WO2021244552A1

WO2021244552A1 - Anti-pdl1×kdr bispecific antibody

Info

Publication number: WO2021244552A1
Application number: PCT/CN2021/097783
Authority: WO
Inventors: 朱祯平; 黄浩旻; 顾昌玲; 祝海霞
Original assignee: 三生国健药业（上海）股份有限公司
Priority date: 2020-06-02
Filing date: 2021-06-01
Publication date: 2021-12-09
Also published as: TW202146461A; CN115698087A; CN113754772A

Abstract

Provided in the present invention is an anti-PDL1×KDR bispecific antibody. The experimental results show that the bispecific antibody can better maintain the activity of each monoclonal antibody, can specifically bind to both PD-L1 and KDR targets, and has good physical and chemical properties.

Description

Anti-PDL1×KDR bispecific antibody

Technical field

The present invention relates to the field of antibodies. More specifically, the present invention discloses bispecific antibodies against PDL1×KDR.

Background technique

Human Programmed Cell Death Receptor-1 (PD-1) is a type I membrane protein with 288 amino acids. It is one of the major known immune checkpoints (Blank et al, 2005, Cancer Immunotherapy) , 54: 307-314). PD-1 is expressed on activated T lymphocytes, and it interacts with the ligand PD-L1 (programmed cell death-Ligand 1) and PD-L2 (programmed cell death receptor- 1). Ligand 2, programmed cell death-Ligand 2) The combination can inhibit the activity of T lymphocytes and related cellular immune responses in the body. PD-L2 is mainly expressed in macrophages and dendritic cells, while PD-L1 is widely expressed in B, T lymphocytes and peripheral cells such as microvascular epithelial cells, lung, liver, heart and other tissue cells. A large number of studies have shown that the interaction of PD-1 and PD-L1 is not only necessary to maintain the balance of the immune system in the body, but also the main mechanism and reason that causes PD-L1 positive tumor cells to evade immune surveillance. By blocking the negative regulation of cancer cells on the PD-1/PD-L1 signaling pathway and activating the immune system, it can promote T cell-related tumor-specific cellular immune responses, thereby opening the door to a new tumor treatment method-- Tumor immunotherapy.

PD-1 (encoded by the gene Pdcd1) is a member of the immunoglobulin superfamily related to CD28 and CTLA-4. Research results show that when PD-1 binds to its ligands (PD-L1 and/or PD-L2), it negatively regulates antigen receptor signal transduction. The structure of mouse PD-1 and the co-crystal structure of mouse PD-1 and human PD-L1 have been clarified (Zhang, X. et al. Immunity 20: 337-347 (2004); Lin et al., Proc. Natl. Acad. Sci. USA 105: 3011-6 (2008)). PD-1 and similar family members are type I transmembrane glycoproteins, which contain an Ig variable (V-type) domain responsible for ligand binding and a cytoplasmic tail region responsible for binding signal transduction molecules. The cytoplasmic tail of PD-1 contains two tyrosine-based signal transduction motifs, ITIM (Immunoreceptor Tyrosine Inhibition Motif) and ITSM (Immune Receptor Tyrosine Switch Motif).

PD-1 plays an important role in the immune evasion mechanism of tumors. Tumor immunotherapy, which uses the body’s own immune system to fight cancer, is a breakthrough tumor treatment method, but the tumor microenvironment can protect tumor cells from effective immune destruction. Therefore, how to break the tumor microenvironment has become an anti-tumor research Focus. Existing research results have determined the role of PD-1 in the tumor microenvironment: PD-L1 is expressed in many mouse and human tumors (and can be induced by IFN-γ in most PD-L1-negative tumor cell lines), It is presumed to be an important target for mediating tumor immune evasion (Iwai Y. et al., Proc. Natl. Acad. Sci. USA 99: 12293-12297 (2002); Strome SE et al., Cancer Res., 63: 6501-6505) (2003)). Through immunohistochemical assessment of biopsies, the expression of PD-1 (on tumor infiltrating lymphocytes) and/or PD-L1 on tumor cells has been found in many primary human tumors. Such tissues include lung cancer, liver cancer, ovarian cancer, cervical cancer, skin cancer, colon cancer, glioma, bladder cancer, breast cancer, kidney cancer, esophageal cancer, gastric cancer, oral squamous cell carcinoma, urothelial cell carcinoma and Pancreatic cancer and head and neck tumors. It can be seen that blocking the interaction of PD-1/PD-L1 can improve the immune activity of tumor-specific T cells and help the immune system to clear tumor cells. Therefore, PD-L1 has become a popular target for the development of tumor immunotherapy drugs. .

