WO2022166987A1

WO2022166987A1 - Antibodies binding lag-3 and use thereof

Info

Publication number: WO2022166987A1
Application number: PCT/CN2022/075525
Authority: WO
Inventors: 王荣娟; 焦莎莎; 张畅; 王双
Original assignee: 迈威(上海)生物科技股份有限公司
Priority date: 2021-02-08
Filing date: 2022-02-08
Publication date: 2022-08-11
Also published as: CN114907478A

Abstract

Provided are antibodies and antibody fragments specifically binding LAG-3, and pharmaceutical compositions containing the antibodies or the antibody fragments. A nucleic acid encoding the antibodies, a host cell comprising the nucleic acid, and a method for preparing the antibodies are provided. Further provided are therapeutic and diagnostic uses of the antibodies binding LAG-3.

Description

Antibodies that bind to LAG-3 and uses thereof

technical field

The present invention relates to the field of antibody drugs. In particular, the present invention relates to novel antibodies and antibody fragments that specifically bind to LAG-3 and compositions containing the antibodies or antibody fragments. Furthermore, the present invention relates to nucleic acids encoding said antibodies and host cells comprising said nucleic acids, and methods of making said antibodies. The present invention also relates to the therapeutic and diagnostic uses of these LAG-3 binding antibodies.

Background technique

Immune cells are regulated by a variety of inhibitory immune checkpoint molecules that act as "security brakes" at various stages of the immune response. These regulations are important in preventing tissue damage due to inappropriate immune responses to invading pathogens. Cancer cells, however, use these inhibitory immune checkpoint molecules to evade the immune response of immune cells that specifically destroy tumors.

In the prior art, by using cytotoxic T lymphocyte-associated protein 4 (CTLA-4, CD152), programmed death 1 (PD-1, CD279) and its ligand PD-L1 (CD274, B7-H1) Cancer therapy that uses monoclonal antibodies (mAbs) to block immune checkpoint molecules to restore tumor-specific immune cell function has significantly improved patient survival in some cancer patients. However, due to tumor drug resistance, the absence of tumor-infiltrating lymphocytes (TILs), and the presence of suppressive myeloid cells, the frequency of patient responses to the use of monoclonal antibodies against these immune checkpoint molecules is suboptimal (Awad, R.M. et al., Turn Back the TIMe: Targeting Tumor Infiltrating Myeloid Cells to Revert Cancer Progression. Front. Immunol. 2018, 9, 1977). In addition, the occurrence of immune-related adverse events is also a reason why patients discontinue mAbs that block the immune checkpoint. In order to improve patient response rates and prevent immune-related adverse events, the researchers also investigated other inhibitory immune checkpoint molecules that play a role in cancer cells evading anti-cancer immunity, among them lymphocyte activation gene-3. 3 protein, LAG-3) is emerging as an important target for cancer therapy and assessment of prognosis.

LAG-3, also known as CD223, is a member of the immunoglobulin superfamily and consists of three parts: the extracellular domain, the transmembrane domain and the cytoplasmic domain. The gene of LAG-3 is located on chromosome 12 (12P13), which is similar to CD4 in chromosomal location and structure, but the amino acid sequence of the two is only 20% homologous.

LAG-3 is mainly expressed on the surface of T cells (especially activated T cells), natural killer cells, B cells and plasmacytoid dendritic cells. It has been shown that LAG-3 is an inhibitory receptor with functions of maintaining homeostasis and participating in immune regulation, and is closely related to the occurrence and development of tumors. The major ligand of LAG-3 is histocompatibility complex class II (MHC-II), in addition, galectin-3 (Galectin-3), hepatic sinusoidal endothelial cell lectin (LSECtin) and fibrinogen-like protein 1 (FGL-1) is also its ligand. In the tumor microenvironment, LSECtin, galectin-3 and FGL-1 can interact with LAG-3 to inhibit the antitumor activity of CD8+ T cells.

In cancer, LAG-3-expressing regulatory T cells (Treg) have enhanced suppressive antitumor activity, whereas LAG-3-expressing cytotoxic CD8+ T cells have reduced proliferation rate and effector cytokine production (Scurr M. et al., Highly prevalent colorectal cancer-infiltrating LAP ⁺ Foxp3 ^- T cells exhibit more potent immunosuppressive activity than Foxp3 ⁺ regulatory T cells, Mucosal Immunol. 2014, 7(2): 428-439). LAG-3 is expressed at elevated levels in many tumor-infiltrating Treg cells, which are also expressed in peripheral blood and tumor tissues of patients with melanoma and colorectal cancer, and MHC-II expressed on antigen-presenting cells (APCs) is associated with The interaction between LAG-3 on Treg cells can produce immunosuppressive cytokines, thereby inhibiting the activity of CD8+ TILs.

Since LAG-3 expression levels and LAG-3+ cell infiltration in tumors are associated with tumor progression and poor prognosis (Maruhashi T, Sugiura D, Okazaki I et al. Journal for ImmunoTherapy of Cancer 2020;8:e001014), there is still a need in the art Development of new anti-LAG-3 antibodies, in particular humanized antibodies, that modulate LAG-3 activity to activate the immune system, for example, for cancer immunotherapy and treatment of other diseases such as infectious diseases.

SUMMARY OF THE INVENTION

Through research, the inventors developed a new group of anti-LAG-3 antibodies (eg, humanized antibody molecules) that bind to LAG-3 with high affinity and activate the immune system by blocking the activity of LAG-3, so as to satisfy the meet the above requirements. The antibodies or antigen-binding fragments of the invention that specifically bind to LAG-3 have one or more of the following properties:

(a) Binds LAG-3 with high affinity, eg, human LAG-3 and cynomolgus LAG-3, eg, the anti-LAG-3 antibody or antigen-binding fragment thereof has a _K for binding to LAG-3 of about 10 ^-7 M to about ^10-12 M, preferably, about ^10-8 M to about ^10-12 M, as measured by the ForteBio kinetic binding assay;

(b) specifically blocking the binding of LAG-3 to FGL-1;

(c) specifically blocking the binding of LAG-3 to cell surface MHC class II molecules (eg, human HLA);

(d) keeping T cells in an activated state;

(e) has antitumor efficacy in vivo.

Accordingly, in a first aspect, the present invention provides an isolated antibody or antigen-binding fragment that specifically binds LAG-3, comprising

(a) 3 CDRs in the heavy chain variable region amino acid sequence shown in SEQ ID NO: 1 and 3 CDRs in the light chain variable region amino acid sequence shown in SEQ ID NO: 2; Variants in which the CDR regions have a single CDR or multiple CDRs with no more than 2 or 1 amino acid change per CDR region;

(b) 3 CDRs in the heavy chain variable region amino acid sequence shown in SEQ ID NO: 3 and 3 CDRs in the light chain variable region amino acid sequence shown in SEQ ID NO: 4; Variants in which the CDR regions have a single CDR or multiple CDRs with no more than 2 or 1 amino acid change per CDR region;

(c) 3 CDRs in the heavy chain variable region amino acid sequence shown in SEQ ID NO: 5 and 3 CDRs in the light chain variable region amino acid sequence shown in SEQ ID NO: 6; Variants in which the CDR regions have a single CDR or multiple CDRs with no more than 2 or 1 amino acid change per CDR region;

(d) 3 CDRs in the heavy chain variable region amino acid sequence shown in SEQ ID NO: 7 and 3 CDRs in the light chain variable region amino acid sequence shown in SEQ ID NO: 8; Variants in which the CDR regions have a single CDR or multiple CDRs with no more than 2 or 1 amino acid change per CDR region;

(e) 3 CDRs in the heavy chain variable region amino acid sequence shown in SEQ ID NO: 9 and 3 CDRs in the light chain variable region amino acid sequence shown in SEQ ID NO: 10; Variants in which the CDR regions have a single CDR or multiple CDRs with no more than 2 or 1 amino acid change per CDR region;

(f) 3 CDRs in the heavy chain variable region amino acid sequence shown in SEQ ID NO: 11 and 3 CDRs in the light chain variable region amino acid sequence shown in SEQ ID NO: 12; CDR regions have variants in which a single CDR or multiple CDRs do not exceed 2 or 1 amino acid change per CDR region.

In some embodiments, the invention provides an isolated antibody or antigen-binding fragment that specifically binds LAG-3, comprising a heavy chain variable region and a light chain variable region, wherein

(a) the heavy chain variable region comprises HCDR1 shown in SYGIS (SEQ ID NO: 31), or a variant of said HCDR1 with no more than 2 amino acid changes or no more than 1 amino acid change; EIYPRSDNTYYNGKFKG (SEQ ID NO: 31) NO: 32), or a variant of said HCDR2 with no more than 2 amino acid changes or no more than 1 amino acid change; and HCDR3 shown in RAFYYYGSNYYAMDY (SEQ ID NO: 33), or a variant of said HCDR3 A variant with no more than 2 amino acid changes or no more than 1 amino acid change; the light chain variable region comprises LCDR1 shown in RSSQSIVHSNGDTYLE (SEQ ID NO: 34), or no more than 2 amino acid changes of said LCDR1 or Variants with no more than 1 amino acid change; LCDR2 shown in KVSNRFS (SEQ ID NO: 35), or a variant of said LCDR2 with no more than 2 amino acid changes or no more than 1 amino acid change; and FQGSHVPWT (SEQ ID NO: 35) NO:36) LCDR3, or a variant of the LCDR3 with no more than 2 amino acid changes or no more than 1 amino acid change;

(b) the heavy chain variable region comprises HCDR1 shown in NYAMS (SEQ ID NO: 37), or a variant of said HCDR1 with no more than 2 amino acid changes or no more than 1 amino acid change; TITYGTTYTFYSDNVKG (SEQ ID NO: 37) NO: 38), or a variant of said HCDR2 with no more than 2 amino acid changes or no more than 1 amino acid change; and HCDR3 shown in GEYGSSFAY (SEQ ID NO: 39), or a variant of said HCDR3 A variant with no more than 2 amino acid changes or no more than 1 amino acid change; the light chain variable region comprises LCDR1 as shown in KASQNVRTAVA (SEQ ID NO: 40), or no more than 2 amino acid changes of said LCDR1 or Variants with no more than 1 amino acid change; LCDR2 shown in LASNRHT (SEQ ID NO: 41), or a variant of said LCDR2 with no more than 2 amino acid changes or no more than 1 amino acid change; and LQHWNYPLT (SEQ ID NO: 41) NO:42) LCDR3, or a variant of the LCDR3 with no more than 2 amino acid changes or no more than 1 amino acid change;

(c) the variable region of the heavy chain comprises HCDR1 shown in DYAVS (SEQ ID NO: 43), or a variant of the HCDR1 with no more than 2 amino acid changes or no more than 1 amino acid change; VVWGDGSTNYHSALIS (SEQ ID NO: 43) NO: 44) shown in HCDR2, or a variant of said HCDR2 with no more than 2 amino acid changes or no more than 1 amino acid change; and HCDR3 shown in GGGGMDY (SEQ ID NO: 45), or a variant of said HCDR3 A variant with no more than 2 amino acid changes or no more than 1 amino acid change; the light chain variable region comprises LCDR1 shown in RASSSVSYMH (SEQ ID NO: 46), or no more than 2 amino acid changes of said LCDR1 or Variants with no more than 1 amino acid change; LCDR2 shown in ATSNLAS (SEQ ID NO: 47), or a variant of said LCDR2 with no more than 2 amino acid changes or no more than 1 amino acid change; and QHYNTNPPTWT (SEQ ID NO: 47) NO:48) LCDR3, or a variant of the LCDR3 with no more than 2 amino acid changes or no more than 1 amino acid change;

(d) the variable region of the heavy chain comprises HCDR1 shown in DDYMH (SEQ ID NO: 49), or a variant of the HCDR1 with no more than 2 amino acid changes or no more than 1 amino acid change; RIDPEDVETKYDPKFQG (SEQ ID NO: 49) NO:50), or a variant of said HCDR2 with no more than 2 amino acid changes or no more than 1 amino acid change; and HCDR3 shown in SFYSNYVNYFDQ (SEQ ID NO:51), or a variant of said HCDR3 A variant with no more than 2 amino acid changes or no more than 1 amino acid change; the light chain variable region comprises LCDR1 shown in KASENVGTYVS (SEQ ID NO: 52), or no more than 2 amino acid changes of said LCDR1 or Variants with no more than 1 amino acid change; LCDR2 shown in GASNRYT (SEQ ID NO: 53), or a variant of said LCDR2 with no more than 2 amino acid changes or no more than 1 amino acid change; and GQSYSYPYT (SEQ ID NO: 53) NO:54) LCDR3, or a variant of the LCDR3 with no more than 2 amino acid changes or no more than 1 amino acid change;

For example, the heavy chain variable region comprises HCDR1 set forth in DDYMH (SEQ ID NO: 49); HCDR2 set forth in RIX ₁ PEDVETKYDPKFQG (SEQ ID NO: 64), preferably, wherein X ₁ is D or N; and HCDR3 shown in SFYSNYVNYFDQ (SEQ ID NO: 51);

The light chain variable region comprises LCDR1 shown by X ₂ ASENVGTYVS (SEQ ID NO: 68), preferably, wherein X ₂ is K or R; LCDR2 shown by X ₃ ASX ₄ RYT (SEQ ID NO: 69) , preferably; wherein X ₃ is G or A; X ₄ is N or T; and LCDR3 shown in GQSYSYPYT (SEQ ID NO: 54);

For example, the heavy chain variable region comprises HCDR1 shown in DDYMH (SEQ ID NO:49); HCDR2 shown in RIDPEDVETKYDPKFQG (SEQ ID NO:50); and HCDR3 shown in SFYSNYVNYFDQ (SEQ ID NO:51); The light chain variable region comprises LCDR1 shown in KASENVGTYVS (SEQ ID NO: 52), LCDR2 shown in GASNRYT (SEQ ID NO: 53), and LCDR3 shown in GQSYSYPYT (SEQ ID NO: 54);

The heavy chain variable region comprises HCDR1 shown in DDYMH (SEQ ID NO: 49); HCDR2 shown in RIDPEDVETKYDPKFQG (SEQ ID NO: 50), HCDR3 shown in SFYSNYVNYFDQ (SEQ ID NO: 51); The chain variable region comprises LCDR1 shown in KASENVGTYVS (SEQ ID NO: 52), LCDR2 shown in AASNRYT (SEQ ID NO: 66), and LCDR3 shown in GQSYSYPYT (SEQ ID NO: 54);

The heavy chain variable region comprises HCDR1 shown in DDYMH (SEQ ID NO: 49); HCDR2 shown in RIDPEDVETKYDPKFQG (SEQ ID NO: 50), HCDR3 shown in SFYSNYVNYFDQ (SEQ ID NO: 51); The chain variable region comprises LCDR1 represented by RASENVGTYVS (SEQ ID NO:65), LCDR2 represented by GASNRYT (SEQ ID NO:53), and LCDR3 represented by GQSYSYPYT (SEQ ID NO:54);

The heavy chain variable region comprises HCDR1 shown in DDYMH (SEQ ID NO: 49); HCDR2 shown in RIDPEDVETKYDPKFQG (SEQ ID NO: 50), HCDR3 shown in SFYSNYVNYFDQ (SEQ ID NO: 51); The chain variable region comprises LCDR1 shown by KASENVGTYVS (SEQ ID NO:52), LCDR2 shown by GASTRYT (SEQ ID NO:67), and LCDR3 shown by GQSYSYPYT (SEQ ID NO:54); the heavy chain can be The variable region comprises HCDR1 represented by DDYMH (SEQ ID NO: 49); HCDR2 represented by RINPEDVETKYDPKFQG (SEQ ID NO: 63), HCDR3 represented by SFYSNYVNYFDQ (SEQ ID NO: 51); the light chain variable region comprises LCDR1 shown by KASENVGTYVS (SEQ ID NO:52), LCDR2 shown by GASNRYT (SEQ ID NO:53), and LCDR3 shown by GQSYSYPYT (SEQ ID NO:54);

The heavy chain variable region comprises HCDR1 shown in DDYMH (SEQ ID NO: 49); HCDR2 shown in RINPEDVETKYDPKFQG (SEQ ID NO: 63), HCDR3 shown in SFYSNYVNYFDQ (SEQ ID NO: 51); The chain variable region comprises LCDR1 represented by KASENVGTYVS (SEQ ID NO:52), LCDR2 represented by AASNRYT (SEQ ID NO:66), and LCDR3 represented by GQSYSYPYT (SEQ ID NO:54); or

The heavy chain variable region comprises HCDR1 shown in DDYMH (SEQ ID NO: 49); HCDR2 shown in RINPEDVETKYDPKFQG (SEQ ID NO: 63), HCDR3 shown in SFYSNYVNYFDQ (SEQ ID NO: 51); The chain variable region comprises LCDR1 shown by KASENVGTYVS (SEQ ID NO:52), LCDR2 shown by GASTRYT (SEQ ID NO:67), and LCDR3 shown by GQSYSYPYT (SEQ ID NO:54);

(e) the heavy chain variable region comprises HCDR1 shown in DDYMH (SEQ ID NO: 49), or a variant of said HCDR1 with no more than 2 amino acid changes or no more than 1 amino acid change; WIDPENGETEYASKFQG (SEQ ID NO: 49) NO: 55) shown in HCDR2, or a variant of said HCDR2 with no more than 2 amino acid changes or no more than 1 amino acid change; and HCDR3 shown in FDY (SEQ ID NO: 56), or a variant of said HCDR3 A variant with no more than 2 amino acid changes or no more than 1 amino acid change; the light chain variable region comprises LCDR1 as shown in KSSQSLLDSDGKTYLN (SEQ ID NO: 57), or no more than 2 amino acid changes of said LCDR1 or Variants with no more than 1 amino acid change; LCDR2 shown in LVSKLDF (SEQ ID NO: 58), or a variant of said LCDR2 with no more than 2 amino acid changes or no more than 1 amino acid change; and WQGTHFPQT (SEQ ID NO: 58) NO:59), or a variant of said LCDR3 with no more than 2 amino acid changes or no more than 1 amino acid change; or

(f) the heavy chain variable region comprises HCDR1 shown in NYGIS (SEQ ID NO: 60), or a variant of said HCDR1 with no more than 2 amino acid changes or no more than 1 amino acid change; EIYPRGGNTYYNGKFKG (SEQ ID NO: 60) NO: 61), or a variant of said HCDR2 with no more than 2 amino acid changes or no more than 1 amino acid change; and HCDR3 shown in RAFYYFGSNYYAMDY (SEQ ID NO: 62), or a variant of said HCDR3 A variant with no more than 2 amino acid changes or no more than 1 amino acid change; the light chain variable region comprises LCDR1 shown in RSSQSIVHSNGDTYLE (SEQ ID NO: 34), or no more than 2 amino acid changes of said LCDR1 or Variants with no more than 1 amino acid change; LCDR2 shown in KVSNRFS (SEQ ID NO: 35), or a variant of said LCDR2 with no more than 2 amino acid changes or no more than 1 amino acid change; and FQGSHVPWT (SEQ ID NO: 35) NO:36), or a variant of said LCDR3 with no more than 2 amino acid changes or no more than 1 amino acid change.

(a) the heavy chain variable region comprises or is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO: 1 and the light chain variable region comprises or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the sequence of SEQ ID NO: 2 % sequence of identity;

(b) the heavy chain variable region comprises or is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO:3 and the light chain variable region comprises or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the sequence of SEQ ID NO:4 % sequence of identity;

(c) the heavy chain variable region comprises or is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO:5 and the light chain variable region comprises or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the sequence of SEQ ID NO:6 % sequence of identity;

(d) the heavy chain variable region comprises or is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO:7 and the light chain variable region comprises or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the sequence of SEQ ID NO: 8 % sequence of identity;

The heavy chain variable region comprises the sequence of SEQ ID NO: 15 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto , and the light chain variable region comprises or is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO: 16 sequence; or

The heavy chain variable region comprises the sequence of SEQ ID NO: 15 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto , and the light chain variable region comprises or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the sequence of SEQ ID NO: 17 the sequence of;

(e) the heavy chain variable region comprises or is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO:9 and the light chain variable region comprises or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the sequence of SEQ ID NO: 10 % sequence of identity;

(f) the heavy chain variable region comprises or is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO: 11 and the light chain variable region comprises or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the sequence of SEQ ID NO: 12 % sequence of identity.

In some embodiments, the isolated antibody or antigen-binding fragment of the invention is a humanized antibody.

In some embodiments, the isolated antibody or antigen-binding fragment of the invention is an IgGl, IgG2, IgG3 or IgG4 antibody; preferably, an IgGl or IgG4 antibody; more preferably, a humanized IgGl or humanized IgG4 antibody . In some embodiments, the antigen-binding fragment of the invention is a Fab, Fab', F(ab') ₂ , Fv, single-chain Fv, single-chain Fab, or diabody.

