CN113621061A

CN113621061A - LAG-3 monoclonal antibodies or antigen binding fragments thereof and related uses

Info

Publication number: CN113621061A
Application number: CN202010387491.5A
Authority: CN
Inventors: 马一明; 李家鹏
Original assignee: Suzhou Genhouse Pharmaceutical Co ltd
Current assignee: Suzhou Genhouse Pharmaceutical Co ltd
Priority date: 2020-05-09
Filing date: 2020-05-09
Publication date: 2021-11-09

Abstract

Monoclonal antibodies or antigen-binding fragments thereof that inhibit the immunosuppressive function of LAG-3 and related uses are provided. Further, a mouse derived antibody comprising the CDR regions of the LAG-3 antibody, a chimeric antibody, and use thereof as an antitumor agent are provided.

Description

LAG-3 monoclonal antibodies or antigen binding fragments thereof and related uses

The present invention relates to monoclonal antibodies and antigen-binding fragments thereof that specifically bind to immunoregulatory receptor lymphocyte activation gene-3 (LAG-3), chimeric antibodies to CDR regions of the anti-LAG-3 antibodies, and the use of pharmaceutical compositions of the antibodies and antigen-binding fragments thereof of the present invention in the treatment and diagnosis of immune-related diseases, such as tumors.

Background

The human body clears tumor cells and inhibits tumor formation through the immune system, where T cell-mediated cellular immunity plays a key role. In general, antigen presenting cells degrade antigen processing into antigenic peptide fragments and present the antigenic peptides to T cells via MHC molecules, priming and activating effector T cells, promoting T cell migration to tumor tissue, recognizing and eliminating tumor cells. When the tumor cells inhibit the immune system of the human body, the tumor cells cannot be normally killed, so that the tumor cells can survive. Tumor immunotherapy is a therapeutic method for controlling or eliminating tumors by activating the immune system and restoring the body's anti-tumor immune response. T cell costimulatory and costimulatory molecules, including PD-1 and CTLA-4, etc., are known to play important roles in regulating T cell activation (Chen et al, Nature Rev. Immunol. 2013, 13: 227-242).

Lymphocyte activation gene 3 (LAG-3) was first discovered by Triebel et al in 1990 as a member of the immunoglobulin superfamily (IgSF) and is an immune negative regulator. LAG-3 is predominantly expressed in activated T cells and NK cells (Triebel et al, J Exp Med 1990, 171: 1393-. Studies have shown that LAG-3 acts by down-regulating the CD3/TCR activation pathway. Known LAG-3 ligands include MHCII, Galectin-3 and FGL-1. In addition, LAG-3 plays a role in Treg cell function (Pardol et al 2012, Nature Reviews Cancer 12: 252-264).

In vitro studies show that LAG-3 can inhibit antigen-dependent T cell proliferation, activation and homeostasis, which plays an important role in the development of immune-related diseases such as tumors. After blocking the binding of LAG-3 to MHCII with anti-LAG-3 antibody, the proliferation and activation of T cells and the secretion of Th1 cytokine are all improved, contributing to the restoration of T cell immune function, and thus being useful for the treatment of diseases such as tumors (Grosso et al, J Clin Invest 2007, 117: 3383-.

Disclosure of Invention

The present invention provides antibodies (murine or chimeric) and corresponding antigen-binding fragments that specifically bind to human and/or cynomolgus monkey LAG-3. The antibodies of the invention can be used for various applications, such as detecting LAG-3 protein, monitoring expression of LAG-3 by immune cells for diagnosis of disease, targeting immune cells expressing LAG-3 molecules and modulating LAG-3 activity, stimulating antigen-specific T cell responses in subjects with neoplastic disease, and thus for treatment of tumors.

