WO2024140863A1

WO2024140863A1 - Antibody targeting crtam and pd-l1 and use thereof

Info

Publication number: WO2024140863A1
Application number: PCT/CN2023/142554
Authority: WO
Inventors: 潘新荣; 周冠月; 张小猛; 蒋美玲; 严帅; 陈虹宇; 谢美娟
Original assignee: 南京圣和药业股份有限公司
Priority date: 2022-12-29
Filing date: 2023-12-28
Publication date: 2024-07-04
Also published as: CN118271451A

Abstract

The present invention relates to the field of antibody drugs, and particularly relates to an anti-CRTAM/anti-PD-L1 antibody, a drug combination comprising same and the use thereof. The anti-CRTAM/anti-PD-L1 antibody has significant anti-tumor activity and can be used in the preparation of anti-tumor drugs.

Description

Antibodies targeting CRTAM and PD-L1 and their applications

Technical Field

The present invention relates to the field of antibody drugs, and in particular to anti-CRTAM/anti-PD-L1 antibodies, pharmaceutical compositions comprising anti-CRTAM/anti-PD-L1 antibodies, and uses thereof.

Background technique

Programmed death-ligand 1 (PD-L1), also known as B7-H1 or CD274, is a type I transmembrane protein belonging to the immunoglobulin superfamily. It has a molecular weight of 40 kDa and contains an Ig-V and Ig-C-like extracellular domain, a transmembrane domain, and a short cytoplasmic tail that does not contain a typical signal motif. PD-L1 is widely expressed on different types of cells, including hepatocytes, vascular endothelial cells, and immune cells such as B cells and macrophages. It is one of the PD-1 ligands involved in immunosuppression. IFN-γ can induce upregulation of PD-L1 expression. Normally, upregulated PD-L1 binds to PD-1 on the surface of T cells, inhibits T cell activation to prevent autoimmune attack, and maintains cellular immune tolerance. PD-L1 is highly expressed in a variety of solid tumors, including non-small cell lung cancer, colorectal cancer, ovarian cancer, and renal cell carcinoma. Tumors use the signaling axis of PD-L1 and PD-1 to inhibit the activity and proliferation of CD8 ⁺ T cells, induce T cell anergy and exhaustion, build a local immune-tolerant tumor microenvironment, and promote tumor immune escape.

PD-L1 is expressed in human non-small cell lung cancer and promotes cancer cell proliferation; knockout of PD-L1 inhibits tumor cell proliferation and induces apoptosis in HCC827 and PC9 cell lines.

CRTAM (Class-I-restricted T cell associated molecule), or CD355, is a member of the immunoglobulin superfamily. CRTAM is a type I transmembrane protein composed of 393 amino acids with a molecular weight of 42.7 kDa. The expression of CRTAM is strictly regulated by NK cell activation receptors and T cell receptor (TCR) activation, and is restricted to activated immune cells, such as activated CD8 ⁺ T cells and NK cells. It is lowly expressed in PBMCs of normal people and highly expressed in PBMCs of asthmatic patients. The CRTAM gene promoter is positively regulated by AP-1 transcription factors, and TCR activation signals control the expression of CRTAM on CD8 + T cells through AP-1 regulatory elements. CRTAM's ligand Necl-2 belongs to the Nectin family of adhesion molecules, also known as TSLC1 (The tumor suppressor in lung cancer-1) or CADM1. The binding of Necl-2 to CRTAM can promote the cytotoxicity of NK cells and the release of IFN-γ by CD8 ⁺ T cells in vitro, and promote the rejection of NK cells to tumors in vivo. Existing literature shows that the expression of Necl-2 in tumor cells will inhibit the growth and proliferation of tumor cells, but the expression of Necl-2 in tumor cells will gradually decrease to silence, providing a possible mechanism for cancer cells to escape the surveillance of the immune system.

Necl-2 and CRTAM pathways have different signaling mechanisms from the PD-1-PD-L1 pathway and have complementary effects. By developing a bifunctional antibody targeting CRTAM and PD-L1, the bispecific antibody contains a PD-L1 binding portion and a CRTAM binding portion, which can not only block the binding of PD-L1 to PD-1, but also stimulate CRTAM, relieve the inhibitory effect of target cells on T cells, activate T cells, promote the release of IFN-γ, and enhance the killing effect of immune cells on tumor cells. It can double block the immune escape mechanism of tumors and regenerate immune-tolerant immune cells in the tumor microenvironment, thus making up for the poor treatment of patients with low PD-L1 expression tumors, and has better anti-tumor activity, targeting specificity and/or higher safety.

Summary of the invention

The present invention provides an anti-CRTAM/anti-PD-L1 antibody, which includes an anti-CRTAM antibody or an antigen-binding fragment thereof and an anti-PD-L1 antibody or an antigen-binding fragment thereof, wherein the anti-CRTAM antibody or the antigen-binding fragment thereof comprises a first heavy chain variable region and a first light chain variable region, wherein the first heavy chain variable region comprises a complementarity determining region 1 (H1CDR1) of the first heavy chain variable region, a complementarity determining region 2 (H1CDR2) of the first heavy chain variable region and/or a complementarity determining region 3 (H1CDR3) of the first heavy chain variable region, and the first light chain variable region comprises a complementarity determining region 1 (L1CDR1) of the first light chain variable region, a complementarity determining region 2 (L1CDR2) of the first light chain variable region and/or a complementarity determining region 3 (L1CDR3) of the first light chain variable region; and the anti-PD-L1 antibody or the antigen-binding fragment thereof is an antibody or the antigen-binding fragment thereof that specifically binds to PD-L1.

In some embodiments, the present invention provides an anti-CRTAM/anti-PD-L1 antibody, comprising an anti-CRTAM antibody or an antigen-binding fragment thereof and an anti-PD-L1 antibody or an antigen-binding fragment thereof, wherein the anti-CRTAM antibody or the antigen-binding fragment thereof comprises a first heavy chain variable region and a first light chain variable region, wherein:

(1) the first heavy chain variable region comprises H1CDR1, H1CDR2 and H1CDR3, whose amino acid sequences are SEQ ID NOs: 1, 2 and 3, respectively, or amino acid sequences having at least 85% sequence identity with the amino acid sequences shown in SEQ ID NOs: 1, 2 and 3; and

(2) The first light chain variable region comprises L1CDR1, L1CDR2 and L1CDR3, whose amino acid sequences are SEQ ID NO: 4, 5 and 6, respectively, or amino acid sequences that have at least 85% sequence identity with the amino acid sequences shown in SEQ ID NO: 4, 5 and 6.

In some embodiments, according to the anti-CRTAM/anti-PD-L1 antibody of the present invention, the anti-CRTAM antibody or antigen-binding fragment thereof comprises a first heavy chain variable region and a first light chain variable region, wherein:

(1) The amino acid sequence of the first heavy chain variable region is selected from:

(A1) the amino acid sequence shown in SEQ ID NO:17;

(A2) an amino acid sequence obtained by substituting, deleting or adding one or more amino acids to the amino acid sequence shown in (A1) and having the same or similar functions as the amino acid sequence shown in (A1); and

(A3) an amino acid sequence having at least 80% sequence identity with the amino acid sequence shown in (A1); and

(2) The amino acid sequence of the first light chain variable region is selected from:

(A4) the amino acid sequence shown in SEQ ID NO:19;

(A5) An amino acid sequence obtained by substituting, deleting or adding one or more amino acids to the amino acid sequence shown in (A4), and having the same or similar functions as the amino acid sequence shown in (A4); and

(A6) An amino acid sequence having at least 80% sequence identity with the amino acid sequence shown in (A4).

The amino acid sequence of the first heavy chain variable region is the amino acid sequence shown in SEQ ID NO:17, an amino acid sequence obtained by substituting, deleting or adding one or more amino acids in SEQ ID NO:17 and having at least 85%, or at least 90%, or at least 95%, or at least 98% sequence identity with SEQ ID NO:17, and the H1CDR1, H1CDR2 and H1CDR3 are the amino acid sequences shown in SEQ ID NO:1, 2 and 3, and the amino acid sequence of the first light chain variable region is SEQ ID NO:19, an amino acid sequence obtained by substituting, deleting or adding one or more amino acids in SEQ ID NO:19 and having at least 85%, or at least 90%, or at least 95%, or at least 98% sequence identity with SEQ ID NO:19, and the L1CDR1, L1CDR2 and L1CDR3 are the amino acid sequences shown in SEQ ID NO:4, 5 and 6.

In some embodiments, the present invention provides an anti-CRTAM/anti-PD-L1 antibody, which includes an anti-CRTAM antibody or an antigen-binding fragment thereof and an anti-PD-L1 antibody or an antigen-binding fragment thereof, wherein the anti-CRTAM antibody or its antigen-binding fragment is as defined in the above embodiments; and the anti-PD-L1 antibody or its antigen-binding fragment comprises a second heavy chain variable region and/or a second light chain variable region, wherein the second heavy chain variable region comprises a complementarity determining region 1 (H2CDR1) of a second heavy chain variable region, a complementarity determining region 2 (H2CDR2) of a second heavy chain variable region, and/or a complementarity determining region 3 (H2CDR3) of a second heavy chain variable region, and the second light chain variable region comprises a complementarity determining region 1 (L2CDR1) of a second light chain variable region, a complementarity determining region 2 (L2CDR2) of a second light chain variable region, and/or a complementarity determining region 3 (L2CDR3) of a second light chain variable region.

In some specific embodiments, the present invention provides an anti-CRTAM/anti-PD-L1 antibody, which comprises an anti-CRTAM antibody or an antigen-binding fragment thereof and an anti-PD-L1 antibody or an antigen-binding fragment thereof, wherein the anti-CRTAM antibody or an antigen-binding fragment thereof is as defined in the above embodiments, and the anti-PD-L1 antibody or an antigen-binding fragment thereof comprises a second heavy chain variable region and a second light chain variable region, wherein:

(1) the second heavy chain variable region comprises H2CDR1, H2DR2 and H2CDR3, whose amino acid sequences are SEQ ID NOs: 7, 8 and 9, respectively, or amino acid sequences having at least 85% sequence identity to the amino acid sequences shown in SEQ ID NOs: 7, 8 and 9; and

(2) The second light chain variable region comprises L2CDR1, L2CDR2 and L2CDR3, whose amino acid sequences are SEQ ID NO: 10, 11 and 12, respectively, or amino acid sequences that have at least 85% sequence identity with the amino acid sequences shown in SEQ ID NO: 10, 11 and 12.