In a second aspect, the present invention provides a nucleic acid encoding the antibody or antigen-binding fragment of the first aspect above, a vector (preferably, an expression vector) comprising the nucleic acid, and a host cell comprising the nucleic acid or the vector . In some embodiments, the host cell is prokaryotic or eukaryotic, eg, selected from E. coli cells, yeast cells, mammalian cells, or other cells suitable for the production of antibodies or antigen-binding fragments, multispecific antibodies. In some embodiments, the host cell is a 293 cell or a CHO cell.

In a third aspect, the invention provides a method of making an antibody or antigen-binding fragment of the invention, the method comprising culturing a host cell of the invention under conditions suitable for expression of the antibody or antigen-binding fragment encoding the invention, optionally The antibody or antigen-binding fragment of the invention is recovered from the host cell or from the culture medium.

In a fourth aspect, the present invention provides a pharmaceutical composition comprising an antibody or antigen-binding fragment of the present invention and a pharmaceutically acceptable carrier.

In some embodiments, the present invention provides pharmaceutical compositions comprising an antibody of the present invention and other therapeutic agents, and optionally a pharmaceutically acceptable carrier; preferably, the other therapeutic agents are selected from chemotherapeutic agents, other antibodies ( For example, anti-PD-1 antibody or anti-PD-L1 antibody), cytotoxic agent.

In some embodiments, the present invention provides a combination product comprising an antibody of the present invention, and one or more other therapeutic agents, such as chemotherapeutic agents, cytotoxic agents, other antibodies, preferably anti-PD-1 antibodies or anti-PD-1 antibodies PD-L1 antibody.

In a fifth aspect, the present invention provides a method of preventing or treating a tumor or infectious disease in a subject or individual, comprising administering to the subject an effective amount of an antibody, pharmaceutical composition, or combination of the present invention product.

In some embodiments, the tumor prevented or treated by the antibody, pharmaceutical composition, or combination product of the invention is a cancer, such as a cancer with elevated expression levels of LAG-3; or the antibody, pharmaceutical composition, or combination product of the invention, The infectious disease that is prevented or treated by the combination product is, for example, a bacterial infection, a viral infection, a fungal infection or a protozoal infection, preferably the infectious disease is a chronic infection in which elevated LAG-3 expression levels lead to The subject or individual is immunocompromised.

In a sixth aspect, the present invention provides a kit for detecting LAG-3 in a sample, the kit comprising the antibody of the present invention, for implementing the following steps:

(a) contacting the sample with an antibody of the invention; and

(b) detecting the formation of a complex between the LAG-3 antibody and LAG-3; optionally, the LAG-3 antibody is detectably labeled,

Thus, it is determined whether there is an elevated LAG-3 expression level in a sample from a subject or individual.

Brief Description of Drawings

Preferred embodiments of the present invention, which are described in detail below, will be better understood when read in conjunction with the accompanying drawings. For the purpose of illustrating the invention, there are shown in the drawings embodiments which are presently preferred. It should be understood, however, that the invention is not limited to the precise arrangements and instrumentalities of the embodiments shown in the drawings.

Figure 1: shows the results of detecting the specific binding activity of the anti-human LAG-3 chimeric antibody to LAG-3 on the surface of 293/rhLAG-3 cells by the FACS method. The negative control in the figure is the isotype control antibody, which is a purified monoclonal antibody of the human IgG4 isotype that does not bind to the LAG-3 molecule, and MFI represents the mean fluorescence intensity.

Figure 2: shows the results of blocking the binding of 293/rhHLA cells to hLAG-3 by anti-human LAG-3 chimeric antibody detected by FACS method.

Figure 3: shows the results of detecting the binding of anti-human LAG-3 humanized antibody to recombinant human LAG-3 by ELISA.

Figure 4: shows the results of detecting the binding of anti-human LAG-3 humanized antibody to recombinant cynomolgus monkey LAG-3 by ELISA.

Figure 5: shows the results of detecting the binding of anti-human LAG-3 humanized antibody to recombinant mouse LAG-3 by ELISA.

Figure 6: Graph showing the results of the affinity of the anti-human LAG-3 humanized antibodies of the present invention for human LAG-3 extracellular domain recombinant protein measured by ForteBio kinetic binding assay.

Figure 7: Graph showing the results of the affinity of BMS-986016 for human LAG-3 extracellular domain recombinant protein measured by ForteBio kinetic binding assay. BMS-986016 is a monoclonal antibody targeting LAG-3 developed by BMS.

Figure 8: shows the results of detecting the specific binding of the anti-human LAG-3 humanized antibody of the present invention to LAG-3 on the surface of 293/rhLAG-3 cells by FACS method.

FIG. 9 : shows the result of blocking the binding of human FGL-1 to LAG-3 by the anti-human LAG-3 humanized antibody of the present invention by ELISA.

Figure 10: shows the results of blocking the binding of 293/rhHLA cells to hLAG-3 by anti-human LAG-3 humanized antibody detected by FACS.

Figure 11: shows the inhibitory effect of the anti-human LAG-3 humanized antibody of the present invention on tumor growth in a B6-huLAG3 humanized mouse subcutaneous tumor model.

Figure 12: shows the effect of the anti-human LAG-3 humanized antibody of the present invention on the body weight of mice in the B6-huLAG3 humanized mouse subcutaneous tumor model.

Detailed description of the invention

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. Furthermore, the materials, methods and examples described herein are illustrative only and not intended to be limiting. Other features, objects and advantages of the present invention will be apparent from the description and drawings, and from the appended claims.

I. Definitions

For the purpose of interpreting this specification, the following definitions will be used and where appropriate, terms used in the singular may also include the plural, and vice versa. It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

The term "about" when used in conjunction with a numerical value is intended to encompass the numerical value within a range having a lower limit that is 5% less than the specified numerical value and an upper limit that is 5% greater than the specified numerical value.

As used herein, the term "and/or" means any one of the alternatives or two or more of the alternatives.

Herein, when the term "comprising" or "comprising" is used, unless otherwise indicated, it also encompasses situations consisting of the recited elements, integers or steps. For example, reference to an antibody variable region that "comprises" a particular sequence is also intended to encompass antibody variable regions that consist of that particular sequence.

The term "antibody" is used herein in the broadest sense to refer to a protein comprising an antigen-binding site, and encompasses natural and artificial antibodies of various structures, including but not limited to monoclonal, polyclonal, multispecific ( For example, bispecific antibodies), single chain antibodies, whole antibodies and antibody fragments. Preferably, the antibody of the present invention is a single domain antibody or a heavy chain antibody.

"Antibody fragment" or "antigen-binding fragment" are used interchangeably herein to refer to a molecule other than an intact antibody that comprises a portion of the intact antibody and binds the antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, Fv, single chain Fv, single chain Fab, or diabodies.

An antibody that exhibits the same or similar binding affinity and/or specificity as a reference antibody refers to an antibody capable of binding at least 50%, 60%, 70%, 80%, 90% or 95% or more of the reference antibody Affinity and/or specificity. This can be determined by any method known in the art for determining binding affinity and/or specificity. In one embodiment, the reference antibody is a monoclonal antibody targeting LAG-3 developed by BMS Corporation: BMS-986016.

"Complementarity determining regions" or "CDR regions" or "CDRs" are loops in the variable domains of antibodies that are hypervariable in sequence and form structurally defined loops ("hypervariable loops") and/or contain antigen-contacting residues ( "antigen contact point"). The CDRs are mainly responsible for binding to antigenic epitopes. The CDRs of the heavy chain are commonly referred to as CDR1, CDR2 and CDR3, numbered sequentially from the N-terminus. In a given heavy chain variable region amino acid sequence, the precise amino acid sequence boundaries of each CDR can be determined using any one or a combination of many well-known antibody CDR assignment systems, including, for example, antibody-based three-dimensional Structure and topology of CDR loops in Chothia (Chothia et al. (1989) Nature 342:877-883, Al-Lazikani et al, "Standard conformations for the canonical structures of immunoglobulins", Journal of Molecular Biology, 273, 927-948 ( 1997)), Kabat based on antibody sequence variability (Kabat et al., Sequences of Proteins of Immunological Interest, 4th edition, U.S. Department of Health and Human Services, National Institutes of Health (1987)), AbM (University of Bath) , Contact (University College London), International ImMunoGeneTics database (IMGT) (http://imgt.cines.fr/), and North CDR definitions based on affinity propagation clustering using a large number of crystal structures.

Unless otherwise stated, in the present invention, the term "CDR" or "CDR sequence" encompasses CDR sequences determined in any of the ways described above.

A CDR can also be determined based on having the same Kabat numbering position as a reference CDR sequence (eg, any of the CDRs exemplified by the invention). In the present invention, references to antibody variable regions and specific CDR sequences (including heavy chain variable region residues) refer to the numbering positions according to the Kabat numbering system.

Although CDRs vary from antibody to antibody, only a limited number of amino acid positions within CDRs are directly involved in antigen binding. Using at least two of the Kabat, Chothia, AbM and Contact methods, the region of minimum overlap can be determined, thereby providing the "minimum binding unit" for antigen binding. The minimal binding unit can be a sub-portion of a CDR. The residues of the remainder of the CDR sequence can be determined by the structure and protein folding of the antibody, as will be apparent to those skilled in the art. Accordingly, the present invention also contemplates variants of any of the CDRs presented herein. For example, in a variant of a CDR, the amino acid residues of the smallest binding unit may remain unchanged, while the remaining CDR residues as defined by Kabat or Chothia or AbM may be replaced by conservative amino acid residues.

The term "chimeric antibody" is an antibody molecule in which (a) a constant region or portion thereof is altered, substituted or exchanged such that the antigen binding site is linked to a constant region of a different or altered class and/or species or confers chimeric or (b) use variable domains or portions thereof with variable domains having different or altered antigen specificities Alter, replace or exchange. For example, murine antibodies can be modified by replacing their constant regions with those from human immunoglobulins. Due to the replacement of human constant regions, the chimeric antibody can retain its specificity in recognizing the antigen while having reduced antigenicity in humans as compared to the original murine antibody.

A "humanized" antibody refers to a chimeric antibody comprising amino acid residues from non-human CDRs and amino acid residues from human FRs. In some embodiments, all or substantially all of the CDRs (eg, CDRs) in the humanized antibody correspond to those of a non-human antibody, and all or substantially all of the FRs correspond to those of a human antibody. A humanized antibody may optionally contain at least a portion of an antibody constant region derived from a human antibody. A "humanized form" of an antibody (eg, a non-human antibody) refers to an antibody that has been humanized.

"Human antibody" refers to an antibody having an amino acid sequence corresponding to the amino acid sequence of an antibody produced by a human or human cell or derived from a non-human source, utilizing a library of human antibodies or other human Antibody coding sequences. This definition of human antibody specifically excludes humanized antibodies comprising non-human antigen-binding residues.

The term "Fc region" is used herein to define the C-terminal region of an immunoglobulin heavy chain comprising at least a portion of the constant region. The term includes native sequence Fc regions and variant Fc regions. In certain embodiments, the human IgG heavy chain Fc region extends from Cys226 or Pro230 to the carbonyl terminus of the heavy chain. However, the C-terminal lysine (Lys447) of the Fc region may or may not be present. Unless otherwise stated, the numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, which is also known as the EU index, as in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National As described in Institutes of Health, Bethesda, MD, 1991.

The term "variable region" or "variable domain" refers to the domain of an antibody heavy or light chain that is involved in antibody binding to an antigen. The variable domains of the heavy and light chains of native antibodies generally have similar structures, with each domain comprising four conserved framework regions (FRs) and three complementarity determining regions (CDRs). (See, eg, Kindt et al. ^Kuby Immunology, 6th ed., WH Freeman and Co. p. 91 (2007)). A single VH or VL domain may be sufficient to confer antigen binding specificity.

As used herein, the term "binding" or "specific binding" means that binding is selective for the antigen and can be distinguished from unwanted or non-specific interactions. The ability of an antibody to bind to a particular antigen can be determined by enzyme-linked immunosorbent assay (ELISA), SPR or biofilm interferometry, or other conventional binding assays known in the art.

The term "immune checkpoint molecule" means a class of inhibitory signaling molecules present in the immune system that avoid tissue damage by regulating the persistence and strength of immune responses in peripheral tissues and are involved in maintaining tolerance to self-antigens (Pardoll DM. , The blockade of immune checkpoints in cancer immunotherapy. Nat Rev Cancer, 2012, 12(4):252-264). Studies have found that one of the reasons why tumor cells can escape the immune system and proliferate out of control is the use of the inhibitory signaling pathway of immune checkpoint molecules, thereby inhibiting the activity of T lymphocytes, so that T lymphocytes cannot effectively kill tumors. Effects (Yao S, Zhu Y and Chen L., Advances in targeting cell surface signaling molecules for immune modulation. Nat Rev Drug Discov, 2013, 12(2):130-146). Immune checkpoint molecules include, but are not limited to, LAG-3, programmed death 1 (PD-1), PD-L2, CTLA-4, TIM-3.

The term "costimulatory molecule" refers to a corresponding binding partner on a cell that specifically binds to a costimulatory ligand to mediate a co-stimulatory response (such as, but not limited to, proliferation) of the cell. Costimulatory molecules are cell surface molecules other than antigen receptors or their ligands that contribute to an effective immune response. Costimulatory molecules include, but are not limited to, MHC class I molecules, TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocyte activation molecules (SLAM proteins), activating NK cell receptors, OX40 , CD40, GITR, 4-1BB (ie CD137), CD27 and CD28. In some embodiments, a "costimulatory molecule" is CD28, OX40, GITR, 4-1BB (ie, CD137) and/or CD27.

An "immunoconjugate" is an antibody conjugated to one or more other substances, including but not limited to cytotoxic agents or labels.

The terms "inhibit" or "block" refer to the reduction of some parameter (eg, activity) of a given molecule (eg, an immune checkpoint molecule). For example, this term includes substances that cause the activity of a given molecule (eg, LAG-3) to be inhibited by at least 5%, 10%, 20%, 30%, 40%, or more. Therefore, inhibition does not have to be 100%.

The terms "individual" or "subject" are used interchangeably and include mammals. Mammals include, but are not limited to, domesticated animals (eg, cattle, sheep, cats, dogs, and horses), primates (eg, humans and non-human primates such as monkeys), rabbits, and rodents (eg, mice and large mouse). In particular, the individual or subject is a human.

The terms "tumor" and "cancer" are used interchangeably herein to encompass both solid and liquid tumors.

The terms "cancer" and "cancerous" refer to a physiological disorder in mammals in which cell growth is unregulated.

The term "tumor" refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues. The terms "cancer," "cancerous," and "tumor" are not mutually exclusive when referred to herein.

"Tumor immune escape" refers to the process by which tumors evade immune recognition and clearance. Thus, as a therapeutic concept, tumor immunity is "treated" when such escape is diminished, and the tumor is recognized and attacked by the immune system. Examples of tumor recognition include tumor binding, tumor shrinkage, and tumor clearance.

The term "infectious disease" refers to a disease caused by a pathogen, including, for example, a viral infection, a bacterial infection, a fungal infection, or an infection of a protozoan such as a parasite.

The term "label" as used herein refers to a compound or composition that is conjugated or fused, directly or indirectly, to an agent, such as an antibody, and facilitates detection of the agent to which it is conjugated or fused. The label can itself be detectable (eg, a radioisotope label or a fluorescent label) or, in the case of an enzymatic label, can catalyze a detectable chemical change of a substrate compound or composition. The term is intended to encompass direct labeling of a probe or antibody by coupling (ie, physically linking) a detectable substance to the probe or antibody, as well as indirect labeling of a probe or antibody by reaction with another reagent that is directly labeled. Examples of indirect labeling include detection of a primary antibody using a fluorescently labeled secondary antibody and end-labeling of a DNA probe with biotin so that it can be detected with fluorescently labeled streptavidin.

An "isolated" antibody means that it has been separated from components of its natural environment. In some embodiments, the LAG-3 antibody is purified to greater than 95% or 99% purity, such as by, eg, electrophoresis (eg, SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (eg, ion exchange or reversed-phase HPLC). For a review of methods for assessing antibody purity, see, eg, Flatman et al., J. Chromatogr. B848:79-87 (2007).

"Isolated" nucleic acid refers to a nucleic acid molecule that has been separated from components of its natural environment. An isolated nucleic acid includes a nucleic acid molecule contained in a cell that normally contains the nucleic acid molecule, but the nucleic acid molecule is present extrachromosomally or at a chromosomal location different from its natural chromosomal location. "Isolated LAG-3 antibody-encoding nucleic acid" refers to one or more nucleic acid molecules encoding a LAG-3 antibody-encoding chain or fragment thereof, including such nucleic acid molecules in a single vector or separate vectors, and present in Such nucleic acid molecules at one or more locations in a host cell.

Calculation of sequence identity between sequences is performed as follows.

To determine the percent identity of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., between the first and second amino acid sequences or nucleic acid sequences for optimal alignment. Gaps are introduced in one or both or non-homologous sequences can be discarded for comparison purposes). In a preferred embodiment, the length of the reference sequences aligned for comparison purposes is at least 30%, preferably at least 40%, more preferably at least 50%, 60% and even more preferably at least 70%, 80% , 90%, 100% of the reference sequence length. The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide at the corresponding position in the second sequence, then the molecules are identical at that position.

Sequence comparisons and calculation of percent identity between two sequences can be accomplished using mathematical algorithms. In a preferred embodiment, the Needlema and Wunsch ((1970) J. Mol. Biol. 48:444-453) algorithm (at http://www.gcg.com) is used that has been integrated into the GAP program of the GCG software package available), using the Blossum 62 matrix or the PAM250 matrix and

gap weights

16, 14, 12, 10, 8, 6, or 4 and

length weights

1, 2, 3, 4, 5, or 6, to determine the distance between two amino acid sequences percent identity. In yet another preferred embodiment, the GAP program in the GCG software package (available at http://www.gcg.com) is used, using the NWSgapdna.CMP matrix and gap weights 40, 50, 60, 70 or 80 and A length weight of 1, 2, 3, 4, 5, or 6 determines the percent identity between two nucleotide sequences. A particularly preferred set of parameters (and one that should be used unless otherwise specified) is the Blossum 62 scoring matrix with a gap penalty of 12, a gap extension penalty of 4, and a frameshift gap penalty of 5.

It is also possible to use the PAM120 weighted remainder table, gap length penalty of 12, gap penalty of 4), using the E. Meyers and W. Miller algorithm that has been incorporated into the ALIGN program (version 2.0), ((1989) CABIOS, 4:11- 17) Determining the percent identity between two amino acid sequences or nucleotide sequences.

Additionally or alternatively, the nucleic acid sequences and protein sequences described herein can be further used as "query sequences" to perform searches against public databases, eg, to identify other family member sequences or related sequences.

As used herein, the term "hybridizes under conditions of low stringency, moderate stringency, high stringency or very high stringency" describes hybridization and washing conditions. Guidance for performing hybridization reactions can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6, incorporated by reference. Aqueous and non-aqueous methods are described in this reference and either method can be used. In some embodiments, the specific hybridization conditions referred to herein are as follows: 1) low stringency hybridization conditions are at about 45°C in 6X sodium chloride/sodium citrate (SSC) followed by at least 50°C (for Low stringency conditions, the temperature of the wash can be increased to 55°C) twice in 0.2X SSC, 0.1% SDS; 2) medium stringency hybridization conditions are at about 45°C in 6X SSC followed by 60°C in One or more washes in 0.2X SSC, 0.1% SDS; 3) High stringency hybridization conditions are one or more washes in 6X SSC at about 45°C followed by one or more washes in 0.2X SSC, 0.1% SDS at 65°C and preferably 4) very high stringency hybridization conditions are one or more washes in 0.5M sodium phosphate, 7% SDS at 65°C followed by one or more washes in 0.2X SSC, 0.1% SDS at 65°C. Very high stringency conditions (4) are the preferred conditions and the one that should be used unless otherwise specified.

The term "pharmaceutical composition" refers to a composition that is in a form that allows the biological activity of the active ingredients contained therein to be effective and does not contain additional ingredients.

The term "pharmaceutically acceptable carrier" refers to diluents, adjuvants (eg, Freund's adjuvant (complete and incomplete), excipients, buffers or stabilizers, etc.) with which the active substance is administered.

As used herein, "treating" refers to slowing, interrupting, retarding, alleviating, stopping, reducing, or reversing the progression or severity of an existing symptom, disorder, condition, or disease. Desired therapeutic effects include, but are not limited to, preventing disease occurrence or recurrence, reducing symptoms, reducing any direct or indirect pathological consequences of disease, preventing metastasis, reducing the rate of disease progression, ameliorating or alleviating disease state, and relieving or improving prognosis. In some embodiments, the antibody molecules of the invention are used to delay disease progression or to slow disease progression.

