unique spatial conformation, which can be "linear" or "conformational". See, e.g., epitopic Mapping Protocols in Methods in Molecular Biology, vol 66, g.e. morris, ed. (1996). In a linear epitope, all points of interaction between a protein and an interacting molecule (e.g., an antibody) exist linearly along the primary amino acid sequence of the protein. In conformational epitopes, the point of interaction exists across protein amino acid residues that are separated from each other.

As used herein, the terms "similarity" or "sequence similarity", "identity" refer to the relationship between the sequences of two or more protein or polypeptide molecules, as determined by aligning and comparing the sequences. "percent identity" means the percentage of identical residues between amino acids in the molecules being compared, and can be calculated based on the size of the smallest molecule to be compared. In order to perform these calculations, gaps in the alignment (if any) must be addressed by a particular mathematical model or computer program (i.e., an "algorithm"). The term "substantial identity", when applied to polypeptides, means that two peptide sequences, when optimally aligned, for example using the programs GAP or BESTFIT, using default GAP weights provided by the programs, share at least 70%, 75% or 80% sequence identity, at least 90% or 95% sequence identity, and at least 97%,98% or 99% sequence identity. In some cases, residue positions that are not identical differ by conservative amino acid substitutions. A "conservative amino acid substitution" is one in which an amino acid residue is replaced with another amino acid residue having a side chain R group that possesses similar chemical properties (e.g., charge or aqueous). In general, conservative amino acid substitutions will not substantially alter the functional properties of the protein. In cases where two or more amino acid sequences differ from each other by conservative substitutions, the percent sequence identity may be upregulated to correct for the conservative nature of the substitution. Methods for making this adjustment are well known to those skilled in the art. See, e.g., pearson, methods mol. Biol.243:307-31 (1994). Examples of groups of amino acids having side chains with similar chemical properties include 1) aliphatic hydroxyl side chains: glycine, alanine, valine, leucine, and isoleucine: 2) Aliphatic hydroxyl side chain: serine and threonine: 3) Amide-containing side chain: asparagine and glutamine: 4) Aromatic side chain: phenylalanine, tyrosine and tryptophan: 5) Basic side chain: lysine, arginine and histidine: 6) Acidic side chain: aspartic acid and glutamic acid; and 7) sulfur containing side chains: cysteine and methionine. For example, the conservative amino acid substitution group is valine-leucine-isoleucine-glycine-alanine, phenylalanine-tyrosine, threonine-serine, lysine-arginine, glutamic acid-aspartic acid, and asparagine-glutamine.

Alternatively, conservative substitutions are described in Gonnet et al, science 256:1443-45 (1992) (incorporated herein by reference) has any variation in the PAM250 log-likelihood matrix that has a positive value. A "moderately conservative" substitution is any change that has a non-negative value in the PAM250 log-likelihood matrix.

Drawings

FIG. 1: binding assay of LAG3 monoclonal antibody to CHO cells.

FIG. 2: a binding assay for a LAG3 monoclonal antibody to CHO-hLAG3, wherein FIG. 2A is a binding assay for LAG3-96, LAG3-188, LAG3-283, LAG3-285, and relatlimab to a cell; FIG. 2B is a binding assay for LAG3-172, LAG3-268, and relatlimab to cells.

FIG. 3: a binding assay for the LAG3 monoclonal antibody to CHO-cynoLAG3, wherein FIG. 3A is a binding assay for LAG3-96, LAG3-188, LAG3-283, LAG3-285, and relatlimab to a cell; FIG. 3B is a binding assay for LAG3-172, LAG3-268, and relatlimab to cells.

FIG. 4: a binding assay for a LAG3 monoclonal antibody to PHA-activated hPPMC, wherein FIG. 4A is a binding assay for LAG3-96, LAG3-188, LAG3-283, LAG3-285, and relatlimab to a cell; FIG. 4B is a binding assay for LAG3-172, LAG3-268, and relatlimab to cells.

FIG. 5 is a schematic view of: competitive analysis of binding epitopes, wherein FIG. 5A shows the competitive binding of the antibody to membrane-expressed human LAG3 by adding the anti-human LAG3 antibody after the control antibody is added to bind to the membrane-expressed human LAG 3; FIG. 5B shows the competitive binding of anti-human LAG3 antibody to membrane-expressed human LAG3, followed by the addition of control antibody and detection of the competitive binding of antibody to membrane-expressed human LAG3

FIG. 6: blocking Raji-MHCII-hLAG3 by the LAG3 antibody, wherein, the block effect of the anti-human LAG3 antibody is detected by adding Raji cells after the antibody is combined with human LAG3 protein in figure 6A; FIG. 6B shows the incubation of antibody with Raii cells prior to detection of binding of antibody to human LAG3 protein

FIG. 7 is a schematic view of: in vitro Jurkat-hLAG3-Raii bioassay of LAG3 antibody.

FIG. 8: SEB-activated hPBMC bioassay, otherwise fig. 8A is donor-derived activated PBMC, using anti-human LAG3 antibody alone; FIG. 8B is a donor-derived activated PBMC, anti-human LAG3 antibody in combination with a fixed concentration of anti-human PD-1 antibody; FIG. 8C is a combination of donor-derived activated PBMCs, a fixed concentration of human LAG3 antibody and different concentrations of anti-human PD-1 antibody; FIG. 8D is donor-two derived activated PBMCs using anti-human LAG3 antibody alone; FIG. 8E shows a combination of donor-derived activated PBMC, anti-human LAG3 antibody and fixed concentration anti-human PD-1 antibody; figure 8F is a combination of donor-derived activated PBMCs, fixed concentrations of human LAG3 antibody and different concentrations of anti-human PD-1 antibody.

FIG. 9: SEB activated PBMC bioassay where figure 9A is donor-derived activated PBMC using anti-human LAG3 antibody alone; FIG. 9B is a donor-derived activated PBMC, anti-human LAG3 antibody in combination with a fixed concentration of anti-human PD-1 antibody; FIG. 9C is a combination of donor-derived activated PBMCs, a fixed concentration of human LAG3 antibody and different concentrations of anti-human PD-1 antibody; figure 9D is donor-derived activated PBMCs using anti-human LAG3 antibody alone; FIG. 9E shows a combination of donor-derived activated PBMC, anti-human LAG3 antibody and fixed concentration anti-human PD-1 antibody; figure 9F is a combination of donor-two derived activated PBMCs, a fixed concentration of human LAG3 antibody and different concentrations of anti-human PD-1 antibody.