In some specific embodiments, the present invention provides an anti-CRTAM/anti-PD-L1 antibody, which includes an anti-CRTAM antibody or an antigen-binding fragment thereof and an anti-PD-L1 antibody or an antigen-binding fragment thereof, wherein:

The anti-CRTAM antibody or antigen-binding fragment thereof comprises a first heavy chain variable region and a first light chain variable region, wherein:

(1) the first heavy chain variable region comprises H1CDR1, H1CDR2 and H1CDR3, whose amino acid sequences are SEQ ID NOs: 1, 2 and 3, respectively, or amino acid sequences having at least 85% sequence identity with the amino acid sequences shown in SEQ ID NOs: 1, 2 and 3;

(2) the first light chain variable region comprises L1CDR1, L1CDR2 and L1CDR3, whose amino acid sequences are SEQ ID NOs: 4, 5 and 6, respectively, or amino acid sequences having at least 85% sequence identity with the amino acid sequences shown in SEQ ID NOs: 4, 5 and 6; and

The anti-PD-L1 antibody or antigen-binding fragment thereof comprises a second heavy chain variable region and a second light chain variable region, wherein:

(1) the second heavy chain variable region comprises H2CDR1, H2DR2 and H2CDR3, whose amino acid sequences are SEQ ID NOs: 7, 8 and 9, respectively, or amino acid sequences having at least 85% sequence identity to the amino acid sequences shown in SEQ ID NOs: 7, 8 and 9;

In some specific embodiments, the present invention provides an anti-CRTAM/anti-PD-L1 antibody, wherein the anti-CRTAM antibody or antigen-binding fragment thereof comprises a first heavy chain variable region and a first light chain variable region, wherein:

(A1) the amino acid sequence shown in SEQ ID NO:17;

(A4) the amino acid sequence shown in SEQ ID NO:19;

(A6) an amino acid sequence having at least 80% sequence identity with the amino acid sequence shown in (A4); and

(1) The amino acid sequence of the second heavy chain variable region is selected from:

(B1) the amino acid sequence shown in SEQ ID NO:13;

(B2) an amino acid sequence obtained by substituting, deleting or adding one or more amino acids to the amino acid sequence shown in (B1) and having the same or similar functions as the amino acid sequence shown in (B1); and

(B3) an amino acid sequence having at least 80% sequence identity with the amino acid sequence shown in (B1); and

(2) The amino acid sequence of the second light chain variable region is selected from:

(B4) the amino acid sequence shown in SEQ ID NO:15;

(B5) an amino acid sequence obtained by substituting, deleting or adding one or more amino acids to the amino acid sequence shown in (B4) and having the same or similar functions as the amino acid sequence shown in (B4); and

(B6) An amino acid sequence having at least 80% sequence identity with the amino acid sequence shown in (B4).

In some specific embodiments, the present invention provides an anti-CRTAM/anti-PD-L1 antibody, wherein the amino acid sequence of the first heavy chain variable region is SEQ ID NO:17, and the amino acid sequence of the first light chain variable region is SEQ ID NO:19; and the amino acid sequence of the second heavy chain variable region is SEQ ID NO:13, and the amino acid sequence of the second light chain variable region is SEQ ID NO:15.

In some embodiments, the present invention provides an anti-CRTAM/anti-PD-L1 humanized antibody, wherein the heavy chain comprises a heavy chain constant region of human IgG1, IgG2, IgG3, IgG4 or a variant thereof, and the light chain comprises a light chain constant region of human κ, λ chain or a variant thereof.

In some specific embodiments, according to the anti-CRTAM/anti-PD-L1 antibody of the present invention, the anti-CRTAM humanized antibody or its antigen-binding fragment further comprises a heavy chain constant region of human IgG1, IgG2, IgG3 or IgG4 or a variant thereof, and a light chain constant region of human κ, λ chain or a variant thereof. In some preferred embodiments, the anti-CRTAM humanized antibody or its antigen-binding fragment of the present invention further comprises a heavy chain constant region of human IgG4 or a variant thereof, and a light chain constant region of human κ chain or a variant thereof.

In some specific embodiments, according to the anti-CRTAM/anti-PD-L1 antibody of the present invention, the anti-PD-L1 humanized antibody or its antigen-binding fragment further comprises a heavy chain constant region of human IgG1, IgG2, IgG3 or IgG4 or a variant thereof, and a light chain constant region of human κ, λ chain or a variant thereof. In some preferred embodiments, the anti-PD-L1 humanized antibody or its antigen-binding fragment of the present invention further comprises a heavy chain constant region of human IgG1, IgG2, IgG4 or a variant thereof, and a light chain constant region of human κ chain or a variant thereof.

In some embodiments, the present invention provides anti-CRTAM/anti-PD-L1 antibodies, wherein the anti-CRTAM antibody or antigen-binding fragment thereof and the anti-PD-L1 antibody or antigen-binding fragment thereof are Fab, Fv, sFv or F(ab) ₂ , respectively. In a specific embodiment, the anti-CRTAM/anti-PD-L1 antibody provided by the present invention is scF(ab) ₂ .

Preferably, the anti-CRTAM/anti-PD-L1 antibody in the above embodiments of the present invention is an anti-CRTAM/anti-PD-L1 bispecific antibody. In some embodiments, the bispecific antibody is a human antibody or a humanized antibody. In some embodiments, one of the binding specificities is for CRTAM, and the other binding specificity is for any other antigen. In some embodiments, one of the binding specificities is for PD-L1, and the other binding specificity is for CRTAM. The bispecific antibody of the present invention can be prepared as a full-length antibody or an antibody fragment (e.g., F(ab') ₂ bispecific antibody).

Methods for preparing bispecific antibodies are known in the art. Traditionally, the recombinant preparation of bispecific antibodies is based on the co-expression of two immunoglobulin heavy chain-light chain pairs, in which the two heavy chains have different specificities (Millstein and Cuello, Nature 305:537 (1983)). Due to the random distribution of immunoglobulin heavy and light chains, these hybridomas (quadromas) may produce a mixture of 10 different antibody molecules, of which only one molecule has the correct bispecific structure. The purification of the correct molecule is usually carried out by affinity chromatography steps, which is quite cumbersome and the product yield is low. Similar methods are disclosed in WO93/08829 and Traunecker et al., EMBO J. 10:3655 (1991).

According to a different approach, an antibody variable region with a desired binding specificity (antibody-antigen binding site) is fused to an immunoglobulin constant region sequence. In some embodiments, the constant region of an immunoglobulin heavy chain comprising at least a portion of the hinge, CH2, and CH3 regions is fused. In some embodiments, a second heavy chain constant region (CH1) comprising a site necessary for binding to a light chain is present in at least a portion of the fusion. DNA encoding the immunoglobulin heavy chain fusion fragment and the immunoglobulin light chain (if necessary) is inserted into different expression vectors and co-transfected into a suitable host organism. In embodiments where the optimal yield is provided when the three polypeptide chains used to construct are not equal in ratio, this provides great flexibility for adjusting the mutual ratio of the three polypeptide fragments. However, when at least two polypeptide chains are expressed in the same ratio to produce high yields or when the ratio is not particularly meaningful, it is possible to insert the coding sequence of two or all three polypeptide chains into one expression vector.

In one embodiment of the method, the bispecific antibody is composed of a hybrid immunoglobulin heavy chain with a first binding specificity in one arm and a hybrid immunoglobulin heavy chain-light chain pair (providing a second binding specificity) in the other arm. Since the presence of an immunoglobulin light chain in only one half of the bispecific molecule provides a convenient separation pathway, it was found that the asymmetric structure facilitates separation of the desired bispecific substance from the unwanted immunoglobulin chain composition. The method is disclosed in WO94/04690. For further information on the generation of bispecific antibodies, see, for example, Sureshetal., Methods in Enzymology 121:210 (1986).

According to another method, the interface between a pair of antibody molecules can be transformed so that the percentage of the heterodimer recovered from the recombinant cell culture is maximized. The interface comprises at least a portion of the antibody constant region CH3 domain. In this method, one or more small amino acid side chains at the interface of the first antibody molecule are replaced with larger side chains (for example tyrosine or tryptophan). By replacing the large amino acid side chains with smaller amino acid side chains (for example alanine or threonine), a compensatory "cavity" of the same or similar size as the large side chain is produced on the interface of the second antibody molecule. This provides a mechanism to improve the output of heterodimers compared to other undesirable end products such as homodimers.

Bispecific antibodies include cross-linked or "heteroconjugate" antibodies. For example, one heteroconjugate antibody can be coupled to avidin and the other heteroconjugate antibody can be coupled to biotin. Heteroconjugate antibodies can be prepared using any convenient cross-linking method. Suitable cross-linking agents are well known in the art and are disclosed in U.S. Pat. No. 4,676,980, along with a number of cross-linking techniques.

The bispecific antibodies of the present invention can be generated by antibody fragments. For example, chemical connection techniques can be used to prepare bispecific antibodies. Brennen et al., Science 229: 81 (1985) describes a method for generating F(ab') ₂ fragments by proteolytic cleavage of intact antibodies. These fragments are decomposed in the presence of a dithiol complexing agent, sodium arsenite (to stabilize adjacent dithiols and prevent the formation of intermolecular disulfide bonds). The resulting Fab' fragments are then converted into thionitrobenzoate (TNB) derivatives. One of the Fab'-TNB derivatives is then restored to Fab'-thiol by reduction of mercaptoethylamine and mixed with another Fab'-TNB derivative in an equimolar amount to form a bispecific antibody.

Fab'-SH fragments can be directly recovered from E. coli, and these fragments can be chemically coupled to form bispecific antibodies. Shalaby et al., J. Exp. Med. 175: 217-225 (1992) describes the generation of fully humanized bispecific antibody F(ab') ₂ molecules. Each Fab' fragment is secreted separately by E. coli and directed chemically coupled in vitro to form bispecific antibodies.

In some embodiments, the bispecific antibody fragments of the present invention can be generated and isolated directly from recombinant cell culture. For example, bispecific antibodies can be generated using leucine zippers (Kostelny et al., J. Immunol. 148 (5): 1547-1553 (1992)). Leucine zipper peptides from Fos and Jun proteins are connected to the Fab' portions of two different antibodies by gene fusion. Antibody homodimers are decomposed in the hinge region to form monomers and then reoxidized to form antibody heterodimers. This method can also be used to generate antibody homodimers. The bispecific antibody technology provides other mechanisms for preparing bispecific antibody fragments. The bispecific antibody fragment comprises a heavy chain variable region (VH) and a light chain variable region (VL) connected by a linker, and the linker is too short to allow the two domains on the same chain to be paired. Therefore, the VH and VL domains on one fragment are forced to pair with the complementary VL and VH domains on another fragment, thereby forming two antigen binding sites. In another embodiment, bispecific antibody fragments can be constructed through the use of single-chain Fv (sFv) dimers.

The present invention encompasses multivalent antibodies with more than two valencies, for example, trispecific antibodies can be prepared. Multivalent antibodies can be internalized (and/or catabolized) by cells expressing antigens to which the antibodies bind faster than bivalent antibodies. The antibodies of the present invention can be multivalent antibodies with three or more antigen binding sites (e.g., tetravalent antibodies) that can be easily generated by recombinant expression of nucleic acids encoding antibody polypeptide chains. Multivalent antibodies may comprise a dimerization domain and three or more antigen binding sites. In some embodiments, the dimerization domain comprises (or consists of) an Fc region or a hinge region. In this case, the antibody will comprise an Fc region and three or more antigen binding sites at the amino terminus of the Fc region. In some embodiments, the multivalent antibody comprises (or consists of) three to about eight antigen binding sites. In some embodiments, the multivalent antibody comprises four antigen binding sites. The multivalent antibody comprises at least one polypeptide chain (e.g., two polypeptide chains), wherein the polypeptide chain comprises two or more variable regions. The multivalent antibody of the present invention may further comprise at least two (e.g., four) light chain variable region polypeptides. The multivalent antibody of the present invention may comprise, for example, about two to about eight light chain variable region polypeptides.The light chain variable region polypeptide of the present invention comprises a light chain variable region, and optionally further comprises a CL domain.