As used herein, "prevention" includes the inhibition of the occurrence or progression of a disease or disorder or symptoms of a particular disease or disorder. In some embodiments, subjects with a family history of cancer are candidates for preventive regimens. Generally, in the context of cancer, the term "prevention" refers to the administration of a drug prior to the onset of signs or symptoms of cancer, particularly in subjects at risk of cancer.

The term "effective amount" refers to that amount or dose of an antibody or composition of the invention which, after administration to the patient in single or multiple doses, produces the desired effect in a patient in need of treatment or prevention. An effective amount can be readily determined by the attending physician, who is skilled in the art, by considering a variety of factors such as the species of mammal; weight, age, and general health; the specific disease involved; the degree or severity of the disease; the individual The patient's response; the particular antibody administered; the mode of administration; the bioavailability profile of the administered formulation; the chosen dosing regimen; and the use of any concomitant therapy.

A "therapeutically effective amount" refers to an amount effective to achieve the desired therapeutic result, at the required dose and for the required period of time. A therapeutically effective amount of an antibody or antibody fragment or composition thereof may vary depending on factors such as the disease state, the age, sex and weight of the individual and the ability of the antibody or antibody portion to elicit a desired response in the individual. A therapeutically effective amount is also one in which any toxic or detrimental effects of the antibody or antibody fragment or composition thereof are outweighed by the therapeutically beneficial effects. A "therapeutically effective amount" preferably inhibits a measurable parameter (eg, tumor growth rate, tumor volume, etc.) by at least about 20%, more preferably at least about 40%, even more preferably at least about 50%, relative to an untreated subject, 60% or 70% and still more preferably at least about 80% or 90%. The ability of a compound to inhibit a measurable parameter (eg, cancer) can be evaluated in an animal model system predictive of efficacy in human tumors.

A "prophylactically effective amount" refers to an amount effective to achieve the desired prophylactic result, at the required dose and for the required period of time. Typically, a prophylactically effective amount will be less than a therapeutically effective amount because a prophylactic dose is administered in a subject prior to or at an earlier stage of the disease.

The term "therapeutic agent" as used herein encompasses any substance that is effective in preventing or treating tumors (eg, cancer) and infections (eg, chronic infections), including chemotherapeutic agents, cytotoxic agents, other antibodies, anti-infective agents, small molecules Drugs or immunomodulators.

The term "immunomodulator" as used herein refers to a natural or synthetic active agent or drug that inhibits or modulates an immune response. The immune response can be a humoral response or a cellular response. Immunomodulators include immune checkpoint molecular inhibitors and costimulatory molecular activators.

The term "small molecule drug" refers to low molecular weight organic compounds capable of modulating biological processes.

As used herein, the term "cytotoxic agent" refers to a substance that inhibits or prevents cell function and/or causes cell death or destruction. Examples of cytotoxic agents are found in those disclosed in WO2015/153513, WO2016/028672 or WO2015/138920.

The term "anti-infective active agent" includes any molecule that specifically inhibits or eliminates the growth of microorganisms, such as viruses, bacteria, fungi, or protozoa, eg, parasites, at the concentration and interval of administration, but is not lethal to the host. As used herein, the term anti-infective active agent includes antibiotics, antibacterial agents, antiviral agents, antifungal agents, and antiprotozoal agents. In a specific aspect, the anti-infective active agent is nontoxic to the host at the concentration and interval of administration.

The term "combination product" refers to a fixed or non-fixed combination or kit of parts for combined administration in one dosage unit form, wherein two or more therapeutic agents can be administered independently at the same time or at a certain time. Administration is administered separately at time intervals, especially when these time intervals allow the combined therapeutic agents to exhibit synergy, eg, a synergistic effect. The term "fixed combination" refers to the simultaneous administration of an antibody of the invention and a combination partner (eg, other therapeutic agent, eg, anti-PD-1 antibody or anti-PD-L1 antibody) to a patient in the form of a single entity or dose. The term "non-fixed combination" means that a LAG-3 antibody of the invention and a combination partner (eg, other therapeutic agent, eg, an anti-PD-1 antibody or an anti-PD-L1 antibody) are administered to a patient simultaneously, concurrently or sequentially as separate entities, There is no particular time limit, wherein such administration provides therapeutically effective levels of both therapeutic agents in the patient. The latter also applies to cocktail therapy, eg, the administration of three or more therapeutic agents. In a preferred embodiment, the drug combination is a non-fixed combination.

The term "combination therapy" or "combination therapy" refers to the administration of two or more therapeutic agents to treat cancer or infection as described in the present disclosure. Such administration includes co-administration of the therapeutic agents in a substantially simultaneous manner, eg, in a single capsule having a fixed ratio of active ingredients. Alternatively, such administration includes co-administration or separate administration or sequential administration of each active ingredient in multiple or separate containers (eg, tablets, capsules, powders, and liquids). Powders and/or liquids can be reconstituted or diluted to the desired dose prior to administration. In some embodiments, administering also includes administering each type of therapeutic agent at approximately the same time, or in a sequential manner at different times. In either case, the treatment regimen will provide the beneficial effect of the drug combination in the treatment of the disorders or conditions described herein.

The term "vector" as used herein when referring to a nucleic acid refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes vectors that are self-replicating nucleic acid structures as well as vectors that are incorporated into the genome of the host cell into which they have been introduced. Some vectors are capable of directing the expression of nucleic acids to which they are operably linked. Such vectors are referred to herein as "expression vectors".

The term "host cell" refers to a cell into which an exogenous polynucleotide has been introduced, including progeny of such cells. Host cells include "transformants" and "transformed cells," which include the primary transformed cell and progeny derived therefrom, regardless of the number of passages. The progeny may not be identical in nucleic acid content to the parent cell, but may contain mutations. Included herein are mutant progeny screened or selected for the same function or biological activity in the originally transformed cell. A host cell is any type of cellular system that can be used to produce the antibody molecules of the invention, including eukaryotic cells, eg, mammalian cells, insect cells, yeast cells; and prokaryotic cells, eg, E. coli cells. Host cells include cultured cells and also include transgenic animals, transgenic plants, or cells within cultured plant or animal tissue.

A "subject/patient sample" refers to a collection of cells, tissues or bodily fluids obtained from a patient or subject. The source of a tissue or cell sample can be solid tissue like from a fresh, frozen and/or preserved organ or tissue sample or biopsy or biopsy; blood or any blood component; body fluids such as cerebrospinal fluid, amniotic fluid (amniotic fluid); ), peritoneal fluid (ascites), or interstitial fluid; cells from any time of pregnancy or development of the subject. Tissue samples may contain compounds that are not naturally associated with tissue in nature, such as preservatives, anticoagulants, buffers, fixatives, nutrients, antibiotics, and the like. Examples of tumor samples herein include, but are not limited to, tumor biopsies, fine needle aspirate, bronchial lavage fluid, pleural fluid (pleural fluid), sputum, urine, surgical specimens, circulating tumor cells, serum, plasma, circulating tumor cells plasma proteins in ascites fluid, primary cell cultures or cell lines derived from tumors or exhibiting tumor-like properties, and preserved tumor samples such as formalin-fixed, paraffin-embedded tumor samples, or frozen tumors sample.

The term "package insert" is used to refer to instructions for use typically contained in commercial packaging of therapeutic products, which contain information on indications, usage, dosage, administration, combination therapy, contraindications and/or warnings related to the use of such therapeutic products .

II. Antibodies of the invention that specifically bind to LAG-3

The antibody that specifically binds to LAG-3 of the present invention comprises a heavy chain variable region and a light chain variable region, wherein:

(f) the heavy chain variable region comprises HCDR1 shown in NYGIS (SEQ ID NO: 60), or a variant of said HCDR1 with no more than 2 amino acid changes or no more than 1 amino acid change; EIYPRGGNTYYNGKFKG (SEQ ID NO: 60) NO: 61), or a variant of said HCDR2 with no more than 2 amino acid changes or no more than 1 amino acid change; and HCDR3 shown in RAFYYFGSNYYAMDY (SEQ ID NO: 62), or a variant of said HCDR3 A variant with no more than 2 amino acid changes or no more than 1 amino acid change; the light chain variable region comprises LCDR1 shown in RSSQSIVHSNGDTYLE (SEQ ID NO: 34), or no more than 2 amino acid changes of said LCDR1 or Variants with no more than 1 amino acid change; LCDR2 shown in KVSNRFS (SEQ ID NO: 35), or a variant of said LCDR2 with no more than 2 amino acid changes or no more than 1 amino acid change; and FQGSHVPWT (SEQ ID NO: 35) NO:36) LCDR3, or a variant of the LCDR3 with no more than 2 amino acid changes or no more than 1 amino acid change;

Wherein the amino acid change is an addition, deletion or substitution of an amino acid, eg, the amino acid change is a conservative amino acid substitution. The CDRs are CDRs according to the Kabat numbering scheme.

In some embodiments, the antibodies of the invention bind mammalian LAG-3, eg, human LAG-3, monkey LAG-3. In some embodiments, the LAG-3 antibodies of the invention bind to one or more extracellular domains of LAG-3.

In some embodiments, the antibodies of the invention have one or more of the following properties:

(1) Binds LAG-3 with high affinity, eg, human LAG-3 and cynomolgus monkey LAG-3, eg, the KD for binding between the anti-LAG-3 antibody or antigen-binding fragment thereof and _LAG -3 is about 10 ^-7 M to about ^10-12 M, preferably, about ^10-8 M to about ^10-12 M, as measured by the ForteBio kinetic binding assay;

(2) specifically blocking the binding of LAG-3 to FGL-1;

(3) specifically blocking the binding of LAG-3 to cell surface HLA;

(4) Keep T cells in an activated state;

(5) It has anti-tumor effect in vivo.

In one embodiment, the antibody or antigen-binding fragment of the invention that specifically binds LAG-3 comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises SYGIS (SEQ ID NO:31 HCDR1 shown in ); HCDR2 shown in EIYPRSDNTYYNGKFKG (SEQ ID NO: 32); and HCDR3 shown in RAFYYYGSNYYAMDY (SEQ ID NO: 33); the light chain variable region comprises RSSQSIVHSNGDTYLE (SEQ ID NO: 34) LCDR1 shown in KVSNRFS (SEQ ID NO: 35); LCDR3 shown in FQGSHVPWT (SEQ ID NO: 36).

In one embodiment, the antibody or antigen-binding fragment of the invention that specifically binds LAG-3 comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises NYAMS (SEQ ID NO:37 HCDR1 shown in ); HCDR2 shown in TITYGTTYTFYSDNVKG (SEQ ID NO: 38); and HCDR3 shown in GEYGSSFAY (SEQ ID NO: 39); the light chain variable region comprises KASQNVRTAVA (SEQ ID NO: 40) LCDR1 shown in LASNRHT (SEQ ID NO: 41); LCDR3 shown in LQHWNYPLT (SEQ ID NO: 42).

In one embodiment, the antibody or antigen-binding fragment of the invention that specifically binds LAG-3 comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises DYAVS (SEQ ID NO:43 HCDR1 represented by ); HCDR2 represented by VVWGDGSTNYHSALIS (SEQ ID NO: 44); and HCDR3 represented by GGGGMDY (SEQ ID NO: 45); the light chain variable region comprises RASSSVSYMH (SEQ ID NO: 46) LCDR1 shown in ATSNLAS (SEQ ID NO: 47); LCDR3 shown in QHYNTNPPTWT (SEQ ID NO: 48).

In some embodiments, the antibody or antigen-binding fragment of the invention that specifically binds LAG-3 comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises DDYMH (SEQ ID NO: 49 HCDR1 shown in ); HCDR2 shown in RIX ₁ PEDVETKYDPKFQG (SEQ ID NO: 64), preferably, wherein X ₁ is D or N; and HCDR3 shown in SFYSNYVNYFDQ (SEQ ID NO: 51);

The light chain variable region comprises LCDR1 shown by X ₂ ASENVGTYVS (SEQ ID NO: 68), preferably, wherein X ₂ is K or R; LCDR2 shown by X ₃ ASX ₄ RYT (SEQ ID NO: 69) , preferably; wherein X ₃ is G or A; X ₄ is N or T; and LCDR3 shown in GQSYSYPYT (SEQ ID NO: 54).

In a specific embodiment, the antibody or antigen-binding fragment of the invention that specifically binds LAG-3 comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises DDYMH (SEQ ID NO: HCDR1 shown in 49); HCDR2 shown in RIDPEDVETKYDPKFQG (SEQ ID NO: 50); and HCDR3 shown in SFYSNYVNYFDQ (SEQ ID NO: 51); the light chain variable region comprises KASENVGTYVS (SEQ ID NO: 52) LCDR1 shown, LCDR2 shown by GASNRYT (SEQ ID NO:53), and LCDR3 shown by GQSYSYPYT (SEQ ID NO:54).

In a specific embodiment, the antibody or antigen-binding fragment of the invention that specifically binds LAG-3 comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises DDYMH (SEQ ID NO: HCDR1 shown in 49); HCDR2 shown in RIDPEDVETKYDPKFQG (SEQ ID NO: 50), HCDR3 shown in SFYSNYVNYFDQ (SEQ ID NO: 51); the light chain variable region comprises KASENVGTYVS (SEQ ID NO: 52). LCDR1 shown by AASNRYT (SEQ ID NO: 66), LCDR2 shown by GQSYSYPYT (SEQ ID NO: 54), and LCDR3 shown by GQSYSYPYT (SEQ ID NO: 54).

In a specific embodiment, the antibody or antigen-binding fragment of the invention that specifically binds LAG-3 comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises DDYMH (SEQ ID NO: HCDR1 shown in 49); HCDR2 shown in RIDPEDVETKYDPKFQG (SEQ ID NO: 50), HCDR3 shown in SFYSNYVNYFDQ (SEQ ID NO: 51); the light chain variable region comprises RASENVGTYVS (SEQ ID NO: 65). LCDR1 shown by GASNRYT (SEQ ID NO: 53), LCDR2 shown by GQSYSYPYT (SEQ ID NO: 54), and LCDR3 shown by GQSYSYPYT (SEQ ID NO: 54).

In a specific embodiment, the antibody or antigen-binding fragment of the invention that specifically binds LAG-3 comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises DDYMH (SEQ ID NO: HCDR1 shown in 49); HCDR2 shown in RIDPEDVETKYDPKFQG (SEQ ID NO: 50), HCDR3 shown in SFYSNYVNYFDQ (SEQ ID NO: 51); the light chain variable region comprises KASENVGTYVS (SEQ ID NO: 52). LCDR1 shown by GASTRYT (SEQ ID NO:67), LCDR2 shown by GQSYSYPYT (SEQ ID NO:54), and LCDR3 shown by GQSYSYPYT (SEQ ID NO:54).

In a specific embodiment, the antibody or antigen-binding fragment of the invention that specifically binds LAG-3 comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises DDYMH (SEQ ID NO: HCDR1 shown in 49); HCDR2 shown in RINPEDVETKYDPKFQG (SEQ ID NO: 63), HCDR3 shown in SFYSNYVNYFDQ (SEQ ID NO: 51); the light chain variable region comprises KASENVGTYVS (SEQ ID NO: 52). LCDR1 shown by GASNRYT (SEQ ID NO: 53), LCDR2 shown by GQSYSYPYT (SEQ ID NO: 54), and LCDR3 shown by GQSYSYPYT (SEQ ID NO: 54).

In a specific embodiment, the antibody or antigen-binding fragment of the invention that specifically binds LAG-3 comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises DDYMH (SEQ ID NO: HCDR1 shown in 49); HCDR2 shown in RINPEDVETKYDPKFQG (SEQ ID NO: 63), HCDR3 shown in SFYSNYVNYFDQ (SEQ ID NO: 51); the light chain variable region comprises KASENVGTYVS (SEQ ID NO: 52). LCDR1 shown by AASNRYT (SEQ ID NO: 66), LCDR2 shown by GQSYSYPYT (SEQ ID NO: 54), and LCDR3 shown by GQSYSYPYT (SEQ ID NO: 54).

In a specific embodiment, the antibody or antigen-binding fragment of the invention that specifically binds LAG-3 comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises DDYMH (SEQ ID NO: HCDR1 shown in 49); HCDR2 shown in RINPEDVETKYDPKFQG (SEQ ID NO: 63), HCDR3 shown in SFYSNYVNYFDQ (SEQ ID NO: 51); the light chain variable region comprises KASENVGTYVS (SEQ ID NO: 52). LCDR1 shown by GASTRYT (SEQ ID NO:67), LCDR2 shown by GQSYSYPYT (SEQ ID NO:54), and LCDR3 shown by GQSYSYPYT (SEQ ID NO:54).

In one embodiment, the antibody or antigen-binding fragment of the invention that specifically binds LAG-3 comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises DDYMH (SEQ ID NO:49 HCDR1 shown in ); HCDR2 shown in WIDPENGETEYASKFQG (SEQ ID NO: 55); and HCDR3 shown in FDY (SEQ ID NO: 56); the light chain variable region comprises KSSQSLLDSDGKTYLN (SEQ ID NO: 57) LCDR1 shown in LVSKLDF (SEQ ID NO: 58); LCDR2 shown in WQGTHFPQT (SEQ ID NO: 59); or

In one embodiment, the antibody or antigen-binding fragment of the invention that specifically binds LAG-3 comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises NYGIS (SEQ ID NO:60 HCDR1 represented by ); HCDR2 represented by EIYPRGGNTYYNGKFKG (SEQ ID NO:61); and HCDR3 represented by RAFYYFGSNYYAMDY (SEQ ID NO:62); the light chain variable region comprises the LCDR1 shown in KVSNRFS (SEQ ID NO: 35); LCDR3 shown in FQGSHVPWT (SEQ ID NO: 36).

In some embodiments, the antibody or antigen-binding fragment of the invention that specifically binds LAG-3 is a humanized LAG-3 antibody or antigen-binding fragment thereof, comprising N-FR1-CDR1- Heavy chain variable region and light chain variable region of FR2-CDR2-FR3-CDR3-FR4-C, wherein the 6 CDRs of each antibody are as defined above, and the FRs are FRs derived from human antibodies. For example, in some embodiments, the heavy chain variable region of a humanized LAG-3 antibody or antigen-binding fragment thereof comprises the amino acid sequences QVQLVQSGAEVKKPGASVKVSCKASGYTFT (SEQ ID NO:80), EVQLVQSGAEVKKPGASVKVSCKASGYTFT (SEQ ID NO:81), EVQLVQSGAEVKKPGASVKVSCKASGYTIK (SEQ ID NO:81) ID NO:82), FR1 of EVQLVQSGAEVKKPGASVKVSCKASGVNFT (SEQ ID NO:83) or EVQLVQSGAEVKKPGASVKVSCKASGVNIK (SEQ ID NO:84), FR2 containing the amino acid sequence WVRQAPGQGLEWMG (SEQ ID NO:85) or WVRQAPGQGLEWIG (SEQ ID NO:86), FR3 of amino acid sequence RVTTRDTSTSTVYMELSSLRSEDTAVYYCAR (SEQ ID NO:87), RVTITRDTSTSTVYMELSSLRSEDTAVYYCAR (SEQ ID NO:88) or RVTITADSTSTAYMELSSLRSEDTAVYYCAR (SEQ ID NO:89) and FR4 containing amino acid sequence WGQGTLVTVSS (SEQ ID NO:90); light chain variable region FR1 containing the amino acid sequence DIQMTQSPSSLSASVGDRVTITC (SEQ ID NO:70) or EIVMTQSPATLSLSPGERATLSC (SEQ ID NO:71), containing the amino acid sequence WYQQKPGKAPKLLIY (SEQ ID NO:72), WFQQKPGKAPKLLIY (SEQ ID NO:73), WYQQKPGQAPRLLIY (SEQ ID NO:71) 74) or FR2 of WFQQKPGQAPRLLIY (SEQ ID NO:75), FR3 containing the amino acid sequence GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC (SEQ ID NO:76), GIPARFSGSGSGTDFTLTISSLQPEDFAVYYC (SEQ ID NO:77) or GIPDRFSGSGSGTDFTLTISSLQPEDFAVYYC (SEQ ID NO:78) and FR3 containing the amino acid sequence FGQGTKLEIK FR4 of (SEQ ID NO:79).

In one embodiment, the antibody or antigen-binding fragment of the invention that specifically binds LAG-3 is a humanized LAG-3 antibody or antigen-binding fragment thereof, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain The chain variable region comprises four framework regions from N-terminal to C-terminal, namely FR1, FR2, FR3 and FR4, and contains FR1 of amino acid sequence SEQ ID NO:80, FR2 containing amino acid sequence of SEQ ID NO:85, and FR2 containing amino acid sequence SEQ ID NO:85. FR3 of amino acid sequence SEQ ID NO: 87 and FR4 containing amino acid sequence SEQ ID NO: 90; wherein the light chain variable region comprises four framework regions from the N-terminus to the C-terminus, namely FR1, FR2, FR3 and FR4, and FR1 containing the amino acid sequence SEQ ID NO:70, FR2 containing the amino acid sequence SEQ ID NO:72, FR3 containing the amino acid sequence SEQ ID NO:76, and FR4 containing the amino acid sequence SEQ ID NO:79.

