CN113004407A - LAG3 antibodies and uses thereof - Google Patents
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- CN113004407A CN113004407A CN201911344025.2A CN201911344025A CN113004407A CN 113004407 A CN113004407 A CN 113004407A CN 201911344025 A CN201911344025 A CN 201911344025A CN 113004407 A CN113004407 A CN 113004407A
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Abstract
本发明涉及抗人LAG3抗体及其应用。所述抗体能阻断细胞表面表达的LAG‑3蛋白与组织相容性复合物II的相互作用，并且在体外细胞功能实验中，显示出刺激细胞因子产生的生物学功能活性。The present invention relates to anti-human LAG3 antibodies and applications thereof. The antibody can block the interaction between the LAG-3 protein expressed on the cell surface and the histocompatibility complex II, and in the in vitro cell function experiment, shows the biological function activity of stimulating the production of cytokines.
Description
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 CD223) is a negative co-stimulatory receptor that regulates T-cell homeostasis, proliferation and activation (Sierro S et al (2010), Exper)t opin. ther. targets, 15: 91-101) belonging to the type I transmembrane immunoglobulin supergene family. The LAG-3 gene was successfully cloned in 1990 by Triebel (Triebel et al (1990) J.exp.Med.171: 1393-. The gene encoding LAG-3 protein is located on chromosome 12 and is structurally and genetically related to the CD4 molecule. LAG-3 proteins are commonly found in activated T cells (Huard et al (1994) immunology 39: 213), memory CD4+And CD8+T cells, regulatory T cells (Huang et al (2004) Immunity 21: 503-513; Camisaschi et al (2010) J.Immunol.184: 6545-655 l; Gagliani et al (2013) nat. Med.19: 739-746), NK cells and plasmacytoid dendritic cells DCs (Workman et al (2009) J.Immunol.182: 1885-1891). LAG-3, as an immune checkpoint negative regulator, negatively regulates the proliferation and activity of CD4+ and CD8+ T cells. Together with PD-1, immune tolerance of CD8+ T cells is co-mediated during lentivirus infection. LAG-3 molecules in activated CD4+CD25+Selective up-regulation on regulatory T cells inhibits DC cell maturation by binding to MHC class II molecules on the surface of DC cells, 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 antibody or antigen-binding fragment thereof against human LAG3, characterized by comprising a heavy chain CDR1-3 and a light chain CDR1-3 selected from the group consisting of:
(1) HCDR 1: consisting of SEQ ID NO: 86 in seq id no; HCDR 2: consisting of SEQ ID NO: 87 by sequence number; HCDR 3: consisting of SEQ ID NO: 88;
LCDR 1: consisting of SEQ ID NO: 89 sequence representation; LCDR 2: consisting of SEQ ID NO: 90, sequence representation; LCDR 3: consisting of SEQ ID NO: 91 sequence representation;
(2) HCDR 1: consisting of SEQ ID NO: 92; HCDR 2: consisting of SEQ ID NO: 93 with a sequence representation; HCDR 3: consisting of SEQ ID NO: 94, sequence representation;
LCDR 1: consisting of SEQ ID NO: 95 in seq id no; LCDR 2: consisting of SEQ ID NO: 96 is represented by the sequence; LCDR 3: consisting of SEQ ID NO: 97, sequence representation;
(3) HCDR 1: consisting of SEQ ID NO: 98 in sequence; HCDR 2: consisting of SEQ ID NO: 99, sequence representation; HCDR 3: consisting of SEQ ID NO: 100 in sequence;
LCDR 1: consisting of SEQ ID NO: 101, sequence representation; LCDR 2: consisting of SEQ ID NO: 102, sequence representation; LCDR 3: consisting of SEQ ID NO: 103;
(4) HCDR 1: consisting of SEQ ID NO: 104; HCDR 2: consisting of SEQ ID NO: 105 in sequence; HCDR 3: consisting of SEQ ID NO: 106;
LCDR 1: consisting of SEQ ID NO: 107; LCDR 2: consisting of SEQ ID NO: 108; LCDR 3: consisting of SEQ ID NO: 109, or a sequence representation of;
(5) HCDR 1: consisting of SEQ ID NO: 110; HCDR 2: consisting of SEQ ID NO: 111; HCDR 3: consisting of SEQ ID NO: 112;
LCDR 1: consisting of SEQ ID NO: 113, sequence representation; LCDR 2: consisting of SEQ ID NO: 114; LCDR 3: consisting of SEQ ID NO: 115, sequence representation;
(6) HCDR 1: consisting of SEQ ID NO: 116; HCDR 2: consisting of SEQ ID NO: 117; HCDR 3: consisting of SEQ ID NO: 118;
LCDR 1: consisting of SEQ ID NO: 119 with sequence representation; LCDR 2: consisting of SEQ ID NO: 120, sequence representation; LCDR 3: consisting of SEQ ID NO: 121 in sequence representation;
(7) HCDR 1: consisting of SEQ ID NO: 122; HCDR 2: consisting of SEQ ID NO: 123 is represented by the sequence; HCDR 3: consisting of SEQ ID NO: 124;
LCDR 1: consisting of SEQ ID NO: 125; LCDR 2: consisting of SEQ ID NO: 126 with a sequence representation; LCDR 3: consisting of SEQ ID NO: 127, sequence representation;
(8) HCDR 1: consisting of SEQ ID NO: 128 in sequence; HCDR 2: consisting of SEQ ID NO: 129, sequence representation; HCDR 3: consisting of SEQ ID NO: 130;