FIG. 10: in vitro T-DC MLR bioassay, wherein FIG. 10A is secretory expression of IL-2; fig. 10B shows secretion of interferon γ.

Detailed Description

Embodiments of the present invention will be described in detail below with reference to examples, but it will be understood by those skilled in the art that the following examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention. The examples, in which specific conditions are not specified, were carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.

EXAMPLE 1 Generation of anti-human LAG3 antibodies

1.1 immunogens

Recombinant human LAG3 fusion proteins were used as immunogens for the production of human LAG3 antibodies. In the course of immunization, the his-tagged human LAG3 extracellular domain fusion protein hLAG3-his (Acrobiostem, cat. LA3-H5222, also referred to as "human LAG3 recombinant protein" in the examples below) was used as immunogen.

1.2 immunization protocol

Anti-human LAG3 murine mabs were generated using the commonly used mouse hybridoma system. The procedure was as follows, 1.2mg/ml of recombinant human LAG3 recombinant protein (i.e., hLAG 3-his) was mixed in equal volumes with Freund's complete adjuvant (Sigma, cat. F5881), and the resulting oily emulsion was administered subcutaneously to the dorsal site of 6-week-old female BALB/c mice (female center of Guangdong provincial medical laboratory animals) at a dose of 0.2 ml. 14 days after the first immunization, mixing the recombinant human LAG3 recombinant protein with Freund's incomplete adjuvant (Sigma, cat. F5506) in equal volume, performing intraperitoneal immunization, performing booster immunization to 4 needles, collecting tail blood for titer detection, and performing spleen fusion when the titer reaches a set titer.

1.3 preparation of hybridoma cells

The mouse tumor cells and immune spleen cells are mixed according to the cell number ratio of 1: 10, transferred into a 50ml centrifuge tube, and washed once by RPMI1640 basic culture medium. Discard the supernatant, mix the cells well, add slowly 1ml 50% PEG1500 fusion. 1min after confluency, cell confluency was terminated by addition of 15ml RPMIl640 basal medium. Centrifuge at 1000rpm for 5min and discard the supernatant. Gently suspending the selection medium in 50ml of RPMI1640 medium, dividing equally into 96-well plates, 10 pieces, 50. Mu.l/well, 37 ℃ and 5% CO ₂ And (5) standing and culturing the cells in a cell culture box. The culture was continued until the sixth day, and the HT medium (HT-containing RPMI1640 complete medium) was replaced twice.

1.4 detection of anti-human LAG3 antibodies

Human Fc-tagged human LAG3 recombinant protein (Acrobiosystem, cat. LA3-H5255) was diluted with 0.05M carbonate buffer to a final concentration of 2. Mu.g/ml, added at 100. Mu.l/well to a 96-well ELISA assay plate, and incubated at 37 ℃ for 2H or coated overnight at 2-4 ℃. Discard the supernatant, add blocking solution (1xPBS +1% BSA) at 200. Mu.l/well, block for 1h at 37 ℃. On day 7 after the recombinant fusion, cell supernatants were added at 100. Mu.l/well and incubated at 37 ℃ for 30min. Wash 3 times with 1 xPBS. HRP-labeled goat anti-mouse IgG (sigma A0168-1 ML) was added to 100. Mu.l/well, and the mixture was incubated at 37 ℃ for 30min and washed 3 times with 1xPBS to carry out a color reaction.

1.5 screening for subclones

The fusions with OD450 of 1 or more were selected and cloned by limiting dilution at least 2 times. Detection was performed using 1.4. The obtained positive subclones are cultured in vitro for seed preservation and expression.

1.6 expression and purification of anti-human LAG3 antibodies

When the clone producing anti-human LAG3 antibody grows to reach 80% of confluence rate, the clone is divided into two partsCell blowing, centrifugation at 1000rpm for 5min, cell suspension with 30ml 5 FBS-containing SFM complete medium and transfer to 150ml SF shake flask, 37 ℃,8 CO% ₂ Culturing at 120rpm for 2-3 days. When the cell density reaches 5x10 ⁵ Centrifugation at 1000rpm for 5 min/ml, resuspension of the cells in 50ml of serum-free SFM basal medium and transfer to 250ml SF shake flasks, 37 ℃,8% CO ₂ Culturing at 120rpm for 4-5 days, centrifuging at 9000rpm for 20min, collecting supernatant, and purifying. The purification was performed by ProA affinity chromatography. The process is as follows, using an AKTA avant 150 chromatographic apparatus, with at least 5CV of equilibration buffer (10 mM PBS) to equilibrate a chromatography column (e.g., mabSelectSuRe LX, GE), loading the sample onto the column, allowing the target protein to adsorb onto the column while other impurities are separated by breakthrough. After the loading was completed, the column was washed again with at least 5CV of equilibration buffer (10 mM PBS), followed by elution of the target protein with elution buffer (20mM NaAc, pH = 3.4), and a neutralization buffer (1M Tris, pH 8.0) was previously added to the collection tube, and the addition volume of the neutralization buffer was determined according to the estimated content of the eluted sample, and 10% of the elution volume was generally added.

1.7 determination of anti-human LAG3 antibody concentration

The sample is subjected to concentration measurement by using Biotek-Epoch-Take-3, the antibody concentration is detected by using an A280 method, namely, the extinction coefficient is E.C =1.37, the optical path is =0.05mm (the optical paths of different holes of a Take-3 plate are slightly different and can be automatically corrected), the light absorption value of the sample is detected by using equipment, and the concentration of the antibody to be detected is calculated according to Lambert-Beer 1 aw. If the sample concentration is too low, ultrafiltration concentration is required, and an ultrafiltration concentration tube (

Ultra-15Centrifugal Filter devices, 30kD) the sample concentration was concentrated to > 0.5mg/ml following the general protocol provided in the specification; the concentrated end samples were collected and sterile filtered through a 0.22um sterile pillow filter (Li Baite, PES,0.22um, 13mm diameter).