The protein encoded by KDR is also named Vascular Endothelial Growth Factor Receptor-2 (VEGFR-2), which is an important receptor for vascular growth factor important signal transduction. VEGFR2 is mainly expressed in vascular endothelial cells, especially tumor vascular endothelial cells, and mainly combines with VEGF-C, D, and A to promote the survival of blood vessels. VEGF specifically binds to the extracellular domain of VEGFR2, which can activate multiple downstream signaling pathways such as MAPK, PI3K, PKC, FAK, etc., and participate in endothelial cell budding, migration, vascular permeability, and tumor cell survival. VEGFR-2 is closely related to many diseases such as tumors, psoriasis, rheumatoid arthritis, diabetic retinopathy, etc. Especially in tumor growth, metastasis and tumor multi-drug resistance. Therefore, VEGFR-2 has become an ideal target for the treatment of these diseases, especially tumors. At present, tumors are known to be rich in blood vessels, and clinical trial data show that targeting VEGFR-2 is essential to inhibit tumor angiogenesis. However, anti-VEGFR-2 drugs used alone are often very short-lived, which may be closely related to excessive vascular pruning and extreme hypoxia. In 2018, the relationship between vascular normalization and immune response has been revealed. There are a large number of immunosuppressive cells and dysfunctional effector T cells in the tumor microenvironment. The combined use of immune checkpoint inhibitors and anti-angiogenesis drugs can significantly extend the treatment window for tumor vascular normalization, and the normalization of blood vessels weakens the tumor The immunosuppressive process in the microenvironment increases the infiltration of T cells and ultimately promotes tumor regression.

Bispecific antibodies are gradually becoming a new class of therapeutic antibodies that can be used to treat various inflammatory diseases, cancers and other diseases.

There is an urgent need in the art to develop bispecific antibodies with excellent activity for the treatment of cancer and other diseases.

Summary of the invention

The invention provides a bispecific antibody against PDL1×KDR and its application.

Therefore, the first object of the present invention is to provide a bispecific antibody against PDL1×KDR.

The second object of the present invention is to provide an isolated nucleotide encoding the bispecific antibody.

The third object of the present invention is to provide an expression vector containing the nucleotide.

The fourth object of the present invention is to provide a host cell containing the expression vector.

The fifth object of the present invention is to provide a method for preparing the bispecific antibody.

The sixth object of the present invention is to provide a pharmaceutical composition containing the bispecific antibody.

The seventh object of the present invention is to provide the use of the bispecific antibody or the pharmaceutical composition in the preparation of drugs for the treatment of cancer.

The eighth object of the present invention is to provide a method for treating cancer with the bispecific antibody or the pharmaceutical composition.

In order to achieve the above objective, the present invention provides the following technical solutions:

The first aspect of the present invention provides an anti-PDL1×KDR bispecific antibody, comprising two polypeptide chains and two light chains selected from the group consisting of:

(a) The polypeptide chain from N-terminus to C-terminus contains VH-PDL1-CH1-CH2-CH3-linker2-VL-KDR-linker1-VH-KDR or VH-PDL1-CH1-CH2-CH3-linker2-VH -KDR-linker1-VL-KDR, the light chain includes VL-PDL1-CL from N-terminus to C-terminus; or

(b) The polypeptide chain includes VL-PDL1-linker1-VH-PDL1-linker2-VH-KDR-CH1-CH2-CH3 from N-terminus to C-terminus, and the light chain includes VL-PDL1-linker1-VH-PDL1-linker2-VH-KDR-CH1-CH2-CH3 from N-terminus to C-terminus. -KDR-CL; or