LCDR 1: consisting of SEQ ID NO: 131, or a sequence representation thereof; LCDR 2: consisting of SEQ ID NO: 132; LCDR 3: consisting of SEQ ID NO: 133;
(9) HCDR 1: consisting of SEQ ID NO: 134; HCDR 2: consisting of SEQ ID NO: 135, sequence representation; HCDR 3: consisting of SEQ ID NO: 136;
LCDR 1: consisting of SEQ ID NO: 137; LCDR 2: consisting of SEQ ID NO: 138; LCDR 3: consisting of SEQ ID NO: 139 sequence representation;
(10) HCDR 1: consisting of SEQ ID NO: 140; HCDR 2: consisting of SEQ ID NO: 141; HCDR 3: consisting of SEQ ID NO: 142;
LCDR 1: consisting of SEQ ID NO: 143; LCDR 2: consisting of SEQ ID NO: 144, sequence representation; LCDR 3: consisting of SEQ ID NO: 145;
(11) HCDR 1: consisting of SEQ ID NO: 146; HCDR 2: consisting of SEQ ID NO: 147; HCDR 3: consisting of SEQ ID NO: 148;
LCDR 1: consisting of SEQ ID NO: 149 in seq id no; LCDR 2: consisting of SEQ ID NO: 150; LCDR 3: consisting of SEQ ID NO: 151, sequence representation;
(12) HCDR 1: consisting of SEQ ID NO: 152 sequence representation; HCDR 2: consisting of SEQ ID NO: 153, sequence representation; HCDR 3: consisting of SEQ ID NO: 154;
LCDR 1: consisting of SEQ ID NO: 155; LCDR 2: consisting of SEQ ID NO: 156; LCDR 3: consisting of SEQ ID NO: 157, sequence representation;
(13) HCDR 1: consisting of SEQ ID NO: 158; HCDR 2: consisting of SEQ ID NO: 159 in seq id no; HCDR 3: consisting of SEQ ID NO: 160;
LCDR 1: consisting of SEQ ID NO: 161 sequence representation; LCDR 2: consisting of SEQ ID NO: 162; LCDR 3: consisting of SEQ ID NO: 163;
(14) HCDR 1: consisting of SEQ ID NO: 164 in sequence representation; HCDR 2: consisting of SEQ ID NO: 165, sequence representation; HCDR 3: consisting of SEQ ID NO: 166, sequence representation;
LCDR 1: consisting of SEQ ID NO: 167, sequence representation; LCDR 2: consisting of SEQ ID NO: 168 sequence representation; LCDR 3: consisting of SEQ ID NO: 169 sequence representation;
(15) HCDR 1: consisting of SEQ ID NO: 170; HCDR 2: consisting of SEQ ID NO: 171 sequence representation; HCDR 3: consisting of SEQ ID NO: 172;
LCDR 1: consisting of SEQ ID NO: 173, sequence number; LCDR 2: consisting of SEQ ID NO: 174; LCDR 3: consisting of SEQ ID NO: 175;
(16) HCDR 1: consisting of SEQ ID NO: 176; HCDR 2: consisting of SEQ ID NO: 177 of the sequence representation; HCDR 3: consisting of SEQ ID NO: 178;
LCDR 1: consisting of SEQ ID NO: 179; LCDR 2: consisting of SEQ ID NO: 180, sequence representation; LCDR 3: consisting of SEQ ID NO: 181 in seq id no;
(17) HCDR 1: consisting of SEQ ID NO: 182, a sequence representation; HCDR 2: consisting of SEQ ID NO: 183 sequence representation; HCDR 3: consisting of SEQ ID NO: 184;
LCDR 1: consisting of SEQ ID NO: 185 of the sequence; LCDR 2: consisting of SEQ ID NO: 186; LCDR 3: consisting of SEQ ID NO: 187, sequence representation;
(18) HCDR 1: consisting of SEQ ID NO: 188; HCDR 2: consisting of SEQ ID NO: 189 a sequence representation; HCDR 3: consisting of SEQ ID NO: 190;
LCDR 1: consisting of SEQ ID NO: 191, sequence representation; LCDR 2: consisting of SEQ ID NO: 192 of seq id no; LCDR 3: consisting of SEQ ID NO: 193 is represented by the sequence;
(19) HCDR 1: consisting of SEQ ID NO: 194, sequence representation; HCDR 2: consisting of SEQ ID NO: 195; HCDR 3: consisting of SEQ ID NO: 196, sequence representation;
LCDR 1: consisting of SEQ ID NO: 197 sequence representation; LCDR 2: consisting of SEQ ID NO: 198 by sequence representation; LCDR 3: consisting of SEQ ID NO: 199 for a sequence representation; or
(20) HCDR 1: consisting of SEQ ID NO: 200 in sequence; HCDR 2: consisting of SEQ ID NO: 201 in sequence; HCDR 3: consisting of SEQ ID NO: 202;
LCDR 1: consisting of SEQ ID NO: 203 of the sequence; LCDR 2: consisting of SEQ ID NO: 204; LCDR 3: 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, 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: 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, 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: 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:
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:
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:
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:
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:
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, 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: 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, 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: 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:
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 of any one of above 1-2, 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 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.