1.8 identification of anti-human LAG3 antibody subtypes

A recombinant human LAG 3-carrying Fc tag recombinant protein (Acrobiosystem, cat. LA3-H5255) was diluted with 0.05M pH9.5 carbonate buffer to a final concentration of 2. Mu.g/mL. Add 100. Mu.l/well to 96-well ELISA assay plates and incubate at 37 ℃ for 2 hours or 4 ℃ overnight. Discard the supernatant, add blocking solution (1xPBS +1% BSA) at 200. Mu.l/well, block for 1h at 37 ℃. The antibody was diluted to 1. Mu.g/ml with 1xPBS containing 1% BSA, and anti-human LAG3 antibody was added at 100. Mu.l/well, followed by incubation at 37 ℃ for 30 minutes. Wash 3 times with 1 xPBS. HRP-labeled goat anti-mouse IgG (sigma A0168-1 ML), HRP-labeled goat anti-mouse IgG2a (thermo fisher M32207), HRP-labeled goat anti-mouse IgG1 (thermo fisher PA 1-74421), HRP-labeled goat anti-mouse IgG2b (thermo fisher M32407), HRP-labeled goat anti-mouse IgG3 (thermo fisher M32607), and HRP-labeled goat anti-mouse IgM (thermo fisher 31440) were added to 100. Mu.l/well, and after incubation at 37 ℃ for 30min, washing was performed 3 times with 1xPBS, a color development reaction was performed.

Example 2 sequence identification of anti-human LAG3 antibody and construction of chimeric antibody

2.1 identification of anti-human LAG3 antibody sequences

Anti-human LAG3 murine monoclonal antibody was selected for sequence analysis. The procedure was as follows, using the RNA extraction kit MagExtractor-RNA- (Toyobo, cat. NPK-201F), from 5X10 ⁶ Total RNA was extracted from each mouse hybridoma. The cDNA sequence was obtained by the 5'RACE method flow using SMARTer RACE 5' kit (Takara Bio, cat.634858). Using 5'RACE Universal primer and 3' murine specific conserved region primer, antibody variable region sequences were extended and PCR cloning sequences containing variable region sequences were ligated to pcDNA3.4 ^TM On the A vector (Gibco) ^TM pcDNA ^TM 3.4TOPO ^TM TA Cloning Kit, fisher scientific, cat. A14697), e.coli DH5 d competent cells (NEB, cat. C29871). Plasmids were extracted using endotoxin-free plasmid macroextraction kit (TIANGEN, cat. DP117) and then subjected to sequencing (Thermofeisher Co.).

The nucleotide sequence of 20 anti-human LAG3 antibodies is shown in Table 1 and the amino acid sequence is shown in Table 2, according to the numbering system of Kabat, as follows:

TABLE 1 nucleotide sequences of anti-human LAG3 antibodies

TABLE 2 amino acid sequence of anti-human LAG3 antibody

2.2 construction and expression of human LAG 3-resistant human murine chimeric antibody

The full-length human murine chimeric antibody sequences were constructed using standard recombinant DNA techniques. Comprises grafting heavy chain variable region of anti-human LAG3 antibody onto human IgG4 constant region (amino acid sequence SEQ ID N0.84) by superposition PCR technique, grafting light chain variable region of anti-human LAG3 antibody onto human Kappa constant region (amino acid sequence SEQ ID N0.85), connecting cloned PCR product onto vector pcDNA3.4, purifying the cell supernatant by ProA affinity chromatography technique with Expi293 cell (ThermoFisher Scientific, cat.A14635) as host cell, concentrating the purified sample by ultrafiltration tube (ultrafiltration tube)

Ultra-15Centrifugal Filter devices, 30kD) to > 0.5mg/ml; collecting concentrated end sample, sterilizing and filtering with 0.22um sterile pillow filter (Kebaite, PES,0.22um, diameter 13 mm), and controlling endotoxin in sample to be less than or equal to 1.0EU/mg in the whole process.

Example 3 binding Activity of anti-human LAG3 antibodies

6 anti-human LAG3 antibodies were selected for evaluation of binding activity, and the 6 antibodies were LAG3-96, LAG3-172, LAG3-188, LAG3-268, LAG3-283 and LAG3-285.

3.1 binding Activity of Chinese hamster cells stably expressing full-Length human LAG3, kiwi LAG3 and mouse LAG3

To evaluate the binding activity of anti-human LAG3 antibodies to human LAG3, cynomolgus LAG3 and mouse LAG3, a recombinant construct was screenedChinese hamster CHO stable cell strains (denoted CHO-hLAG3, CHO-mLAG3 and CHO-cynoLAG3 respectively) stably expressing the full-length protein of human LAG3 (Genbank No. NP-002277, SEQ ID N0.1), macaque LAG3 (Macaca mulatta LAG3 pa23-5, SEQ ID N0.2) and mouse LAG3 (Genbank No. NP-032505, SEQ ID N0.3) were constructed. The binding activity of anti-human LAG3 antibodies was evaluated as follows. Resuspending the CHO stabilized cell lines to 2x10 with 1xFCM buffer (1xPBS +3% BSA) ⁶ Perml, 100. Mu.l/well into a 96-well V-plate. Anti-human LAG3 antibody was diluted with 1XFCM buffer to a concentration of 20. Mu.g/ml. The cells were added to 100. Mu.l/well, incubated on ice for 30min, centrifuged at 250Xg for 5min, and the supernatant was aspirated. After washing 2 times with 1XFCM, PE-labeled goat anti-human Fc fluorescent secondary antibody (dilution factor 1: 500) (Biolegend, cat. 409304) diluted with 1XFCM buffer was added at 100. Mu.l/well and incubated on ice for 30min. After centrifugation at 250Xg for 5min, the supernatant was aspirated and washed with 1XFCM for 2 times, and then 1xPBS was added at 100. Mu.l/well to resuspend the cells, which were then detected by flow cytometry (Beckman, cytoFLEX).

As shown in FIG. 1, the 6 anti-LAG 3 antibodies did not bind non-specifically to CHO null cells and did not cross-react with membrane-expressed murine LAG3 protein, while the binding activity to CHO-hLAG3 and CHO-cynoLAG3 was comparable to BMS relatlimab (Poissolk, inc., igG4 subtype).

3.2 binding Activity of CHO-hLAG3 stably expressing human LAG3 EC50

To characterize the binding affinity of anti-human LAG3 antibody to human LAG3, anti-human LAG3 antibody was diluted 3-fold with 1XFCM buffer (1xPBS +3% BSA) at an initial concentration of 100. Mu.g/ml and then co-incubated with the CHO-hLAG3 stable cell line. The specific experimental procedure is as in 2.1.

As shown in FIG. 2 (A and B), the binding activity of 5 anti-human LAG3 antibodies was comparable to that of BMSrelatimab, except that LAG3-285 had a weaker binding activity to CHO-hIAG 3.

3.3 binding Activity of CHO-cynoLAG3 stably expressing macaque LAG3 EC50

To characterize the binding affinity of anti-human LAG3 antibody to cynomolgus LAG3, anti-human LAG3 antibody was diluted 3-fold with 1XFCM buffer (1xPBS +3% BSA) at an initial concentration of 100. Mu.g/ml and then co-incubated with the CHO-cynoLAG3 stable cell line. The specific experimental procedure is as in 2.1.