The anti-LAG-3 antibody or antigen-binding fragment thereof of the present invention comprises a heavy chain variable region and a light chain variable region, wherein:

the heavy chain variable region comprises SEQ ID NO: 5, the heavy chain CDR1 amino acid sequence of SEQ ID NO: 7, and the heavy chain CDR2 amino acid sequence of SEQ ID NO: 9 heavy chain CDR3 amino acid sequence; and the light chain variable region comprises SEQ ID NO: 11, the light chain CDR1 amino acid sequence of SEQ ID NO: 13, and the light chain CDR2 amino acid sequence of SEQ ID NO: 15, a light chain CDR3 amino acid sequence; or the heavy chain variable region comprises SEQ ID NO: 21, the heavy chain CDR1 amino acid sequence of SEQ ID NO: 23, and the heavy chain CDR2 amino acid sequence of SEQ ID NO: 25, the heavy chain CDR3 amino acid sequence; and the light chain variable region comprises SEQ ID NO: 27, light chain CDR1 amino acid sequence of SEQ ID NO: 29, and the light chain CDR2 amino acid sequence of SEQ ID NO: 31 a light chain CDR3 amino acid sequence; or

The heavy chain variable region comprises SEQ ID NO: 37, the heavy chain CDR1 amino acid sequence of SEQ ID NO: 39, and the heavy chain CDR2 amino acid sequence of SEQ ID NO: 41, heavy chain CDR3 amino acid sequence; and the light chain variable region comprises SEQ ID NO: 43, light chain CDR1 amino acid sequence of SEQ ID NO: 45, and the light chain CDR2 amino acid sequence of SEQ ID NO: 47 light chain CDR3 amino acid sequence; or the heavy chain variable region comprises SEQ ID NO: 53, the heavy chain CDR1 amino acid sequence of SEQ ID NO: 55, and the heavy chain CDR2 amino acid sequence of SEQ ID NO: the heavy chain CDR3 amino acid sequence of 57; and the light chain variable region comprises SEQ ID NO: 59, the light chain CDR1 amino acid sequence of SEQ ID NO: 61, and the light chain CDR2 amino acid sequence of SEQ ID NO: 63, light chain CDR3 amino acid sequence.

In some embodiments of the invention, the antibody is a full length antibody, such as an IgG4 antibody.

In some embodiments of the invention, the antigen binding fragment is a Fab.

In some embodiments of the invention, the antibody is a chimeric antibody.

In some embodiments of the invention, the heavy chain variable region comprises a heavy chain variable region that differs from SEQ ID NO: 1. SEQ ID NO: 17. SEQ ID NO: 33 or SEQ ID NO: 49 has at least 75% identity to the sequence shown in seq id no.

In some embodiments of the invention, the light chain variable region comprises a sequence identical to SEQ ID NO: 3. SEQ ID NO: 19. SEQ ID NO: 35 or SEQ ID NO: 51 has an amino acid sequence that is at least 75% identical.

In some embodiments of the invention, the heavy chain variable region comprises SEQ ID NO: 1 and the light chain variable region comprises the sequence shown in SEQ ID NO: 3, and (b) is shown in the specification.

In some embodiments of the invention, the heavy chain variable region comprises SEQ ID NO: 17 and the light chain variable region comprises the sequence shown in SEQ ID NO: 19, and (b) the sequence shown in the specification.

In some embodiments of the invention, the heavy chain variable region comprises SEQ ID NO: 33 and the light chain variable region comprises the sequence shown in SEQ ID NO: 35, and (b) a sequence shown in the specification.

In some embodiments of the invention, the heavy chain variable region comprises SEQ ID NO: 49 and the light chain variable region comprises the sequence shown in SEQ ID NO: 51, or a sequence shown in SEQ ID NO.

In some embodiments of the invention, the anti-LAG-3 antibody or antigen-binding fragment thereof comprises a heavy chain constant region and/or a light chain constant region; wherein the antibody heavy chain constant region is a heavy chain constant region of a mouse antibody or a heavy chain constant region of a human antibody, and/or the antibody light chain constant region is a light chain constant region of a mouse antibody or a light chain constant region of a human antibody.

The present invention provides pharmaceutically effective antibodies that specifically bind human LAG-3. In particular, the pharmaceutically effective human antibodies can bind to LAG-3 on the surface of T cells and other immune cells.

In some embodiments of the invention, the anti-LAG-3 antibody, or antigen-binding fragment thereof, inhibits binding of LAG-3 to human MHC class II molecules.