(c) The polypeptide chain includes VH-KDR-linker1-VL-KDR-linker2-VH-PDL1-CH1-CH2-CH3 or VL-KDR-linker1-VH-KDR-linker2-VH from N-terminus to C-terminus -PDL1-CH1-CH2-CH3, the light chain includes VL-PDL1-CL from N-terminus to C-terminus; or

(d) The polypeptide chain includes VH-KDR-CH1-CH2-CH3-linker2-VL-PDL1-linker1-VH-PDL1 or VH-KDR-CH1-CH2-CH3-linker2-VH from N-terminus to C-terminus -PDL1-linker1-VL-PDL1, the light chain includes VL-KDR-CL from N-terminus to C-terminus; or

(e) The polypeptide chain includes VH-PDL1-linker1-VL-PDL1-linker2-VH-KDR-CH1-CH2-CH3 from N-terminus to C-terminus, and the light chain includes VL from N-terminus to C-terminus -KDR-CL;

Wherein, the VH-PDL1 is a heavy chain variable region that binds PD-L1,

The VL-PDL1 is the light chain variable region that binds to PD-L1,

The VH-KDR is a heavy chain variable region that binds to KDR,

The VL-KDR is the light chain variable region that binds to KDR,

The linker1 and linker2 are each independently a flexible peptide linker,

The CH1-CH2-CH3 is the heavy chain constant region, the CL is the light chain constant region, and the VH-PDL1 and the VL-PDL1 form an antigen binding site that specifically binds to PD-L1, The VH-KDR and the VL-KDR form an antigen binding site that specifically binds to the KDR.

In another preferred embodiment, the bispecific antibody has the activity of simultaneously binding to KDR and binding to PD-L1.

In another preferred embodiment, the flexible peptide linker includes 6-30 amino acids, preferably 10-25 amino acids.

In another preferred embodiment, the flexible peptide linker includes 2-6 G4S and/or G3S.

In another preferred example, the Linker1 is 3-5 G4S.

In another preferred example, the Linker2 is 2-4 G4S.

In another preferred example, the linker1 is 4 G4S, and the linker2 is 3 G4S.

In another preferred embodiment, the anti-PDL1×KDR bispecific antibody comprises two polypeptide chains and two light chains, wherein:

(b) The polypeptide chain includes VL-PDL1-linker1-VH-PDL1-linker2-VH-KDR-CH1-CH2-CH3 from N-terminus to C-terminus, and the light chain includes VL-PDL1-linker1-VH-PDL1-linker2-VH-KDR-CH1-CH2-CH3 from N-terminus to C-terminus. -KDR-CL;

Wherein, the VH-PDL1 is a heavy chain variable region that binds PD-L1,

The VL-PDL1 is the light chain variable region that binds to PD-L1,

The VH-KDR is a heavy chain variable region that binds to KDR,

The VL-KDR is the light chain variable region that binds to KDR,

The linker1 and linker2 are each independently a flexible peptide linker; the CH1-CH2-CH3 is the heavy chain constant region, the CL is the light chain constant region, the VH-PDL1 and the VL- PDL1 forms an antigen binding site that specifically binds to PD-L1, and said VH-KDR and said VL-KDR form an antigen binding site that specifically binds to KDR.

Wherein, the VH-PDL1 is a heavy chain variable region that binds PD-L1,

The VL-PDL1 is the light chain variable region that binds to PD-L1,

The VH-KDR is a heavy chain variable region that binds to KDR,

The VL-KDR is the light chain variable region that binds to KDR,

The linker1 is 4 G4S, the linker2 is 3 G4S, the CH1-CH2-CH3 is the heavy chain constant region, the CL is the light chain constant region, and the VH-PDL1 is the same as the The VL-PDL1 forms an antigen binding site that specifically binds to PD-L1, and the VH-KDR and the VL-KDR form an antigen binding site that specifically binds to KDR.