As shown in FIG. 3 (A and B), the binding activity of the 6-strain anti-human LAG3 antibody to cynomolgus LAG3 stably expressed in CHO was better than that of BMS relatlimab.

3.4 binding Activity to activate PBMCs of healthy humans

Adopts human lymphocyte separation liquid Lymphoprep ^TM (Axis-Shield, cat.07851) peripheral blood lymphocytes PBMC were isolated from healthy human peripheral blood. Resuspend healthy human PBMCs with 10% FBS in X-VIVO15 complete medium (Lonza, cat. 04-418Q) and adjust cell density to 1X10 ⁶ /ml，37℃5％CO ₂ After standing for 2h in the cell culture box, phytohemagglutinin PHA-L (Sigma, cat. L4144) with a final concentration of 1. Mu.g/ml was added, and after mixing, the mixture was continuously stimulated for 3 days. The cell suspension was removed, centrifuged at 250Xg for 5min, the supernatant was aspirated off, and the cell density was adjusted to 2X10 with 1XFCM buffer (1xPBS +3% BSA) ⁶ And/ml, 100. Mu.l/well in 96-well V-plate. Anti-human LAG3 antibody diluted 3-fold with 1XFCM buffer (1xPBS +3% BSA) at an initial concentration of 100. Mu.g/ml was added to the cells at 100. Mu.l/well, incubated on ice for 30min, centrifuged at 250Xg for 5min, and the supernatant was discarded. After washing 2 times with 1XFCM, PE-labeled goat anti-human Fc fluorescent secondary antibody (dilution factor 1: 500) (Biolegend, cat. 409304) diluted with 1XFCM buffer was added at 100. Mu.l/well and incubated on ice for 30min. After centrifugation at 250Xg for 5min, the supernatant was aspirated and washed with 1XFCM for 2 times, and then 1xPBS was added at 100. Mu.l/well to resuspend the cells, which were then detected by flow cytometry (Beckman, cytoFLEX).

As shown in FIG. 4 (A and B), the binding activity of 5 other anti-human LAG3 antibodies was comparable to BMS relaglimab, except that LAG3-285 had a weaker binding activity to activated healthy human PBMC.

3.5 binding kinetics KD analysis of human LAG3

Using MD ForteBIO QK ^e The platform performed an analysis of the binding kinetic constants of anti-human LAG3 antibodies to human LAG 3. Experimental method As follows, the his-tagged human LAG3 extracellular region recombinant protein hLAG3-his (Acrobiosystem, cat. LA3-H5222) was diluted with equilibration buffer (1xPBS +0.02% Tween 20) to a final concentration of 4. Mu.g/ml. Balance buffer solution (1xPBS) 0.02% tween 20) 2-fold diluted anti-human LAG3 antibody at an initial concentration of 200nM for a total of 7 concentrations. After the anti-Penta-HIS biosensor (ForteBIO, cat.18-5122) is fully hydrated, hLAG3-HIS recombinant protein is solidified for 120 seconds, and is combined with the anti-human LAG3 antibody after being balanced for 90 seconds, wherein the combination time is 300 seconds, and the dissociation time is 600 seconds. The whole reaction was carried out at 25 ℃ and 1000 rpm. Finally, curve fitting is carried out by using Octet analysis software, and the binding kinetic constant KD of the anti-human LAG3 antibody is obtained.

As shown in table 2, the binding kinetic constants of the 6-strain anti-human LA3 antibody and the human LAG3 recombinant protein were all on the pM level.

Table 2:

name of antibody	KD，x10 ^-10 (M)
		LAG3-96	＜0.01
LAG3-172	＜0.01
		LAG3-188	2.87
LAG3-268	0.056
		LAG3-283	1.88
LAG3-285	0.28
		relatlimab	3.68

Example 4 epitope analysis of anti-human LAG3 antibody

4.1 epitopbinding Using MD forteBIO QK ^e The platform performs epitope competition analysis of anti-human LAG3 antibodies. Experimental method As follows, the his-tagged human LAG3 extracellular region recombinant protein hLAG3-his (Acrobiosystem, cat. LA3-H5222) was diluted with equilibration buffer (1xPBS +0.02% Tween 20) to a final concentration of 3. Mu.g/ml. Anti-human LAG3 antibody was diluted with equilibration buffer (1xPBS +0.02% Tween 20) to a final concentration of 30. Mu.g/ml. After the anti-Penta-HIS biosensor (ForteBIO, cat.18-5122) is fully hydrated, hLAG3-HIS recombinant protein is solidified for 180 seconds, after 30 seconds of equilibrium, the anti-human LAG3 antibody is solidified for 180 seconds, and after 30 seconds of equilibrium, the anti-human LAG3 antibody is combined with the anti-human LAG3 antibody, wherein the combination time is 180 seconds. The whole reaction was carried out at 25 ℃ and 1000 rpm. Finally, the epitope binding analysis was performed using Octet analysis software.

As a result of experiments, the 6-strain anti-human LAG3 antibody and BMS relatlimab epitope are different, wherein, LAG3-188 and LAG3-285 are independent antigen binding epitopes respectively, and LAG3-96, LAG3-172, LAG3-268 and LAG3-283 have epitope competition.

4.2 flow assay of antibody epitope Competition at cellular level

The anti-LAG 3 antibody was diluted with 1xFCM buffer (1xFBS +3% BSA), wherein the first antibody comprised BMS relalatimab-CH 1 (mIgG 1 subtype, which was produced by transient transfection expression from Expi293 cells, and mIgG1 isotype control antibody (Biolegend, cat. 401411) at a concentration of 10. Mu.g/ml, and the second antibody comprised 6 anti-human LAG3 antibody, BMS relalatimab (hIgG 4 subtype), and hIgG4 control antibody (Biolegend, cat. 403702) at a concentration of 1. Mu.g/ml. Resuspending the CHO-hLAG3 stable cell line in 1XFCM buffer, adjusting the cell density to 2X10 ⁶ The samples were divided into 96-well plates at 100. Mu.l/well. After centrifugation at 250Xg for 5min, the supernatant was aspirated and addedAfter incubating one antibody 100. Mu.l on ice for 30min, 100. Mu.l of the second antibody was added and incubated on ice for 30min. After centrifugation at 250Xg for 5min, the supernatant was aspirated. After washing 2 times with 1XFCM, PE-labeled goat anti-human Fc fluorescent secondary antibody (dilution factor 1: 500) (Biolegend, cat. 409304) diluted with 1XFCM buffer was added at 100. Mu.l/well and incubated on ice for 30min. After centrifugation at 250Xg for 5min, the supernatant was aspirated off, 1XFCM was added thereto to wash the cells for 2 times, and 1xPBS was added to the cells at a concentration of 100. Mu.l/well to resuspend the cells, followed by detection using a flow cytometer (Beckman, cytoFLEX). In contrast, the first antibody included 6 anti-human LAG3 antibodies, BMS relatlumab (hIgG 4 subtype), and hIgG4 isotype control antibody at a concentration of 10. Mu.g/ml, and the second antibody included relatlumab-CH 1 (mIgG 1 subtype), mIgG1 isotype control antibody at a concentration of 1. Mu.g/ml. The fluorescent secondary antibody was a PE-labeled goat anti-mouse Fc fluorescent secondary antibody (dilution factor 1.