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')2Fv 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 of any one of 1 to 5 above.
7. The polynucleotide of 6 above selected from the group consisting of SEQ ID NO: 4 and 5; SEQ ID NO: 8 and 9; 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; 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 neoplastic disease or for the manufacture of a medicament for the prophylactic and/or therapeutic and/or adjunctive therapeutic and/or diagnostic treatment of a neoplastic disease, preferably a tumor expressing LAG3, preferably a cancer, or for the manufacture of a medicament for modulating an immune response, preferably for stimulating 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 VLAnd constant region Domain CL. 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 VHAnd 3 constant region domains CH1、CH2And CH3。VHThe structural domain is at the amino terminal end of the polypeptide, and CHThe structural domain is at the carboxyl terminal, CH3Closest 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 the variable region of CH1 and 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 CsH1And CH2A heavy chain fragment of a domain. Two heavy chain fragments through two or more disulfide bonds and through CH3The 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)HDomains and CH1Domains and alsoCH1And CH2Part 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')2A molecule.
As used herein, the term "F (ab')2A fragment "comprises two light chains and two heavy chains comprising part 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')2The fragment thus consists of two Fab' fragments held together by the 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 herein, the term "Fd" fragment means a fragment consisting of VHAnd CH1Antibody fragments consisting of domains (Ward et al, Nature 341: 544-546 (1989)).
As used herein, the term "dAb" fragment (Ward et al, Nature 341: 544-546(1989)) is derived from VHDomain 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 diffusion capacity and slow metabolic clearance in vivo, while maintaining high affinity (Liujian Jun, J. cell & 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: 423-58426 (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 attachment of Fab' fragments. See, e.g., Songsivilai and Lachmann, 1990, clin. exp. immunol.79: 315- > 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: 522525 (1986); reichmann et al, Nature, 332: 323329 (1988); presta, curr, op.struct.biol., 2: 593596 (1992); and Clark, immunol. today 21: 397402(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, the points of all interactions between a protein and an interacting molecule (e.g., an antibody) are linearly present 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 specific 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 positive values in the PAM250 log-likehood matrix (PAM250 log-likehood matrix). 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 of the LAG3 monoclonal antibody to CHO-hLAG3, wherein fig. 2A is a binding assay of LAG3-96, LAG3-188, LAG3-283, LAG3-285, and relatlimab to cells; FIG. 2B is a binding assay for LAG3-172, LAG3-268, and relatlimab to cells.
FIG. 3: binding assay of LAG3 monoclonal antibody to CHO-cynoLAG3, wherein FIG. 3A is a binding assay of LAG3-96, LAG3-188, LAG3-283, LAG3-285, and relatlimab to cells; FIG. 3B is a binding assay for LAG3-172, LAG3-268, and relatlimab to cells.
FIG. 4: a binding assay for the 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 cells; FIG. 4B is a binding assay for LAG3-172, LAG3-268, and relatlimab to cells.
FIG. 5: competitive analysis of binding epitopes, wherein FIG. 5A shows that after a control antibody is added to bind to membrane-expressed human LAG3, an anti-human LAG3 antibody is added to detect the competitive binding between the antibody and 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 the antibody to membrane-expressed human LAG3
FIG. 6: blocking Raji-MHCII-hLAG3 by an LAG3 antibody, wherein in a picture 6A, after the antibody is combined with human LAG3 protein, Raji cells are added, and the blocking effect of the anti-human LAG3 antibody is detected; FIG. 6B shows the antibody incubated with Raii cells prior to detection of binding of the antibody to human LAG3 protein
FIG. 7: in vitro Jurkat-hLAG3-Raii bioassay of LAG3 antibody.
FIG. 8: SEB-activated hPBMC bioassay, otherwise figure 8A activated PBMC from donor-source, 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; figure 8D is donor-two derived activated PBMCs using anti-human LAG3 antibody alone; FIG. 8E is 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, a fixed concentration 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-two derived activated PBMCs using anti-human LAG3 antibody alone; FIG. 9E is 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-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 those skilled in the art will appreciate 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 conducted under 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 protein was used as an immunogen for the production of human LAG3 antibodies. In the course of immunization, hLAG3-his (Acrobiosystem, Cat. LA3-H5222, also referred to as "human LAG3 recombinant protein" in the examples below) human LAG3 extracellular domain fusion protein carrying his tag was used as immunogen.