In one embodiment, the antibody or antigen-binding fragment of the invention that specifically binds LAG-3 is a humanized LAG-3 antibody or antigen-binding fragment thereof, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain The chain variable region comprises four framework regions from N-terminal to C-terminal, namely FR1, FR2, FR3 and FR4, and contains FR1 of amino acid sequence SEQ ID NO:80, FR2 containing amino acid sequence of SEQ ID NO:85, and FR2 containing amino acid sequence SEQ ID NO:85. FR3 of amino acid sequence SEQ ID NO: 87 and FR4 containing amino acid sequence SEQ ID NO: 90; wherein the light chain variable region comprises four framework regions from the N-terminus to the C-terminus, namely FR1, FR2, FR3 and FR4, and FR1 containing the amino acid sequence SEQ ID NO:71, FR2 containing the amino acid sequence SEQ ID NO:74, FR3 containing the amino acid sequence SEQ ID NO:77, and FR4 containing the amino acid sequence SEQ ID NO:79.

In one embodiment, the antibody or antigen-binding fragment of the invention that specifically binds LAG-3 is a humanized LAG-3 antibody or antigen-binding fragment thereof, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain The chain variable region comprises four framework regions from N-terminal to C-terminal, namely FR1, FR2, FR3 and FR4, and contains FR1 of amino acid sequence SEQ ID NO: 81, FR2 containing amino acid sequence of SEQ ID NO: 86, containing FR3 of amino acid sequence SEQ ID NO: 87 and FR4 containing amino acid sequence SEQ ID NO: 90; wherein the light chain variable region comprises four framework regions from the N-terminus to the C-terminus, namely FR1, FR2, FR3 and FR4, and FR1 containing the amino acid sequence SEQ ID NO:70, FR2 containing the amino acid sequence SEQ ID NO:73, FR3 containing the amino acid sequence SEQ ID NO:76, and FR4 containing the amino acid sequence SEQ ID NO:79.

In one embodiment, the antibody or antigen-binding fragment of the invention that specifically binds LAG-3 is a humanized LAG-3 antibody or antigen-binding fragment thereof, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain The chain variable region comprises four framework regions from N-terminal to C-terminal, namely FR1, FR2, FR3 and FR4, and contains FR1 of amino acid sequence SEQ ID NO: 81, FR2 containing amino acid sequence of SEQ ID NO: 86, containing FR3 of amino acid sequence SEQ ID NO: 87 and FR4 containing amino acid sequence SEQ ID NO: 90; wherein the light chain variable region comprises four framework regions from the N-terminus to the C-terminus, namely FR1, FR2, FR3 and FR4, and FR1 containing the amino acid sequence SEQ ID NO:70, FR2 containing the amino acid sequence SEQ ID NO:72, FR3 containing the amino acid sequence SEQ ID NO:76, and FR4 containing the amino acid sequence SEQ ID NO:79.

In one embodiment, the antibody or antigen-binding fragment of the invention that specifically binds LAG-3 is a humanized LAG-3 antibody or antigen-binding fragment thereof, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain The chain variable region comprises four framework regions from N-terminal to C-terminal, namely FR1, FR2, FR3 and FR4, and contains FR1 of amino acid sequence SEQ ID NO:81, FR2 containing amino acid sequence of SEQ ID NO:85, and FR2 containing amino acid sequence SEQ ID NO:85. FR3 of amino acid sequence SEQ ID NO: 88 and FR4 containing amino acid sequence SEQ ID NO: 90; wherein the light chain variable region comprises four framework regions from the N-terminus to the C-terminus, namely FR1, FR2, FR3 and FR4, and FR1 containing the amino acid sequence SEQ ID NO:70, FR2 containing the amino acid sequence SEQ ID NO:73, FR3 containing the amino acid sequence SEQ ID NO:76, and FR4 containing the amino acid sequence SEQ ID NO:79.

In one embodiment, the antibody or antigen-binding fragment of the invention that specifically binds LAG-3 is a humanized LAG-3 antibody or antigen-binding fragment thereof, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain The chain variable region comprises four framework regions from N-terminal to C-terminal, namely FR1, FR2, FR3 and FR4, and contains FR1 of amino acid sequence SEQ ID NO:81, FR2 containing amino acid sequence of SEQ ID NO:85, and FR2 containing amino acid sequence SEQ ID NO:85. FR3 of amino acid sequence SEQ ID NO: 88 and FR4 containing amino acid sequence SEQ ID NO: 90; wherein the light chain variable region comprises four framework regions from the N-terminus to the C-terminus, namely FR1, FR2, FR3 and FR4, and FR1 containing the amino acid sequence SEQ ID NO:70, FR2 containing the amino acid sequence SEQ ID NO:72, FR3 containing the amino acid sequence SEQ ID NO:76, and FR4 containing the amino acid sequence SEQ ID NO:79.

In one embodiment, the antibody or antigen-binding fragment of the invention that specifically binds LAG-3 is a humanized LAG-3 antibody or antigen-binding fragment thereof, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain The chain variable region comprises four framework regions from N-terminal to C-terminal, namely FR1, FR2, FR3 and FR4, and contains FR1 of amino acid sequence SEQ ID NO: 81, FR2 containing amino acid sequence of SEQ ID NO: 86, containing FR3 of amino acid sequence SEQ ID NO: 87 and FR4 containing amino acid sequence SEQ ID NO: 90; wherein the light chain variable region comprises four framework regions from the N-terminus to the C-terminus, namely FR1, FR2, FR3 and FR4, and FR1 containing the amino acid sequence SEQ ID NO:71, FR2 containing the amino acid sequence SEQ ID NO:74, FR3 containing the amino acid sequence SEQ ID NO:77, and FR4 containing the amino acid sequence SEQ ID NO:79.

In one embodiment, the antibody or antigen-binding fragment of the invention that specifically binds LAG-3 is a humanized LAG-3 antibody or antigen-binding fragment thereof, comprising a heavy chain variable region and a light chain variable region, wherein the heavy The chain variable region comprises four framework regions from N-terminal to C-terminal, namely FR1, FR2, FR3 and FR4, and contains FR1 of amino acid sequence SEQ ID NO:81, FR2 containing amino acid sequence of SEQ ID NO:85, and FR2 containing amino acid sequence SEQ ID NO:85. FR3 of amino acid sequence SEQ ID NO: 88 and FR4 containing amino acid sequence SEQ ID NO: 90; wherein the light chain variable region comprises four framework regions from the N-terminus to the C-terminus, namely FR1, FR2, FR3 and FR4, and FR1 containing the amino acid sequence SEQ ID NO:71, FR2 containing the amino acid sequence SEQ ID NO:74, FR3 containing the amino acid sequence SEQ ID NO:77, and FR4 containing the amino acid sequence SEQ ID NO:79.

In one embodiment, the antibody or antigen-binding fragment of the invention that specifically binds LAG-3 is a humanized LAG-3 antibody or antigen-binding fragment thereof, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain The chain variable region comprises four framework regions from N-terminal to C-terminal, namely FR1, FR2, FR3 and FR4, and contains FR1 of amino acid sequence SEQ ID NO:81, FR2 containing amino acid sequence of SEQ ID NO:85, and FR2 containing amino acid sequence SEQ ID NO:85. FR3 of amino acid sequence SEQ ID NO: 87 and FR4 containing amino acid sequence SEQ ID NO: 90; wherein the light chain variable region comprises four framework regions from the N-terminus to the C-terminus, namely FR1, FR2, FR3 and FR4, and FR1 containing the amino acid sequence SEQ ID NO:70, FR2 containing the amino acid sequence SEQ ID NO:73, FR3 containing the amino acid sequence SEQ ID NO:76, and FR4 containing the amino acid sequence SEQ ID NO:79.

In one embodiment, the antibody or antigen-binding fragment of the invention that specifically binds LAG-3 is a humanized LAG-3 antibody or antigen-binding fragment thereof, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain The chain variable region comprises four framework regions from N-terminal to C-terminal, namely FR1, FR2, FR3 and FR4, and contains FR1 of amino acid sequence SEQ ID NO:81, FR2 containing amino acid sequence of SEQ ID NO:85, and FR2 containing amino acid sequence SEQ ID NO:85. FR3 of amino acid sequence SEQ ID NO: 87 and FR4 containing amino acid sequence SEQ ID NO: 90; wherein the light chain variable region comprises four framework regions from the N-terminus to the C-terminus, namely FR1, FR2, FR3 and FR4, and FR1 containing the amino acid sequence SEQ ID NO:70, FR2 containing the amino acid sequence SEQ ID NO:72, FR3 containing the amino acid sequence SEQ ID NO:76, and FR4 containing the amino acid sequence SEQ ID NO:79.

In one embodiment, the antibody or antigen-binding fragment of the invention that specifically binds LAG-3 is a humanized LAG-3 antibody or antigen-binding fragment thereof, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain The chain variable region comprises four framework regions from N-terminal to C-terminal, namely FR1, FR2, FR3 and FR4, and contains FR1 of amino acid sequence SEQ ID NO:81, FR2 containing amino acid sequence of SEQ ID NO:85, and FR2 containing amino acid sequence SEQ ID NO:85. FR3 of amino acid sequence SEQ ID NO: 87 and FR4 containing amino acid sequence SEQ ID NO: 90; wherein the light chain variable region comprises four framework regions from the N-terminus to the C-terminus, namely FR1, FR2, FR3 and FR4, and FR1 containing the amino acid sequence SEQ ID NO:71, FR2 containing the amino acid sequence SEQ ID NO:74, FR3 containing the amino acid sequence SEQ ID NO:77, and FR4 containing the amino acid sequence SEQ ID NO:79.

In one embodiment, the antibody or antigen-binding fragment of the invention that specifically binds LAG-3 is a humanized LAG-3 antibody or antigen-binding fragment thereof, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain The chain variable region comprises four framework regions from N-terminal to C-terminal, namely FR1, FR2, FR3 and FR4, and contains FR1 of amino acid sequence SEQ ID NO:81, FR2 containing amino acid sequence of SEQ ID NO:85, and FR2 containing amino acid sequence SEQ ID NO:85. FR3 of amino acid sequence SEQ ID NO: 87 and FR4 containing amino acid sequence SEQ ID NO: 90; wherein the light chain variable region comprises four framework regions from the N-terminus to the C-terminus, namely FR1, FR2, FR3 and FR4, and FR1 containing the amino acid sequence SEQ ID NO:71, FR2 containing the amino acid sequence SEQ ID NO:75, FR3 containing the amino acid sequence SEQ ID NO:78, and FR4 containing the amino acid sequence SEQ ID NO:79.

In one embodiment, the antibody or antigen-binding fragment of the invention that specifically binds LAG-3 is a humanized LAG-3 antibody or antigen-binding fragment thereof, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain The chain variable region comprises four framework regions from the N-terminus to the C-terminus, namely FR1, FR2, FR3 and FR4, and contains FR1 of the amino acid sequence SEQ ID NO:82, FR2 of the amino acid sequence of SEQ ID NO:85, and FR2 of the amino acid sequence of SEQ ID NO:85. FR3 of amino acid sequence SEQ ID NO: 87 and FR4 containing amino acid sequence SEQ ID NO: 90; wherein the light chain variable region comprises four framework regions from the N-terminus to the C-terminus, namely FR1, FR2, FR3 and FR4, and FR1 containing the amino acid sequence SEQ ID NO:70, FR2 containing the amino acid sequence SEQ ID NO:73, FR3 containing the amino acid sequence SEQ ID NO:76, and FR4 containing the amino acid sequence SEQ ID NO:79.

In one embodiment, the antibody or antigen-binding fragment of the invention that specifically binds LAG-3 is a humanized LAG-3 antibody or antigen-binding fragment thereof, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain The chain variable region comprises four framework regions from the N-terminus to the C-terminus, namely FR1, FR2, FR3 and FR4, and contains FR1 of the amino acid sequence SEQ ID NO:82, FR2 of the amino acid sequence of SEQ ID NO:85, and FR2 of the amino acid sequence of SEQ ID NO:85. FR3 of amino acid sequence SEQ ID NO: 87 and FR4 containing amino acid sequence SEQ ID NO: 90; wherein the light chain variable region comprises four framework regions from the N-terminus to the C-terminus, namely FR1, FR2, FR3 and FR4, and FR1 containing the amino acid sequence SEQ ID NO:70, FR2 containing the amino acid sequence SEQ ID NO:72, FR3 containing the amino acid sequence SEQ ID NO:76, and FR4 containing the amino acid sequence SEQ ID NO:79.

In one embodiment, the antibody or antigen-binding fragment of the invention that specifically binds LAG-3 is a humanized LAG-3 antibody or antigen-binding fragment thereof, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain The chain variable region comprises four framework regions from the N-terminus to the C-terminus, namely FR1, FR2, FR3 and FR4, and contains FR1 of the amino acid sequence SEQ ID NO:82, FR2 of the amino acid sequence of SEQ ID NO:85, and FR2 of the amino acid sequence of SEQ ID NO:85. FR3 of amino acid sequence SEQ ID NO: 87 and FR4 containing amino acid sequence SEQ ID NO: 90; wherein the light chain variable region comprises four framework regions from the N-terminus to the C-terminus, namely FR1, FR2, FR3 and FR4, and FR1 containing the amino acid sequence SEQ ID NO:71, FR2 containing the amino acid sequence SEQ ID NO:74, FR3 containing the amino acid sequence SEQ ID NO:77, and FR4 containing the amino acid sequence SEQ ID NO:79.

In one embodiment, the antibody or antigen-binding fragment of the invention that specifically binds LAG-3 is a humanized LAG-3 antibody or antigen-binding fragment thereof, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain The chain variable region comprises four framework regions from the N-terminus to the C-terminus, namely FR1, FR2, FR3 and FR4, and contains FR1 of the amino acid sequence SEQ ID NO:82, FR2 of the amino acid sequence of SEQ ID NO:85, and FR2 of the amino acid sequence of SEQ ID NO:85. FR3 of amino acid sequence SEQ ID NO: 87 and FR4 containing amino acid sequence SEQ ID NO: 90; wherein the light chain variable region comprises four framework regions from the N-terminus to the C-terminus, namely FR1, FR2, FR3 and FR4, and FR1 containing the amino acid sequence SEQ ID NO:71, FR2 containing the amino acid sequence SEQ ID NO:75, FR3 containing the amino acid sequence SEQ ID NO:78, and FR4 containing the amino acid sequence SEQ ID NO:79.

In one embodiment, the antibody or antigen-binding fragment of the invention that specifically binds LAG-3 is a humanized LAG-3 antibody or antigen-binding fragment thereof, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain The chain variable region comprises four framework regions from the N-terminus to the C-terminus, namely FR1, FR2, FR3 and FR4, and contains the FR1 of the amino acid sequence of SEQ ID NO:83, the FR2 of the amino acid sequence of SEQ ID NO:85, the FR3 of amino acid sequence SEQ ID NO: 87 and FR4 containing amino acid sequence SEQ ID NO: 90; wherein the light chain variable region comprises four framework regions from the N-terminus to the C-terminus, namely FR1, FR2, FR3 and FR4, and FR1 containing the amino acid sequence SEQ ID NO:70, FR2 containing the amino acid sequence SEQ ID NO:73, FR3 containing the amino acid sequence SEQ ID NO:76, and FR4 containing the amino acid sequence SEQ ID NO:79.

In one embodiment, the antibody or antigen-binding fragment of the invention that specifically binds LAG-3 is a humanized LAG-3 antibody or antigen-binding fragment thereof, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain The chain variable region comprises four framework regions from the N-terminus to the C-terminus, namely FR1, FR2, FR3 and FR4, and contains the FR1 of the amino acid sequence of SEQ ID NO:83, the FR2 of the amino acid sequence of SEQ ID NO:85, the The FR3 of the amino acid sequence SEQ ID NO:87 and the FR4 containing the amino acid sequence of SEQ ID NO:90; wherein the light chain variable region comprises four framework regions from the N-terminus to the C-terminus, namely FR1, FR2, FR3 and FR4, and FR1 containing the amino acid sequence SEQ ID NO:70, FR2 containing the amino acid sequence SEQ ID NO:72, FR3 containing the amino acid sequence SEQ ID NO:76, and FR4 containing the amino acid sequence SEQ ID NO:79.

In one embodiment, the antibody or antigen-binding fragment of the invention that specifically binds LAG-3 is a humanized LAG-3 antibody or antigen-binding fragment thereof, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain The chain variable region comprises four framework regions from the N-terminus to the C-terminus, namely FR1, FR2, FR3 and FR4, and contains the FR1 of the amino acid sequence of SEQ ID NO:83, the FR2 of the amino acid sequence of SEQ ID NO:85, the FR3 of amino acid sequence SEQ ID NO: 87 and FR4 containing amino acid sequence SEQ ID NO: 90; wherein the light chain variable region comprises four framework regions from the N-terminus to the C-terminus, namely FR1, FR2, FR3 and FR4, and FR1 containing the amino acid sequence SEQ ID NO:71, FR2 containing the amino acid sequence SEQ ID NO:74, FR3 containing the amino acid sequence SEQ ID NO:77, and FR4 containing the amino acid sequence SEQ ID NO:79.

In one embodiment, the antibody or antigen-binding fragment of the invention that specifically binds LAG-3 is a humanized LAG-3 antibody or antigen-binding fragment thereof, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain The chain variable region comprises four framework regions from the N-terminus to the C-terminus, namely FR1, FR2, FR3 and FR4, and contains the FR1 of the amino acid sequence of SEQ ID NO:83, the FR2 of the amino acid sequence of SEQ ID NO:85, the FR3 of amino acid sequence SEQ ID NO: 87 and FR4 containing amino acid sequence SEQ ID NO: 90; wherein the light chain variable region comprises four framework regions from the N-terminus to the C-terminus, namely FR1, FR2, FR3 and FR4, and FR1 containing the amino acid sequence SEQ ID NO:71, FR2 containing the amino acid sequence SEQ ID NO:75, FR3 containing the amino acid sequence SEQ ID NO:78, and FR4 containing the amino acid sequence SEQ ID NO:79.

In one embodiment, the antibody or antigen-binding fragment of the invention that specifically binds LAG-3 is a humanized LAG-3 antibody or antigen-binding fragment thereof, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain The chain variable region comprises four framework regions from the N-terminus to the C-terminus, namely FR1, FR2, FR3 and FR4, and contains the FR1 of the amino acid sequence of SEQ ID NO:84, the FR2 of the amino acid sequence of SEQ ID NO:86, the FR3 of amino acid sequence SEQ ID NO: 89 and FR4 containing amino acid sequence SEQ ID NO: 90; wherein the light chain variable region comprises four framework regions from N-terminus to C-terminus, namely FR1, FR2, FR3 and FR4, and FR1 containing the amino acid sequence SEQ ID NO:70, FR2 containing the amino acid sequence SEQ ID NO:73, FR3 containing the amino acid sequence SEQ ID NO:76, and FR4 containing the amino acid sequence SEQ ID NO:79.

In one embodiment, the antibody or antigen-binding fragment of the invention that specifically binds LAG-3 is a humanized LAG-3 antibody or antigen-binding fragment thereof, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain The chain variable region comprises four framework regions from the N-terminus to the C-terminus, namely FR1, FR2, FR3 and FR4, and contains the FR1 of the amino acid sequence of SEQ ID NO:84, the FR2 of the amino acid sequence of SEQ ID NO:86, the FR3 of amino acid sequence SEQ ID NO: 89 and FR4 containing amino acid sequence SEQ ID NO: 90; wherein the light chain variable region comprises four framework regions from N-terminus to C-terminus, namely FR1, FR2, FR3 and FR4, and FR1 containing the amino acid sequence SEQ ID NO:70, FR2 containing the amino acid sequence SEQ ID NO:72, FR3 containing the amino acid sequence SEQ ID NO:76, and FR4 containing the amino acid sequence SEQ ID NO:79.

In one embodiment, the antibody or antigen-binding fragment of the invention that specifically binds LAG-3 is a humanized LAG-3 antibody or antigen-binding fragment thereof, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain The chain variable region comprises four framework regions from the N-terminus to the C-terminus, namely FR1, FR2, FR3 and FR4, and contains the FR1 of the amino acid sequence of SEQ ID NO:84, the FR2 of the amino acid sequence of SEQ ID NO:86, the FR3 of amino acid sequence SEQ ID NO: 89 and FR4 containing amino acid sequence SEQ ID NO: 90; wherein the light chain variable region comprises four framework regions from N-terminus to C-terminus, namely FR1, FR2, FR3 and FR4, and FR1 containing the amino acid sequence SEQ ID NO:71, FR2 containing the amino acid sequence SEQ ID NO:75, FR3 containing the amino acid sequence SEQ ID NO:76, and FR4 containing the amino acid sequence SEQ ID NO:79.