Yet another aspect of the invention relates to an isolated nucleic acid encoding the heavy chain variable region, the light chain variable region, and the CDR regions of an anti-LAG-3 antibody according to the invention.

Yet another aspect of the invention relates to a vector molecule encoding the heavy chain of the anti-LAG-3 antibody or antigen-binding fragment thereof of the invention, the light chain of the anti-LAG-3 antibody or antigen-binding fragment thereof of the invention, the heavy chain and the light chain of the anti-LAG-3 antibody or antigen-binding fragment thereof of the invention.

Yet another aspect of the invention relates to a cell comprising a host cell for the nucleotide or the vector (e.g., an expression vector), which may be, but is not limited to, a bacterial cell (including, but not limited to, e.coli) or an animal cell (including, but not limited to, CHO cells and HEK293 cells).

The present invention provides a method for preparing an anti-LAG-3 antibody or antigen-binding fragment thereof, comprising expressing an anti-LAG-3 antibody or antigen-binding fragment thereof in a host cell as described above, and isolating the anti-LAG-3 antibody or antigen-binding fragment thereof from the host cell.

The murine antibody provided by the invention specifically binds human LAG-3. (see example 2)

The murine antibody provided by the invention can block the combination of human LAG-3 and ligand MHCII molecules thereof. (see example 3)

The chimeric antibody provided by the invention has no species cross reaction with mice. (see example 4)

The chimeric antibodies P03G10, P05C07 and P11D12 provided by the invention have species cross reaction with cynomolgus monkeys, and the P05E03 has no species cross reaction with cynomolgus monkeys. (see example 4)

The chimeric antibodies provided by the invention specifically bind human LAG-3. (see example 5)

The chimeric antibody provided by the invention can block the binding of human LAG-3 and ligand MHCII molecules thereof. (see example 6)

The chimeric antibody provided by the invention has stronger in vitro biological activity. In a superantigen-stimulated PBMC cell system, the antibodies of the invention can promote IL-2 secretion from primary T cells (see example 7).

The antibody provided by the invention has the following functions in vivo and in vitro: blocking binding of LAG-3 to MHCII, modulating (e.g., down-regulating) LAG-3 activity or LAG-3 levels, relieving immunosuppression of LAG-3 to the body, activating T lymphocytes, or increasing expression of cytokines (such as IL-2) by T lymphocytes.

In some embodiments, the present invention provides methods for inhibiting tumor growth. Examples of cancer include, but are not limited to, primary and/or recurrent cancers, including melanoma, skin cancer, breast cancer, mesothelioma, brain cancer (e.g., glioblastoma multiforme), head and neck squamous cell carcinoma, thyroid cancer, oral cancer, esophageal cancer, gastric cancer, lung cancer (e.g., non-small cell lung cancer), liver cancer, kidney cancer, membrane adenocarcinoma, uterine cancer, cervical cancer, ovarian cancer, testicular cancer, prostate cancer, intestinal cancer, bladder cancer, bone cancer, and hematological malignancies (e.g., lymphoma, myeloma, or leukemia).

Drawings

FIG. 1A FACS detection of anti-LAG-3 Fabs binding to HEK 293T/humanLAG-3;

FIG. 1B FACS detection of anti-LAG-3 Fabs blocking binding of human LAG-3 recombinant protein to MHCII;

FIG. 2A ELISA for detection of binding of anti-LAG-3 chimeric antibodies to human LAG-3 recombinant protein;

FIG. 2B ELISA for detection of binding of anti-LAG-3 chimeric antibody to mouse LAG-3 recombinant protein;

FIG. 2C ELISA detection of anti-LAG-3 chimeric antibody binding to cynomolgus monkey LAG-3 recombinant protein;

FIG. 3A binding of LAG-3 chimeric antibody to 293T/hLAG-3 cells;

FIG. 3B. LAG-3 chimeric antibody blocks binding of LAG-3 to MHCII;

FIG. 4 shows that LAG-3 chimeric antibody promotes IL-2 secretion from PBMC.

Detailed Description

Specific embodiments of the present invention will be described in detail below with reference to examples to assist understanding of the present invention, and should not be construed as limiting the scope of the present invention in any way.