As shown in fig. 5 (a and B), the antigen-binding epitope of the 6-strain anti-human LAG3 antibody was different from that of the BMS-targeting antibody, and there was no competition relationship.

Example 5 anti-human LAG3 antibody-mediated ligand blockade assay

LAG3 protein expressed on the surface of lymphocytes inhibits the stimulatory activity of lymphocytes by binding to the histocompatibility complex molecule, MHCII. The blocking anti-human LAG3 antibody was evaluated by the following experimental methods: anti-human LAG3 antibody was diluted 3-fold in 1XFCM buffer at an initial concentration of 100. Mu.g/ml. Recombinant human LAG3-mFc protein (Acrobiosystem, cat. LA3-H52 Aa) was diluted with 1XFCM buffer to a concentration of 20. Mu.g/ml. Resuspending Raji cells (from Tokyo, zhongko, beijing) to 1X10 with 1XFCM buffer ⁶ Cell density per ml, 100. Mu.l/well divided in 96-well V-plates. The experiment was carried out in 2 modes, (1) adding 100. Mu.l/well of the antibody to Raji cells, incubating on ice for 30min, adding 100. Mu.l/well of recombinant human LAG3-mFc protein, and incubating on ice for 30min. After centrifugation at 250Xg for 5min, the supernatant was aspirated and washed 1 time with 1XFCM, 100. Mu.l/well of PE-labeled goat-anti-mouse Fc fluorescent secondary antibody (dilution 1: 500) (Biolegend, cat. 405307) diluted with 1XFCM buffer was added and incubated on ice for 30min. Centrifuging at 250Xg for 5min, removing supernatant, washing with 1XFCM for 2 times, and washing at a volume of 100. Mu.l/well1xPBS was added to resuspend the cells and detection was performed using a flow cytometer (Beckman, cytoFLEX). (2) Recombinant human LAG3-mFc protein was added at 100. Mu.l/well and incubated on ice for 30min, then antibody was added at 100. Mu.l/well to Raji cells and incubated on ice for 30min. After centrifugation at 250Xg for 5min, the supernatant was aspirated, washed 1 time with 1XFCM, 100. Mu.l/well of PE-labeled goat-anti-mouse Fc fluorescent secondary antibody (dilution l: 500) (Biolegend, cat. 405307) diluted with 1XFCM buffer was added, and incubated on ice for 30min. After centrifugation at 250Xg for 5min, the supernatant was aspirated off, 1XFCM was added thereto to wash the cells for 2 times, and 1xPBS was added to the cells at a concentration of 100. Mu.l/well to resuspend the cells, followed by detection using a flow cytometer (Beckman, cytoFLEX).

As shown in fig. 6 (a and B), method (1) anti-human LAG3 antibody showed better blocking activity compared to method (2). Among them, LAG3-172, LAG3-188 and LAG3-268 showed better blocking activity.

Example 6 in vitro Activity characterization of anti-human LAG3 antibodies

6.1 stimulation of IL-2 production in Jurkat-hLAG3 and Raji Co-incubation systems

Since Jurkat cells do not express endogenous human LAG3 protein, jurkat cells (ATCC YB-ATCC-2237) stably expressing human LAG3 protein (NCBI Reference Sequence: NP-002277.4), a Jurkat-hLAG3 stable cell strain, were constructed by a lentiviral packaging system using conventional methods. On the day of the experiment, raji cells were resuspended to 5X10 by complete medium with RPMIl640 containing 10% FBS ⁶ 2.5. Mu.g/ml mitomycin c (Selleck, cat. S8146) was added to the cells, and after treatment at 37 ℃ for 30min, the cells were washed 2 times with complete medium and then resuspended in Raji cells. At the same time, jurkat-hLAG3 cells were resuspended in complete medium. Mixing Jurkat-hLAG3 and Raji cells at a ratio of 1:5, wherein the Raji cell density is 2x10 ⁴ A hole. The mixed cells were dispensed in a 96-well flat bottom plate at 150. Mu.l/well. Diluting anti-human LAG3 antibody with complete medium at a 3-fold ratio to an initial concentration of 40 μ g/ml, sucking the antibody at 50 μ l/well into Jurkat-hLAG3 and Raji mixed cells, mixing well, and 5% CO at 37 ℃% ₂ Culturing in a cell culture box for 1 day. The supernatant was aspirated and IL-2 expression was detected using IL-2Human Uncoated ELISAKit (eBioscience, cat. 88-7025).

As shown in FIG. 7, among the 6 anti-LAG 3 antibodies, LAG3-188 and LAG3-285 showed good IL-2 expression-stimulating activity.