In some embodiments, an antibody or antigen-binding fragment of the invention that specifically binds LAG-3 comprises or consists of a heavy chain variable region and a light chain variable region of the following sequences:

(i) any paired heavy chain variable region selected from the group consisting of SEQ ID NOs: 1/2, 3/4, 5/6, 7/8, 9/10, 11/12, 15/16 and 15/17 sequence/light chain variable region sequence;

(ii) variable with any paired heavy chain selected from the group consisting of SEQ ID NOs: 1/2, 3/4, 5/6, 7/8, 9/10, 11/12, 15/16 and 15/17 A sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to any of the region sequences/light chain variable region sequences ;or

(iii) variable with any paired heavy chain selected from the group consisting of SEQ ID NOs: 1/2, 3/4, 5/6, 7/8, 9/10, 11/12, 15/16 and 15/17 1 or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2, 1) amino acid changes (preferably amino acids) substitutions, more preferably amino acid conservative substitutions) of the amino acid sequence or consisting of, preferably, the amino acid changes do not occur in the CDR regions.

In some embodiments, the antibodies of the invention have an Fc region, eg, an Fc region from an IgG, eg, IgGl, IgG2, IgG3, or IgG4. In some embodiments, the Fc region is from IgG4. In some embodiments, the Fc region is from human IgG4.

In some embodiments of the invention, the amino acid changes described herein include amino acid substitutions, insertions or deletions. Preferably, the amino acid changes described herein are amino acid substitutions, preferably conservative substitutions.

In preferred embodiments, the amino acid changes described herein occur in regions outside the CDRs (eg, in FRs). More preferably, the amino acid changes described in the present invention occur in regions outside the heavy chain variable region and the light chain variable region. In some embodiments, the substitutions are conservative substitutions. Conservative substitution refers to the substitution of one amino acid by another amino acid within the same class, e.g., substitution of an acidic amino acid by another acidic amino acid, substitution of a basic amino acid by another basic amino acid, or substitution of a neutral amino acid by another neutral amino acid replace. Exemplary substitutions are shown in Table 1 below:

Table 1

原始残基original residue	示例性取代Exemplary substitution	优选的取代Preferred substitution
Ala(A)Ala(A)	Val；Leu；IleVal; Leu; Ile	ValVal
Arg(R)Arg(R)	Lys；Gln；AsnLys; Gln; Asn	LysLys
Asn(N)Asn(N)	Gln；His；Asp、Lys；ArgGln; His; Asp, Lys; Arg	GlnGln
Asp(D)Asp(D)	Glu；AsnGlu; Asn	GluGlu
Cys(C)Cys(C)	Ser；AlaSer; Ala	SerSer
Gln(Q)Gln(Q)	Asn；GluAsn;Glu	AsnAsn
Glu(E)Glu(E)	Asp；GlnAsp;Gln	AspAsp
Gly(G)Gly(G)	AlaAla	AlaAla
His(H)His(H)	Asn；Gln；Lys；ArgAsn; Gln; Lys; Arg	ArgArg
Ile(I)Ile(I)	Leu，Val；Met；Ala；Phe；正亮氨酸Leu, Val; Met; Ala; Phe; Norleucine	LeuLeu
Leu(L)Leu(L)	正亮氨酸；Ile；Val；Met；Ala；PheNorleucine; Ile; Val; Met; Ala; Phe	IleIle
Lys(K)Lys(K)	Arg；Gln；AsnArg; Gln; Asn	ArgArg
Met(M)Met(M)	Leu；Phe；IleLeu; Phe; Ile	LeuLeu

原始残基original residue	示例性取代Exemplary substitution	优选的取代Preferred substitution
Phe(F)Phe(F)	Trp；Leu；Val；Ile；Ala；TyrTrp; Leu; Val; Ile; Ala; Tyr	TyrTyr
Pro(P)Pro(P)	AlaAla	AlaAla
Ser(S)Ser(S)	ThrThr	ThrThr
Thr(T)Thr(T)	Val；SerVal; Ser	SerSer
Trp(W)Trp(W)	Tyr；PheTyr; Phe	TyrTyr
Tyr(Y)Tyr(Y)	Trp；Phe；Thr；SerTrp; Phe; Thr; Ser	PhePhe
Val(V)Val(V)	Ile；Leu；Met；Phe；Ala；正亮氨酸Ile; Leu; Met; Phe; Ala; Norleucine	LeuLeu

In certain embodiments, the LAG-3 antibodies provided herein are altered to increase or decrease the degree of glycosylation thereof. Glycosylation sites are added or deleted to the LAG-3 antibody by altering the amino acid sequence to create or remove one or more glycosylation sites. When the LAG-3 antibody comprises an Fc region, the carbohydrate attached to the Fc region can be altered. In some applications, modifications to remove unwanted glycosylation sites are advantageous, such as removal of fucose moieties to enhance antibody-dependent cellular cytotoxicity (ADCC) function. In other applications, galactosylation modifications can be made to modulate complement-dependent cytotoxicity (CDC). In certain embodiments, one or more amino acid modifications can be introduced into the Fc region of the LAG-3 antibodies provided herein, thereby generating Fc region variants, in order to enhance, for example, the treatment of cancer or infectious diseases by the antibodies of the invention effectiveness.

III. Nucleic Acids of the Invention and Host Cells Containing the Same

In one aspect, the present invention provides nucleic acids encoding any of the above LAG-3 antibodies, or antigen-binding fragments thereof, or any chain thereof. In one embodiment, a vector comprising the nucleic acid is provided. In one embodiment, the vector is an expression vector. In one embodiment, a host cell comprising the nucleic acid or the vector is provided. In one embodiment, the host cell is eukaryotic. In another embodiment, the host cell is selected from yeast cells, mammalian cells (eg, CHO cells or 293 cells) or other cells suitable for the production of antibodies or antigen-binding fragments thereof. In another embodiment, the host cell is prokaryotic.

For example, the nucleic acid of the present invention comprises a nucleic acid encoding an amino acid sequence selected from any one of SEQ ID NOs: 1-12, 15-28, or a nucleic acid encoding an amino acid sequence selected from any one of SEQ ID NOs: 1-12, 15-28 A stated amino acid sequence has an amino acid sequence of at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity.

The present invention also encompasses nucleic acids that hybridize under stringent conditions to nucleic acids that hybridize under stringent conditions or that encode polypeptide sequences having one or more amino acid substitutions (eg, conservative substitutions), deletions or insertions compared to nucleic acids comprising encoding selected A nucleic acid comprising a nucleic acid sequence selected from the amino acid sequence shown in any one of SEQ ID NOs: 1-12, 15-28; The amino acid sequence has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity of the nucleic acid sequence of the nucleic acid sequence of the amino acid sequence.

In one embodiment, one or more vectors are provided comprising the nucleic acid. In one embodiment, the vector is an expression vector, such as a eukaryotic expression vector. Vectors include, but are not limited to, viruses, plasmids, cosmids, lambda phage, or yeast artificial chromosomes (YACs). In one embodiment, the vector is a pCDNA3.1 vector.

Once the expression vector or DNA sequence for expression has been prepared, the expression vector can be transfected or introduced into a suitable host cell. Various techniques can be used to achieve this, eg, protoplast fusion, calcium phosphate precipitation, electroporation, transduction of retroviruses, viral transfection, biolistic, lipid-based transfection, or other conventional techniques. In the case of protoplast fusion, cells are grown in culture medium and screened for appropriate activity. Methods and conditions for culturing the transfected cells produced and for recovering the antibody molecules produced are known to those skilled in the art and can be based on methods known in this specification and in the prior art, depending on the particular expression vector used and Mammalian host cell modification or optimization.

Additionally, cells that have stably incorporated DNA into their chromosomes can be selected by introducing one or more markers that allow selection of transfected host cells. Markers can, for example, provide prototrophy, biocidal resistance (eg, antibiotics), or heavy metal (eg, copper) resistance, etc. to an auxotrophic host. The selectable marker gene can be introduced into the same cell by direct ligation to the DNA sequence to be expressed or by co-transformation. Additional elements may also be required for optimal mRNA synthesis. These elements can include splicing signals, as well as transcriptional promoters, enhancers, and termination signals.

In one embodiment, host cells comprising the polynucleotides of the present invention are provided. In some embodiments, host cells comprising the expression vectors of the present invention are provided. In some embodiments, the host cell is selected from yeast cells, mammalian cells, or other cells suitable for producing antibodies. Suitable host cells include prokaryotic microorganisms such as E. coli. Host cells can also be eukaryotic microorganisms such as filamentous fungi or yeast, or various eukaryotic cells such as insect cells and the like. Vertebrate cells can also be used as hosts. For example, mammalian cell lines engineered for growth in suspension can be used. Examples of useful mammalian host cell lines include SV40 transformed monkey kidney CV1 line (COS-7); human embryonic kidney lines (HEK 293 or 293F cells), 293 cells, baby hamster kidney cells (BHK), monkey kidney cells ( CV1), African green monkey kidney cells (VERO-76), human cervical cancer cells (HELA), canine kidney cells (MDCK), Buffalo rat liver cells (BRL 3A), human lung cells (W138), human liver cells (Hep G2), Chinese hamster ovary cells (CHO cells), CHOS cells, NSO cells, myeloma cell lines such as Y0, NSO, P3X63 and Sp2/0, etc. For a review of suitable mammalian host cell lines for protein production see, e.g., Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (B.K.C. Lo, ed., Humana Press, Totowa, NJ), pp. 255-268 (2003). In a preferred embodiment, the host cells are CHO cells or 293 cells.

IV. Production and Purification of Antibodies of the Invention

In one embodiment, the invention provides a method of making a LAG-3 antibody, wherein the method comprises culturing the nucleic acid comprising the LAG-3 antibody under conditions suitable for expression of the nucleic acid encoding the LAG-3 antibody or a host cell containing an expression vector for the nucleic acid, and optionally the LAG-3 antibody isolated. In a certain embodiment, the method further comprises recovering the LAG-3 antibody from the host cell (or host cell culture medium).

For recombinant production of the antibodies of the invention, nucleic acid encoding the LAG-3 antibody of the invention is first isolated and inserted into a vector for further cloning and/or expression in host cells. Such nucleic acids are readily isolated and sequenced using routine procedures, eg, by using oligonucleotide probes capable of binding specifically to nucleic acids encoding LAG-3 antibodies of the invention.

Antibodies of the invention prepared as described herein can be purified by known prior art techniques such as high performance liquid chromatography, ion exchange chromatography, gel electrophoresis, affinity chromatography, size exclusion chromatography, and the like. The actual conditions used to purify a particular protein will also depend on factors such as net charge, hydrophobicity, hydrophilicity, etc., and these will be apparent to those skilled in the art. The purity of the antibodies of the invention can be determined by any of a variety of well-known analytical methods, including size exclusion chromatography, gel electrophoresis, high performance liquid chromatography, and the like.

V. Activity Assays for Antibodies of the Invention and Activity of Antibodies of the Invention

The LAG-3 antibodies provided herein can be identified, screened, or characterized for their physical/chemical properties and/or biological activity by a variety of assays known in the art. In one aspect, the antibodies of the invention are tested for their antigen-binding activity, eg, by known methods such as ELISA, Western blotting, and the like. Binding to LAG-3 can be determined using methods known in the art, exemplary methods are disclosed herein. In some embodiments, SPR or ForteBio kinetic binding assays are used to determine binding of an antibody of the invention to LAG-3.

The present invention also provides assays for identifying biologically active LAG-3 antibodies. Biological activities may include, for example, binding to cell surface LAG-3 (eg, human LAG-3, monkey LAG-3), binding to LAG-3/MHC class II molecules, or inhibition of binding of LAG-3/FGL-1 role, etc.

Cells for use in any of the above in vitro assays include cell lines that naturally express LAG-3 or are engineered to express LAG-3. The LAG-3 cell line engineered to express LAG-3 is a cell line that does not normally express LAG-3 and expresses LAG-3 after transfection of DNA encoding LAG-3 into the cells.

In one embodiment, the anti-LAG-3 humanized antibody hz7F10 of the invention specifically binds human LAG-3 protein with an affinity (K _D ) of about 4.17E- 11M, while the control antibody BMS-986016 binds human LAG-3 protein with an affinity (KD) of about _1.61E -10M. Therefore, the LAG-3 antibody of the present invention can specifically bind to the LAG-3 protein with high affinity.

In one embodiment, the anti-LAG-3 humanized antibody hz7F10 of the present invention can effectively bind human LAG-3 recombinant protein with a median effective binding concentration (EC50) value of about 0.034 as measured by an ELISA assay nM.

In one embodiment, the anti-LAG-3 humanized antibody hz7F10 of the present invention can effectively bind to the human LAG-3 protein on the cell surface, as measured by FACS, with a half effective binding concentration (EC50) value of about 1.6 nM, which is comparable to the binding level of the control antibody BMS-986016 (EC50: 1.5 nM) to human LAG-3 protein on the cell surface.

In one embodiment, the anti-LAG-3 humanized antibody hz7F10 of the present invention can specifically block the binding of cell surface HLA to recombinant LAG-3 protein, as measured by FACS method, and its corresponding half effective inhibitory concentration The IC50 value was 4.8 nM, and the blocking ability was better than that of the control antibody BMS-986016 (IC50: 9.9 nM).

In one embodiment, the anti-LAG-3 humanized antibody hz7F10 of the invention is capable of blocking the binding of human LAG-3 to FGL-1, as measured by an ELISA assay, with a corresponding half effective inhibitory concentration IC50 value was 0.038nM, which was comparable to the blocking activity of the control antibody BMS-986016 (IC50: 0.038nM).

In one embodiment, the anti-LAG-3 humanized antibody hz7F10 of the present invention has good in vivo efficacy in mice as determined by experimental animal models. In the B6-huPD1huLAG3 humanized mouse subcutaneously transplanted tumor model, the anti-LAG-3 humanized antibody hz7F10 of the present invention has better anti-tumor efficacy than the control antibody BMS-986016, and the body weight of animals in each experimental group increased steadily without significant toxic effects.

VI. PHARMACEUTICAL COMPOSITIONS AND PHARMACEUTICAL FORMULATIONS

In some embodiments, the present invention provides compositions comprising any of the LAG-3 antibodies described herein or immunoconjugates thereof, preferably the compositions are pharmaceutical compositions. In one embodiment, the composition further comprises a pharmaceutically acceptable carrier. In one embodiment, a composition (eg, a pharmaceutical composition) comprises an antibody of the invention or an immunoconjugate thereof, and one or more other therapeutic agents (eg, chemotherapeutic agents, cytotoxic agents, other antibodies, anti-infective agents) combination of active agents, small molecule drugs or immunomodulators, preferably anti-PD-1 antibodies or anti-PD-L1 antibodies).

In some embodiments, the composition is used to prevent or treat tumors. In some embodiments, the tumor is cancer. In some embodiments, the compositions are used to prevent or treat infections, eg, chronic infections, eg, bacterial infections, viral infections, fungal infections, protozoal infections, and the like.

The present invention also includes compositions (including pharmaceutical compositions or pharmaceutical formulations) comprising LAG-3 antibodies or immunoconjugates thereof and/or compositions (including pharmaceutical compositions or medicaments) comprising polynucleotides encoding LAG-3 antibodies preparation). These compositions may also contain suitable pharmaceutically acceptable carriers such as those known in the art, pharmaceutically acceptable excipients, including buffers.

As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, isotonic and absorption delaying agents, and the like that are physiologically compatible. Pharmaceutically acceptable carriers suitable for use in the present invention may be sterile liquids such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is the preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerin , propylene, glycol, water, ethanol, etc. See also "Handbook of Pharmaceutical Excipients", Fifth Edition, R.C. Rowe, P.J. Seskey and S.C. Owen, Pharmaceutical Press, London, Chicago, for the use of excipients and their uses. The compositions may also contain minor amounts of wetting or emulsifying agents, or pH buffering agents, if desired. These compositions can take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained release formulations and the like. Oral formulations may contain standard pharmaceutical carriers and/or excipients, such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, saccharin.

The compounds described herein can be prepared by mixing an antibody of the invention of the desired purity with one or more optional pharmaceutically acceptable carriers (Remington's Pharmaceutical Sciences, 16th Ed., Osol, A. ed. (1980)). The pharmaceutical formulation of the LAG-3 antibody is preferably in the form of a lyophilized formulation or an aqueous solution.

The pharmaceutical compositions or formulations of the present invention may also contain more than one active ingredient required for the particular indication being treated, preferably those having complementary activities that do not adversely affect each other. For example, it would be desirable to also provide other anti-cancer active ingredients or anti-infective active ingredients such as chemotherapeutic agents, cytotoxic agents, other antibodies, anti-infective active agents, small molecule drugs or immunomodulatory agents such as anti-PD-1 antibodies, anti- PD-L1 antibody, etc. The active ingredients are suitably combined in amounts effective for the intended use.

Sustained release formulations can be prepared. Suitable examples of sustained release formulations include semipermeable matrices of solid hydrophobic polymers containing the antibodies of the invention in the form of shaped articles such as films or microcapsules.

VII. Combination products or kits

In some embodiments, the invention also provides a combination product comprising an antibody or antigen-binding fragment thereof of the invention, or an immunoconjugate thereof, and one or more other therapeutic agents (eg, chemotherapeutic agents, other antibodies, cytotoxic agents, anti-infective agents, small molecule drugs or immunomodulators, etc.). In some embodiments, other antibodies such as anti-PD-1 antibodies, anti-PD-L1 antibodies.

In some embodiments, the combination product is used to prevent or treat tumors. In some embodiments, the tumor is cancer or the like. In some embodiments, the combination product is used to prevent or treat an infection, eg, a chronic infection, eg, bacterial infection, viral infection, fungal infection, protozoal infection, and the like.

In some embodiments, the two or more components of the combination product may be administered to the subject in combination sequentially, separately, or simultaneously.

In some embodiments, the present invention also provides kits comprising the antibodies, pharmaceutical compositions, or combination products of the present invention, and optional package inserts directing administration.

In some embodiments, the present invention also provides a pharmaceutical product comprising the antibody, pharmaceutical composition combination product of the invention, optionally further comprising a package insert to direct administration.

VIII. Uses of the Antibodies of the Invention

In one aspect, the present invention relates to a method of modulating an immune response in an individual. The method comprises administering to a subject an effective amount of a LAG-3 antibody disclosed herein, or a pharmaceutical composition or combination comprising the LAG-3 antibody, thereby modulating an immune response in the subject. In one embodiment, a therapeutically effective amount of a LAG-3 antibody or pharmaceutical composition or combination disclosed herein restores, enhances, stimulates or increases an immune response in a subject.

In some embodiments, the present invention relates to methods of inhibiting the activity of LAG-3, blocking the binding of LAG-3 to MHC class II molecules, blocking the binding of LAG-3 to FGL-1 molecules in an individual, the methods comprising An effective amount of a LAG-3 antibody disclosed herein, or a pharmaceutical composition or combination product comprising the same, is administered to the subject.

In another aspect, the present invention relates to a method of preventing or treating a tumor (eg, cancer) in a subject, the method comprising administering to the subject an effective amount of a LAG-3 antibody disclosed herein, or a pharmaceutical combination comprising the same substance or combination product. In some embodiments, the tumor is an immune escaped tumor.

In another aspect, the present invention relates to a method of preventing or treating an infectious disease in a subject, the method comprising administering to the subject an effective amount of a LAG-3 antibody disclosed herein or a pharmaceutical composition comprising the same or Combination products.

In another aspect, the invention relates to a method of inducing antibody-dependent cell-mediated cytotoxicity in a subject, the method comprising administering to the subject an effective amount of a LAG-3 antibody disclosed herein or comprising the same pharmaceutical compositions or combination products.

The subject can be a mammal, eg, a primate, preferably a higher primate, eg, a human (eg, a patient having or at risk of having a disease described herein). In one embodiment, the subject has or is at risk of having a disease described herein (eg, a tumor or an infectious disease as described herein). In certain embodiments, the subject receives or has received other treatments, such as chemotherapy treatment and/or radiation therapy. Alternatively or in combination, the subject is or is at risk of being immunocompromised due to the infection.

In some embodiments, tumors, eg, cancers, described herein, include, but are not limited to, solid tumors, hematological cancers, soft tissue tumors, and metastatic lesions.

Examples of solid tumors include malignancies, eg, sarcomas and carcinomas (including adenocarcinomas and squamous cell carcinomas) of multiple organ systems, such as those that invade the liver, lung, breast, lymph, gastrointestinal tract (eg, colon), reproductive Those of the urinary tract (eg, kidney, bladder epithelial cells), prostate and pharynx. Adenocarcinomas include malignancies such as most colon cancers, rectal cancers, renal cell carcinomas, liver cancers, non-small cell lung cancers in lung cancers, small bowel cancers, and esophageal cancers. Squamous cell carcinoma includes malignant tumors such as those in the lung, esophagus, skin, head and neck region, mouth, anus, and cervix. In one embodiment, the cancer is melanoma, eg, advanced melanoma. In one embodiment, the cancer is renal cell carcinoma. Metastatic lesions of the aforementioned cancers can also be treated or prevented using the methods and compositions of the present invention.