Examples

Example 1: generation of anti-human LAG-3 antibodies

Mice were immunized.

Anti-human LAG-3 antibodies were obtained by construction and screening of immune Fab phage display libraries. First, mice were immunized with hLAG-3-hFc fusion protein. After the third immunization, mouse spleen lymphocytes are separated, and a Fab phage antibody display library is constructed.

And (3) constructing and screening a phage display antibody library.

The isolated mouse lymphocytes were lysed with Trizol and total cellular RNA was extracted. After the total RNA is reversely transcribed into cDNA, the variable region of the mouse antibody gene is amplified by PCR to construct a Fab library. The amplicons of the heavy and light chains were pooled and cloned into a phagemid vector. Subsequently, three rounds of screening were performed using the recombinant protein LAG-3-hFc and LAG-3 overexpressing cells to identify LAG-3 specific Fab positive clones. The variable region sequences of the antibodies were then sequenced.

Example 2: binding of anti-human LAG-3 Fabs to human LAG-3.

Method of assessing binding of anti-human LAG-3 Fabs to human LAG-3 overexpressing cells

FACS methods were used to assess binding of anti-human LAG-3 Fabs to 293T-hLAG-3 cells. 293T-hLAG-3 cells were resuspended in FACS buffer and cell density adjusted to 4X 10⁶cell/mL, 50. mu.L of cell suspension was added to each well of a 96-well round bottom plate, giving a 2X 10 cell count per well⁵. Subsequently 50. mu.L of the antibody to be tested at different concentrations were added to each well and incubated at 4 ℃ for 1 hour. After the cells were washed three times with FACS buffer, PE-labeled goat anti-mouse IgG was added and incubated at 4 ℃ for 0.5 hours. Cells were washed three times with FACS buffer, 200 μ Ι _ of FACS buffer resuspended cells, fluorescence signal detected by flow cytometry and data analyzed. The results are shown in FIG. 1A, where anti-LAG-3 Fab was tested for binding to 293T/hLAG-3.

Example 3: ligand blocking Activity against human LAG-3 Fabs

Blocking activity of anti-human LAG-3 Fabs on hLAG-3 and MHC class II molecule binding was evaluated using FACS method. First, the hLAG-3 recombinant protein was biotinylated. Subsequently, in 96-well round bottom plates, anti-human LAG-3 Fabs or negative control Fab were added at an initial concentration of 10 μ g/mL, 1: after 6 gradients of 4-fold dilution, 50. mu.L of biotin-labeled hLAG-3 recombinant protein (2. mu.g/mL) was incubated with 100. mu.L of the above antibody at 4 ℃ for 60 min. The above-mentioned mixed system is subsequently mixed with 5X 10⁴Individual Raji cells were mixed and incubated at 4 ℃ for an additional 60 minutes. The above mixed system was washed 2 times with FACS buffer and incubated with PE labeled Streptavidin for 30 minutes at 4 ℃. Cells were washed three times with FACS buffer, fluorescence signal was detected by flow cytometry and data was analyzed. The anti-human LAG-3 Fabs tested blocked the binding of LAG-3 recombinant protein to MHCII positive cells Raji. The variable regions of the selected Fabs were then cloned into the human heavy and light chain constant regions, expressed as the entire IgG4 chimeric antibody, and evaluated for ligand blocking activity in the above system. As a result, as shown in FIG. 1B, the entire length was engagedThe antibody blocks binding of recombinant LAG-3 protein to MHCII-expressed Raji cells.

Example 4: species cross-reactivity of anti-LAG-3 chimeric antibodies.