According to a preferred embodiment of the present invention, the VH-PDL1 includes the amino acid sequence of the heavy chain CDR shown in SEQ ID NO: 1-3, and the VL-PDL1 includes the amino acid sequence of the amino acid sequence shown in SEQ ID NO: 4-6. The VH-KDR includes the heavy chain CDR shown in SEQ ID NO: 7-9, and the VL-KDR includes the amino acid sequence shown in SEQ ID NO: 10-12. Light chain CDR.

According to a preferred embodiment of the present invention, the VH-PDL1 has the amino acid sequence shown in SEQ ID NO: 13, the VL-PDL1 has the amino acid sequence shown in SEQ ID NO: 14, and the VH -KDR has the amino acid sequence shown in SEQ ID NO: 15, and the VL-KDR has the amino acid sequence shown in SEQ ID NO: 16.

According to a preferred embodiment of the present invention, the heavy chain constant region includes an IgG1, IgG2, IgG3 or IgG4 heavy chain constant region, and the light chain constant region includes a kappa or lambda light chain constant region.

According to a preferred embodiment of the present invention, the polypeptide chain has an amino acid sequence as shown in SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 24 or SEQ ID NO: 25, and the light chain has The amino acid sequence shown in SEQ ID NO: 19; or the polypeptide chain has the amino acid sequence shown in SEQ ID NO: 20, SEQ ID NO: 26, SEQ ID NO: 27 or SEQ ID NO: 28, so The light chain has an amino acid sequence as shown in SEQ ID NO:21.

The second aspect of the present invention provides an isolated nucleotide, which encodes the bispecific antibody.

The third aspect of the present invention provides an expression vector, which contains the above-mentioned nucleotides.

The fourth aspect of the present invention provides a host cell, which contains the expression vector as described above.

The fifth aspect of the present invention provides a method for preparing the bispecific antibody, the method comprising the following steps:

(a) Culturing the host cell as described above under expression conditions to express the bispecific antibody;

(b) Isolation and purification of the bispecific antibody described in (a).

The sixth aspect of the present invention provides a pharmaceutical composition containing the bispecific antibody as described above and a pharmaceutically acceptable carrier.

In another preferred embodiment, the pharmaceutical composition also contains an anti-tumor agent.

In another preferred embodiment, the pharmaceutical composition is in a unit dosage form.

In another preferred embodiment, the anti-tumor agent and the bispecific antibody may be separately present in a separate package, or the anti-tumor agent may be coupled to the bispecific antibody.

In another preferred embodiment, the dosage form of the pharmaceutical composition includes a dosage form for gastrointestinal administration or a dosage form for parenteral administration.

In another preferred embodiment, the parenteral administration dosage form includes intravenous injection, intravenous drip, subcutaneous injection, local injection, intramuscular injection, intratumoral injection, intraperitoneal injection, intracranial injection, or intracavity injection.

The seventh aspect of the present invention provides the use of the above-mentioned bispecific antibody or the above-mentioned pharmaceutical composition in the preparation of a medicament for the treatment of cancer.

According to the present invention, the cancer is selected from the group consisting of: colorectal cancer, non-small cell lung cancer, gastric cancer, gastroesophageal junction adenocarcinoma, melanoma, lung cancer, liver cancer, lymphoma, leukemia, prostate cancer, bone marrow cancer and Other neoplastic malignant diseases, etc.

The eighth aspect of the present invention provides a method of treating cancer, which comprises administering the above-mentioned bispecific antibody, or the immunoconjugate thereof, or the above-mentioned pharmaceutical composition to a subject in need.

The ninth aspect of the present invention provides an immunoconjugate, the immunoconjugate comprising:

(a) The bispecific antibody as described in the first aspect of the present invention; and

(b) A coupling moiety selected from the group consisting of detectable markers, drugs, toxins, cytokines, radionuclides, or enzymes.

In another preferred embodiment, the conjugate part is selected from: fluorescent or luminescent markers, radioactive markers, MRI (magnetic resonance imaging) or CT (electronic computed tomography technology) contrast agents, or can produce Detect enzymes, radionuclides, biotoxins, cytokines (such as IL-2, etc.) of the product.