1.2 immunization protocol
The commonly used mouse hybridoma system was used to generate anti-human LAG3 murine mab. The procedure was as follows, mixing 1.2mg/ml of recombinant human LAG3 recombinant protein (i.e., hLAG3-his) with Freund's complete adjuvant (Sigma, Cat. F5881) in equal volume, and the resulting oily emulsion was subcutaneously administered 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 recombinant human LAG3 recombinant protein and Freund's incomplete adjuvant (Sigma, Cat. F5506) in equal volume, performing intraperitoneal immune enhancement, performing booster immunization until 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 the immune spleen cells were mixed according to the cell number ratio of 1: 10, transferred to a 50ml centrifuge tube, and washed once with RPMI1640 basic medium. The supernatant was discarded, the cells were mixed well and 1ml of 50% PEG1500 was added slowly for fusion. 1min after fusion, cell fusion was terminated by the addition of 15ml RPMIl640 basal medium. Centrifuge at 1000rpm for 5min and discard the supernatant. The suspension was gently suspended in 50ml of RPMI1640 screening medium and divided equally into 10 aliquots in 96-well plates at 37 ℃ in 5% CO2And (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 ℃. The supernatant was discarded, and blocking solution (1xPBS + 1% BSA) was added at 200. mu.l/well and blocked at 37 ℃ for 1 hour. On day 7 after the recombinant fusion, cell supernatants were added at 100. mu.l/well and incubated at 37 ℃ for 30 min. Wash 3 times with 1 xPBS. HRP-labeled goat anti-mouse IgG (sigma A0168-1ML) was added to 100. mu.l/well, and the mixture was incubated at 37 ℃ for 30min, washed 3 times with 1xPBS, and then subjected to color development reaction.
1.5 screening for subclones
The fusions with OD 450. gtoreq.1 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 antibody
When the clone producing anti-human LAG3 antibody grew to 80% confluence, the cells were blown down, centrifuged at 1000rpm for 5min, resuspended in 30ml of SFM containing 5% FBS, and transferred to 150ml SF shake flasks at 37 ℃ with 8% CO2Culturing at 120rpm for 2-3 days. When the cell density reaches 5x105Centrifugation at 1000rpm for 5 min/ml, resuspension of cells in 50ml serum-free SFM basal medium and transfer to 250ml SF shake flasks at 37 ℃ with 8% CO2Culturing 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 (10mM 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 (10mM PBS), followed by elution of the target protein with elution buffer (20mM NaAc, pH 3.4), and a neutralization buffer (1M Tris, pH8.0) was added to the collection tube in advance, the volume of the neutralization buffer added being determined according to the estimated content of the eluted sample, and typically 10% of the elution volume was added.
1.7 determination of anti-human LAG3 antibody concentration
The sample is subjected to concentration measurement by using Biotek-Epoch-Take-3, the concentration of the antibody is detected by using an A280 method, namely, the extinction coefficient E.C. is 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) following the general protocol provided in the specification, the sample concentration was concentrated to > 0.5 mg/ml; the concentrated end samples were collected and sterile filtered through a 0.22um sterile pillow filter (Libert, PES, 0.22um, 13mm diameter).
1.8 identification of anti-human LAG3 antibody subtypes
Recombinant human LAG3 recombinant protein carrying the human Fc tag (Acrobiosystem, Cat. LA3-H5255) was diluted with 0.05M carbonated buffer solution at pH9.5 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. The supernatant was discarded, and blocking solution (1xPBS + 1% BSA) was added at 200. mu.l/well and blocked at 37 ℃ for 1 hour. 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, and incubated at 37 ℃ for 30 minutes. Wash 3 times with 1 xPBS. HRP-labeled goat anti-mouse IgG (sigma A0168-1ML), HRP-labeled goat anti-mouse IgG2a (thermo fisher M32207), HRP-labeled goat anti-mouse IgG1(thermo fisher PA1-74421), 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 mab was selected for sequence analysis. The method is as follows, using RNA extraction kit MagExtractor-RNA- (Toyobo, Cat. NPK-201F),from 5x106Total RNA was extracted from the individual murine hybridoma cells. The cDNA sequence was obtained by the 5 'RACE procedure using SMARTer RACE 5' Kit (Takara Bio, Cat.634858). Using 5 'RACE universal primer and 3' mouse specific conserved region primer, the variable region sequence of antibody is expanded and the PCR cloning sequence containing the variable region sequence is connected to pcDNA3.4TMOn the A vector (Gibco)TMpcDNATM3.4TOPOTMTA Cloning Kit, Fisher scientific, cat. a14697), e.coli DH5 d competent cells (NEB, cat. c 29871). 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 according to the kabat numbering system is shown in Table 1, and the amino acid sequence is shown in Table 2, as follows:
TABLE 1 nucleotide sequence of anti-human LAG3 antibody
TABLE 2 amino acid sequence of anti-human LAG3 antibody
2.2 construction and expression of human LAG3 chimeric human murine antibody
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), grafting light chain variable region of anti-human LAG3 antibody onto human Kappa constant region (amino acid sequence SEQ ID N0.85), connecting clone PCR product onto vector pcDNA3.4, and performing PEI-mediated transient transfection with Expi293 cells (ThermoFisher Scientific, Cat. A14635) as host cells, expressing anti-human LAG3 antibody, purifying the cell supernatant by ProA affinity chromatography, and ultrafiltering and concentrating the purified sample
Ultra-15Centrifugal Filter Devices, 30kD) to > 0.5 mg/ml; collecting concentrated end sample, sterilizing and filtering with 0.22um sterile pillow filter (Kebaite, PES, 0.22um, diameter 13mm), 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 antibody
6 anti-human LAG3 antibodies are selected to evaluate the binding activity, and the 6 antibodies are LAG3-96, LAG3-172, LAG3-188, LAG3-268, LAG3-283 and LAG 3-285.