6.2 stimulation of IL-2 and IFN gamma production in healthy human PBMC systems by SEB activation

The method of activating healthy human PBMC by using superantigen SEB stimulates the production of IL-2 and IFN gamma. Human lymphocyte separation liquid Lymphoprep is adopted in experiment ^TM (Axis-Shield, cat.07851) peripheral blood lymphocytes PBMC were isolated from healthy human peripheral blood. Resuspending healthy human PBMCs with 10% FBS-containing X-VIVO15 complete medium (Lonza, cat. 04-418Q), transferring to T75 cell culture flasks, 5% CO at 37 ℃% ₂ The cell incubator was allowed to stand overnight. The next day, healthy human PBMC were diluted with X-VIV015 complete medium to a cell density of 1X10 ⁶ 0.15. Mu.g/ml staphylococcal enterotoxin SEB (toxin technology, cat. BT202) was added to each well, mixed well and dispensed into 96-well plates at 100. Mu.l/well. Adding the antibody to be detected, mixing, and adding CO at 37 deg.C and 5% ₂ The cells were cultured in a cell incubator for 3 days. Supernatants were aspirated and IL-2 and IFN gamma expression was detected using an IL-2Human Uncoated ELISA Kit (eBioscience, cat. 88-7025-88) and a Human IFN gamma unocoated ELISA Kit (eBioscience, cat. 88-7316-88). Antibodies were formulated as follows for 3 groups. (1) Anti-human LAG3 antibody was used alone, and anti-human LAG3 antibody was diluted with X-VIVO15 complete medium to a final concentration of 10. Mu.g/ml; (2) Anti-human LAG3 antibody and anti-human PD1 antibody (Opdivo, poison's purse) were used in combination, wherein the final concentration of the fixed anti-human PD1 antibody was 0.1. Mu.g/ml, and the final concentrations of the anti-human LAG3 antibody were 10, 2 and 0.4. Mu.g/ml; (3) Anti-human LAG3 antibody and anti-human PD1 antibody were used in combination, wherein the final concentration of the fixed anti-human LAG3 antibody was 10 μ g/ml, and the concentrations of the anti-human PD1 antibody were 1, 0.2, 0.04, 0.008, and 0.0016 μ g/ml.

The experimental results are shown in FIGS. 8A, 8D, 9A and 9D, and when anti-human LAG3 antibody is used alone, LAG3-188 shows better activity in inducing SEB activated healthy human PBMC to produce IL-2 and IFN gamma; as shown in fig. 8B, 8E, 9B and 9E, fixing anti-human PD1 antibody Opdivo at 0.1 μ g/ml, LAG3-188 and LAG3-285 showed better cytokine secretion stimulating activity when combined with anti-human LAG3 antibody at different concentrations; as shown in FIGS. 8C, 8F, 9C and 9F, LAG3-188, LAG3-283 and LAG3-285 showed better cytokine secretion stimulating activity when the anti-human LAG3 antibody concentration was fixed at 10. Mu.g/ml and different concentrations of anti-human PDl antibody Opdivo were used in combination.

6.3 Stimulation of IL-2 and IFN gamma production in T-DC allo-mixed lymphoreactive systems

Anti-human LAG3 antibodies were evaluated for their ability to stimulate the cytokines IL-2 and IFN gamma activity by allogeneic T-DC MLR assays. The experimental process is as follows, using human lymphocyte separation liquid Lymphoprep ^TM (Axis-Shield, cat.07851) peripheral blood lymphocytes PBMC were isolated from healthy human peripheral blood. Wherein, the PBMC of the donor 1 is positively screened by human CDl4Microbeads (Miltenyi, cat.130-050-201) to obtain CD14 ⁺ A monocyte. Monocytes were plated at 2x10 ⁶ And/ml, inoculating the cells into a T25 culture bottle, supplementing the cytokines human GM-CSF and IL-4 to a final concentration of 50ng/ml, continuously stimulating for 6 days, supplementing TNF alpha to a final concentration of 50ng/ml, and continuously inducing and differentiating for 3 days to obtain mature DC cells. Donor 2 PBMC via EasySep ^TM CD3 is obtained by negative screening of Human T Cell Enrichment Kit (Stemcell, cat.19051) ⁺ T cells. The ratio of DC to T cells was 1: 10, the amount of DC cells was 2X10 ⁴ For each well, DC and T cells were mixed well and distributed in a 96-well U-plate system of 150. Mu.l/well. Anti-human LAG3 antibody was diluted with X-VIVO15 complete medium and added to the cells at 50. Mu.l/well. Two experiments were performed in total, (1) anti-human LAG3 antibody was used alone at a final concentration of 10 μ g/ml; (2) The anti-human PD1 antibody is combined, namely, the concentration of the anti-human PD1 antibody Opdivo is 0.05 mu g/ml, and the concentration of the anti-human LAG3 antibody is 10 mu g/ml. After 3-5 days of reaction in the mixed lymph experiment, the expression of IL-2 and IFN gamma in the cell supernatant was examined.

As shown in fig. 10A, the anti-human LAG3 antibody alone did not show a significant effect of increasing cytokine secretion in the T-DC allomixis reaction experiment. When the human PD1 antibody Opdivo is used in combination, the IL-2 secretion improving effect of the LAG3-96, the LAG3-188, the LAG3-283 and the LAG3-285 is better, and the IFN gamma secretion improving effect of the LAG3-172 is more obvious.

Claims

1. An anti-human LAG3 antibody or antigen-binding fragment thereof, characterized in that the anti-human LAG3 antibody comprises heavy chain CDRs 1-3 and light chain CDRs 1-3 selected from the group consisting of:

(1) HCDR1: represented by the sequence of SEQ ID NO 122; HCDR2: represented by the sequence of SEQ ID NO 123; HCDR3: represented by the sequence of SEQ ID NO: 124;

LCDR1: represented by the sequence of SEQ ID NO 125; LCDR2: represented by the sequence of SEQ ID NO: 126; LCDR3: represented by the sequence of SEQ ID NO: 127;

(2) HCDR1: represented by the sequence SEQ ID NO: 152; HCDR2: represented by the sequence of SEQ ID NO 153; HCDR3: represented by the sequence of SEQ ID NO 154;

LCDR1: represented by the sequence of SEQ ID NO: 155; LCDR2: represented by the sequence of SEQ ID NO: 156; LCDR3: represented by the sequence of SEQ ID NO: 157;

(3) HCDR1: represented by the sequence of SEQ ID NO: 164; HCDR2: represented by the sequence of SEQ ID NO 165; HCDR3: represented by the sequence of SEQ ID NO 166;

LCDR1: represented by the sequence of SEQ ID NO: 167; LCDR2: represented by the sequence of SEQ ID NO: 168; LCDR3: represented by the sequence of SEQ ID NO 169;

(4) HCDR1: represented by the sequence of SEQ ID NO 176; HCDR2: represented by the sequence of SEQ ID NO: 177; HCDR3: 178 by SEQ ID NO;

LCDR1: represented by the sequence of SEQ ID NO 179; LCDR2: represented by the sequence of SEQ ID NO 180; LCDR3: represented by the sequence of SEQ ID NO. 181;

(5) HCDR1: represented by the sequence of SEQ ID NO. 182; HCDR2: represented by the sequence of SEQ ID NO 183; HCDR3: represented by the sequence of SEQ ID NO 184;

LCDR1: 185, as represented by the sequence of SEQ ID NO; LCDR2: represented by the sequence of SEQ ID NO: 186; LCDR3: represented by the sequence of SEQ ID NO: 187;

(6) HCDR1: represented by the sequence of SEQ ID NO 188; HCDR2: represented by the sequence of SEQ ID NO: 189; HCDR3: represented by the sequence of SEQ ID NO 190;

LCDR1: represented by the sequence of SEQ ID NO. 191; LCDR2: represented by the sequence of SEQ ID NO 192; LCDR3: represented by the sequence of SEQ ID NO: 193.