In another preferred embodiment, the immunoconjugate includes an antibody-drug conjugate (ADC).

In another preferred embodiment, the immunoconjugate is used to prepare a pharmaceutical composition for treating tumors.

Beneficial effects: The present invention provides a bispecific antibody against PDL1×KDR. Experimental results show that the biantibody can better maintain the activity of the respective monoclonal antibodies, and can specifically bind to the two targets of PD-L1 and KDR at the same time. Point, has good physical and chemical properties.

Description of the drawings

Figure 1 is a schematic diagram of the structure of the anti-PDL1×KDR double antibody molecule of the present invention. Figure 1A shows the anti-KDR scFv series connected to the C-terminal of anti-PDL1 mAb, and Figure 1B shows the anti-PDL1 scFv series connected to the anti-KDR mAb. N-terminal.

Figure 2 shows the HPLC detection spectrum and SDS-PAGE detection results of the anti-PDL1×KDR double antibody, among which Figure 2A is the HPLC detection spectrum and Figure 2B is the SDS-PAGE detection result.

Figure 3 shows the binding results of anti-PDL1×KDR double antibodies to PD-L1 and KDR, respectively, by ELISA. Figure 3A shows the binding results to PD-L1, and Figure 3B shows the binding results to KDR.

Figure 4 shows the results of dual-specific ELISA detecting the binding of anti-PDL1×KDR double antibodies to PD-L1 and KDR at the same time.

Figure 5 shows the results of FACS detection of the binding of anti-PDL1×KDR double antibodies to N87-PDL1 cells.

Figure 6 shows the results of anti-PDL1×KDR double antibodies blocking the activity of PD1/PD-L1 on cells.

Figure 7 shows the results of anti-PDL1×KDR double antibodies blocking the binding of KDR and VEGF on cells.

Figure 8 shows the anti-PDL1×KDR double antibody (anti-PDL1×KDR rev3, rev4, rev5, rev6, rev7) blocking the activity of PD1/PD-L1 at the cellular level.

Figure 9 shows the anti-PDL1×KDR double antibody (anti-PDL1×KDR rev3, rev4, rev5, rev6, rev7) blocking the activity of KDR and VEGF binding on cells; where Figure 9A shows the resistance of anti-PDL1×KDR rev3, rev4 Figure 9B shows the blocking activity of anti-PDL1×KDR rev5 and rev6; Figure 9C shows the blocking activity of anti-PDL1×KDR rev7.

detailed description

Through extensive and in-depth research and a large number of screenings, the inventors obtained a bispecific antibody composed of an anti-KDR antibody and an anti-PD-L1 antibody. The bispecific antibody of the present invention belongs to a homodimer. The bispecific antibody of the present invention not only maintains the activities of the anti-KDR antibody and the anti-PD-L1 antibody, but can also bind to KDR and PD-L1 at the same time. The bispecific antibody of the present invention can be developed as an antitumor drug with superior curative effect. On this basis, the inventor completed the present invention.

the term

In the present invention, the terms "Antibody (Ab)" and "Immunoglobulin G (Abbreviation IgG)" are heterotetrameric glycoproteins with the same structural characteristics, which are composed of two identical light chains (L ) And two identical heavy chains (H). Each light chain is connected to the heavy chain by a covalent disulfide bond, and the number of disulfide bonds between the heavy chains of different immunoglobulin isotypes (isotype) is different. Each heavy and light chain also has regularly spaced intrachain disulfide bonds. Each heavy chain has a variable region (VH) at one end, followed by a constant region. The heavy chain constant region is composed of three structural domains, CH1, CH2, and CH3. Each light chain has a variable region (VL) at one end and a constant region at the other end. The light chain constant region includes a structural domain CL; the light chain constant region is paired with the CH1 domain of the heavy chain constant region, and the light chain can be The variable region is paired with the variable region of the heavy chain. Constant regions are not directly involved in the binding of antibodies and antigens, but they exhibit different effector functions, such as participating in antibody-dependent cell-mediated cytotoxicity (ADCC, antibody-dependent cell-mediated cytotoxicity) and so on. The heavy chain constant region includes IgG1, IgG2, IgG3, and IgG4 subtypes; the light chain constant region includes kappa (Kappa) or lambda (Lambda). The heavy and light chains of the antibody are covalently linked together by the disulfide bond between the CH1 domain of the heavy chain and the CL domain of the light chain. The two heavy chains of the antibody are covalently linked together by the inter-polypeptide disulfide formed between the hinge regions. The bonds are linked together covalently.