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 monkey LAG3 and mouse LAG3, chinese hamster CHO stable cell strains (CHO-hLAG 3, CHO-mLAG3 and CHO-cynomolgus monkey LAG3, respectively) stably expressing the full-length human LAG3 protein (Genbank No. np _002277, SEQ ID N0.1), cynomolgus monkey LAG3(Macaca mulatta LAG3 pa23-5, SEQ ID N0.2) and mouse LAG3(Genbank No. np _032505, SEQ ID N0.3) were screened and constructed. The binding activity of anti-human LAG3 antibody was evaluated as follows. Resuspend CHO Stable cell line to 2X10 with 1XFCM buffer (1xPBS + 3% BSA)6Perml, 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 30 min. 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. 1, the 6 strain anti-LAG 3 antibody did not bind nonspecifically 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 (Behcet-Miss-S.A., 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 μ g/ml and then co-incubated with 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 weak 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 μ g/ml and then co-incubated with 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 in healthy humans
Adopts human lymphocyte separation liquid LymphoprepTM(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 1X106/ml，37℃5％CO2After 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)6And/ml, 100. mu.l/well in 96-well V-plate. Anti-human LAG3 antibody diluted 3-fold in 1XFCM buffer (1xPBS + 3% BSA) at an initial concentration of 100. mu.gMl, 100. mu.l/well into cells, ice incubation for 30min, 250Xg centrifugation for 5min, and aspiration of the supernatant. 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 30 min. 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. 4 (a and B), the binding activity of 5 other anti-human LAG3 antibodies was comparable to BMS relatlimab, except that LAG3-285 had a weaker binding activity to activated healthy human PBMCs.
3.5 binding kinetics KD analysis of human LAG3
By MD ForteBIO QKePlatform performed analysis of binding kinetic constants of anti-human LAG3 antibody to human LAG 3. Experimental method the his-tagged human LAG3 extracellular domain 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. Anti-human LAG3 antibody was diluted 2-fold in equilibration buffer (1xPBS + 0.02% tween 20) 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 after 90 seconds of equilibrium, the hLAG3-HIS recombinant protein is combined with an anti-human LAG3 antibody, 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 was performed by Octet analysis software to obtain the binding kinetic constant KD of the anti-human LAG3 antibody.
As shown in table 2, the binding kinetic constants of the 6-strain anti-human LA3 antibody and the human LAG3 recombinant protein are all in 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 QKeThe platform performs epitope competition analysis of anti-human LAG3 antibody. Experimental method the his-tagged human LAG3 extracellular domain 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 balance, the anti-human LAG3 antibody is solidified for 180 seconds, and after 30 seconds of balance, the anti-human LAG3 antibody is combined, wherein the combination time is 180 seconds. The whole reaction was carried out at 25 ℃ and 1000 rpm. Finally, the product was analyzed by OctetThe software performs an epitope binding analysis.
The experimental result shows that 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
anti-LAG 3 antibody was diluted with 1XFCM buffer (1xFBS + 3% BSA), wherein the first antibody comprised BMS relalatimab-CH 1(mIgG1 subtype, which was produced by transient transfection and expression from Expi293 cells, 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 (hIgG4 subtype), and hIgG4 isotype control antibody (Biolegend, Cat.403702) at a concentration of 1. mu.g/ml. Resuspending the CHO-hLAG3 stabilized cell line in 1XFCM buffer, adjusting the cell density to 2X106The samples were divided into 96-well plates at 100. mu.l/well. After centrifugation at 250Xg for 5min, the supernatant was aspirated off, 100. mu.l of the first antibody was added, and after incubation on ice for 30min, 100. mu.l of the second antibody was added, and incubation on ice for 30 min. 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 30 min. 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 antibody, BMS relatlimab (hIgG4 subtype), and hIgG4 isotype control antibody at a concentration of 10. mu.g/ml, and the second antibody included relatlimab-CH1(mIgG1 subtype), and 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: 500) (Biolegend, Cat. 405307).
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 counterpart antibody, and there was no competition relationship.