In a bispecific antibody comprising a CRTAM targeting portion and a PD-L1 targeting portion, one of the CRTAM targeting portion and the PD-L1 targeting portion can be a full-length antibody, and the other can be an antigen-binding fragment (e.g., scFv) comprising a heavy chain CDR, a light chain CDR, or a combination thereof. A full-length antibody targeting one of the CRTAM and PD-L1 proteins and an antigen-binding fragment targeting another protein can be chemically linked (e.g., covalently linked) directly or through a peptide linker. An antigen-binding fragment (e.g., scFv) can be directly or through a peptide linker to the N-terminus of a full-length antibody (e.g., the N-terminus of a light chain or a heavy chain of a full-length antibody), the C-terminus of a full-length antibody (e.g., the C-terminus of a heavy chain (or Fc or CH3 domain) of a full-length antibody), or both.

In one embodiment, the bispecific antibody may comprise a full-length anti-CRTAM antibody, an antigen-binding fragment of an anti-PD-L1 antibody (e.g., scFab, scFv), and a peptide linker therebetween. In other embodiments, the bispecific antibody may comprise a full-length anti-CRTAM antibody, an antigen-binding fragment of an anti-PD-L1 antibody (e.g., scFab, scFv), and a peptide linker therebetween.

In one embodiment, the scFv contained in the bispecific antibody may contain the heavy chain variable region and the light chain variable region in any order. For example, the scFv contained in the bispecific antibody may contain the heavy chain variable region and the light chain variable region in the direction from the N-terminus to the C-terminus and optionally a peptide linker therebetween, or alternatively, the scFv contained in the bispecific antibody may contain the light chain variable region and the heavy chain variable region in the direction from the N-terminus to the C-terminus and optionally a peptide linker therebetween.

In some embodiments, the peptide linker can include, for example, Gly, Asn and/or Ser residues, and can also include neutral amino acids, such as Thr and/or Ala. The amino acid sequence applicable to the peptide linker can be those known in the relevant art. Meanwhile, the length of the peptide linker can be determined differently within such limits that the fusion protein function is not affected. For example, the peptide linker can be formed by including a total of about 1 to about 100, about 2 to about 50, or about 5 to about 25 groups selected from by Gly, Asn, Ser, Thr and Ala composition. In one embodiment, the peptide linker can be expressed as (GmSl)n (m, l and n are independently about 1 to about 10 integers, particularly about 2 to about 5 integers).

In another embodiment, the CRTAM targeting moiety and the PD-L1 targeting moiety can both be full-length antibodies or antigen-binding fragments comprising heavy chain CDRs, light chain CDRs, or a combination thereof.

In another embodiment, the bispecific antibody can be in the form of a heterodimer comprising a first arm including a pair of a second heavy chain and a second light chain targeting one of CRTAM and PD-L1, and a second arm including a pair of a first heavy chain and a first light chain targeting the other.

In one embodiment, the full-length antibody may be in the form of a full-length immunoglobulin (e.g., IgG, IgM, IgA, IgE, or IgD, such as human IgG, human IgM, human IgA, human IgE, or human IgD), and the antigen-binding fragment may be selected from the group consisting of Fab, Fab', F(ab') ₂ , Fd, Fv, scFv, scFab, single-chain antibody, sdFv, etc. For example, the full-length antibody may be in the form of a full-length human IgG (human IgG1, human IgG2, human IgG3, or human IgG4), and the antigen-binding fragment may be a scFv.

For example, the antibodies described herein may contain a flexible linker sequence, or may be modified to add a functional moiety (eg, PEG, a drug, a toxin, or a label).

In some specific embodiments, according to the anti-CRTAM/anti-PD-L1 bispecific antibody of the present invention, the structure of the anti-PD-L1 antibody or its antigen-binding fragment is (VL-CL)-peptide linker-(VH)-IgG4CH, and the structure of the anti-CRTAM antibody or its antigen-binding fragment is self-assembled by (VL-CL) and (VH)-IgG4CH. In some specific embodiments, the peptide linker is in the form of (GGGGS)n, wherein n is 1-12, preferably 3-10, and more preferably 6-8, for example 6, 7, or 8 GGGGS repeats. In other specific embodiments, the IgG4CH in the (VL-CL)-peptide linker-(VH)-IgG4CH targeting the PD-L1 portion is an IgG4CH segment containing S228P, L235E, T366W, and S354C mutations to form a "Knob" structure, and the IgG4CH in the (VH)-IgG4CH targeting the CRTAM portion is an IgG4CH segment containing S228P, L235E, Y349C, T366S, L368A, and Y407V mutations to form a "Hole" structure. In some specific embodiments, the amino acid sequence of VL in the (VL-CL)-peptide linker-(VH)-IgG4CH targeting the PD-L1 portion is SEQ ID NO: 15, the amino acid sequence of CL is SEQ ID NO: 16, the amino acid sequence of VH is SEQ ID NO: 13, and the amino acid sequence of IgG4CH is SEQ ID NO: 14; and/or the amino acid sequence of VL in the (VL-CL) and (VH)-IgG4CH targeting the CRTAM portion is SEQ ID NO: 19, the amino acid sequence of CL is SEQ ID NO: 16, the amino acid sequence of VH is SEQ ID NO: 17, and the amino acid sequence of IgG4CH is SEQ ID NO: 18.

In some embodiments, the anti-PD-L1 antibody or antigen-binding fragment thereof according to the present invention, wherein the antibody is a humanized antibody or a fully human antibody.

Another aspect of the present invention provides isolated nucleic acids. In some embodiments, the isolated nucleic acids according to the present invention encode the anti-PD-L1/anti-CRTAM antibodies of the present invention. In some embodiments, the isolated nucleic acids according to the present invention encode the anti-CRTAM antibodies of the present invention or their antigen-binding fragments. In other embodiments, the isolated nucleic acids according to the present invention encode the anti-PD-L1 antibodies of the present invention or their antigen-binding fragments.

In a specific embodiment, according to the isolated nucleic acid of the present invention, the nucleotide sequence encoding the first heavy chain variable region SEQ ID NO: 17 is shown as SEQ ID NO: 22, and the nucleotide sequence encoding the first light chain variable region SEQ ID NO: 19 is shown as SEQ ID NO: 23. In another specific embodiment, according to the isolated nucleic acid of the present invention, the nucleotide sequence encoding the second heavy chain variable region SEQ ID NO: 13 is shown as SEQ ID NO: 20, and the nucleotide sequence encoding the second light chain variable region SEQ ID NO: 15 is shown as SEQ ID NO: 21.

Another aspect of the present invention provides an expression vector. In some embodiments, the expression vector of the present invention expresses the anti-CRTAM/anti-PD-L1 bispecific antibody of the present invention. In some embodiments, the expression vector of the present invention expresses the anti-CRTAM antibody of the present invention or its antigen-binding fragment. In other embodiments, the expression vector of the present invention expresses the anti-PD-L1 antibody of the present invention or its antigen-binding fragment. In some embodiments, according to the expression vector of the present invention, the vector expressing the anti-CRTAM antibody of the present invention or its antigen-binding fragment and the vector expressing the anti-PD-L1 antibody or its antigen-binding fragment are the same expression vectors. According to the expression vector of the present invention, it comprises an isolated nucleic acid molecule of the present invention.

Another aspect of the present invention provides a host cell transformed with the expression vector as described above.

In some embodiments, the host cell according to the present invention is selected from prokaryotic cells and eukaryotic cells. In some embodiments, the host cell is a bacterium, preferably Escherichia coli. In another preferred embodiment, the host cell is a mammalian cell.

Another aspect of the present invention provides a method for preparing the anti-CRTAM/anti-PD-L1 bispecific antibody of the present invention, comprising the steps of expressing the antibody in the host cell and isolating the antibody from the host cell.

Another aspect of the present invention provides a pharmaceutical composition comprising an anti-CRTAM/anti-PD-L1 bispecific antibody of the present invention and a pharmaceutically acceptable carrier. In some embodiments, the present invention provides a pharmaceutical composition comprising an anti-CRTAM/anti-PD-L1 bispecific antibody of the present invention, and further comprising other active components, such as other antibodies, targeted drugs, etc. In some embodiments, the pharmaceutically acceptable carrier is selected from antioxidants, polypeptides, proteins, hydrophilic polymers, amino acids, sugars, chelating agents, sugar alcohols, ions and surfactants. In a specific embodiment, the pharmaceutically acceptable carrier is a buffered aqueous solution. In another specific embodiment, the pharmaceutically acceptable carrier is in the form of liposomes.

Another aspect of the present invention provides a chimeric antigen receptor (CAR) fusion protein, which comprises an anti-CRTAM antibody or antigen binding fragment thereof and/or an anti-PD-L1 antibody or antigen binding fragment thereof of the present invention. In some embodiments, the chimeric antigen receptor fusion protein comprises an anti-CRTAM antibody or antigen binding fragment thereof of the present invention, which is a single-chain variable fragment (scFv) of V _H and V _L for CRTAM antigen. In other embodiments, the chimeric antigen receptor fusion protein comprises an anti-PD-L1 antibody or antigen binding fragment thereof of the present invention, which is a single-chain variable fragment (scFv) of V _H and V _L for PD-L1 antigen. In other embodiments, the chimeric antigen receptor fusion protein comprises a first single-chain variable fragment (scFv) of V _H and V _L for CRTAM antigen and a second single-chain variable fragment (scFv) of V _H and V _L for PD-L1 antigen. The first scFv of _VH and _VL against CRTAM antigen has H1CDR1, H1CDR2 and H1CDR3 of the first heavy chain variable region and L1CDR1, L1CDR2 and L1CDR3 of the first light chain variable region described in the above embodiment. The second scFv of _VH and _VL against PD-L1 antigen has H2CDR1, H2DR2 and H2CDR3 of the second heavy chain variable region and L2CDR1, L2CDR2 and L2CDR3 of the second light chain variable region described in the above embodiment.

The anti-CRTAM/anti-PD-L1 bispecific antibody of the present invention can be mixed with a pharmaceutically acceptable carrier, diluent or excipient to prepare a pharmaceutical preparation suitable for oral or parenteral administration. The method of administration includes, but is not limited to oral, intradermal, intramuscular, intraperitoneal, intravenous, intracerebral, intraocular, intratracheal, subcutaneous, and intranasal routes. The preparation can be administered by any route, such as by infusion or push injection, and by a route of absorption through the epithelium or skin mucosa (such as oral mucosa or rectum, etc.). Administration can be systemic or local. The preparation can be prepared by methods known in the art and includes carriers, diluents or excipients conventionally used in the field of pharmaceutical preparations.

Another aspect of the present invention provides a method for treating and/or preventing a disease associated with CRTAM, PD-L1 or both, comprising administering the anti-CRTAM/anti-PD-L1 bispecific antibody of the present invention or the pharmaceutical composition of the present invention to an individual in need thereof.