Non-limiting examples of preferred cancers for treatment include lymphoma (eg, diffuse large B-cell lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma), breast cancer (eg, metastatic breast cancer), Liver cancer (eg, hepatocellular carcinoma (HCC)), lung cancer (eg, non-small cell lung cancer (NSCLC), eg, stage IV or recurrent non-small cell lung cancer, NSCLC adenocarcinoma, or NSCLC squamous cell carcinoma), myeloma (eg, multiple myeloma), leukemia (eg, chronic myeloid leukemia), skin cancer (eg, melanoma (eg, stage III or IV melanoma) or Merkel cell carcinoma), head and neck cancer (eg, head and neck squamous cell carcinoma) Squamous cell carcinoma (HNSCC), myelodysplastic syndrome, bladder cancer (eg, transitional cell carcinoma), kidney cancer (eg, renal cell carcinoma, eg, clear cell renal cell carcinoma, eg, advanced or metastatic clear cell renal cell carcinoma) cell carcinoma) and colon cancer. Additionally, refractory or relapsed malignancies can be treated using the LAG-3 antibodies described herein or a pharmaceutical composition or combination product comprising the same.

In one embodiment, the disease is a disease with elevated (nucleic acid or protein) levels of LAG-3. In some embodiments, the tumor is a tumor, such as cancer, that can be inhibited by inhibiting the binding of LAG-3 to MHC class II molecules and/or FGL-1 molecules. In some embodiments, the tumor or infection is a disease that would benefit from inhibition of LAG-3 at the nucleic acid or protein level.

In some embodiments, the infection is acute or chronic. In some embodiments, the chronic infection is a persistent infection, a latent infection, or a slow infection. In some embodiments, the chronic infection is caused by a pathogen selected from bacteria, viruses, fungi and protozoa.

In some embodiments, an antibody of the invention, or a composition or combination product comprising the same, delays the onset of the disorder and/or symptoms associated with the disorder.

In some embodiments, the methods of prevention or treatment described herein further comprise administering to said subject or individual a combination of a LAG-3 antibody or pharmaceutical composition or combination product disclosed herein, and one or more other therapies, For example, therapeutic modalities and/or other therapeutic agents.

In some embodiments, treatment modalities include surgery (eg, tumor resection); radiation therapy (eg, external particle beam therapy, which involves three-dimensional conformal radiation therapy in which the irradiation area is designed), localized irradiation (eg, directed to a preselected target) or organ irradiation) or focused irradiation), etc. The focused irradiation may be selected from stereotactic radiosurgery, segmented stereotactic radiosurgery, and intensity-modulated radiotherapy. Focused irradiation may have a radiation source selected from particle beams (protons), cobalt-60 (photons) and linear accelerators (X-rays), eg, as described in WO 2012/177624.

Radiation therapy can be administered by one or a combination of several methods including, but not limited to, external particle beam therapy, internal radiation therapy, implant irradiation, stereotactic radiosurgery, systemic radiation therapy, radiotherapy, and permanent or transient Interstitial brachytherapy.

In some embodiments, the therapeutic agent is selected from chemotherapeutic agents, cytotoxic agents, other antibodies, anti-infective agents, small molecule drugs, or immunomodulatory agents (eg, activators of co-stimulatory molecules or inhibitors of immune checkpoint molecules).

Exemplary other antibodies include, but are not limited to, inhibitors of immune checkpoint molecules (eg, anti-PD-1, anti-PD-L1, anti-TIM-3, or anti-CEACAM); antibodies that stimulate immune cells (eg, agonistic GITR antibodies) or CD137 antibody) and so on. Preferably, the other antibodies are selected from anti-PD-1 antibodies and/or anti-PD-L1 antibodies. More preferably, the anti-PD-1 antibody is Nivolumab of Bristol-Myers Squibb (BMS) and Pembrolizumab of Merck; the anti-PD-L1 The antibodies are atezolizumab developed by Roche, avelumab developed by Merck KGaA in Germany and Pfizer in the United States, and durvalumab developed by AstraZeneca.

In some embodiments, the immunomodulatory agent is an agonist of a costimulatory molecule. In one embodiment, the agonist of the costimulatory molecule is selected from the group consisting of agonists (eg, agonistic antibodies or antigen-binding fragments thereof, or soluble fusions) of the following molecules: OX40, CD2, CD27, CD28, CDS, ICAM-1 , LFA-1(CD11a/CD18), ICOS(CD278), 4-1BB(CD137), GITR, CD30, CD40, BAFFR, HVEM, CD7, LIGHT, NKG2C, SLAMF7, NKp80, CD160, B7-H3 or CD83 body.

Combination therapy of the present invention encompasses combined administration (wherein two or more therapeutic agents are contained in the same formulation or separate formulations) and separate administration. In the case of separate administration, administration of the antibodies or immunoconjugates, etc. of the invention may be performed before, concurrently with, and/or after administration of the other therapy.

In one embodiment, the administration of the LAG-3 antibody and the administration of the other therapy (eg, treatment modality or therapeutic agent) are within about one month of each other, or within about one, two or three weeks, or about 1, 2, 3, occur within 4, 5, or 6 days.

The antibodies of the invention (and pharmaceutical compositions or immunoconjugates comprising the same) can be administered by any suitable method, including parenteral, intrapulmonary, and intranasal administration, and, if desired for topical treatment, Intralesional administration. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal or subcutaneous administration. Depending in part on whether the administration is short-term or long-term, administration may be by any suitable route, eg, by injection, eg, intravenously or subcutaneously. Various dosing schedules are contemplated herein, including, but not limited to, single administration or multiple administrations at multiple time points, bolus administration, and pulse infusion.

For the prevention or treatment of disease, the appropriate dosage of the antibodies of the invention (when used alone or in combination with one or more other therapeutic agents) will depend on the type of disease to be treated, the type of LAG-3 antibody, the severity of the disease sex and course, whether the LAG-3 antibody was administered for prophylactic or therapeutic purposes, previous treatments, the patient's clinical history and response to the LAG-3 antibody, and the judgment of the attending physician. The LAG-3 antibody is suitably administered to the patient in a single treatment or over a series of treatments. Dosages and treatment regimens of LAG-3 antibodies can be determined by the skilled artisan.

It will be appreciated that the compositions or combination products of the invention can be used in place of LAG-3 antibodies for any of the prophylaxis or treatments described above.

IX. Methods and compositions for diagnosis and detection

In certain embodiments, any of the LAG-3 antibodies provided herein can be used to detect the presence of LAG-3 in a biological sample. As used herein, the term "detection" includes quantitative or qualitative detection, and exemplary detection methods may involve immunohistochemistry, immunocytochemistry, flow cytometry (eg, FACS), magnetic beads complexed with antibody molecules, ELISA assays Law. In certain embodiments, the biological sample is blood, serum, or other bodily fluid sample of biological origin. In certain embodiments, the biological sample comprises cells or tissues. In some embodiments, the biological sample is from a hyperproliferative or cancerous lesion.

In one embodiment, LAG-3 antibodies for use in a diagnostic or detection method are provided. In another aspect, a method of detecting the presence of LAG-3 in a biological sample is provided. In certain embodiments, the method comprises detecting the presence of LAG-3 protein in a biological sample. In certain embodiments, the LAG-3 is human LAG-3. In certain embodiments, the method comprises contacting a biological sample with a LAG-3 antibody as described herein under conditions that allow binding of the LAG-3 antibody to LAG-3, and detecting the presence of LAG-3 antibody and LAG-3 Whether a complex is formed between 3. Formation of the complex indicates the presence of LAG-3. The method can be in vitro or in vivo. In one embodiment, the LAG-3 antibody is used to select subjects suitable for treatment with the LAG-3 antibody, eg, wherein LAG-3 is the biomarker used to select the subject.

In one embodiment, the antibodies of the invention can be used to diagnose cancer or tumors, eg, to evaluate (eg, monitor) a subject for treatment or progression of a disease described herein (eg, a hyperproliferative or cancerous disease), its diagnosis and/or or installment. In certain embodiments, labeled LAG-3 antibodies are provided. Labels include, but are not limited to, labels or moieties that are detected directly (such as fluorescent labels, chromophore labels, electron-dense labels, chemiluminescent labels, and radioactive labels), and moieties that are detected indirectly, such as enzymes or ligands, for example, through enzymatic reactions or molecular interactions. Exemplary labels include, but are not limited to, radioisotopes32P, ^14C , ^125I , ^3H ^and131I ^, fluorophores such as rare earth chelates or fluorescein and derivatives thereof, rhodamine and derivatives thereof, dansyl ( dansyl), umbelliferone, luceriferase, eg, firefly luciferase and bacterial luciferase (US Pat. No. 4,737,456), luciferin, 2,3-dihydrophthalazine dihydrogen Ketones, horseradish peroxidase (HR), alkaline phosphatase, beta-galactosidase, glucoamylase, lysozyme, carbohydrate oxidase, e.g., glucose oxidase, galactose oxidase, and glucose -6-phosphate dehydrogenase, heterocyclic oxidases such as uricase and xanthine oxidase, and enzymes that utilize hydrogen peroxide dye precursors such as HR, lactoperoxidase, or microperoxidase ), biotin/avidin, spin tags, phage tags, stabilized free radicals, etc.

In some embodiments of any of the inventions provided herein, the sample is obtained prior to treatment with the LAG-3 antibody. In some embodiments, the sample is obtained after the cancer has metastasized. In some embodiments, the sample is a formalin-fixed, paraffin-coated (FFPE) sample. In some embodiments, the sample is a biopsy (eg, a core biopsy), a surgical specimen (eg, a specimen from a surgical resection), or a fine needle aspirate.

In some embodiments, LAG-3 is detected prior to treatment, eg, prior to initiation of treatment or prior to a treatment following a treatment interval.

In some embodiments, there is provided a method of treating a tumor or infection, the method comprising: testing a subject (eg, a sample) (eg, a subject sample comprising cancer cells) for the presence of LAG-3, The LAG-3 value is thus determined, the LAG-3 value is compared to a control value (eg, the value of LAG-3 in a sample from a healthy individual), and if the LAG-3 value is greater than the control value, a therapeutically effective amount is administered to the subject of LAG-3 antibodies (eg, LAG-3 antibodies described herein), optionally in combination with one or more other therapies, thereby treating tumors or infections.

It will be appreciated that various embodiments described in various sections of the present invention, eg, diseases, therapeutic agents, treatment modalities, and administrations, are equally applicable to, or may be combined with, embodiments of other sections of the present invention. Embodiments of the properties, uses, and methods described in various sections of the present invention that are applicable to LAG-3 antibodies are equally applicable to compositions, conjugates, combination products, kits, etc. comprising LAG-3 antibodies.

Example

Example 1: Preparation and screening of anti-human LAG-3 antibody hybridoma cells

1. Hybridoma Preparation

The human LAG-3 extracellular region (NCBI accession number: NP_002277.4, 1aa-450aa) was fused to mouse Fc to obtain hLAG-3-mFc fusion protein, which was used to immunize Balb/c mice to stimulate the immune response.

The human LAG-3 extracellular region (NCBI accession number: NP_002277.4, 1aa-450aa) was fused to human Fc to obtain hLAG-3-hFc fusion protein, and the hLAG-3-hFc fusion protein was used to coat a 96-well microtiter plate , the anti-hLAG-3 antibody titer in mouse serum was detected by enzyme-linked immunosorbent assay (ELISA) after immunizing Balb/c mice with hLAG-3-mFc fusion protein.

When Balb/c mice had a high titer of anti-hLAG-3 antibody and produced the desired immune response, the mice were immunized with hLAG-3-mFc fusion protein, and the mice were aseptically removed 3 days later. rat spleen. The spleen cells of Balb/c mice were collected to prepare a cell suspension and fused with SP2/0 myeloma cells. After the fused cells were resuspended in HAT medium, they were dispensed into 96-well cell culture plates. Incubate in a 37°C, 5% CO ₂ incubator to form hybridoma cell lines.

2. Positive Hybridoma Binding Screening

10-14 days after fusion of Balb/c mouse splenocytes and SP2/0 myeloma cells, hLAG-3-hFc fusion protein, cynomolgus monkey LAG-3 recombinant protein (catalog number: 90841-C08H, Beijing Yiqiao Shenzhou Technology Co., Ltd.) (20ng/ml) were coated with ELISA plates, 4°C, overnight; washed three times with PBS, blocked with 4% nonfat dry milk-PBS, room temperature, 1hr; washed three times with PBS, added hybridoma clones Culture supernatant, room temperature, 1hr. The following controls were set: (1) positive control (PC): mouse serum after immunization with hLAG-3-mFc fusion protein (1:1000 dilution with PBS); (2) negative control (NC): hLAG-3-mFc fusion protein Pre-immune mouse serum (diluted 1:1000 in PBS).

Washed three times with PBST (0.05%Tween-PBS), washed twice with PBS, added HRP-goat anti-mouse IgG (Fcγ), 37°C, 0.5hr; then washed three times with PBST (0.05%Tween-PBS), Add TMB color developing solution, protect from light for 15-30 min, add ELISA stop solution to terminate the reaction; take 630 nm as the reference wavelength, read and record the absorbance A450nm-630nm of the well plate at the wavelength of 450nm. Select the top 15 clones with high absorbance readings from high to low. The clone culture supernatant was taken every other day for secondary ELISA confirmation, and the positive hybridoma clone number was determined.

Example 2: Sequence determination of murine anti-human LAG-3 antibody

Murine anti-human LAG-3 antibody clones 7C5, 4C8, 7F1, 7F10, 3A10 and 10B4 were selected as exemplary candidate clones for sequence determination.

Specifically, after the exemplary hybridoma cells 7C5, 4C8, 7F1, 7F10, 3A10, and 10B4 secreting anti-human LAG-3 antibodies were expanded and cultured, total cellular RNA was extracted and reverse transcribed into cDNA; amplified using degenerate primers Antibody light chain variable region VL and heavy chain variable region VH sequences; and sequenced, the results are shown in Table 2 below, wherein, the 7C5 mouse antibody heavy chain variable region (VH) amino acid sequence is shown in SEQ ID NO: 1, light The amino acid sequence of the chain variable region (VL) is shown in SEQ ID NO:2; the amino acid sequence of the heavy chain variable region of the 4C8 mouse antibody is shown in SEQ ID NO:3, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:4; the 7F1 mouse antibody The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:5, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:6; the amino acid sequence of the heavy chain variable region of the 7F10 mouse antibody is shown in SEQ ID NO:7, and the light chain variable region is shown in SEQ ID NO:7. The amino acid sequence is shown in SEQ ID NO: 8; the amino acid sequence of the 3A10 mouse antibody heavy chain variable region amino acid sequence is shown in SEQ ID NO: 9, and the light chain variable region amino acid sequence is shown in SEQ ID NO: 10; 10B4 mouse antibody heavy chain variable region amino acid sequence See SEQ ID NO: 11, and the light chain variable region amino acid sequence is shown in SEQ ID NO: 12.

Table 2 The variable region amino acid sequence of murine anti-LAG-3 antibody

Example 3: Preparation of anti-human LAG-3 chimeric antibody and control antibody

The 7C5, 4C8, 7F1, 7F10, 3A10 and 10B4 murine antibody light chain variable region and heavy chain variable region nucleotide sequences obtained in Example 2 were cloned into a heavy chain containing human-kappa light chain constant region and human IgG4, respectively. Human-mouse chimeric light chains (ch7C5L, ch4C8L, ch7F1L, ch7F10L, ch3A10L and ch10B4L) and human-mouse chimeric light chains (ch7C5L, ch4C8L, ch7F1L, ch7F10L, ch3A10L and ch10B4L) and Chimeric heavy chain (ch7C5H, ch4C8H, ch7F1H, ch7F10H, ch3A10H and ch10B4H) expression plasmids were transformed into HEK293 cells (ATCC) for recombinant expression. 5-6 days after HEK293 cells were transfected, the culture supernatant was taken, and the expression supernatant was purified by ProteinA affinity chromatography. The obtained chimeric antibodies (ch7C5, ch4C8, ch7F1, ch7F10, ch3A10 and ch10B4).

Similarly, the control antibody BMS-986016 light and heavy chain sequences were fully synthesized, cloned into a eukaryotic transient expression vector, and recombinantly expressed in HEK293 cells. 5-6 days after the cells were transfected, the culture supernatant was taken, and the expression supernatant was purified by ProteinA affinity chromatography to obtain BMS-986016 recombinant protein. Among them, the amino acid sequence of the control antibody BMS-986016 is derived from WHO Drug Information (Vol.32, No.2, 2018), the amino acid sequence of the heavy chain is shown in Sequence 13, and the amino acid sequence of the light chain is shown in Sequence 14.

SEQ ID NO: 13: Control antibody BMS-986016 heavy chain amino acid sequence

Example 4: Chimeric Antibody Affinity Analysis

Using the Octet QKe system instrument of Fortebio Company, the affinity of the chimeric antibody in Example 3 was determined by the method of capturing the Fc segment of the antibody with the capture antibody (AHC) biological probe against the Fc segment of the human antibody. The specific operations are as follows.

The ch7C5, ch4C8, ch7F1, ch7F10, ch3A10, and ch10B4 chimeric antibodies were diluted with PBS buffer to 4 μg/ml, respectively, and flowed over the surface of AHC probe (Cat: 18-0015, PALL) for 120 s. The hLAG-3-mFc fusion protein (60nM) obtained by fusion of human LAG-3 extracellular domain (NCBI accession number: NP_002277.4, 1aa-450aa) to mouse Fc was used as mobile phase, the binding time was 300s, and the dissociation time was 300s. for 300s. After the experiment was completed, the response value of the blank control was deducted, and the 1:1 Langmuir binding mode was fitted with the software to calculate the kinetic constant of antigen-antibody binding.

The kinetic constants are shown in Table 3 below. The results showed that all clones were combined with human LAG-3 recombinant protein, and the sequence clones were correct.

Table 3. Affinity determination results of chimeric antibody and human LAG-3 recombinant protein

抗体名称Antibody name	K _D(M) K _D (M)
ch7C5ch7C5	3.65E-103.65E-10
ch4C8ch4C8	4.27E-104.27E-10
ch7F1ch7F1	5.52E-095.52E-09
ch7F10ch7F10	1.17E-101.17E-10
ch3A10ch3A10	1.28E-101.28E-10
ch10B4ch10B4	8.84E-118.84E-11

Example 5: FACS detection of binding of anti-human LAG-3 chimeric antibody to LAG-3 on HEK293 cell surface

Construction of HEK293 cell line transiently expressing human LAG-3: The transient expression plasmid encoding full-length human LAG-3 (NCBI accession number: NP_002277.4) was transferred into HEK293 cells by liposome method, and transfected After 48 hours, the obtained cells (hereinafter also referred to as 293/rhLAG-3 cells) were used for FACS detection.

The 293/rhLAG-3 cell suspension was incubated with different concentrations of ch7C5, ch4C8, ch7F1, ch7F10, ch3A10, and ch10B4 chimeric antibodies for 30 min at room temperature. After washing the cells three times with PBS, 1:200 diluted goat anti-human was added. IgG-FITC (Cat: F9512, Sigma) and incubated for 30 min.

The cells were washed with PBS for 3 times by flow cytometry, and the mean fluorescence intensity (MFI) of the cells was detected to detect the binding ability of the chimeric antibody to LAG-3 on the cell surface. Through FACS detection, ch7C5, ch4C8, ch7F1, ch7F10, ch3A10, ch10B4 can all bind to LAG-3 on the surface of 293 cells, and the specific binding map is shown in Figure 1.

As can be seen from Figure 1, the ch7C5, ch4C8, ch7F1, ch7F10, ch3A10, ch10B4 chimeric antibodies showed specific binding to human LAG-3 on the surface of HEK293 cells compared to the isotype control.

Example 6: FACS detection of blocking activity of anti-human LAG-3 chimeric antibody on the binding of HLA to LAG-3 on the surface of 293 cells

Construction of 293 cell line transiently expressing human HLA: encoding full-length human HLA-A (Cat: HG13263-UT, Beijing Yiqiao Shenzhou Technology Co., Ltd.) and HLA-B (Cat: HG16629-UT, Beijing Yiqiao Shenzhou Co., Ltd.) Technology Co., Ltd.) was co-transfected into HEK293 cells by liposome method, and 48 hours after transfection, the obtained cells (hereinafter also referred to as 293/rhHLA cells) were used for FACS detection.