Example 5 anti-human LAG3 antibody-mediated ligand blockade experiments
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 method: 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-H52Aa) was diluted with 1XFCM buffer to a concentration of 20. mu.g/ml. Resuspending Raji cells (from Hospital, Beijing, Zhongkou) to 1X10 with 1XFCM buffer6Cell density per ml, 100. mu.l/well divided in 96-well V-plates. The experiment was performed in 2 modes, (1) adding 100. mu.l/well of 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 30 min. 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 30 min. 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). (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 30 min. 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 30 min. 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 antibody
6.1 stimulation of IL-2 production in Jurkat-hLAG3 and Raji Co-incubation System
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, was constructed by a lentiviral packaging system using conventional methods. On the day of the experiment, Raji cells were resuspended at 5X10 in complete media with RPMIl640 containing 10% FBS62.5. 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 2x104A 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 culture medium at 3 times ratio to 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 cooling to 37 deg.C with 5% CO2Culturing 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 better activity of stimulating IL-2 expression.
6.2SEB activation of IL-2 and IFN gamma production in healthy human PBMC systems
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 experimentTM(Axis-Shield, Cat.07851) peripheral blood lymphocytes PBMC were isolated from healthy human peripheral blood. Resuspending healthy human PBMC in 10% FBS-containing X-VIVO15 complete medium (Lonza, Cat.04-418Q), transferring to T75 cell culture flask, 5% CO at 37 deg.C2The cell incubator was left standing overnight. The next day, healthy human PBMC were diluted with X-VIV015 complete medium to a cell density of 1X1060.15. 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, adding 5% CO at 37 deg.C2The cells were cultured in a cell incubator for 3 days. Sucking the supernatant, and treating with IL-2Human UncoatedELISA Kit (eBioscience, Cat.88-7025-88) and Human IFN gamma Uncoated ELISA Kit (eBioscience, Cat.88-7316-88) were used to detect the expression of IL-2 and IFN gamma. 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 wort) 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, with anti-human LAG3 antibody fixed at a final concentration of 10 μ g/ml and anti-human PD1 antibody fixed at concentrations of 1, 0.2, 0.04, 0.008 and 0.0016 μ g/ml.
The experimental results are shown in fig. 8A, 8D, 9A and 9D, and LAG3-188 showed better activity in inducing IL-2 and IFN gamma production by SEB-activated healthy human PBMCs when anti-human LAG3 antibody alone was used; as shown in fig. 8B, 8E, 9B and 9E, 0.1 μ g/ml of fixed anti-human PD1 antibody Opdivo, when combined with anti-human LAG3 antibody at different concentrations, LAG3-188 and LAG3-285 showed better cytokine secretion stimulating activity; as shown in FIGS. 8C, 8F, 9C and 9F, when the concentration of the fixed anti-human LAG3 antibody is 10 μ g/ml and different concentrations of anti-human PDl antibody Opdivo are combined, LAG3-188, LAG3-283 and LAG3-285 show better activity of stimulating cytokine secretion.
6.3 stimulation of IL-2 and IFN gamma production in T-DC allomixis lymphoreactive systems
Anti-human LAG3 antibody was evaluated for its ability to stimulate the cytokines IL-2 and IFN gamma by allogeneic T-DC MLR assay. The experimental procedure is as follows, using human lymphocyte separation liquid LymphoprepTM(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 2x106And/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 EasySepTMHuman T Cell Enrichment Kit(Stemcell，Cat.19051) Negative selection to obtain CD3+T cells. The ratio of DC to T cells was 1: 10, and the amount of DC cells was 2X104For 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 used together, namely the concentration of Opdivo of the anti-human PD1 antibody 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, anti-human LAG3 antibody alone did not show significant cytokine secretion enhancing effect in T-DC allomixis lymphoreaction experiments. When the combined use with anti-human PD1 antibody Opdivo, the secretion effect of IL-2 is better improved by the LAG3-96, the LAG3-188, the LAG3-283 and the LAG3-285, and the secretion effect of IFN gamma is more obvious by the LAG 3-172.