Species cross-reactivity of chimeric antibodies. Species cross-reactivity of the chimeric antibody was detected using ELISA methods. mu.g/mL hLAG-3-mFc, mLAG-3-mFc, or cynoLAG-3 (SinoBiological, catalog # 90841-C08H) recombinant protein was coated onto 96-well clear enzyme plates (Corning, catalog # 9018) and incubated overnight at 4 ℃. Plates were washed three times with PBST (1 XPBS, 0.05% Tween-20) and blocked with 5% skim milk powder (Oxoid, Cat. No. LP 0031B) for 2 hours at 37 ℃. After three PBST washes, the test antibody was added and incubated for 1 hour at room temperature. After washing the plates with PBST, add 1: a10000-diluted HRP-labeled goat anti-human IgG secondary antibody (Jackson Immuno, catalog No. 109-035-088) was incubated at room temperature for 1 hour. After washing the plates with PBST, TMB (Life Technologies, Cat. No. 002023) was added for color development, 1M concentrated sulfuric acid (national drug, Cat. No. 10021618) was added to terminate the reaction, and OD was added₄₅₀And (4) reading the value. As a result, as shown in FIG. 2, the anti-LAG-3 chimeric antibody bound to human-derived LAG-3 and cynomolgus monkey-derived LAG-3, and did not bind to mouse-derived LAG-3.

Example 5: binding of anti-LAG-3 chimeric antibodies to 293T/hLAG-3 cells

FACS was used to detect binding of LAG-3 chimeric antibodies to 293T/hLAG-3 cells. 293T/hLAG-3 cells were resuspended in FACS buffer (1 XPBS, 1% BSA) and cell density adjusted to 4X 10⁶cells/mL, 96-well round bottom plate (Corning, Cat. No. 3795) 50. mu.L of cell suspension was added per well, giving a 2X 10 cell count per well⁵. Subsequently 50. mu.L of different concentrations of the antibody to be tested (starting concentration 10. mu.g/mL, 1: 5 fold dilution, 8 gradients) were added to each well and incubated for 1 hour at 4 ℃. After washing the cells three times with FACS buffer, a PE-labeled goat anti-mouse IgG secondary antibody (Biolegend, cat 405307) was added and incubated for 0.5 hours at 4 ℃. Cells were washed three times with FACS buffer, 200 μ Ι _ FACS buffer was used to resuspend the cells, fluorescence signals were detected using an Attune nxt flow cytometer (Thermo Fisher), and data were analyzed with Graphpad Prism 6.01 software. The results are shown in FIG. 3A.

Example 6: anti-LAG-3 chimeric antibodies block binding between MHC class II molecules and LAG-3

FACS was used to evaluate whether anti-LAG-3 chimeric antibodies could block the binding between LAG-3 and its ligand MHC class II molecules. The hLAG-3mFc recombinant protein was first biotinylated using the biotin labeling kit (Dojindo, cat # LK 03) according to the product instructions. Different concentrations of anti-LAG-3 antibody (maximum concentration 10 μ g/mL, 1: 4 fold dilution, total 6 concentrations) were incubated with biotinylated hLAG-3-mFc recombinant protein for 15 min at room temperature, followed by Raji cells for 40 min at room temperature, FACS buffer (1 × PBS, 1% BSA) washed three times, and 1: PE at 200 dilution labeled Streptavidin (BD, cat # 554061) was incubated at room temperature for 1 hour. After three washes with FACS buffer, the cells were resuspended in 200 μ L FACS buffer, the fluorescence signal was detected using an Attune nxt flow cytometer (Thermo Fisher), and the data was analyzed with Graphpad Prism 6.01 software. The results are shown in FIG. 3B.

Example 7: anti-human LAG-3 chimeric antibodies enhance immune function of primary T cells

The present invention evaluates the activation function of primary human T cells by anti-human LAG-3 antibody in a mixed Peripheral Blood Mononuclear Cell (PBMC) and SED system. Specifically, the method comprises freezing human PBMC (Allcells) at a ratio of 1X 10 per well⁵The PBMC cells were plated on a 96-well round bottom plate and subsequently SED (final concentration 400 ng/mL) and anti-human LAG-3 antibody or Isotype antibody (final concentration 10. mu.g/mL) were added to each well. Placing the mixed system at 37 ℃ and 5% CO₂Culturing in an incubator. After 5 days, an appropriate amount of the medium was taken and the IL-2 level in the medium was measured by ELISA. The results are shown in FIG. 4.