Another aspect of the present invention provides the use of the anti-CRTAM/anti-PD-L1 bispecific antibody or anti-CRTAM antibody of the present invention or the pharmaceutical composition of the present invention in the preparation of a drug for treating and/or preventing a disease associated with CRTAM, PD-L1 or both. In some embodiments, the disease associated with CRTAM, PD-L1 or both includes hematological tumors, lymphomas, breast cancer, lung cancer, gastric cancer, intestinal cancer, esophageal cancer, ovarian cancer, cervical cancer, kidney cancer, bladder cancer, pancreatic cancer, glioma and/or melanoma. The tumor can be any tumor expressing CRTAM protein, such as bladder cancer, liver cancer, colon cancer, rectal cancer, endometrial cancer, leukemia, lymphoma, pancreatic cancer, lung cancer (such as small cell lung cancer, non-small cell lung cancer, etc.), breast cancer, urethral cancer, head and neck cancer, gastrointestinal cancer, gastric cancer, esophageal cancer, ovarian cancer, kidney cancer, melanoma, prostate cancer, thyroid cancer, etc. The tumor can be a primary or metastatic tumor. In some embodiments, the present invention provides the use of the above-mentioned anti-CRTAM/anti-PD-L1 bispecific antibody or the pharmaceutical composition of the present invention in the preparation of anti-tumor drugs, for example, the tumor is selected from blood tumors, lymphomas, breast cancer, lung cancer, gastric cancer, intestinal cancer, esophageal cancer, ovarian cancer, cervical cancer, kidney cancer, bladder cancer, pancreatic cancer, glioma and melanoma.

The anti-CRTAM/anti-PD-L1 bispecific antibody provided by the present invention has a significant anti-tumor effect and can significantly inhibit tumor growth. The immunogenicity of the humanized antibody is greatly reduced, and the rejection reaction of the human immune system to exogenous monoclonal antibodies is effectively eliminated. It can be used in the preparation of drugs for the treatment of various tumor diseases and has broad market prospects.

definition

Unless otherwise defined, the meaning of the scientific and technical terms used herein is the meaning commonly understood by those skilled in the art. The nomenclature and techniques used in cell and tissue culture, molecular biology, and protein and oligo- or polynucleotide chemistry and hybridization described herein are well known and commonly used in the art. For recombinant DNA, oligonucleotide synthesis and tissue culture and transformation (such as electroporation, lipofection), standard techniques are used. Enzymatic reactions and purification techniques are performed according to the manufacturer's instructions or methods commonly used in the art or described herein. The aforementioned techniques and methods are generally used as described in a number of comprehensive and more specific documents that are well known in the art and cited and discussed in this specification. See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual (2nd Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (1989)). The nomenclature and laboratory methods and techniques used in analytical chemistry, synthetic organic chemistry, and medical and pharmaceutical chemistry described herein are well known and commonly used in the art.

In the present invention, the term "at least 80% sequence identity" refers to at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% sequence identity. In the present invention, the term "at least 85% sequence identity" refers to at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% sequence identity. In some preferred embodiments, the sequence identity described in the present invention can be at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%. Sequence comparison and identity percentage determination between two sequences can be performed by BLASTN/BLASTP algorithm on the website of National Center For Biotechnology Instutute.

In an antibody molecule, three hypervariable regions of the light chain and three hypervariable regions of the heavy chain are arranged relative to each other in three-dimensional space to form an antigen-binding surface. The antigen-binding surface is complementary to the three-dimensional surface of the bound antigen, and the three hypervariable regions of each heavy and light chain are called "complementarity determining regions" or "CDRs". The assignment of amino acids to each domain is based on Kabat, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Maryland (1987 and 1991)) or Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987), Chothia et al., Nature 342:878-883 (1989).

The "antibody" of the present invention refers to a polypeptide or polypeptide complex that specifically recognizes and binds to an antigen. The antibody can be a complete antibody and any antigen-binding fragment or single chain thereof. The "antibody" of the present invention includes any protein or peptide that contains at least a portion of an Ig molecule that has a biological activity of binding to an antigen. Examples of the "antibody" of the present invention include, but are not limited to, a heavy chain or light chain CDR or a ligand-binding portion thereof, a heavy chain or light chain variable region, a heavy chain or light chain constant region, a framework region or any portion thereof.

The "antigen-binding fragment" described in the present invention includes Fab fragments, Fab' fragments, F(ab')2 fragments, and Fv fragments and scFv fragments that bind to human CRTAM or PD-L1 with antigen binding activity. The Fv fragment contains the second heavy chain variable region and the second light chain variable region of the antibody, but has no constant region, and is the smallest antibody fragment with all antigen binding sites. Generally, the Fv antibody also contains a polypeptide linker between the VH and VL domains, and is capable of forming the structure required for antigen binding. The two antibody variable regions can also be connected into a polypeptide chain using different connectors, which is called a single-chain antibody or single-chain Fv (scFv). The anti-CRTAM or anti-PD-L1 antibody of the present invention may be a single-chain variable region fragment (scFv), which is derived from a single-chain polypeptide of an antibody and retains the ability to bind to an antigen. Examples of scFv include antibody polypeptides formed by recombinant DNA technology, in which the Fv regions of immunoglobulin heavy chain (H chain) and light chain (L chain) fragments are connected via spacer sequences. Various methods for preparing scFv are well known to those skilled in the art.

The antibody of the present invention refers to an immunoglobulin molecule or an immunologically active portion thereof, i.e., a molecule comprising an antigen binding site that specifically binds to (immunoreacts with) an antigen. "Specific binding" means that the antibody reacts with one or more antigenic determinants of an antigen but does not react with other polypeptides or binds to other polypeptides with very low affinity (Kd> ^10-6 ). Antibodies include, but are not limited to, polyclonal, monoclonal, chimeric, dAb (domain antibodies), single chain, Fab, Fab' and F(ab')2 fragments, Fv, scFv and Fab expression libraries. Monoclonal antibodies (mAbs) are antibodies obtained from a single clonal cell line, and the cell line is not limited to eukaryotic, prokaryotic or phage clonal cell lines. Monoclonal antibodies or antigen-binding fragments can be obtained by recombination using, for example, hybridoma technology, recombinant technology, phage display technology, and synthetic technology such as CDR grafting or other existing technologies.

The "bispecific antibody" described in the present invention refers to a monoclonal antibody that has binding specificity for at least two different antigens.

The "peptide linkers" described in the present invention may be those including 1 to 10, particularly 2 to 50, any amino acids, and may include any kind of amino acids without any limitation.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 is a schematic diagram of the structure of the bispecific ScFab (HuPL721-C110-HK).

Detailed ways

The following representative examples are intended to better illustrate the present invention, but are not intended to limit the scope of protection of the present invention. The experimental methods in the following examples where the conditions are not specified are usually carried out under conventional conditions, such as the Cold Spring Harbor Antibody Technology Laboratory Manual, Molecular Cloning Manual, etc., or under the conditions recommended by the raw material or commodity manufacturer. The materials and reagents used in the examples were commercially available unless otherwise specified.

Example 1: Preparation of CRTAM antibody-related antigen protein and positive control antibody

1. Construction of expression vectors for antigen protein and positive control antibody

(1) Construction of expression vector for antigen protein

A gene fragment encoding the full length of human CRTAM protein was synthesized, and the amino acid sequence was designed as shown in SEQ ID NO: 26. The nucleotide sequence was cloned into the eukaryotic expression plasmid pTargetT to obtain its expression plasmid pT-hCRTAM.

A gene fragment encoding the full-length monkey CRTAM protein was synthesized, and the amino acid sequence was designed as shown in SEQ ID NO: 27. The nucleotide sequence was cloned into the eukaryotic expression plasmid pTargetT to obtain its expression plasmid pT-cCRTAM.

The amino acid sequence of the fused human CRTAM protein extracellular region and mIgG1-Fc tag is shown in SEQ ID NO: 28. After codon optimization of the human CRTAM protein extracellular region sequence, the nucleotide sequence of hCRTAM-mFc with the tag was synthesized and cloned into the eukaryotic expression plasmid pHR to obtain its expression plasmid pHR-hCRTAM-mFc.

The amino acid sequences of the fused extracellular region of human CRTAM protein and hIgG1-Fc or His tag are shown in SEQ ID NO:29 and SEQ ID NO:30. After codon optimization of the above amino acid sequences, the nucleotide sequences of hCRTAM-hFc and hCRTAM-His with tags were synthesized and cloned into the eukaryotic expression plasmid pHR, respectively, to obtain the expression plasmids pHR-hCRTAM-hFc and pHR-hCRTAM-His.

The amino acid sequence of the fused monkey CRTAM protein extracellular region and mIgG1-Fc tag is shown in SEQ ID NO: 31. After codon optimization of the monkey CRTAM protein extracellular region sequence, the nucleotide sequence of cCRTAM-mFc with the tag was synthesized and cloned into the eukaryotic expression plasmid pHR to obtain its expression plasmid pHR-cCRTAM-mFc.

The amino acid sequences of the fused monkey CRTAM protein extracellular region and hIgG1-Fc or His tag are shown in SEQ ID NO:32 and SEQ ID NO:33. After codon optimization of the above amino acid sequences, the nucleotide sequences of cCRTAM-hFc and cCRTAM-His with tags were synthesized and cloned into the eukaryotic expression plasmid pHR, respectively, to obtain the expression plasmids pHR-cCRTAM-hFc and pHR-cCRTAM-His.

The amino acid sequence of the fused mouse CRTAM protein extracellular region and mIgG1-Fc tag is shown in SEQ ID NO: 34. After codon optimization of the mouse CRTAM protein extracellular region sequence, the nucleotide sequence of mCRTAM-mFc with the tag was synthesized and cloned into the eukaryotic expression plasmid pHR to obtain its expression plasmid pHR-mCRTAM-mFc.

The amino acid sequences of the fused mouse CRTAM protein extracellular region and hIgG1-Fc or His tag are shown in SEQ ID NO:35 and SEQ ID NO:36. After codon optimization of the above amino acid sequences, the nucleotide sequences of mCRTAM-hFc and mCRTAM-His with tags were synthesized and cloned into the eukaryotic expression plasmid pHR, respectively, to obtain the expression plasmids pHR-mCRTAM-hFc and pHR-mCRTAM-His.

(2) Construction of expression vector for ligand protein

The amino acid sequence of the fused human CADM1 protein extracellular region and mIgG1-Fc tag is shown in SEQ ID NO: 37. The above amino acid sequence was codon-optimized to synthesize the nucleotide sequence of CADM1-mFc with the tag. It was cloned into the eukaryotic expression plasmid pHR to obtain its expression plasmid pHR-CADM1-mFc.

The amino acid sequences of the fused extracellular region of human CADM1 protein and hIgG1-Fc or His tag are shown in SEQ ID NO:38 and SEQ ID NO:39. After codon optimization of the above amino acid sequences, the nucleotide sequences of CADM1-hFc and CADM1-His with tags were synthesized and cloned into the eukaryotic expression plasmid pHR, respectively, to obtain the expression plasmids pHR-CADM1-hFc and pHR-CADM1-His.

(3) Construction of expression vector for positive control antibody

The humanized antibody 5A11 disclosed in PCT application WO2019/086878 (hereinafter referred to as 5A11) was used as a positive control antibody. 5A11 was prepared according to the method disclosed in WO2019/086878. The amino acid sequence of 5A11 is as follows:

5A11 heavy chain amino acid sequence: SEQ ID NO:40;

5A11 light chain amino acid sequence: SEQ ID NO:41;

The codons of the amino acid sequence corresponding to the 5A11 antibody were artificially optimized, and its light and heavy chain gene fragments were cloned into the eukaryotic expression plasmid pHR, respectively, to obtain the heavy chain eukaryotic expression plasmid pHR-5A11-hG1m and the light chain expression plasmid pHR-5A11-hλ of 5A11.