Claims (10)
1. An antibody or antigen-binding fragment thereof against human LAG3, characterized by comprising a heavy chain CDR1-3 and a light chain CDR1-3 selected from the group consisting of:
(1) HCDR 1: consisting of SEQ ID NO: 86 in seq id no; HCDR 2: consisting of SEQ ID NO: 87 by sequence number; HCDR 3: consisting of SEQ ID NO: 88;
LCDR 1: consisting of SEQ ID NO: 89 sequence representation; LCDR 2: consisting of SEQ ID NO: 90, sequence representation; LCDR 3: consisting of SEQ ID NO: 91 sequence representation;
(2) HCDR 1: consisting of SEQ ID NO: 92; HCDR 2: consisting of SEQ ID NO: 93 with a sequence representation; HCDR 3: consisting of SEQ ID NO: 94, sequence representation;
LCDR 1: consisting of SEQ ID NO: 95 in seq id no; LCDR 2: consisting of SEQ ID NO: 96 is represented by the sequence; LCDR 3: consisting of SEQ ID NO: 97, sequence representation;
(3) HCDR 1: consisting of SEQ ID NO: 98 in sequence; HCDR 2: consisting of SEQ ID NO: 99, sequence representation; HCDR 3: consisting of SEQ ID NO: 100 in sequence;
LCDR 1: consisting of SEQ ID NO: 101, sequence representation; LCDR 2: consisting of SEQ ID NO: 102, sequence representation; LCDR 3: consisting of SEQ ID NO: 103;
(4) HCDR 1: consisting of SEQ ID NO: 104; HCDR 2: consisting of SEQ ID NO: 105 in sequence; HCDR 3: consisting of SEQ ID NO: 106;
LCDR 1: consisting of SEQ ID NO: 107; LCDR 2: consisting of SEQ ID NO: 108; LCDR 3: consisting of SEQ ID NO: 109, or a sequence representation of;
(5) HCDR 1: consisting of SEQ ID NO: 110; HCDR 2: consisting of SEQ ID NO: 111; HCDR 3: consisting of SEQ ID NO: 112;
LCDR 1: consisting of SEQ ID NO: 113, sequence representation; LCDR 2: consisting of SEQ ID NO: 114; LCDR 3: consisting of SEQ ID NO: 115, sequence representation;
(6) HCDR 1: consisting of SEQ ID NO: 116; HCDR 2: consisting of SEQ ID NO: 117; HCDR 3: consisting of SEQ ID NO: 118;
LCDR 1: consisting of SEQ ID NO: 119 with sequence representation; LCDR 2: consisting of SEQ ID NO: 120, sequence representation; LCDR 3: consisting of SEQ ID NO: 121 in sequence representation;
(7) HCDR 1: consisting of SEQ ID NO: 122; HCDR 2: consisting of SEQ ID NO: 123 is represented by the sequence; HCDR 3: consisting of SEQ ID NO: 124;
LCDR 1: consisting of SEQ ID NO: 125; LCDR 2: consisting of SEQ ID NO: 126 with a sequence representation; LCDR 3: consisting of SEQ ID NO: 127, sequence representation;
(8) HCDR 1: consisting of SEQ ID NO: 128 in sequence; HCDR 2: consisting of SEQ ID NO: 129, sequence representation; HCDR 3: consisting of SEQ ID NO: 130;
LCDR 1: consisting of SEQ ID NO: 131, or a sequence representation thereof; LCDR 2: consisting of SEQ ID NO: 132; LCDR 3: consisting of SEQ ID NO: 133;
(9) HCDR 1: consisting of SEQ ID NO: 134; HCDR 2: consisting of SEQ ID NO: 135, sequence representation; HCDR 3: consisting of SEQ ID NO: 136;
LCDR 1: consisting of SEQ ID NO: 137; LCDR 2: consisting of SEQ ID NO: 138; LCDR 3: consisting of SEQ ID NO: 139 sequence representation;
(10) HCDR 1: consisting of SEQ ID NO: 140; HCDR 2: consisting of SEQ ID NO: 141; HCDR 3: consisting of SEQ ID NO: 142;
LCDR 1: consisting of SEQ ID NO: 143; LCDR 2: consisting of SEQ ID NO: 144, sequence representation; LCDR 3: consisting of SEQ ID NO: 145;
(11) HCDR 1: consisting of SEQ ID NO: 146; HCDR 2: consisting of SEQ ID NO: 147; HCDR 3: consisting of SEQ ID NO: 148;
LCDR 1: consisting of SEQ ID NO: 149 in seq id no; LCDR 2: consisting of SEQ ID NO: 150; LCDR 3: consisting of SEQ ID NO: 151, sequence representation;
(12) HCDR 1: consisting of SEQ ID NO: 152 sequence representation; HCDR 2: consisting of SEQ ID NO: 153, sequence representation; HCDR 3: consisting of SEQ ID NO: 154;
LCDR 1: consisting of SEQ ID NO: 155; LCDR 2: consisting of SEQ ID NO: 156; LCDR 3: consisting of SEQ ID NO: 157, sequence representation;
(13) HCDR 1: consisting of SEQ ID NO: 158; HCDR 2: consisting of SEQ ID NO: 159 in seq id no; HCDR 3: consisting of SEQ ID NO: 160;
LCDR 1: consisting of SEQ ID NO: 161 sequence representation; LCDR 2: consisting of SEQ ID NO: 162; LCDR 3: consisting of SEQ ID NO: 163;
(14) HCDR 1: consisting of SEQ ID NO: 164 in sequence representation; HCDR 2: consisting of SEQ ID NO: 165, sequence representation; HCDR 3: consisting of SEQ ID NO: 166, sequence representation;
LCDR 1: consisting of SEQ ID NO: 167, sequence representation; LCDR 2: consisting of SEQ ID NO: 168 sequence representation; LCDR 3: consisting of SEQ ID NO: 169 sequence representation;
(15) HCDR 1: consisting of SEQ ID NO: 170; HCDR 2: consisting of SEQ ID NO: 171 sequence representation; HCDR 3: consisting of SEQ ID NO: 172;
LCDR 1: consisting of SEQ ID NO: 173, sequence number; LCDR 2: consisting of SEQ ID NO: 174; LCDR 3: consisting of SEQ ID NO: 175;
(16) HCDR 1: consisting of SEQ ID NO: 176; HCDR 2: consisting of SEQ ID NO: 177 of the sequence representation; HCDR 3: consisting of SEQ ID NO: 178;