In the present invention, the term "bispecific antibody (or dual antibody)" refers to an antibody molecule that can specifically bind to two antigens (targets) or two epitopes at the same time. According to symmetry, bispecific antibodies can be divided into structurally symmetric and asymmetric molecules. According to the number of binding sites, bispecific antibodies can be divided into bivalent, trivalent, tetravalent and multivalent molecules.

In the present invention, the term "monoclonal antibody (monoclonal antibody)" refers to an antibody obtained from a substantially homogeneous population, that is, the single antibodies contained in the population are the same, except for a few naturally occurring mutations that may exist. Monoclonal antibodies are highly specific to a single antigenic site. Moreover, unlike conventional polyclonal antibody preparations (usually a mixture of different antibodies directed against different antigenic determinants), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, the advantage of monoclonal antibodies is that they can be synthesized by culturing hybridomas without being contaminated by other immunoglobulins. The modifier "monoclonal" indicates the characteristics of the antibody, which is obtained from a substantially uniform antibody population, which should not be interpreted as requiring any special method to produce the antibody.

In the present invention, the terms "Fab" and "Fc" mean that papain can cleave an antibody into two identical Fab segments and one Fc segment. The Fab segment is composed of the VH and CH1 of the heavy chain of the antibody and the VL and CL domains of the light chain. The Fc segment can be a fragment crystallizable (Fc), which is composed of the CH2 and CH3 domains of the antibody. The Fc segment has no antigen binding activity and is the site where the antibody interacts with effector molecules or cells.

In the present invention, the term "scFv" refers to a single chain antibody (single chain antibody fragment, scFv), which is formed by connecting the variable region of the heavy chain and the variable region of the light chain of the antibody through a linker of 15-25 amino acids. become.

In the present invention, the term "variable" means that certain parts of the variable region of the antibody are different in sequence, which forms the binding and specificity of various specific antibodies to their specific antigens. However, the variability is not evenly distributed throughout the variable regions of antibodies. It is concentrated in three fragments called the complementarity-determining region (CDR) or hypervariable region in the variable region of the heavy chain and the variable region of the light chain. The more conservative part of the variable region is called the frame region (FR). The variable regions of the natural heavy chain and light chain each contain four FR regions, which are roughly in a β-sheet configuration, connected by three CDRs forming a connecting loop, and in some cases can form a partial β-sheet structure. The CDRs in each chain are closely placed together through the FR region and form the antigen binding site of the antibody together with the CDRs of the other chain (see Kabat et al., NIH Publ. No. 91-3242, Volume I, pages 647-669 (1991)).

As used herein, the term "framework region" (FR) refers to the amino acid sequence inserted between CDRs, that is, those parts of the light chain and heavy chain variable regions of immunoglobulins that are relatively conserved among different immunoglobulins in a single species . The light chain and heavy chain of an immunoglobulin each have four FRs, which are called FR1-L, FR2-L, FR3-L, FR4-L and FR1-H, FR2-H, FR3-H, FR4-H, respectively. Accordingly, the light chain variable domain can therefore be referred to as (FR1-L)-(CDR1-L)-(FR2-L)-(CDR2-L)-(FR3-L)-(CDR3-L)-( FR4-L) and the heavy chain variable domain can therefore be expressed as (FR1-H)-(CDR1-H)-(FR2-H)-(CDR2-H)-(FR3-H)-(CDR3-H) -(FR4-H). Preferably, the FR of the present invention is a human antibody FR or a derivative thereof, and the derivative of the human antibody FR is basically the same as the naturally-occurring human antibody FR, that is, the sequence identity reaches 85%, 90%, 95%, 96% , 97%, 98% or 99%.