2. Expression and purification of antigen protein

(1) Construction of stable transfected cell lines expressing antigenic proteins

The eukaryotic expression plasmid pT-hCRTAM was electrotransfected into CHO-K1 cells (Shanghai Institute of Cell Biology, Chinese Academy of Sciences) at 160V voltage and 15msec square pulses, and cultured in an incubator at 37°C and 5% CO ₂ concentration. After 24h, the cells were cultured under pressure in a medium containing 1000μg/mL G418 (Gibco, #10131-027). 16 days after transfection, the positive rate of the transfected pool was detected by flow cytometry, and the cells of the pool with a higher positive rate were plated (according to a cell density of 1×10 ⁶ /mL, 100μL/well, 96-well plate), and the cells were incubated with 5A11 antibody and Goat pAb to Hu IgG (PE) (Abcam, ab98596) antibody, and the mean value at a wavelength of 392nm was detected by flow cytometry (ACEABIO, Novocyte 2060R), and data analysis was generated using GraphPad. The positive cell lines were subcloned, and the cloned CHO-K1 cell line with high expression of hCRTAM protein was selected and named CHO-K1-hCRTAM.

The eukaryotic expression plasmid pT-cCRTAM was electrotransfected into CHO-K1 cells (Shanghai Institute of Cell Biology, Chinese Academy of Sciences) at 160V voltage and 15msec square pulses, and cultured in an incubator at 37°C and 5% CO ₂ concentration. After 24h, the cells were cultured under pressure in a medium containing 1000μg/mL G418 (Gibco, #10131-027). 16 days after transfection, the positive rate of the transfected pool was detected by flow cytometry, and the cells of the pool with a higher positive rate were plated (according to a cell density of 1×10 ⁶ /mL, 100μL/well, 96-well plate), and the cells were incubated with 5A11 antibody and Goat pAb to Hu IgG (PE) (Abcam, ab98596) antibody, and the mean value at a wavelength of 392nm was detected by flow cytometry (ACEABIO, Novocyte 2060R), and data analysis was generated using GraphPad. The positive cell lines were subcloned, and the cloned CHO-K1 cell line with high expression of cCRTAM protein was selected and named CHO-K1-cCRTAM.

(2) Expression of tagged antigen protein

In a 1L cell culture flask, inoculate 293E cells (from ATCC) at a density of 1×10 ⁶ cells/ml, add fresh preheated FreeStyle293 medium to a total volume of 250 mL, and culture overnight at 37°C, 8% CO ₂ , and 100 rpm in a humidified cell shaker. Take 7.5 mL of FreeStyle293 medium, add 500 μL of 1 mg/mL PEI solution, mix well, let stand for 5 minutes, and take 250 μg of the plasmid to be transfected and add 8 mL The cells were mixed evenly in FreeStyle293 culture medium and allowed to stand for 5 minutes. The label antigen protein plasmids pHR-hCRTAM-mFc, pHR-hCRTAM-hFc, pHR-hCRTAM-His, pHR-cCRTAM-mFc, pHR-cCRTAM-hFc, pHR-cCRTAM-His, pHR-mCRTAM-mFc, pHR-mCRTAM-hFc, pHR-mCRTAM-His, pHR-CADM1-mFc, pHR-CADM1-hFc, and pHR-CADM1-His were transfected respectively, and the positive control antibody 5A11 heavy chain plasmid pHR-5A11-hG1m and light chain plasmid pHR-5A11-hλ were co-transfected at a mass ratio of 1.1:1. The mixed solution of PEI and FreeStyle293 medium was added to the plasmid, mixed evenly, and allowed to stand at room temperature for 20 minutes, then added to the cell culture, and cultured at 37°C, 8% _CO2 , and 100rpm in a humidified cell shaker. On the first and third days after cell transfection, the cells were fed, and 12.5mL of OPM-CHO PFF05 (Shanghai Aopuma Biotechnology Co., Ltd., F81279), 5mL of glucose (mother liquor concentration of 180g/L) and 2.5mL of glutamine (mother liquor concentration of 200mM) were added to each bottle. When the cell viability dropped to 75%, the cell supernatant was collected. The cell culture was centrifuged at 2000rpm for 5min, the supernatant was collected, and then centrifuged at 6000rpm for 20min, the supernatant was collected, and filtered using 0.45μm and 0.22μm filter cups, respectively, and the collected filtrate was stored in a 4°C refrigerator for purification.

(3) Affinity chromatography column purification

Use AKTA (GE, AKTA pure-150) to purify the protein using affinity chromatography column according to the properties of the protein.

Example 2: Preparation of anti-CRTAM monoclonal antibodies

1. Preparation of hybridoma monoclones

(1) Animal immunization

The experimental animals were immunized with hCRTAM antigen proteins with different labels and adjuvants, or CHO-K1-hCRTAM cell lines were used for cell immunization. The experimental animals included Balb/c mice, SJL mice and SD rats. Protein immunization: For mouse immunization, 50 μg of antigen was used to immunize one mouse for the first immunization, and 25 μg of antigen protein was used per mouse in the later period; for SD rat immunization, 100 μg of antigen was used to immunize one rat for the first immunization, and 50 μg of antigen protein was used per rat in the later period. The immune adjuvant can be Freund's adjuvant (Sigma) or Quick Antibody-Mouse5W (Q5W, Beijing Biolong Immunotechnology Co., Ltd.). Freund's adjuvant was used to emulsify the antigen, and the hCRTAM antigen protein samples with different labels were added dropwise to the adjuvant solution, and vortexed while adding to mix thoroughly. The dosage of the adjuvant was based on the instructions. After mixing evenly to form an oil-in-water emulsion, immunization was performed. Quick Antibody-Mouse5W was used as an adjuvant, and hCRTAM antigen protein samples with different labels were mixed with Quick Antibody-Mouse5W at a volume ratio of 1:1. After mixing, SD rats were immunized by intramuscular injection. Cellular immunization: CHO-K1-hCRTAM cells were resuspended in PBS, and mice were immunized at 1×10 ⁷ cells/mouse; SD rats were immunized at 2×10 ⁷ cells/mouse.

(2) Hybridoma fusion

Obtaining and preparing spleen cells: After boosting immunization, mice/rats were killed and soaked in 75% alcohol. The spleen was dissected and removed. After grinding with a grinding rod, it was filtered through a cell screen to prepare a single cell suspension. The spleen cell suspension was centrifuged at 2000rpm for 5min and the supernatant was discarded. 2mL of red blood cell lysis buffer was added to lyse the red blood cells at room temperature for 2min, PBS was added to 20mL, and centrifuged at 1500rpm for 7min, the supernatant was discarded, and the viable cells were counted after resuspending. Sp2/0 cells in the culture flask were collected, centrifuged at 1000rpm for 5min, the supernatant was discarded, and the viable cells were counted after resuspending. The cells were mixed at a ratio of spleen cells: Sp2/0 cells = 1.6:1, and the supernatant was discarded after centrifugation at 1500rpm for 7min. The cells were resuspended with 20mL of electrotransfer buffer and centrifuged at 1500rpm for 7min. The supernatant was discarded and repeated once. The cells were resuspended with an appropriate amount of electrotransfer buffer to ensure that the cell concentration was about 2×10 ⁷ cells/mL. The cell suspension was added to a 9mL electrotransfer fusion tank for fusion. After fusion, transfer the cell suspension to 15mL RPMI 1640 complete medium containing 20% FBS and place at room temperature for 20 minutes. Resuspend the fused cells in RPMI 1640 medium containing 1×HAT, 1×BIOMYC3, and 20% FBS. Add the cell suspension to several 96-well cell culture plates at 100μL/well to ensure that the cell volume per well is about 4×10 ⁴ cells/well, and culture in a 37℃ cell culture incubator. After 5 days, add 100μL/well RPMI 1640 complete medium (containing 20% FBS, 1×HAT, 1×BIOMYC-3).

(3) Hybridoma and subclone screening

One week after fusion, the cell culture supernatant of the hybridoma mother clone was taken, and the hybridoma mother clones binding to hCRTAM protein and cCRTAM protein were screened by ELISA, and the mother clones that could bind to CHO-K1-hCRTAM cell line were further screened by flow cytometry.

The mother clones with strong binding ability were screened by ELISA and FACS, and the positive mother clones were subcloned by limiting dilution method. After one week of culture, the binding activity of the subclone supernatant with hCRTAM protein was detected by ELISA to obtain the monoclonal cell line secreting anti-hCRTAM antibody. A better monoclonal cell line was selected and marked as C110.

2. Preparation of monoclonal antibodies

The monoclonal antibody cell line was determined based on the results of the subclone supernatant activity analysis and expanded. The culture conditions were 1640 medium containing 10% FBS, 1×NAEE, 1× sodium pyruvate, and 1% penicillin-streptomycin double antibody. When the cell confluence was greater than >80%, the cells were subcultured and expanded. When the culture reached about 50 mL, the supernatant was collected and the antibody was purified. The obtained antibody was determined to have good purity by SDS-PAGE gel electrophoresis.

3. Monoclonal Antibody Sequencing

The positive hybridoma cells after subcloning were expanded and cultured. An appropriate amount of cells were taken to extract total RNA according to the instructions of the RNeasy Plus Mini Kit (Qiagen, 74134) kit, and the first strand of cDNA was synthesized using the Prime Script 1st strand cDNA Synthesis Kit (Takara, 6110A) reverse transcription kit.

Universal primers were designed based on the conserved sequences at both ends of the antibody variable region (the 5' end contained a homology arm sequence for homologous recombination with a eukaryotic expression vector), and cDNA was used as a template for PCR amplification of the antibody variable region gene, thereby obtaining the gene fragments of the mouse antibody light chain and heavy chain variable regions respectively; primers were designed (references: 1. Anke Krebber, Susanne Bornhauser, Jorg Burmester et al. Reliable cloning of functional antibody variable domains from hybridomas and spleen cell repertoires employing a reengineered phage display system. Journal of Immunological Methods, 1997, 201: 35-55; 2. Simon Antibody variable-region sequencing as a method for hybridoma cell-line authentication, 2008, 78: 1071-1078), DNA sequencing was performed to obtain the sequence. The amino acid sequence of the heavy chain variable region of mouse anti-C110 is SEQ ID NO: 42, and the amino acid sequence of the light chain variable region is SEQ ID NO: 43.

Example 3: Construction of anti-CRTAM chimeric antibody

The purified mouse antibody heavy chain and light chain variable region gene fragments were co-transformed with the linearized eukaryotic expression plasmid pHR-hG1m/pHR-hλ containing the human antibody heavy chain or light chain constant region into E. coli DH5α competent cells. The mixture was evenly spread on the surface of an agar plate containing ampicillin antibiotics, and several single colonies were picked for DNA sequencing after inverted overnight culture in a 37°C constant temperature incubator. The correctly sequenced chimeric antibody was labeled C110CHI. The amino acid sequence of the heavy chain variable region and the amino acid sequence of the light chain variable region of the antibody C110CHI are the same as those of the corresponding mouse antibody C110.

The chimeric antibody heavy and light chain plasmids were co-transfected into HEK293E cells, and the chimeric antibody was expressed and purified, and then subjected to purity detection, activity analysis and affinity determination.