2. The antibody or antigen-binding fragment thereof of claim 1, wherein the antibody comprises:

(1) A heavy chain variable region comprising or consisting of the sequence:

the amino acid sequence shown as SEQ ID NO. 30, or

A sequence having at least 60% sequence identity to the sequence shown as SEQ ID NO. 30, and

a light chain variable region comprising or consisting of the sequence:

the amino acid sequence shown as SEQ ID NO. 31, or

A sequence having at least 60% sequence identity to the sequence shown as SEQ ID NO. 31;

(2) A heavy chain variable region comprising or consisting of the sequence:

the amino acid sequence shown as SEQ ID NO. 50, or

A sequence having at least 60% sequence identity to the sequence shown as SEQ ID NO. 50, and

a light chain variable region comprising or consisting of the sequence:

51, or of the amino acid sequence shown in SEQ ID NO, or

A sequence having at least 60% sequence identity to the sequence shown as SEQ ID NO. 51;

(3) A heavy chain variable region comprising or consisting of the sequence:

the amino acid sequence shown as SEQ ID NO:58, or

A sequence having at least 60% sequence identity to the sequence shown as SEQ ID NO:58, and

a light chain variable region comprising or consisting of the sequence:

the amino acid sequence shown as SEQ ID NO. 59, or

A sequence having at least 60% sequence identity to the sequence shown as SEQ ID NO. 59;

(4) A heavy chain variable region comprising or consisting of the sequence:

66, or

A sequence having at least 60% sequence identity to the sequence shown as SEQ ID NO. 66, and

a light chain variable region comprising or consisting of the sequence:

67 of the amino acid sequence shown in SEQ ID NO, or

A sequence having at least 60% sequence identity to the sequence shown as SEQ ID NO. 67;

(5) A heavy chain variable region comprising or consisting of the sequence:

70, or

A sequence having at least 60% sequence identity to the sequence shown as SEQ ID NO. 70, and

a light chain variable region comprising or consisting of the sequence:

the amino acid sequence shown as SEQ ID NO. 71, or

A sequence having at least 60% sequence identity to the sequence shown as SEQ ID NO 71;

(6) A heavy chain variable region comprising or consisting of the sequence:

74, or

A sequence having at least 60% sequence identity to the sequence shown as SEQ ID NO. 74, and

a light chain variable region comprising or consisting of the sequence:

the amino acid sequence shown as SEQ ID NO. 75, or

A sequence having at least 60% sequence identity to the sequence shown as SEQ ID NO. 75.

3. The antibody or antigen-binding fragment thereof of claim 2, wherein the sequence of the heavy chain variable region has at least 70% sequence identity to the sequence set forth in SEQ ID NO 30, 50, 58, 66, 70, or 74.

4. The antibody or antigen-binding fragment thereof of claim 2, wherein the sequence of the heavy chain variable region has at least 80% sequence identity to the sequence set forth in SEQ ID No. 30, 50, 58, 66, 70, or 74.

5. The antibody or antigen-binding fragment thereof of claim 2, wherein the sequence of the heavy chain variable region has at least 85% sequence identity to the sequence set forth in SEQ ID NO 30, 50, 58, 66, 70, or 74.

6. The antibody or antigen-binding fragment thereof of claim 2, wherein the sequence of the heavy chain variable region has at least 86% sequence identity to the sequence set forth in SEQ ID NO 30, 50, 58, 66, 70, or 74.

7. The antibody or antigen-binding fragment thereof of claim 2, wherein the sequence of the heavy chain variable region has at least 87% sequence identity to the sequence set forth in SEQ ID No. 30, 50, 58, 66, 70, or 74.

8. The antibody or antigen-binding fragment thereof of claim 2, wherein the sequence of the heavy chain variable region has at least 88% sequence identity to the sequence set forth in SEQ ID NO 30, 50, 58, 66, 70, or 74.

9. The antibody or antigen-binding fragment thereof of claim 2, wherein the sequence of the heavy chain variable region has at least 89% sequence identity to the sequence set forth in SEQ ID NO 30, 50, 58, 66, 70, or 74.

10. The antibody or antigen-binding fragment thereof of claim 2, wherein the sequence of the heavy chain variable region has at least 90% sequence identity to the sequence set forth in SEQ ID NO 30, 50, 58, 66, 70, or 74.

11. The antibody or antigen-binding fragment thereof of claim 2, wherein the sequence of the heavy chain variable region has at least 91% sequence identity to the sequence set forth in SEQ ID NO 30, 50, 58, 66, 70, or 74.

12. The antibody or antigen-binding fragment thereof of claim 2, wherein the sequence of the heavy chain variable region has at least 92% sequence identity to the sequence set forth in SEQ ID NO 30, 50, 58, 66, 70, or 74.

13. The antibody or antigen-binding fragment thereof of claim 2, wherein the sequence of the heavy chain variable region has at least 93% sequence identity to the sequence set forth in SEQ ID NO 30, 50, 58, 66, 70, or 74.

14. The antibody or antigen-binding fragment thereof of claim 2, wherein the sequence of the heavy chain variable region has at least 94% sequence identity to the sequence set forth in SEQ ID No. 30, 50, 58, 66, 70, or 74.

15. The antibody or antigen-binding fragment thereof of claim 2, wherein the sequence of the heavy chain variable region has at least 95% sequence identity to the sequence set forth in SEQ ID NO 30, 50, 58, 66, 70, or 74.

16. The antibody or antigen-binding fragment thereof of claim 2, wherein the sequence of the heavy chain variable region has at least 96% sequence identity to the sequence set forth in SEQ ID NO 30, 50, 58, 66, 70, or 74.

17. The antibody or antigen-binding fragment thereof of claim 2, wherein the sequence of the heavy chain variable region has at least 97% sequence identity to the sequence set forth in SEQ ID NO 30, 50, 58, 66, 70, or 74.

18. The antibody or antigen-binding fragment thereof of claim 2, wherein the sequence of the heavy chain variable region has at least 98% sequence identity to the sequence set forth in SEQ ID NO 30, 50, 58, 66, 70, or 74.