Knowing the amino acid sequence of the CDR, those skilled in the art can easily determine the framework regions FR1-L, FR2-L, FR3-L, FR4-L and/or FR1-H, FR2-H, FR3-H, FR4-H.

As used herein, the term "human framework region" is substantially the same (about 85% or more, specifically 90%, 95%, 97%, 99% or 100%) framework region of a naturally occurring human antibody. .

As used herein, the term "linker" refers to the insertion of an immunoglobulin domain to provide sufficient mobility for the light chain and heavy chain domains to fold to exchange one or more amino acid residues of the immunoglobulin with dual variable regions. base. In the present invention, the preferred linker refers to the linker Linker1 and Linker2, where Linker1 connects the VH and VL of a single-chain antibody (scFv), and Linker2 is used to connect the scFv to the heavy chain of another antibody.

Examples of suitable linkers include single glycine (Gly) or serine (Ser) residues, and the identity and sequence of amino acid residues in the linker can vary with the type of secondary structural elements that need to be implemented in the linker.

Bispecific antibody

The bispecific antibody of the present invention is an anti-PDL1×EGFR bispecific antibody, including an anti-PDL1 antibody part and an anti-EGFR antibody part.

Preferably, the sequence of the anti-PD-L1 antibody of the present invention is as described in the patent application PCT/CN2020/090442. Those skilled in the art can also modify or transform the anti-PD-L1 antibody of the present invention through techniques well known in the art, such as adding , Deletion and/or substitution of one or several amino acid residues, thereby further increasing the affinity or structural stability of anti-PD-L1, and obtaining modified or modified results through conventional measurement methods.

In the present invention, "conservative variants of the bispecific antibody of the present invention" refer to at most 10, preferably at most 8, and more preferably at most 5 compared with the amino acid sequence of the bispecific antibody of the present invention. Optimally, at most 3 amino acids are replaced by amino acids with similar or similar properties to form a polypeptide. These conservative variant polypeptides are best produced according to Table A by performing amino acid substitutions.

Table A

最初的残基Initial residues	代表性的取代Representative substitution	优选的取代Preferred substitution
Ala(A)Ala(A)	Val；Leu；IleVal; Leu; Ile	ValVal
Arg(R)Arg(R)	Lys；Gln；AsnLys; Gln; Asn	LysLys
Asn(N)Asn(N)	Gln；His；Lys；ArgGln; His; Lys; Arg	GlnGln
Asp(D)Asp(D)	GluGlu	GluGlu
Cys(C)Cys(C)	SerSer	SerSer
Gln(Q)Gln(Q)	AsnAsn	AsnAsn
Glu(E)Glu(E)	AspAsp	AspAsp
Gly(G)Gly(G)	Pro；AlaPro; Ala	AlaAla
His(H)His(H)	Asn；Gln；Lys；ArgAsn; Gln; Lys; Arg	ArgArg
Ile(I)Ile(I)	Leu；Val；Met；Ala；PheLeu; Val; Met; Ala; Phe	LeuLeu
Leu(L)Leu(L)	Ile；Val；Met；Ala；PheIle; Val; Met; Ala; Phe	IleIle
Lys(K)Lys(K)	Arg；Gln；AsnArg; Gln; Asn	ArgArg
Met(M)Met(M)	Leu；Phe；IleLeu; Phe; Ile	LeuLeu
Phe(F)Phe(F)	Leu；Val；Ile；Ala；TyrLeu; Val; Ile; Ala; Tyr	LeuLeu
Pro(P)Pro(P)	AlaAla	AlaAla
Ser(S)Ser(S)	ThrThr	ThrThr
Thr(T)Thr(T)	SerSer	SerSer