LCDR 1: consisting of SEQ ID NO: 179; LCDR 2: consisting of SEQ ID NO: 180, sequence representation; LCDR 3: consisting of SEQ ID NO: 181 in seq id no;
(17) HCDR 1: consisting of SEQ ID NO: 182, a sequence representation; HCDR 2: consisting of SEQ ID NO: 183 sequence representation; HCDR 3: consisting of SEQ ID NO: 184;
LCDR 1: consisting of SEQ ID NO: 185 of the sequence; LCDR 2: consisting of SEQ ID NO: 186; LCDR 3: consisting of SEQ ID NO: 187, sequence representation;
(18) HCDR 1: consisting of SEQ ID NO: 188; HCDR 2: consisting of SEQ ID NO: 189 a sequence representation; HCDR 3: consisting of SEQ ID NO: 190;
LCDR 1: consisting of SEQ ID NO: 191, sequence representation; LCDR 2: consisting of SEQ ID NO: 192 of seq id no; LCDR 3: consisting of SEQ ID NO: 193 is represented by the sequence;
(19) HCDR 1: consisting of SEQ ID NO: 194, sequence representation; HCDR 2: consisting of SEQ ID NO: 195; HCDR 3: consisting of SEQ ID NO: 196, sequence representation;
LCDR 1: consisting of SEQ ID NO: 197 sequence representation; LCDR 2: consisting of SEQ ID NO: 198 by sequence representation; LCDR 3: consisting of SEQ ID NO: 199 for a sequence representation; or
(20) HCDR 1: consisting of SEQ ID NO: 200 in sequence; HCDR 2: consisting of SEQ ID NO: 201 in sequence; HCDR 3: consisting of SEQ ID NO: 202;
LCDR 1: consisting of SEQ ID NO: 203 of the sequence; LCDR 2: consisting of SEQ ID NO: 204; LCDR 3: consisting of SEQ ID NO: 205.
2. The antibody or antigen-binding fragment thereof of claim, 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, 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: 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, 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: 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:
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:
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:
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:
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:
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, and
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, 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: 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:
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 of any one of claims 1-2, 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 claims 1-2, wherein the antigen-binding fragment is selected from the group consisting of Fab, svFv, Fab ', dAb, F (ab')2Fv or Fab/c.
5. The antibody or antigen-binding fragment thereof of any one of claims 1-2, 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 of any one of claims 1-5.
7. The polynucleotide of claim 6, which is selected from the group consisting of SEQ ID NO: 4 and 5; SEQ ID NO: 8 and 9; 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; 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 claims 1-5; optionally, it further comprises a pharmaceutically acceptable carrier and/or excipient.
9. Use of an antibody or antigen-binding fragment thereof according to any one of claims 1-5 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 a cancer, or for the manufacture of a medicament for modulating an immune response, preferably for stimulating 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 in a form suitable for administration by subcutaneous, intradermal, intravenous, intramuscular or intralesional injection.
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20170022273A1 (en) * | 2015-07-22 | 2017-01-26 | Sorrento Therapeutics, Inc. | Antibody therapeutics that bind lag3 |
| US20170101472A1 (en) * | 2015-10-09 | 2017-04-13 | Regeneron Pharmaceuticals, Inc. | Anti-LAG3 Antibodies and Uses Thereof |
| CN109970856A (en) * | 2017-12-27 | 2019-07-05 | 信达生物制药(苏州)有限公司 | Anti-lag-3 antibody and application thereof |
| US20190225687A1 (en) * | 2017-02-22 | 2019-07-25 | I-Mab | Anti-lag-3 antibodies and uses thereof |
| CN110204614A (en) * | 2018-02-28 | 2019-09-06 | 广州誉衡生物科技有限公司 | Anti-human LAG-3 monoclonal antibody and preparation method and use thereof |
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2019
- 2019-12-20 CN CN201911344025.2A patent/CN113004407B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20170022273A1 (en) * | 2015-07-22 | 2017-01-26 | Sorrento Therapeutics, Inc. | Antibody therapeutics that bind lag3 |
| US20170101472A1 (en) * | 2015-10-09 | 2017-04-13 | Regeneron Pharmaceuticals, Inc. | Anti-LAG3 Antibodies and Uses Thereof |
| US20190225687A1 (en) * | 2017-02-22 | 2019-07-25 | I-Mab | Anti-lag-3 antibodies and uses thereof |
| CN109970856A (en) * | 2017-12-27 | 2019-07-05 | 信达生物制药(苏州)有限公司 | Anti-lag-3 antibody and application thereof |
| CN110204614A (en) * | 2018-02-28 | 2019-09-06 | 广州誉衡生物科技有限公司 | Anti-human LAG-3 monoclonal antibody and preparation method and use thereof |
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