19. The antibody or antigen-binding fragment thereof of claim 2, wherein the sequence of the heavy chain variable region has at least 99% sequence identity to the sequence set forth in SEQ ID No. 30, 50, 58, 66, 70, or 74.

20. The antibody or antigen-binding fragment thereof of claim 2, wherein the sequence of the light chain variable region has at least 70% sequence identity to the sequence set forth in SEQ ID NOs 31, 51, 59, 67, 71, or 75.

21. The antibody or antigen-binding fragment thereof of claim 2, wherein the sequence of the light chain variable region has at least 80% sequence identity to the sequence set forth in SEQ ID NOs 31, 51, 59, 67, 71, or 75.

22. The antibody or antigen-binding fragment thereof of claim 2, wherein the sequence of the light chain variable region has at least 85% sequence identity to the sequence set forth in SEQ ID NO 31, 51, 59, 67, 71, or 75.

23. The antibody or antigen-binding fragment thereof of claim 2, wherein the sequence of the light chain variable region has at least 86% sequence identity to the sequence set forth in SEQ ID NO 31, 51, 59, 67, 71, or 75.

24. The antibody or antigen-binding fragment thereof of claim 2, wherein the sequence of the light chain variable region has at least 87% sequence identity to the sequence set forth in SEQ ID NO 31, 51, 59, 67, 71, or 75.

25. The antibody or antigen-binding fragment thereof of claim 2, wherein the sequence of the light chain variable region has at least 88% sequence identity to the sequence set forth in SEQ ID NO 31, 51, 59, 67, 71, or 75.

26. The antibody or antigen-binding fragment thereof of claim 2, wherein the sequence of the light chain variable region has at least 89% sequence identity to the sequence set forth in SEQ ID NO 31, 51, 59, 67, 71, or 75.

27. The antibody or antigen-binding fragment thereof of claim 2, wherein the sequence of the light chain variable region has at least 90% sequence identity to the sequence set forth in SEQ ID NO 31, 51, 59, 67, 71, or 75.

28. The antibody or antigen-binding fragment thereof of claim 2, wherein the sequence of the light chain variable region has at least 91% sequence identity to the sequence set forth in SEQ ID NOs 31, 51, 59, 67, 71, or 75.

29. The antibody or antigen-binding fragment thereof of claim 2, wherein the sequence of the light chain variable region has at least 92% sequence identity to the sequence set forth in SEQ ID NO 31, 51, 59, 67, 71, or 75.

30. The antibody or antigen-binding fragment thereof of claim 2, wherein the sequence of the light chain variable region has at least 93% sequence identity to the sequence set forth in SEQ ID NO 31, 51, 59, 67, 71, or 75.

31. The antibody or antigen-binding fragment thereof of claim 2, wherein the sequence of the light chain variable region has at least 94% sequence identity to the sequence set forth in SEQ ID NO 31, 51, 59, 67, 71, or 75.

32. The antibody or antigen-binding fragment thereof of claim 2, wherein the sequence of the light chain variable region has at least 95% sequence identity to the sequence set forth in SEQ ID NO 31, 51, 59, 67, 71, or 75.

33. The antibody or antigen-binding fragment thereof of claim 2, wherein the sequence of the light chain variable region has at least 96% sequence identity to the sequence set forth in SEQ ID NOs 31, 51, 59, 67, 71, or 75.

34. The antibody or antigen-binding fragment thereof of claim 2, wherein the sequence of the light chain variable region has at least 97% sequence identity to the sequence set forth in SEQ ID NO 31, 51, 59, 67, 71, or 75.

35. The antibody or antigen-binding fragment thereof of claim 2, wherein the sequence of the light chain variable region has at least 98% sequence identity to the sequence set forth in SEQ ID NO 31, 51, 59, 67, 71, or 75.

36. The antibody or antigen-binding fragment thereof of claim 2, wherein the sequence of the light chain variable region has at least 99% sequence identity to the sequence set forth in SEQ ID NO 31, 51, 59, 67, 71, or 75.

37. The antibody or antigen-binding fragment thereof of claim 1, wherein the antibody further comprises a heavy chain constant region and a light chain constant region.

38. The antibody or antigen-binding fragment thereof of claim 37, wherein the heavy and light chain constant regions are from human IgG or IgM.

39. The antibody or antigen-binding fragment thereof of claim 37, wherein the heavy chain constant region and the light chain constant region are from IgG4.

40. The antibody or antigen-binding fragment thereof of any one of claims 37-39, wherein the amino acid sequence of the heavy chain constant region is the sequence shown in SEQ ID No.84 and the amino acid sequence of the light chain constant region is the sequence shown in SEQ ID No. 85.

41. The antibody or antigen-binding fragment thereof of any one of claims 1-36, wherein the antigen-binding fragment is selected from the group consisting of Fab, scFv, fab ', F (ab') ₂ Fv or Fab/c.

42. The antibody or antigen-binding fragment thereof of any one of claims 1-36, wherein the antibody is a humanized antibody, a chimeric antibody, a multispecific antibody.

43. The antibody or antigen-binding fragment thereof of any one of claims 1-36, wherein the antibody is a bispecific antibody.

44. A polynucleotide encoding the antibody or antigen-binding fragment thereof of any one of claims 1-43.

45. The polynucleotide of claim 44 selected from the group consisting of SEQ ID NO 28 and 29; 48 and 49 in SEQ ID NO; 56 and 57; 64 and 65 for SEQ ID NO; 68 and 69; 72 and 73 in SEQ ID NO.

46. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof of any one of claims 1-43.

47. The pharmaceutical composition of claim 46, further comprising a pharmaceutically acceptable carrier and/or excipient.

48. Use of an antibody or antigen-binding fragment thereof according to any one of claims 1-43, for the manufacture of a medicament for the prevention and/or treatment and/or co-treatment and/or diagnosis of a LAG3 expressing neoplastic disease, or for the manufacture of a medicament for stimulating the secretion of IL-2 or IFN gamma.

49. The use of claim 48, wherein the neoplastic disease is cancer.

50. The pharmaceutical composition of claim 46 or 47, in a form suitable for injection.

51. The pharmaceutical composition of claim 46 or 47, which is in a form suitable for administration by subcutaneous injection, intradermal injection, intravenous injection, intramuscular injection, or intralesional injection.

52. The pharmaceutical composition of claim 50, which is in a form suitable for administration by subcutaneous injection, intradermal injection, intravenous injection, intramuscular injection, or intralesional injection.