Example 4: Construction and production of anti-CRTAM humanized antibodies

Based on the immunological activity analysis of the chimeric antibody, the chimeric antibody C110CHI was transformed into a humanized antibody.

The humanization of antibodies was first confirmed by comparing with the mouse antibody sequence in the immune gene database (IMGT) to confirm the mouse germline of the variable region of the C110CHI antibody. After homology comparison, the FR region of the heavy chain variable region sequence of the C110CHI antibody was most similar to the mouse antibody germline gene (IGHV1-85*01); the FR sequence of the antibody light chain variable region was most similar to the mouse antibody (IGKV1-110*01). Using the C110CHI antibody framework region sequence FR1-FR3 as a template, a full human framework with similar 3D structure but lower immunogenicity was searched in the human framework region library to replace the FR1-FR3 sequence of C110CHI. The full-length sequence of the heavy chain/light chain was 3D modeled and structurally compared with the heavy chain/light chain sequence of the original antibody. Taking into account the antigenicity and 3D structural similarity, 4 humanized heavy chain variable regions and 5 humanized light chain variable regions of C110CHI were selected for the next step of optimization. The non-CDR region sequences of C110CHI's humanized antibodies were more than 95% humanized.

Humanized antibodies with good purity, activity and affinity were selected and labeled as HuC110A. The 34th G gene of the light chain variable region of the humanized antibody HuC110A was mutated to K (see SEQ ID NO: 19) by site-directed mutagenesis to improve the stability of the antibody, which was labeled as HuC110. The sequence is shown in Table 1.

Table 1 Anti-CRTAM humanized antibody sequences

Considering the function and stability of the antibody, the appropriate antibody subtype skeleton was selected to match the optimized humanized antibody variable region to construct a complete humanized antibody. The amino acid sequences of the light chain and heavy chain variable regions of the humanized antibody were reverse transcribed into corresponding nucleotide sequences, codons were optimized according to the expression host, and oligonucleotide fragments containing complementary sequences between adjacent fragments were generated. The oligonucleotide fragments were annealed and connected by Overlap PCR, and then the complete light chain and heavy chain variable region nucleotide fragments were amplified using specific primers (the 5' end contained a homologous arm sequence for homologous recombination with the eukaryotic expression vector); the purified light chain variable region nucleotide fragments and the linearized eukaryotic expression plasmid containing the human κ light chain constant region were co-transformed into Escherichia coli DH5α competent cells, and the purified heavy chain variable region nucleotide fragments and the eukaryotic expression plasmid containing the human IgG heavy chain constant region were co-transformed into Escherichia coli DH5α competent cells, and the competent cells of the transformed plasmids were evenly spread on the surface of agar plates containing ampicillin antibiotics, and several single colonies were picked for DNA sequencing after overnight culture in a 37°C constant temperature incubator.

The positive clones with correct sequencing were inoculated into 2×YT liquid culture medium containing ampicillin antibiotics and cultured at 37°C with shaking for more than 12 hours. The bacteria were then collected for plasmid extraction to obtain humanized antibody light chain and heavy chain expression plasmids. The concentration and purity of the plasmids were detected using a nucleic acid quantitative analyzer.

The plasmid was transfected into HEK293E cells, and a large amount of antibodies were expressed and purified, and then purity, activity and affinity tests were performed. The complete humanized antibody sequence is shown in Table 2.

Table 2 Complete sequence of anti-CRTAM humanized antibody

Example 5: Anti-CRTAM antibody binding activity assay for human CRTAM (ELISA)

The binding activity of the antibody was analyzed by ELISA. The hCRTAM-His protein (1.5 μg/mL, 50 μL/well, prepared in Example 1) was coated onto a 96-well ELISA plate and incubated at 37°C for 2 h. After washing three times with 1x PBST, the plate was blocked with PBST containing 5% skim milk at 37°C for 2 h. After washing three times with 1x PBST, the anti-CRTAM antibody of the present invention was used as the primary antibody starting from 10 μg/mL, and was added to the ELISA plate (50 μL/well) with a 5-fold gradient dilution, with a total of 8 concentrations, namely 10000 ng/mL, 2000 ng/mL, 400 ng/mL, 80 ng/mL, 16 ng/mL, 3.2 ng/mL, 0.64 ng/mL, and 0.128 ng/mL, and incubated at 37°C for 1.5 h. The control antibody was 5A11 (prepared in Example 1). After washing three times with 1x PBST, the plate was patted dry, and Anti-Human IgG HRP (Jackson, 109-035-003, 1:5000, 50 μL/well) was used as the secondary antibody, and the plate was incubated at 37°C for 1 h. After washing with 1xPBST for 5 times, pat dry, add single-component TMB colorimetric solution (Solebol, PR1200, 50μL/well), color development for about 5 minutes in the dark, add 2M sulfuric acid (50μL/well) to terminate, and read OD450 value using a microplate reader (thermo, Multiskan FC). EC50 was generated using GraphPad, and the results are shown in Table 3.

Table 3 Binding activity of anti-CRTAM humanized antibodies to human CRTAM

The experimental results show that the anti-CRTAM antibody of the present invention has a good ability to bind to human CRTAM.

Example 6: Determination of the binding activity of anti-CRTAM antibody to cynomolgus monkey CRTAM on the cell surface (FACS)

The binding activity of the antibody to cCRTAM on the surface of CHO-K1-cCRTAM cells was analyzed by FACS. After digestion, CHO-K1-cCRTAM cells were resuspended in 2% FBS-PBS solution and counted. The above cells were plated on a cell plate at a density of 1.5x10 ⁵ cells per well. The anti-CRTAM antibody of the present invention was used as a primary antibody starting from 20 μg/ml and was added to the cell plate in a 5-fold gradient dilution, with a total of 8 concentrations, namely 20000 ng/mL, 4000 ng/mL, 800 ng/mL, 160 ng/mL, 32 ng/mL, 6.4 ng/mL, 1.28 ng/mL, and 0.26 ng/mL, and incubated at 4°C for 1.5 h; after washing the cells three times with PBS, the secondary antibody was incubated at 4°C for 1 h using PE-Anti-Human IgG (Biolegend, Cat. No. 409303, 1.25 μl/well); after washing with PBS three times, the fluorescence intensity generated by the binding of the antibody to the cell surface was detected by flow cytometry, and the results are shown in Table 4.

Table 4 Binding activity of anti-CRTAM antibodies to cynomolgus monkey CRTAM on cell surface

The experimental results show that the humanized anti-CRTAM antibody of the present invention has a good ability to bind to CRTAM on the cell surface.

Example 7: Determination of epitope competition activity of anti-CRTAM antibody and 5A11 on human CRTAM (ELISA)

The ELISA method was used to analyze the epitope competitive activity of the anti-CRTAM antibody of the present invention and 5A11 on human CRTAM. The hCRTAM-His protein (2 μg/mL, 50 μL/well, prepared in Example 1) was coated onto a 96-well ELISA plate and incubated at 37°C for 2 h. After washing 3 times with 1x PBST, the plate was blocked with PBST containing 5% skim milk at 37°C for 2 h. At the same time, the anti-CRTAM antibody of the present invention was labeled with Biotin-NHS ester ^TM (Biovsion, 2347-50), and the labeled antibody was named HuC110-biotin. The blocked ELISA plate was washed 3 times with 1x PBST, and then HuC110-biotin solution with a concentration of 1 μg/mL was prepared respectively. The primary antibody was diluted with the above solution as the diluent. The anti-CRTAM antibody of the present invention (competitive positive control) and 5A11 were used as the primary antibody starting from 100 μg/mL, and 5-fold gradient dilution was added to the ELISA plate (50 μL/well), with a total of 8 concentrations, namely 100000 ng/mL, 20000 ng/mL, 4000 ng/mL, 800 ng/mL, 160 ng/mL, 32 ng/mL, 6.4 ng/mL, and 1.28 ng/mL, and incubated at 37°C for 1.5 h; after washing 3 times with 1x PBST, patted dry, Streptavidin-HRP (BD, 554066, 1:10000, 50 μL/well) was used as the secondary antibody, and incubated at 37°C for 1 h. After washing with 1xPBST for 5 times, pat dry, add single-component TMB colorimetric solution (Solebol, PR1200, 50 μL/well), color development for about 5 min in the dark, add 2M sulfuric acid (50 μL/well) to terminate, and read OD450 value using a microplate reader (thermo, Multiskan FC). IC50 was generated using GraphPad.

The experimental results show that there is almost no epitope competition between the humanized anti-CRTAM antibody HuC110 of the present invention and 5A11, and it does not block the binding of 5A11 to human CRTAM, indicating that the binding epitopes of the humanized anti-CRTAM antibody of the present invention and 5A11 to CRTAM are completely different.

Example 8: Affinity determination of anti-CRTAM antibodies and human CRTAM protein

The affinity of the humanized anti-CRTAM antibody of the present invention to the antigen hCRTAM-His (prepared in Example 1) was measured using Fortebio Octet. The anti-CRTAM antibody was diluted to a concentration of 10 μg/mL with SD buffer (PBS+0.02% Tween20+0.1% BSA), and the antigen hCRTAM-His was diluted 4 times with SD buffer to a concentration of 12 μg/mL, 3 μg/mL, 0.75 μg/mL, and 0 μg/mL. The AHC sensor was used to solidify the antibody, and the affinity was measured according to the operating procedures of Fortebio Octet RED96. The specific parameters and experimental results are shown in Table 5.

Table 5 Affinity determination of anti-CRTAM antibodies and human CRTAM protein

The experimental results show that the affinity of the humanized anti-CRTAM antibody of the present invention is significantly better than that of 5A11, showing a more persistent ability to bind to the human CRTAM protein.

Example 9: In vitro efficacy testing of anti-CRTAM antibodies

The in vitro efficacy of anti-CRTAM antibodies was detected by stimulating healthy human PBMC with SEB (Staphylococcal enterotoxin B).

Resuscitate PBMC according to the required cell amount, add to 8-9 ml of IMDM complete medium, centrifuge at 900 g for 10 min, and discard the supernatant; resuspend with an appropriate amount of medium, count with a hemocytometer, and resuspend to 1 M/mL, and add 100 μL/well to a 96-well plate; prepare the antibody and Isotype to be tested with IMDM complete medium at 4 times the concentration (i.e., 40 μg/mL), 50 μL/well, mark, and vortex; add the antibody solution to the 96-well plate, add 50 μL/well of medium to the control group, place the 96-well plate in a 37°C incubator, and incubate the cells and antibodies for 1 hour; prepare SEB solution with IMDM complete medium at 4 times the concentration (i.e., 200 ng/mL), and add 50 μL/well to the corresponding wells; after incubating the 96-well plate in a 37°C, 5% CO ₂ incubator for 72 hours, collect 150 μL of cell-free supernatant, dilute it according to a certain ratio, and use the hIFN-γ ELISA detection kit (R&D The concentration of IFN-γ in the supernatant was detected according to the instructions of ELISA kit, R&D system Cat: DY285B. The results are shown in Table 6.

Table 6 Anti-CRTAM antibodies promote IFN-γ release from PBMC

The experimental results show that compared with 5A11, the anti-CRTAM antibody of the present invention has a better ability to promote the release of IFN-γ.