Different concentrations of ch7C5, ch4C8, ch7F1, ch7F10, ch3A10, ch10B4 chimeric antibodies and 2ug/ml hLAG-3-mFc fusion protein were incubated at room temperature for 60min, then added to 293/rhHLA cell suspension and incubated at 4°C For 60 min, after washing the cells three times with PBS, 1:200 diluted goat anti-mouse IgG-FITC (Cat: F9006, Sigma) was added and incubated for 30 min. After washing the cells three times with PBS, the mean fluorescence intensity (MFI) of the cells was detected by flow cytometry to detect the ability of the chimeric antibody to block the binding of HLA and recombinant LAG-3 protein on the cell surface. The FACS test results are shown in Figure 2.

It can be seen from Figure 2 that ch7C5, ch4C8, ch7F1, ch7F10, ch3A10, ch10B4 chimeric antibodies can significantly block the binding of cell surface HLA to recombinant LAG-3 protein, and the corresponding half effective inhibitory concentration IC50 values are shown in Table 4.

Table 4. IC50 of FACS detection of anti-human LAG-3 chimeric antibody for blocking the binding of HLA to LAG-3 on the surface of 293 cells

抗体名称Antibody name	ch4C8ch4C8	ch7C5ch7C5	ch10B4ch10B4	ch3A10ch3A10	ch7F1ch7F1	ch7F10ch7F10
IC50(ug/ml)IC50(ug/ml)	1.2761.276	1.8721.872	2.0032.003	3.3333.333	11.4611.46	1.2121.212

Example 7: Humanization and recombinant expression of anti-human LAG-3 monoclonal antibody

1. Humanization of murine monoclonal antibody 7F10

(1) CDR transplantation

Firstly, the sequence of the heavy chain of the murine antibody was comprehensively analyzed to determine the complementary determinant (CDR) region of the antibody binding to the antigen and the framework region (framework) supporting the conserved three-dimensional conformation of the antibody. Then, according to the homology comparison results, the most similar human antibody template VH1 (1-46) was selected as the basic template, combined with the full sequence blast results, CDR transplantation was performed, and according to the CDR3 sequence, JH4 (WGQGTLVTVSS) was selected as the J region sequence. , CDR transplantation was carried out to realize the humanization of 7F10 heavy chain variable region (VH) in the framework region. Similarly, VK I (O12) and VKIII (L25) were selected as the basic templates, combined with the full sequence blast results, and CDR transplantation was carried out. According to the CDR3 sequence (GQSYSYPYT), JK2 (FGQGTKLEIK) was selected as the JK region sequence to realize the light chain. Humanization of framework regions. The amino acid sequence of the humanized heavy chain variable region hz7F10_VH1 of 7F10 antibody CDR Grafted (CDR Grafted) is shown in SEQ ID NO: 15; the amino acid sequence of the humanized light chain variable region hz7F10_VL1 amino acid sequence is shown in SEQ ID NO: 16, and the humanized light chain The variable region hz7F10_VL2 amino acid sequence is shown in SEQ ID NO:17.

SEQ ID NO: 15: Humanized antibody hz7F10 heavy chain variable region VH1 amino acid sequence

SEQ ID NO: 16: Humanized antibody hz7F10 light chain variable region VL1 amino acid sequence

SEQ ID NO: 17: Humanized antibody hz7F10 light chain variable region VL2 amino acid sequence

(2) Antibody variable region mutation design

According to the sequence characteristics of murine 7F10, mutation design was carried out on the CDR-transplanted humanized light and heavy chain variable region sequences and light chain variable region sequences. The mutation sites are shown in Table 5 below.

Table 5. 7F10 Humanized Sequence Design

NOTE: Humanized hz7F10 heavy chain variable regions are named herein as follows: hz7F10_VH followed by a numerical identification number, eg, 1, 2, 3, 4, 5, 6, 7. Therefore, according to this nomenclature, for example, hz7F10_VH1 represents the first hz7F10 heavy chain variable region, hz7F10_VH2 represents the second hz7F10 heavy chain variable region, and hz7F10_VH7 represents the seventh hz7F10 heavy chain variable region;

Humanized hz7F10 light chain variable regions are named herein as follows: hz7F10_VL followed by a numerical identification number, eg, 1, 2, 3, 4, 5, 6, 7. Therefore, according to this nomenclature, for example, hz7F10_VL1 represents the first hz7F10 light chain variable region, hz7F10_VL2 represents the second hz7F10 light chain variable region, and hz7F10_VL7 represents the seventh hz7F10 light chain variable region;

Q1E in the table represents that the amino acid Q at position 1 of the "CDR-grafted" sequence SEQ ID NO: 15 in the corresponding column is mutated to E;

Y36F represents the mutation of amino acid Y at position 36 of the "CDR-grafted" sequence SEQ ID NO: 16 of the corresponding column to F;

N53T represents the mutation of amino acid N at position 53 of the "CDR-grafted" sequence SEQ ID NO: 17 of the corresponding column to T.

The humanized antibody hz7F10 heavy chain variable region and light chain variable region sequences shown below were obtained.

SEQ ID NO: 18: Humanized antibody hz7F10 heavy chain variable region VH2 amino acid sequence

SEQ ID NO: 19: Humanized antibody hz7F10 heavy chain variable region VH3 amino acid sequence

SEQ ID NO:20: Humanized antibody hz7F10 heavy chain variable region VH4 amino acid sequence

SEQ ID NO: 21: Humanized antibody hz7F10 heavy chain variable region VH5 amino acid sequence

SEQ ID NO: 22: Humanized antibody hz7F10 heavy chain variable region VH6 amino acid sequence

SEQ ID NO: 23: Humanized antibody hz7F10 heavy chain variable region VH7 amino acid sequence

SEQ ID NO: 24: Humanized antibody hz7F10 light chain variable region VL3 amino acid sequence

SEQ ID NO: 25: Humanized antibody hz7F10 light chain variable region VL4 amino acid sequence

SEQ ID NO: 26: Humanized antibody hz7F10 light chain variable region VL5 amino acid sequence

SEQ ID NO: 27: Humanized antibody hz7F10 light chain variable region VL6 amino acid sequence

SEQ ID NO: 28: Humanized antibody hz7F10 light chain variable region VL7 amino acid sequence

2. Recombinant expression of humanized monoclonal antibodies

The humanized designed hz7F10 antibody light and heavy chain variable region sequences (hz7F10_VL1, hz7F10_VL2, hz7F10_VH1) were fully synthesized, and the humanized heavy chain variable regions were cloned into the eukaryotic transient expression vector pCDNA3.1 (catalog number: V79520 , Invitrogen) the upstream of the heavy chain constant region encoding gene of human IgG4, the heavy chain constant region amino acid sequence is shown in sequence 29; the humanized light chain variable region is cloned into the eukaryotic transient expression vector encoding of the human light chain CK Upstream of the gene, the amino acid sequence of the light chain constant region is shown in sequence 30, and the light and heavy chain expression vectors of the humanized hz7F10 antibody are constructed.

SEQ ID NO: 29: Heavy chain constant region amino acid sequence

SEQ ID NO:30: Light chain constant region amino acid sequence

According to the mutation design in Table 6, site-directed mutagenesis was carried out on the light chain and heavy chain expression plasmids pCDNA3.1 respectively, and then transferred into E. coli DH5α for amplification to obtain hz7F10 antibody light and heavy chain mutant expression plasmids; Light and heavy chain plasmids were combined, and then transferred into HEK293 cells for recombinant expression. The combination information is shown in Table 6.

Table 6. hz7F10 light and heavy chain combination information table

5-6 days after the cells were transfected, they were cultured with FreeStyle293 medium (catalog number: 12338-018, Gibco), the culture supernatant was taken, and the expression supernatant was purified by ProteinA affinity chromatography to obtain a humanized antibody.

Using the Octet QKe system instrument of Fortebio Company, the antibody affinity was determined by using the capture antibody (AHC) biological probe against the Fc segment of the human antibody to capture the Fc segment of the antibody. During the measurement, hz7F10 antibody and chimeric antibody ch7F10 were diluted with PBS buffer to 4ug/mL, and flowed through the surface of AHC probe (Cat:18-0015, PALL) for 300S. hLAG-3-mFc recombinant protein was used as mobile phase at a concentration of 60 nM. The binding time was 300s and the dissociation time was 300s. After the experiment, the software was used to fit the 1:1 Langmuir binding mode to calculate the kinetic constant of antigen-antibody binding.

The affinity of hz7F10 combination antibody and chimeric antibody ch7F10 and hLAG-3-mFc recombinant protein was measured by ForteBio, and the affinity measurement data of humanized antibody was obtained (Table 7),

It can be seen from Table 7 that after site mutation of antibody hz7F10-1 or antibody hz7F10-2, the affinity of antibodies hz7F10-3 to hz7F10-25 is improved by about 1-2 orders of magnitude compared with hz7F10-1 or hz7F10-2. Among them, the antibody hz7F10-23 was selected as an example for subsequent experiments.

Table 7. Results of affinity determination of the combination of light and heavy chains of hz7F10

Example 8: Species specificity study of ELISA detection of anti-LAG-3 humanized antibody binding to LAG-3

Human LAG-3-mFc recombinant protein, cynomolgus monkey LAG-3 recombinant protein (Cat: 90841-C08H, Beijing Yiqiao Shenzhou Technology Co., Ltd.), mouse LAG-3 recombinant protein (Cat: 53069-M08H, Beijing Yiqiao Shenzhou Technology Co., Ltd.), coated overnight at 4°C, and the coating concentration was 1 μg/mL; after washing the plate three times with PBS, add 5% BSA PBS, block at 37°C for 60 min, and wash the plate three times with PBST; add different dilution times hz7F10-23 (initial concentration of 10 μg/mL, 3-fold gradient dilution for 12 concentrations), incubated at 37°C for 60 min, washed 4 times with PBST; added 1:5000 diluted HRP-anti-human Fc (Cat: 109- 035-098, Jackson Immuno Research), incubate at 37°C for 45min, wash the plate 4 times with PBST; add TMB substrate to develop color, after incubation at 37°C for 10min, add 2M HCl to stop the reaction; read and record with 630nm as the reference wavelength The absorbance of the well plate at a wavelength of 450nm is A450nm-630nm. The specific binding of hz7F10-23 and control antibody to human, cynomolgus monkey and mouse LAG-3 is shown in Figure 3, Figure 4 and Figure 5, respectively.

The results show that hz7F10-23 can specifically bind to human and cynomolgus monkey LAG-3 recombinant protein, and the corresponding half effective binding concentration EC50 value is shown in Table 8, but it has no binding activity to mouse LAG-3 recombinant protein (Fig. 5), which provides a basis for the selection of subsequent in vivo experimental models.

Table 8. EC50 of anti-human LAG-3 humanized antibody binding to different species of LAG-3 detected by ELISA

抗体名称Antibody name	结合人LAG-3的EC50EC50 for binding to human LAG-3	结合食蟹猴LAG-3的EC50EC50 for binding to cynomolgus monkey LAG-3
hz7F10-23hz7F10-23	0.034nM0.034nM	0.20nM0.20nM

Example 9: Anti-LAG-3 Humanized Antibody Affinity Analysis

Using the Octet QKe system instrument of Fortebio Company, the antibody affinity was determined by using the capture antibody (AHC) biological probe against the Fc segment of the human antibody to capture the Fc segment of the antibody. The specific operations are as follows.

Antibodies (hz7F10-23 and control antibody BMS-986016) were diluted to 4 ug/mL with PBS buffer and flowed over the surface of AHC probe (Cat: 18-0015, PALL) for 120 s. Human LAG-3-mFc recombinant protein was used as mobile phase. The binding time was 300s and the dissociation time was 300s. After the experiment was completed, the response value of the blank control was deducted, and the 1:1 Langmuir binding mode was fitted with the software to calculate the kinetic constant of antigen-antibody binding.

The binding reaction curves of antibody hz7F10-23 and control antibody BMS-986016 to human LAG-3 recombinant protein are shown in Figure 6 and Figure 7, respectively. Curves were fitted and binding affinities were calculated, the hz7F10-23 affinity (KD) was _4.17E -11M and the BMS-986016 affinity (KD) was _1.61E -10M. The specific kinetic parameters are shown in Table 9 below. The results showed that hz7F10-23 had a high affinity for human LAG-3, which was better than that of the control antibody BMS-986016.

Table 9. Affinity measurement results of anti-human LAG-3 humanized antibody and human LAG-3 extracellular domain recombinant protein

	K _D值(M) K _D value (M)	k _on(1/Ms) k _on (1/Ms)	k _dis(1/s) _kdis (1/s)
hz7F10-23hz7F10-23	4.17E-114.17E-11	2.45E+052.45E+05	1.02E-051.02E-05
BMS-986016BMS-986016	1.61E-101.61E-10	2.72E+052.72E+05	4.36E-054.36E-05

Example 10: FACS detection of binding of anti-human LAG-3 humanized antibody to LAG-3 on the surface of 293 cells

Construction of 293 cell line transiently expressing human LAG-3: The transient expression plasmid encoding full-length human LAG-3 (NCBI accession number: NP_002277.4) was transferred into HEK293 cells by liposome method, and transfected After 48 hours, 293/rhLAG-3 cells were used for FACS detection.

The 293/rhLAG-3 cell suspension was incubated with different concentrations of humanized antibody hz7F10-23 and control antibody BMS-986016 for 30 min at room temperature. After washing the cells three times with PBS, 1:200 diluted goat anti-human IgG was added. -FITC (Cat: F9512, Sigma) and incubated for 30 min. After washing the cells three times with PBS, the cells were subjected to flow cytometry to detect the mean fluorescence intensity (MFI) of the cells to detect the binding ability of the humanized antibody to LAG-3 on the cell surface.

Through FACS detection, the antibody hz7F10-23 can bind to LAG-3 on the surface of 293 cells, and it can be seen from Figure 8 that the binding ability of the antibody hz7F10-23 to the cell surface LAG-3 is better than that of the control antibody BMS-986016.

The half effective binding concentration EC50 values of the antibody hz7F10-23 and the control antibody BMS-986016 were 1.6nM and 1.5nM, respectively, and the binding abilities of the two were equivalent.

Example 11: ELISA detection of the blocking activity of anti-LAG-3 humanized antibodies to the binding of LAG-3 to FGL-1

Human FGL-1 recombinant protein (Cat: CW55, Inshore Bio) was coated on a 96-well plate overnight at 4°C with a coating concentration of 0.5 μg/mL; after washing the plate three times with PBS, 5% BSA PBS was added, and the plate was blocked at 37°C for 60 min , PBST washed 3 times; different concentrations of hz7F10-23 and control antibody BMS-986016 and 0.02ug/ml LAG-3-mFc (NCBI accession number: NP_002277.4, 1aa-450aa) were pre-incubated at 37°C for 30min Then, add it to the coated wells, incubate at 37°C for 60min, wash the plate 4 times with PBST; add 1:5000 diluted HRP anti-mouse IgG (Cat: 115-035-071, Jackson Immuno Research), incubate at 37°C for 45min, The plate was washed 4 times with PBST; TMB substrate was added for color development, and after incubation at 37°C for 10 min, 2M HCl was added to terminate the reaction; with 630 nm as the reference wavelength, read and record the absorbance A450nm-630nm of the well plate at a wavelength of 450nm. The blocking of the binding of LAG-3 to FGL-1 by the humanized antibody hz7F10-23 is shown in FIG. 9 .

It can be seen from Figure 9 that both the humanized antibody hz7F10-23 and the control antibody can specifically block the binding of human LAG-3 to FGL-1, and their corresponding half effective inhibitory concentration IC50 values are 0.038nM, 0.038nM, respectively. The blocking activity is comparable.

Example 12: FACS detection of the blocking activity of anti-human LAG-3 humanized antibody on the binding of HLA to LAG-3 on the surface of 293 cells

Construction of 293 cell line transiently expressing human HLA: encoding full-length human HLA-A (Cat: HG13263-UT, Beijing Yiqiao Shenzhou Technology Co., Ltd.) and HLA-B (Cat: HG16629-UT, Beijing Yiqiao Shenzhou Co., Ltd.) Technology Co., Ltd.) expression plasmids were co-transfected into HEK293 cells by liposome method, and 48 hours after transfection, 293/rhHLA cells were used for FACS detection.

Different concentrations of humanized antibody hz7F10-23 and control antibody BMS-986016 were incubated with 3ug/ml LAG-3-mFc recombinant protein at room temperature for 40min, then added to the 293/rhHLA cell suspension and incubated at 4°C for 60min , after washing the cells 3 times with PBS, goat anti-mouse IgG-FITC (Cat: F9006, Sigma) diluted 1:200 was added and incubated for 30 min. After washing the cells three times with PBS, flow cytometry was used to detect the mean fluorescence intensity (MFI) of the cells to detect the ability of the humanized antibody to block the binding of HLA on the cell surface to the recombinant LAG-3 protein. Figure 10 shows the results of blocking the binding of LAG-3 to HLA by the humanized antibody hz7F10-23 detected by FACS.

It can be seen from Figure 10 that the antibody hz7F10-23 can block the binding of cell surface HLA and recombinant LAG-3 protein, and the blocking ability is better than that of the control antibody. The half effective inhibition concentration IC50 values of hz7F10-23 and control antibody BMS-986016 are respectively were 4.8nM and 9.9nM.

Example 13: Pharmacodynamic evaluation of anti-LAG-3 antibody in B6-huPD1huLAG3 humanized mouse subcutaneous xenograft model

6-8-week-old PD-1 and LAG-3 double transgenic mice B6-huPD1huLAG3 (Jicui Yaokang, catalog number: T004621) were taken and subcutaneously inoculated with 3 × 10 ⁶ MC38 mouse colon adenocarcinoma cells (ATCC) , when the tumor grows to about 100mm ³ , random grouping, 6 animals/group, grouping, dosage and frequency are shown in Table 10. Each group is intraperitoneally administered twice a week for a total of 6 times, and the tumor is measured at the same time. Volume and body weight of mice, when the body weight of mice decreased by more than 15%, or when the tumor volume of a single animal exceeded 3000mm ³ or the average tumor volume of a group of animals exceeded 2000mm ³ , the experiments on related mice were stopped, and the mice were euthanized.

Table 10. Grouping, dosage and frequency of B6-huLAG3 mice

组别group	药物drug	给药剂量 Dosage		给药频率Dosing frequency
11	同种型对照isotype control	10mg/kg10mg/kg	Biw×6Biw×6
22	hz7F10-23hz7F10-23	10mg/kg10mg/kg	Biw×6Biw×6
33	BMS-986016BMS-986016	10mg/kg10mg/kg	Biw×6Biw×6

In the B6-huLAG3 humanized mouse subcutaneous tumor model, it can be seen from Figure 11 that the humanized antibody hz7F10-23 can statistically significantly inhibit tumor growth compared with the control mice using the isotype antibody , P=0.0086, has a clear anti-tumor efficacy; and the anti-tumor efficacy of the humanized antibody hz7F10-23 is better than that of the control antibody BMS-986016; from Figure 12, it can be seen that the body weight of the animals in each experimental group increased steadily, which was no different from the control. significant difference.

Exemplary embodiments of the present invention have been described above, and it should be understood by those skilled in the art that these disclosures are merely exemplary and various other substitutions, adaptations and modifications may be made within the scope of the present invention. Therefore, the invention is not limited to the specific embodiments recited herein.