Claims

1. An anti-LAG 3 antibody or antigen-binding fragment thereof that inhibits binding of LAG-3 to human MHCII, the anti-LAG 3 antibody or antigen-binding fragment thereof comprising a Heavy Chain Variable Region (HCVR) and a Light Chain Variable Region (LCVR), wherein:

(a) heavy chain complementarity determining regions of the HCVR domain (HCDR 1, HCDR2, and HCDR 3), the immunoglobulin chain being comprised within SEQ ID NO: 5. 7, 9, 21, 23, 25, 37, 39, 41, 53, 55, 57; and/or

(b) Light chain complementarity determining regions (LCDR 1, LCDR2, and LCDR 3) of the LCVR domain, the immunoglobulin chain being comprised in SEQ ID NO: 11. 13, 15, 27, 29, 31, 43, 45, 47, 59, 61, 63.

2. The anti-LAG-3 antibody, or antigen-binding fragment thereof, of claim 1, which is an antibody comprising:

(1) the HCVR comprises the amino acid sequence as set forth in SEQ ID NO: 5, HCDR1 as set forth in SEQ ID NO: 7, and HCDR2 as set forth in SEQ ID NO: HCDR3 shown at 9; and/or the LCVR comprises the amino acid sequence as set forth in SEQ ID NO: 11, LCDR1 as shown in SEQ ID NO: 13, and LCDR2 as shown in SEQ ID NO: LCDR3 shown at 15; or

(2) The HCVR comprises the amino acid sequence as set forth in SEQ ID NO: 21, HCDR1 as set forth in SEQ ID NO: HCDR2 as shown in SEQ ID NO: HCDR3 shown at 25; and/or the LCVR comprises the amino acid sequence as set forth in SEQ ID NO: LCDR1 as shown in SEQ ID NO: 29, and LCDR2 as set forth in SEQ ID NO: LCDR3 shown at 31; or

(3) The HCVR comprises the amino acid sequence as set forth in SEQ ID NO: 37, HCDR1 as set forth in SEQ ID NO: 39, and HCDR2 as set forth in SEQ ID NO: HCDR3 shown at 41; and/or the LCVR comprises the amino acid sequence as set forth in SEQ ID NO: 43, LCDR1 as set forth in SEQ ID NO: 45, and LCDR2 as shown in SEQ ID NO: LCDR3 shown at 47; or

(4) The HCVR comprises the amino acid sequence as set forth in SEQ ID NO: 53, HCDR1 as set forth in SEQ ID NO: 55, and HCDR2 as set forth in SEQ ID NO: HCDR3 shown at 57; and/or the LCVR comprises the amino acid sequence as set forth in SEQ ID NO: LCDR1 as shown in SEQ ID NO: 61, and LCDR2 as shown in SEQ ID NO: LCDR3 shown at 63.

3. The anti-LAG-3 antibody or antigen-binding fragment thereof of claim 2, wherein the antibody or antigen-binding fragment thereof comprises the amino acid sequence of SEQ ID NO: 1/3, 17/19, 33/35, 49/51.

4. The anti-LAG-3 antibody, or antigen-binding fragment thereof, of claims 1-3, which is a full-length antibody.

5. The anti-LAG-3 antibody of claims 1-3, or antigen-binding fragment thereof, which is a Fab.

6. The anti-LAG-3 antibody, or antigen-binding fragment thereof, of any one of claims 1-5, which is a chimeric antibody.

7. The invention provides antibodies or antigen-binding fragments thereof comprising an HCVR comprising the amino acid sequence of SEQ ID NO: 1. SEQ ID NO: 17. SEQ ID NO: 33 or SEQ ID NO: 49 or a substantially similar sequence having at least 75% sequence identity thereto.

8. The invention provides antibodies or antigen-binding fragments thereof comprising an LCVR comprising the amino acid sequence of SEQ ID NO: 3. SEQ ID NO: 19. SEQ ID NO: 35 or SEQ ID NO: 51 or a substantially similar sequence having at least 75% sequence identity thereto.

9. The anti-LAG-3 antibody or antigen-binding fragment thereof of any one of claims 1-8, having a HCVR comprising the amino acid sequence of SEQ ID NO: 1. SEQ ID NO: 17. SEQ ID NO: 33 or SEQ ID NO: 49.