Example 10: Anti-tumor experiment of HCC827 (del19) human non-small cell lung cancer cell PBMC reconstruction model

1. Experimental Materials

(1) Experimental cells and animals

HCC827 (del19) cells were purchased from the American Type Culture Collection (ATCC);

NSG mice, female, 6-8 weeks old, weighing 18-20 g, were purchased from Biocytogen (Jiangsu) Gene Biotechnology Co., Ltd.;

(2) Test and control samples

The reference substance 5A11 was prepared in Example 1 and used as a positive control; the reference substance PBS was used as a negative control; before the test, the anti-CRTAM antibody of the present invention was prepared with PBS to 1 mg/mL.

2. Experimental methods

HCC827 (del19) human non-small cell lung cancer cell PBMC reconstruction model

50 μL PBS containing 1×10 ⁷ HCC827 (del19) cells was mixed with 50 μL matrix gel and inoculated subcutaneously in the right hind limb of mice. Grouping began when the tumor grew to an average volume of 100 mm ^3. PBMCs were fresh blood from healthy blood donors and were injected into the tail vein 1 hour before antibody administration after grouping on DAY1 (10M PBMC cells were injected into each mouse). Tail vein administration was performed twice a week, and the tumor diameter was measured with a vernier caliper twice a week to calculate the tumor volume. The calculation formula for tumor volume was: V = 0.5a × A2, a and b represent the long diameter and short diameter of the tumor, respectively, and the relative tumor proliferation rate (T/C) was calculated. The tumor growth inhibition rate was calculated using the following formula: TGI (%) = [1-(Ti-T0)/(Vi-V0)] × 100, where Ti is the average tumor volume of a certain dosing group on a certain day, T0 is the average tumor volume of this dosing group at the start of dosing; Vi is the average tumor volume of the vehicle control group on a certain day (the same day as Ti), and V0 is the average tumor volume of the vehicle control group at the start of drug administration. The tumor volume measured for 22 consecutive days of dosing was recorded, and the tumor volume growth curve was drawn using GraphPad Prism. After the experiment, the tumor weight was detected, and the tumor inhibition rate (TGI)% of each group was calculated. The results are shown in Table 7.

Table 7 Anti-tumor results of HCC827 (del19) human non-small cell lung cancer cell PBMC reconstruction model

The experimental results show that compared with 5A11, the anti-CRTAM antibody of the present invention can better inhibit the growth of HCC827 (del19) human non-small cell lung cancer tumors.

Example 11: Construction, expression and purification of anti-CRTAM/anti-PD-L1 bispecific antibodies

1. Construction of anti-CRTAM/anti-PD-L1 bispecific antibody expression vector

(1) Genetic engineering was used to construct an anti-CRTAM/anti-PD-L1 bispecific antibody, the structure of which is shown in Figure 1. The bispecific antibody is formed by two anti-CRTAM/anti-PD-L1 antibody single chains through a heterodimer, wherein the light chain of the anti-PD-L1 antibody is connected to the N-terminus of the antibody heavy chain through an additional flexible connecting peptide, and the connecting peptide is a GGGGS repeating sequence containing glycine (G) and serine (S) residues, containing 8 GGGGS repeating sequences, and the anti-CRTAM antibody is composed of a heavy chain and a light chain paired through an interchain disulfide bond. In addition, to promote the formation of heterodimers, S228P/L235E/S354C/T366W mutations are added to the CH3domain of the anti-PD-L1 antibody single chain, and Y349C/T366S/L368A/Y407V mutations are added to the CH3domain of the anti-CRTAM antibody single chain. According to this structural form, the anti-PD-L1 antibody HuPL7-21 (also known as HuPL721, light chain variable region SEQ ID NO: 15 and constant region SEQ ID NO: 16) sequence is used to obtain a single-chain nucleotide fragment of the anti-PD-L1 antibody, namely ScFabHuPL7-21Ks (see WO2021/170082) by gene synthesis; the anti-CRTAM antibody HuC110 (light chain variable region SEQ ID NO: 19 and constant region SEQ ID NO: 16) sequence is used to obtain a single-chain light chain nucleotide fragment of the anti-CRTAM antibody, namely HuC110L, by gene synthesis; the anti-CRTAM antibody HuC110 (heavy chain variable region SEQ ID NO: 17) sequence is used to obtain a single-chain heavy chain nucleotide fragment of the anti-CRTAM antibody, namely HuC110HHs, by gene synthesis. The ScFabHuPL7-21Ks, HuC110HHs and HuC110L nucleotide fragments (with homology arms of appropriate lengths in both upstream and downstream) were co-transformed into E. coli DH5α competent cells with the linearized eukaryotic expression plasmid pHR, and the competent cells transformed with the plasmids were evenly spread on the surface of agar plates containing corresponding antibiotics, and several single colonies were picked for DNA sequencing after overnight culture in a 37°C constant temperature incubator. The positive clones with correct sequencing were subjected to plasmid extraction to obtain ScFabHuPL7-21Ks, HuC110HHs and HuC110L expression vectors.

The schematic structure of the ScFabHuPL7-21Ks sequence is as follows:

Among them, HuPL721VL-CL: humanized PD-L1 monoclonal antibody HuPL7-21 light chain;

(GGGGS)8: flexible linker peptide with 8 GGGGS repeats;

HuPL721-VH: heavy chain variable region of humanized PD-L1 monoclonal antibody HuPL7-21;

IgG4CH/Ks: IgG4 heavy chain constant region containing S228P/L235E/S354C/T366W mutations forming a "Knobs" structure (specific sequence is shown in SEQ ID NO: 14);

The schematic structure of the HuC110HHs sequence is as follows:

Wherein HuC110-VH: heavy chain variable region of humanized CRTAM monoclonal antibody HuC110;

IgG4CH/Hs: IgG4 heavy chain constant region containing S228P/L235E/Y349C/T366S/L368A/Y407V mutations forming a "HoLe" structure (specific sequence is shown in SEQ ID NO: 18);

The schematic structure of the HuC110L sequence is as follows:

Humanized CRTAM antibody HuC110 light chain.

2. Transient expression of anti-CRTAM/anti-PD-L1 bispecific antibody in Expi-CHO cells

The above-mentioned ScFabHuPL7-21Ks, HuC110HHs and HuC110L expression vectors were co-recombinantly transfected into Expi-CHO cells using the Expi-Fectamine CHO Transfection Kit plasmid transfection kit. After 12 days of culture in serum-free medium, the supernatant of Expi-CHO cells was collected and the expression of bispecific antibodies was detected by immunoblotting. Among them, the bispecific antibody formed by dimerization of three single chains of ScFabHuPL7-21Ks, HuC110HHs and HuC110L was named HuPL721-C110-HK.

3. Purification of anti-CRTAM/anti-PD-L1 bispecific antibodies

After the bispecific antibody of the present invention is expressed and secreted by Expi-CHO cells, it is purified by Protein A affinity chromatography. The specific method is as follows: After the Protein A affinity chromatography column is equilibrated with a buffer, the supernatant of the Expi-CHO cell culture fluid concentrated by the ultrafilter is injected, and monitored by A280 (nm), washed with a cleaning solution until all unbound proteins are eluted, and then eluted with an eluent to obtain the corresponding bispecific antibody. The purified bispecific antibody is used for subsequent pharmaceutical research after SEC-HPLC purity detection and quality identification. The SEC-HPLC identification results show that the purity of the bispecific antibody HuPL721-C110-HK is more than 95%.

Example 12: Construction of a stable cell line with high expression of hPD-L1 and hCRTAM

The eukaryotic expression plasmid pTargeT-hPD-L1 of the hPD-L1 (human PD-L1) sequence of 290 amino acids (Primary accession: Q9NZQ7, SEQ ID NO: 24) was transfected into CHO-K1 cells (from the Shanghai Institute of Cell Biology, Chinese Academy of Sciences) by electroporation and cultured in an incubator at 37°C and 5% CO _2. After 48 hours, the cells were cultured under pressure in a medium containing 500ug/ml G418. After 12 days, FACS was used to detect the pool positivity rate. The cells after electroporation of the plasmid were plated (cell density of ^1x106 cells/ml, 100uL/well). FITC anti-human PD-L1 antibody (SINO BIOLOGICAL, 10084-MMB6-F) was incubated with the cells at 4°C for 60 min. The mean value under the FITC channel was read by flow cytometer. After data analysis of the results, the positive cell lines were selected for subcloning, and the cloned CHO-K1 cell line was selected. This cell line expressed PD-L1 molecules at a high level and was named CHO-K1-hPD-L1.

The eukaryotic expression plasmid pTargeT-hCRTAM containing 393 amino acids of the hCRTAM (human CRTAM) sequence (Primary accession: O95727, SEQ ID NO: 25) was transfected into CHO-K1 cells (from the Shanghai Institute of Cell Biology, Chinese Academy of Sciences) by electroporation and cultured in an incubator at 37°C and 5% CO _2. After 48 hours, the cells were cultured under pressure in a medium containing 500ug/ml G418. After 12 days, the pool positivity rate was detected by FACS, and the cells after electroporation of the plasmid were plated (cell density of 1x10 ⁶ cells/ml, 100uL/well). PE anti-human CRTAM antibody (SINO BIOLOGICAL, 11975-MM12-P) was incubated with the cells at 4°C for 60 min, and the mean value under the PE channel was read by flow cytometer. After data analysis of the results, the positive cell lines were selected for subcloning, and the cloned CHO-K1 cell line was selected. This cell line expressed CRTAM molecules at a high level and was named CHO-K1-hCRTAM.

Example 13: Anti-CRTAM/anti-PD-L1 bispecific antibody ELISA in vitro binding and blocking experiments

1. Anti-CRTAM/anti-PD-L1 bispecific antibody PD-L1 binding ELISA experiment

Human PD-L1-His protein (1 μg/mL, 50 μL/well) was coated onto a 96-well ELISA plate and incubated at 37°C for 2 h. After washing 3 times with 1xPBST, the plate was blocked with 5% skim milk at 4°C overnight and washed 3 times with 1xPBST. The concentration of the double antibody started from 133nmoL/L, and a 5-fold gradient dilution was added to the ELISA plate, incubated at 37°C for 1.5 h. The control antibody was the monoclonal antibody HuPL7-21; after washing 5 times with 1xPBST, HRP-Anti-Human IgG secondary antibody (Jackson, 109-035-003, 1:10000) was added and incubated at 37°C for 40 min. Wash 5 times with 1xPBST, add the colorimetric solution TMB, and read the OD450 value using an ELISA reader (thermo, MuLtiskan FC) after termination. The EC ₅₀ results are shown in Table 8.

Table 8 Binding activity of bispecific antibodies to human PD-L1

Experimental results showed that the bispecific antibody HuPL721-C110-HK has the ability to bind to human PD-L1, and its binding activity is comparable to that of the monoclonal antibody HuPL7-21.

2. Anti-CRTAM/anti-PD-L1 bispecific antibody CRTAM binding ELISA experiment

Human CRTAM protein (2μg/mL, 100μL/well) was coated onto a 96-well ELISA plate and incubated at 37°C for 2h. After washing with 1xPBST 3 times, the plate was blocked with 5% skim milk at 4°C overnight and washed with 1xPBST 3 times. The double antibody concentration started from 200nmoL/L, and was diluted 5-fold to add to the ELISA plate, incubated at 37°C for 1.5h. The control antibody was 5A11 (patent WO2016154341); after washing with 1xPBST 5 times, HRP-Anti-Human IgG secondary antibody (Jackson, 109-035-003, 1:10000) was added and incubated at 37°C for 40min. After washing with 1xPBST 5 times, the color developing solution TMB was added, and the OD450 value was read using an ELISA reader (thermo, MuLtiskan FC) after termination.