Claims

An antibody or antigen-binding fragment that specifically binds LAG-3, comprising

(a) 3 CDRs in the heavy chain variable region amino acid sequence shown in SEQ ID NO: 1 and 3 CDRs in the light chain variable region amino acid sequence shown in SEQ ID NO: 2; Variants in which the CDR regions have a single CDR or multiple CDRs with no more than 2 or 1 amino acid change per CDR region;

(b) 3 CDRs in the heavy chain variable region amino acid sequence shown in SEQ ID NO: 3 and 3 CDRs in the light chain variable region amino acid sequence shown in SEQ ID NO: 4; Variants in which the CDR regions have a single CDR or multiple CDRs with no more than 2 or 1 amino acid change per CDR region;

(c) 3 CDRs in the heavy chain variable region amino acid sequence shown in SEQ ID NO: 5 and 3 CDRs in the light chain variable region amino acid sequence shown in SEQ ID NO: 6; Variants in which the CDR regions have a single CDR or multiple CDRs with no more than 2 or 1 amino acid change per CDR region;

(d) 3 CDRs in the heavy chain variable region amino acid sequence shown in SEQ ID NO: 7 and 3 CDRs in the light chain variable region amino acid sequence shown in SEQ ID NO: 8; Variants in which the CDR regions have a single CDR or multiple CDRs with no more than 2 or 1 amino acid change per CDR region;

(e) 3 CDRs in the heavy chain variable region amino acid sequence shown in SEQ ID NO: 9 and 3 CDRs in the light chain variable region amino acid sequence shown in SEQ ID NO: 10; Variants in which the CDR regions have a single CDR or multiple CDRs with no more than 2 or 1 amino acid change per CDR region;

(f) 3 CDRs in the heavy chain variable region amino acid sequence shown in SEQ ID NO: 11 and 3 CDRs in the light chain variable region amino acid sequence shown in SEQ ID NO: 12; Variants in which the CDR regions have a single CDR or multiple CDRs with no more than 2 or 1 amino acid change per CDR region;

wherein the amino acid change is an amino acid addition, deletion or substitution.
An antibody or antigen-binding fragment that specifically binds LAG-3, comprising a heavy chain variable region and a light chain variable region, wherein:

(a) the heavy chain variable region comprises HCDR1 shown in SYGIS (SEQ ID NO: 31), or a variant of said HCDR1 with no more than 2 amino acid changes or no more than 1 amino acid change; EIYPRSDNTYYNGKFKG (SEQ ID NO: 31) NO: 32), or a variant of said HCDR2 with no more than 2 amino acid changes or no more than 1 amino acid change; and HCDR3 shown in RAFYYYGSNYYAMDY (SEQ ID NO: 33), or a variant of said HCDR3 A variant with no more than 2 amino acid changes or no more than 1 amino acid change; the light chain variable region comprises LCDR1 shown in RSSQSIVHSNGDTYLE (SEQ ID NO: 34), or no more than 2 amino acid changes of said LCDR1 or Variants with no more than 1 amino acid change; LCDR2 shown in KVSNRFS (SEQ ID NO: 35), or a variant of said LCDR2 with no more than 2 amino acid changes or no more than 1 amino acid change; and FQGSHVPWT (SEQ ID NO: 35) NO:36) LCDR3, or a variant of the LCDR3 with no more than 2 amino acid changes or no more than 1 amino acid change;

(b) the heavy chain variable region comprises HCDR1 shown in NYAMS (SEQ ID NO: 37), or a variant of said HCDR1 with no more than 2 amino acid changes or no more than 1 amino acid change; TITYGTTYTFYSDNVKG (SEQ ID NO: 37) NO: 38), or a variant of said HCDR2 with no more than 2 amino acid changes or no more than 1 amino acid change; and HCDR3 shown in GEYGSSFAY (SEQ ID NO: 39), or a variant of said HCDR3 A variant with no more than 2 amino acid changes or no more than 1 amino acid change; the light chain variable region comprises LCDR1 shown in KASQNVRTAVA (SEQ ID NO: 40), or no more than 2 amino acid changes of said LCDR1 or Variants with no more than 1 amino acid change; LCDR2 shown in LASNRHT (SEQ ID NO: 41), or a variant of said LCDR2 with no more than 2 amino acid changes or no more than 1 amino acid change; and LQHWNYPLT (SEQ ID NO: 41) NO:42) LCDR3, or a variant of the LCDR3 with no more than 2 amino acid changes or no more than 1 amino acid change;

(c) the variable region of the heavy chain comprises HCDR1 shown in DYAVS (SEQ ID NO: 43), or a variant of the HCDR1 with no more than 2 amino acid changes or no more than 1 amino acid change; VVWGDGSTNYHSALIS (SEQ ID NO: 43) NO: 44) shown in HCDR2, or a variant of said HCDR2 with no more than 2 amino acid changes or no more than 1 amino acid change; and HCDR3 shown in GGGGMDY (SEQ ID NO: 45), or a variant of said HCDR3 A variant with no more than 2 amino acid changes or no more than 1 amino acid change; the light chain variable region comprises LCDR1 shown in RASSSVSYMH (SEQ ID NO: 46), or no more than 2 amino acid changes of said LCDR1 or Variants with no more than 1 amino acid change; LCDR2 shown in ATSNLAS (SEQ ID NO: 47), or a variant of said LCDR2 with no more than 2 amino acid changes or no more than 1 amino acid change; and QHYNTNPPTWT (SEQ ID NO: 47) NO:48) LCDR3, or a variant of the LCDR3 with no more than 2 amino acid changes or no more than 1 amino acid change;

(d) the variable region of the heavy chain comprises HCDR1 shown in DDYMH (SEQ ID NO: 49), or a variant of the HCDR1 with no more than 2 amino acid changes or no more than 1 amino acid change; RIDPEDVETKYDPKFQG (SEQ ID NO: 49) NO:50), or a variant of said HCDR2 with no more than 2 amino acid changes or no more than 1 amino acid change; and HCDR3 shown in SFYSNYVNYFDQ (SEQ ID NO:51), or a variant of said HCDR3 A variant with no more than 2 amino acid changes or no more than 1 amino acid change; the light chain variable region comprises LCDR1 shown in KASENVGTYVS (SEQ ID NO: 52), or no more than 2 amino acid changes of said LCDR1 or Variants with no more than 1 amino acid change; LCDR2 shown in GASNRYT (SEQ ID NO: 53), or a variant of said LCDR2 with no more than 2 amino acid changes or no more than 1 amino acid change; and GQSYSYPYT (SEQ ID NO: 53) NO:54) LCDR3, or a variant of the LCDR3 with no more than 2 amino acid changes or no more than 1 amino acid change;

(e) the heavy chain variable region comprises HCDR1 shown in DDYMH (SEQ ID NO: 49), or a variant of said HCDR1 with no more than 2 amino acid changes or no more than 1 amino acid change; WIDPENGETEYASKFQG (SEQ ID NO: 49) NO: 55) shown in HCDR2, or a variant of said HCDR2 with no more than 2 amino acid changes or no more than 1 amino acid change; and HCDR3 shown in FDY (SEQ ID NO: 56), or a variant of said HCDR3 A variant with no more than 2 amino acid changes or no more than 1 amino acid change; the light chain variable region comprises LCDR1 as shown in KSSQSLLDSDGKTYLN (SEQ ID NO: 57), or no more than 2 amino acid changes of said LCDR1 or Variants with no more than 1 amino acid change; LCDR2 shown in LVSKLDF (SEQ ID NO: 58), or a variant of said LCDR2 with no more than 2 amino acid changes or no more than 1 amino acid change; and WQGTHFPQT (SEQ ID NO: 58) NO:59), or a variant of said LCDR3 with no more than 2 amino acid changes or no more than 1 amino acid change; or

(f) the heavy chain variable region comprises HCDR1 shown in NYGIS (SEQ ID NO: 60), or a variant of said HCDR1 with no more than 2 amino acid changes or no more than 1 amino acid change; EIYPRGGNTYYNGKFKG (SEQ ID NO: 60) NO: 61), or a variant of said HCDR2 with no more than 2 amino acid changes or no more than 1 amino acid change; and HCDR3 shown in RAFYYFGSNYYAMDY (SEQ ID NO: 62), or a variant of said HCDR3 A variant with no more than 2 amino acid changes or no more than 1 amino acid change; the light chain variable region comprises LCDR1 shown in RSSQSIVHSNGDTYLE (SEQ ID NO: 34), or no more than 2 amino acid changes of said LCDR1 or Variants with no more than 1 amino acid change; LCDR2 shown in KVSNRFS (SEQ ID NO: 35), or a variant of said LCDR2 with no more than 2 amino acid changes or no more than 1 amino acid change; and FQGSHVPWT (SEQ ID NO: 35) NO:36) LCDR3, or a variant of the LCDR3 with no more than 2 amino acid changes or no more than 1 amino acid change;

wherein the amino acid change is an amino acid addition, deletion or substitution.
The antibody or antigen-binding fragment that specifically binds to LAG-3 according to claim 2, comprising a heavy chain variable region and a light chain variable region, wherein

The heavy chain variable region comprises HCDR1 set forth in DDYMH (SEQ ID NO: 49); HCDR2 set forth in RIX 1 PEDVETKYDPKFQG (SEQ ID NO: 64), optionally wherein X 1 is D or N; and SFYSNYVNYFDQ HCDR3 shown in (SEQ ID NO:51);

The variable region of the light chain comprises LCDR1 represented by X 2 ASENVGTYVS (SEQ ID NO: 68), optionally, wherein X 2 is K or R; X 3 ASX 4 RYT (SEQ ID NO: 69) LCDR2, optionally; wherein X3 is G or A; X4 is N or T; and LCDR3 shown by GQSYSYPYT (SEQ ID NO: 54).
The antibody or antigen-binding fragment that specifically binds to LAG-3 according to claim 3, comprising a heavy chain variable region and a light chain variable region, wherein

The heavy chain variable region comprises HCDR1 represented by DDYMH (SEQ ID NO: 49); HCDR2 represented by RIDPEDVETKYDPKFQG (SEQ ID NO: 50); and HCDR3 represented by SFYSNYVNYFDQ (SEQ ID NO: 51); the The light chain variable region comprises LCDR1 shown in KASENVGTYVS (SEQ ID NO: 52), LCDR2 shown in GASNRYT (SEQ ID NO: 53), and LCDR3 shown in GQSYSYPYT (SEQ ID NO: 54);

The heavy chain variable region comprises HCDR1 shown in DDYMH (SEQ ID NO: 49); HCDR2 shown in RIDPEDVETKYDPKFQG (SEQ ID NO: 50), HCDR3 shown in SFYSNYVNYFDQ (SEQ ID NO: 51); The chain variable region comprises LCDR1 shown in KASENVGTYVS (SEQ ID NO: 52), LCDR2 shown in AASNRYT (SEQ ID NO: 66), and LCDR3 shown in GQSYSYPYT (SEQ ID NO: 54);

The heavy chain variable region comprises HCDR1 shown in DDYMH (SEQ ID NO: 49); HCDR2 shown in RIDPEDVETKYDPKFQG (SEQ ID NO: 50), HCDR3 shown in SFYSNYVNYFDQ (SEQ ID NO: 51); The chain variable region comprises LCDR1 represented by RASENVGTYVS (SEQ ID NO:65), LCDR2 represented by GASNRYT (SEQ ID NO:53), and LCDR3 represented by GQSYSYPYT (SEQ ID NO:54);

The heavy chain variable region comprises HCDR1 shown in DDYMH (SEQ ID NO: 49); HCDR2 shown in RIDPEDVETKYDPKFQG (SEQ ID NO: 50), HCDR3 shown in SFYSNYVNYFDQ (SEQ ID NO: 51); The chain variable region comprises LCDR1 shown by KASENVGTYVS (SEQ ID NO:52), LCDR2 shown by GASTRYT (SEQ ID NO:67), and LCDR3 shown by GQSYSYPYT (SEQ ID NO:54);

The heavy chain variable region comprises HCDR1 shown in DDYMH (SEQ ID NO: 49); HCDR2 shown in RINPEDVETKYDPKFQG (SEQ ID NO: 63), HCDR3 shown in SFYSNYVNYFDQ (SEQ ID NO: 51); The chain variable region comprises LCDR1 represented by KASENVGTYVS (SEQ ID NO:52), LCDR2 represented by GASNRYT (SEQ ID NO:53), and LCDR3 represented by GQSYSYPYT (SEQ ID NO:54);

The heavy chain variable region comprises HCDR1 shown in DDYMH (SEQ ID NO: 49); HCDR2 shown in RINPEDVETKYDPKFQG (SEQ ID NO: 63), HCDR3 shown in SFYSNYVNYFDQ (SEQ ID NO: 51); The chain variable region comprises LCDR1 represented by KASENVGTYVS (SEQ ID NO:52), LCDR2 represented by AASNRYT (SEQ ID NO:66), and LCDR3 represented by GQSYSYPYT (SEQ ID NO:54); or

The heavy chain variable region comprises HCDR1 shown in DDYMH (SEQ ID NO: 49); HCDR2 shown in RINPEDVETKYDPKFQG (SEQ ID NO: 63), HCDR3 shown in SFYSNYVNYFDQ (SEQ ID NO: 51); The chain variable region comprises LCDR1 represented by KASENVGTYVS (SEQ ID NO:52), LCDR2 represented by GASTRYT (SEQ ID NO:67), and LCDR3 represented by GQSYSYPYT (SEQ ID NO:54).
The antibody or antigen-binding fragment that specifically binds to LAG-3 according to claim 1 or 2, comprising a heavy chain variable region and a light chain variable region, wherein

(a) the heavy chain variable region comprises or is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO: 1 and the light chain variable region comprises or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the sequence of SEQ ID NO: 2 % sequence of identity;

(b) the heavy chain variable region comprises or is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO:3 and the light chain variable region comprises or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the sequence of SEQ ID NO:4 % sequence of identity;

(c) the heavy chain variable region comprises or is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO:5 and the light chain variable region comprises or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the sequence of SEQ ID NO:6 % sequence of identity;

(d) (i) the heavy chain variable region comprises or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or the same as the sequence of SEQ ID NO:7 A sequence that is 99% identical, and the light chain variable region comprises or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98 the sequence of SEQ ID NO: 8 Sequences of % or 99% identity;

(ii) the heavy chain variable region comprises or is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO: 15 and the light chain variable region comprises or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the sequence of SEQ ID NO: 16 % identical to the sequence; or

(iii) the heavy chain variable region comprises or is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO: 15 and the light chain variable region comprises or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the sequence of SEQ ID NO: 17 % sequence of identity;

(e) the heavy chain variable region comprises or is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO:9 and the light chain variable region comprises or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the sequence of SEQ ID NO: 10 % sequence of identity;

(f) the heavy chain variable region comprises or is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO: 11 and the light chain variable region comprises or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the sequence of SEQ ID NO: 12 % sequence of identity.
The antibody or antigen-binding fragment that specifically binds to LAG-3 according to claim 5, comprising a heavy chain variable region and a light chain variable region, wherein the antibody or antigen-binding fragment comprises

(a) the heavy chain variable region shown in SEQ ID NO:1 and the light chain variable region shown in SEQ ID NO:2;

(b) the heavy chain variable region shown in SEQ ID NO:3 and the light chain variable region shown in SEQ ID NO:4;

(c) the heavy chain variable region shown in SEQ ID NO:5 and the light chain variable region shown in SEQ ID NO:6;

(d) the heavy chain variable region shown in SEQ ID NO:7 and the light chain variable region shown in SEQ ID NO:8;

The heavy chain variable region shown in SEQ ID NO:15 and the light chain variable region shown in SEQ ID NO:16;

The heavy chain variable region shown in SEQ ID NO:15 and the light chain variable region shown in SEQ ID NO:17;

The heavy chain variable region shown in SEQ ID NO:18 and the light chain variable region shown in SEQ ID NO:24;

The heavy chain variable region shown in SEQ ID NO:18 and the light chain variable region shown in SEQ ID NO:25;

The heavy chain variable region shown in SEQ ID NO:19 and the light chain variable region shown in SEQ ID NO:24;

The heavy chain variable region shown in SEQ ID NO:19 and the light chain variable region shown in SEQ ID NO:25;

The heavy chain variable region shown in SEQ ID NO:18 and the light chain variable region shown in SEQ ID NO:26;

The heavy chain variable region shown in SEQ ID NO:18 and the light chain variable region shown in SEQ ID NO:27;

The heavy chain variable region shown in SEQ ID NO:19 and the light chain variable region shown in SEQ ID NO:26;

The heavy chain variable region shown in SEQ ID NO:19 and the light chain variable region shown in SEQ ID NO:27;

The heavy chain variable region shown in SEQ ID NO:20 and the light chain variable region shown in SEQ ID NO:24;

The heavy chain variable region shown in SEQ ID NO:20 and the light chain variable region shown in SEQ ID NO:25;

The heavy chain variable region shown in SEQ ID NO:20 and the light chain variable region shown in SEQ ID NO:27;

The heavy chain variable region shown in SEQ ID NO:20 and the light chain variable region shown in SEQ ID NO:28;

The heavy chain variable region shown in SEQ ID NO:21 and the light chain variable region shown in SEQ ID NO:24;

The heavy chain variable region shown in SEQ ID NO:21 and the light chain variable region shown in SEQ ID NO:25;

The heavy chain variable region shown in SEQ ID NO:21 and the light chain variable region shown in SEQ ID NO:27;

The heavy chain variable region shown in SEQ ID NO:21 and the light chain variable region shown in SEQ ID NO:28;

The heavy chain variable region shown in SEQ ID NO:22 and the light chain variable region shown in SEQ ID NO:24;

The heavy chain variable region shown in SEQ ID NO:22 and the light chain variable region shown in SEQ ID NO:25;

The heavy chain variable region shown in SEQ ID NO:22 and the light chain variable region shown in SEQ ID NO:27;

The heavy chain variable region shown in SEQ ID NO:22 and the light chain variable region shown in SEQ ID NO:28;

The heavy chain variable region shown in SEQ ID NO:23 and the light chain variable region shown in SEQ ID NO:24;

The heavy chain variable region set forth in SEQ ID NO:23 and the light chain variable region set forth in SEQ ID NO:25; or

The heavy chain variable region shown in SEQ ID NO:23 and the amino acid sequence of SEQ ID NO:28;

(e) the heavy chain variable region set forth in SEQ ID NO:9 and the amino acid sequence of SEQ ID NO:10; or

(f) the heavy chain variable region shown in SEQ ID NO:11 and the amino acid sequence of SEQ ID NO:12.
The antibody or antigen-binding fragment that specifically binds LAG-3 according to any one of claims 1 to 6, which is an IgG1, IgG2, IgG3 or IgG4 antibody; optionally, it is an IgG1 or IgG4 antibody; optionally Typically, it is a humanized IgG1 or humanized IgG4 antibody.
The antibody or antigen-binding fragment that specifically binds LAG-3 according to any one of claims 1 to 7, wherein the antigen-binding fragment is Fab, Fab', F(ab')2, Fv, single-chain Fv , single chain Fab or diabody.
The antibody or antigen-binding fragment that specifically binds LAG-3 according to any one of claims 1 to 8, which has one or more of the following properties:

(1) Binds LAG-3 with high affinity, eg, human LAG-3 and cynomolgus monkey LAG-3, eg, the KD for binding between the anti-LAG-3 antibody or antigen-binding fragment thereof and LAG -3 is about 10 -7 M to about 10-12 M, preferably, about 10-8 M to about 10-12 M, as measured by the ForteBio kinetic binding assay;

(2) specifically blocking the binding of LAG-3 to FGL-1;

(3) specifically blocking the binding of LAG-3 to cell surface MHC class II molecules (for example, human HLA);

(4) Keep T cells in an activated state;

(5) It has anti-tumor effect in vivo.
An isolated nucleic acid encoding the anti-LAG-3 antibody or antigen-binding fragment thereof of any one of claims 1 to 9.
A vector comprising the nucleic acid of claim 10, preferably the vector is an expression vector.
A host cell comprising the nucleic acid of claim 10 or the vector of claim 11, preferably, the host cell is prokaryotic or eukaryotic, more preferably selected from Escherichia coli cells, yeast cells, mammalian cells or suitable for producing Other cells for antibodies or antigen-binding fragments thereof, most preferably, the host cells are 293 cells or CHO cells.
A method for preparing the anti-LAG-3 antibody or antigen-binding fragment thereof of any one of claims 1 to 9, said method comprising in a method suitable for expressing the anti-LAG-3 antibody or its Culturing the host cell of claim 12 under conditions of nucleic acid of an antigen-binding fragment, optionally isolating the anti-LAG-3 antibody or antigen-binding fragment thereof, optionally the method further comprises recovering the antibody from the host cell. A LAG-3 antibody or antigen-binding fragment thereof.
A pharmaceutical composition comprising the anti-LAG-3 antibody or antigen-binding fragment thereof of any one of claims 1 to 9, and optionally a pharmaceutically acceptable carrier.
A pharmaceutical composition comprising the anti-LAG-3 antibody or antigen-binding fragment thereof of any one of claims 1 to 9, and other therapeutic agents, and optionally a pharmaceutically acceptable carrier; preferably, the other therapeutic agents are selected from the group consisting of: Autochemotherapeutic agents, other antibodies (eg, anti-PD-1 antibodies or anti-PD-L1 antibodies), cytotoxic agents.
Use of the anti-LAG-3 antibody or antigen-binding fragment thereof of any one of claims 1 to 9, the pharmaceutical composition of claim 14 or 15, for the manufacture of a medicament for preventing or treating tumors in a subject.
The purposes of claim 16, wherein said tumor is cancer, such as blood cancer, brain cancer, renal cell carcinoma, ovarian cancer, bladder cancer, prostate cancer, breast cancer, hepatocellular carcinoma, bone cancer, colon cancer (for example, colon gland cancer) carcinoma), non-small cell lung cancer, head and neck squamous cell carcinoma, colorectal cancer, mesothelioma, B-cell lymphoma, and melanoma.
A kit for detecting LAG-3 in a sample, the kit comprising the anti-LAG-3 antibody or antigen-binding fragment thereof of any one of claims 1 to 9, for carrying out the following steps:

(a) contacting the sample with the anti-LAG-3 antibody or antigen-binding fragment thereof of any one of claims 1 to 9; and

(b) detecting the formation of a complex between the anti-LAG-3 antibody or antigen-binding fragment thereof and LAG-3; optionally, the anti-LAG-3 antibody or antigen-binding fragment thereof is detectably labeled .