Experiments showed that the binding activity of the bispecific antibody HuPL721-C110-HK to CRTAM was comparable to that of the monoclonal antibody HuC110.

Example 14: Determination of the affinity of anti-CRTAM/anti-PD-L1 bispecific antibodies to human PD-L1 and CRTAM (Fortebio)

1. Anti-CRTAM/anti-PD-L1 bispecific antibody affinity test for human PD-L1

The human PD-L1-His was diluted 3 times with SD buffer (0.02% Tween20 + 0.1% BSA solution) to a concentration of 3ug/ml, 1ug/ml, 0.33ug/ml, and 0ug/ml. The bispecific antibody HuPL721-C110-HK was diluted to a concentration of 10ug/ml with SD buffer. The AHC sensor was used to solidify the antibody protein, and the affinity was measured according to the operating procedures of fortebio Octet RED96. The specific parameters and experimental results are shown in Table 10.

[Corrected 11.01.2024 in accordance with Rule 26]
Table 10 Affinity determination of bispecific antibodies binding to human PD-L1 protein

The experimental results show that the bispecific antibody HuPL721-C110-HK has a good affinity with PD-L1. 2. Anti-CRTAM/anti-PD-L1 bispecific antibody and human CRTAM affinity test experiment

Referring to the method of Example 8, the affinity of the bispecific antibody HuPL721-C110-HK of the present invention to the antigen hCRTAM-His (prepared in Example 1) was measured using Fortebio Octet. The affinity was measured according to the operating procedures of Fortebio Octet RED96, and the specific parameters and experimental results are shown in Table 11.

[Corrected 11.01.2024 in accordance with Rule 26]
Table 11 Affinity determination of the bispecific antibody binding to human CRTAM protein

The experimental results showed that the bispecific antibody HuPL721-C110-HK has a good affinity with CRTAM.

Example 15: Anti-CRTAM/anti-PD-L1 bispecific antibody SEB activation PBMC experiment

Resuscitate PBMC, resuspend and count with an appropriate amount of IMDM medium, and add 1M/mL, 100μL/well to a 96-well plate; the anti-CRTAM/anti-PD-L1 bispecific antibody provided by the present invention has an initial concentration of 40μg/mL (the final concentration is reduced by 4 times), 10-fold gradient dilution, a total of 3 concentrations, and 50μL/well is added to a 96-well plate with PBMC, and incubated in a 37°C incubator for 1h; 400ng/mL SEB is configured with culture medium, 50μL/well is added to a 96-well plate, and after incubation in a 37°C, 5% _CO2 incubator for 72h, 120μL of cell-free supernatant is taken for ELISA detection of cytokine IFN-γ secretion. Operate according to the operating instructions of the ELISA detection kit, and then read the OD450 value using an enzyme reader (thermo, Multiskan FC). The results are shown in Table 12.

Table 12 Anti-CRTAM/anti-PD-L1 bispecific antibodies promote PBMC release of IFN-γ

The experimental results showed that the anti-PD-L1//anti-CRTAM bispecific antibody HuPL721-C110-HK has a good effect in promoting SEB to activate PBMC to release cytokine IFN-γ.

Example 16: Anti-CRTAM/anti-PD-L1 bispecific antibody induced PBMC to kill HCC827 cells

Day 1: HCC827 cells were resuspended and counted in 1640 medium, and then plated on a 96-well cell culture plate at 1×10 ⁴ cells/well for overnight culture. PBMCs were revived, counted, and cultured overnight in flasks, with <10M per flask, medium: 1640+10% FBS+IL-2 (10 ng/mL)+1% P/S. Day 2: The old medium in the wells was discarded, 50 μl of fresh medium was added to each well, and the control wells were operated in parallel. Medium: 1640+2% FBS (inactivated)+1% P/S. The anti-CRTAM/anti-PD-L1 bispecific antibody of the present invention and the control antibody Atezolizumab (Sino Biological, Cat: 68049-H001, abbreviated as Ate) were prepared at an initial concentration of 800 nmol/L (the final concentration was reduced by 4 times), 10-fold gradient dilution, a total of 7 concentrations, and added to a cell plate pre-plated with HCC827 at 50 μl/well. Take the PBMC cells resuscitated on the first day, adjust the cells to 4×10 ⁶ /mL with 1640+2% FBS (inactivated)+1% P/S, add 100ul to each well according to the effect-target ratio of 40:1, mix thoroughly, and incubate in a 37°C incubator for 48h. According to the LDH kit operating instructions, take 120μl of the cell supernatant and mix with 60μl of LDH working solution, incubate at room temperature in the dark for 30min, and then use an enzyme reader (thermo, Multiskan FC) to read the OD490 value. Calculate the killing activity (cytotoxicity)% of each group, and the results are shown in Table 13.

Table 13 Dual antibodies induce PBMC to kill HCC827 cells

The experimental results showed that the anti-CRTAM/anti-PD-L1 bispecific antibody HuPL721-C110-HK has a good ability to induce PBMC to kill small cell lung cancer cells.

Although the present invention has been described in detail above, it is understood by those skilled in the art that various modifications and changes can be made to the present invention without departing from the spirit and scope of the present invention. The scope of the present invention is not limited to the detailed description above, but should be attributed to the claims.

Claims

An anti-CRTAM/anti-PD-L1 antibody, comprising an anti-CRTAM antibody or an antigen-binding fragment thereof and an anti-PD-L1 antibody or an antigen-binding fragment thereof, wherein:

The anti-CRTAM antibody or antigen-binding fragment thereof comprises a first heavy chain variable region and a first light chain variable region, wherein:

(1) the first heavy chain variable region comprises H1CDR1, H1CDR2 and H1CDR3, whose amino acid sequences are SEQ ID NOs: 1, 2 and 3, respectively, or amino acid sequences having at least 85% sequence identity with the amino acid sequences shown in SEQ ID NOs: 1, 2 and 3;

(2) The first light chain variable region comprises L1CDR1, L1CDR2 and L1CDR3, whose amino acid sequences are SEQ ID NO: 4, 5 and 6, respectively, or amino acid sequences that have at least 85% sequence identity with the amino acid sequences shown in SEQ ID NO: 4, 5 and 6.
The anti-CRTAM/anti-PD-L1 antibody according to claim 1, wherein

The anti-PD-L1 antibody or antigen-binding fragment thereof comprises a second heavy chain variable region and a second light chain variable region, wherein:

(1) the second heavy chain variable region comprises H2CDR1, H2DR2 and H2CDR3, whose amino acid sequences are SEQ ID NOs: 7, 8 and 9, respectively, or amino acid sequences having at least 85% sequence identity to the amino acid sequences shown in SEQ ID NOs: 7, 8 and 9; and

(2) The second light chain variable region comprises L2CDR1, L2CDR2 and L2CDR3, whose amino acid sequences are SEQ ID NO: 10, 11 and 12, respectively, or amino acid sequences that have at least 85% sequence identity with the amino acid sequences shown in SEQ ID NO: 10, 11 and 12.
The anti-CRTAM/anti-PD-L1 antibody according to claim 1 or 2, wherein:

The anti-CRTAM antibody or antigen-binding fragment thereof comprises a first heavy chain variable region and a first light chain variable region, wherein:

(1) The amino acid sequence of the first heavy chain variable region is selected from:

(A1) the amino acid sequence shown in SEQ ID NO:17;

(A2) an amino acid sequence obtained by substituting, deleting or adding one or more amino acids to the amino acid sequence shown in (A1), and having the same or similar functions as the amino acid sequence shown in (A1); and

(A3) an amino acid sequence having at least 80% sequence identity with the amino acid sequence shown in (A1); and

(2) The amino acid sequence of the first light chain variable region is selected from:

(A4) the amino acid sequence shown in SEQ ID NO:19;

(A5) An amino acid sequence obtained by substituting, deleting or adding one or more amino acids to the amino acid sequence shown in (A4), and having the same or similar functions as the amino acid sequence shown in (A4); and

(A6) An amino acid sequence having at least 80% sequence identity with the amino acid sequence shown in (A4).
The anti-CRTAM/anti-PD-L1 antibody according to any one of claims 1 to 3, wherein the anti-PD-L1 antibody or antigen-binding fragment thereof comprises a second heavy chain variable region and a second light chain variable region, wherein:

(1) The amino acid sequence of the second heavy chain variable region is selected from:

(B1) the amino acid sequence shown in SEQ ID NO:13;

(B2) an amino acid sequence obtained by substituting, deleting or adding one or more amino acids to the amino acid sequence shown in (B1) and having the same or similar functions as the amino acid sequence shown in (B1); and

(B3) an amino acid sequence having at least 80% sequence identity with the amino acid sequence shown in (B1); and

(2) The amino acid sequence of the second light chain variable region is selected from:

(B4) the amino acid sequence shown in SEQ ID NO:15;

(B5) an amino acid sequence obtained by substituting, deleting or adding one or more amino acids to the amino acid sequence shown in (B4) and having the same or similar functions as the amino acid sequence shown in (B4); and

(B6) An amino acid sequence having at least 80% sequence identity with the amino acid sequence shown in (B4).
The anti-CRTAM/anti-PD-L1 antibody according to any one of claims 1 to 4, wherein the antibody is a humanized antibody or a fully human antibody.
The anti-CRTAM/anti-PD-L1 antibody of any one of claims 1 to 5, wherein the antibody is a bispecific antibody.
An isolated nucleic acid encoding the anti-CRTAM/anti-PD-L1 antibody of any one of claims 1-6.
The nucleic acid of claim 7, wherein:

(1) the nucleotide sequence encoding the amino acid sequence of the first heavy chain variable region is shown in SEQ ID NO: 22;

(2) the nucleotide sequence encoding the amino acid sequence of the first light chain variable region is shown in SEQ ID NO: 23;

(3) The nucleotide sequence encoding the amino acid sequence of the second heavy chain variable region is as shown in SEQ ID NO: 20 show; and

(4) The nucleotide sequence encoding the amino acid sequence of the second light chain variable region is shown in SEQ ID NO:21.
An expression vector comprising the nucleic acid according to claim 7 or 8.
A host cell transformed with the expression vector according to claim 9, wherein the host cell is selected from prokaryotic cells and eukaryotic cells, preferably mammalian cells.
A method for preparing the anti-CRTAM/anti-PD-L1 antibody according to any one of claims 1 to 6, comprising the steps of expressing the antibody in the host cell according to claim 10, and isolating the antibody from the host cell.
A pharmaceutical composition comprising the anti-CRTAM/anti-PD-L1 antibody according to any one of claims 1 to 6 and a pharmaceutically acceptable carrier.
Use of the anti-CRTAM/anti-PD-L1 antibody according to any one of claims 1 to 6 or the pharmaceutical composition according to claim 12 in the preparation of a medicament for inhibiting PD-L1 activity and/or stimulating CRTAM activity, preferably the medicament is used to treat blood tumors, lymphomas, breast cancer, lung cancer, gastric cancer, intestinal cancer, esophageal cancer, ovarian cancer, cervical cancer, kidney cancer, bladder cancer, pancreatic cancer, glioma and/or melanoma.