Trp(W)Trp(W)	Tyr；PheTyr; Phe	TyrTyr
Tyr(Y)Tyr(Y)	Trp；Phe；Thr；SerTrp; Phe; Thr; Ser	PhePhe
Val(V)Val(V)	Ile；Leu；Met；Phe；AlaIle; Leu; Met; Phe; Ala	LeuLeu

In the present invention, the terms "anti", "binding", and "specific binding" refer to the non-random binding reaction between two molecules, such as the reaction between an antibody and the antigen it is directed against. Generally, the antibody binds to the antigen with an equilibrium dissociation constant (KD) of ^{less than about 10 -7} M, for example, less than about 10 ^-8 M, 10 ^-9 M, 10 ^-10 M, 10 ^{-11 M or less.} In the present invention, the term "KD" refers to the equilibrium dissociation constant of a specific antibody-antigen interaction, which is used to describe the binding affinity between the antibody and the antigen. The smaller the equilibrium dissociation constant, the tighter the antibody-antigen binding, and the higher the affinity between the antibody and the antigen. For example, Surface Plasmon Resonance (SPR) is used to measure the binding affinity of an antibody to an antigen in a BIACORE instrument or an ELISA is used to measure the relative binding affinity of an antibody to the antigen.

In the present invention, the term "epitope" refers to a polypeptide determinant that specifically binds to an antibody. The epitope of the present invention is a region of an antigen that is bound by an antibody.

The bispecific antibodies of the present invention can be used alone, or can be combined or coupled with detectable markers (for diagnostic purposes), therapeutic agents, or any combination of these substances.

Coding nucleic acid and expression vector

The present invention also provides polynucleotide molecules encoding the above-mentioned antibodies or fragments or fusion proteins thereof. The polynucleotide of the present invention may be in the form of DNA or RNA. DNA forms include cDNA, genomic DNA or synthetic DNA. DNA can be single-stranded or double-stranded. DNA can be a coding strand or a non-coding strand.

In the present invention, the term "expression vector" refers to a vector carrying an expression cassette for expressing a specific target protein or other substances, such as a plasmid, a viral vector (such as adenovirus, retrovirus), a phage, a yeast plasmid or other vectors. Representative examples include, but are not limited to: pTT5, pSECtag series, pCGS3 series, pcDNA series vectors, etc., and other vectors used in mammalian expression systems. The expression vector includes fusion DNA sequences linked to appropriate transcription and translation regulatory sequences.

Once the relevant sequence is obtained, the recombination method can be used to obtain the relevant sequence in large quantities. This is usually done by cloning it into a vector, then transferring it into a cell, and then isolating the relevant sequence from the proliferated host cell by conventional methods.

The present invention also relates to a vector containing the above-mentioned appropriate DNA sequence and an appropriate promoter or control sequence. These vectors can be used to transform appropriate host cells so that they can express proteins.

In the present invention, the term "host cell" refers to a cell suitable for expressing the above-mentioned expression vector. It can be a eukaryotic cell. For example, mammalian or insect host cell culture systems can be used for the expression of the fusion protein of the present invention. CHO (Chinese hamster Ovary, Chinese Hamster Ovary), HEK293, COS, BHK and derived cells of the above-mentioned cells are all suitable for the present invention.

Pharmaceutical composition and application

The invention also provides a composition. Preferably, the composition is a pharmaceutical composition, which contains the aforementioned antibody or active fragment or fusion protein thereof, and a pharmaceutically acceptable carrier. Generally, these substances can be formulated in a non-toxic, inert and pharmaceutically acceptable aqueous carrier medium, where the pH is usually about 5-8, preferably about 6-8, although the pH can be The nature of the formulated substance and the condition to be treated vary. The formulated pharmaceutical composition can be administered by conventional routes, including (but not limited to): intravenous injection, intravenous drip, subcutaneous injection, local injection, intramuscular injection, intratumor injection, intraperitoneal injection (such as intraperitoneal injection) ), intracranial injection, or intracavity injection. In the present invention, the term "pharmaceutical composition" means that the bispecific antibody of the present invention can be combined with a pharmaceutically acceptable carrier to form a pharmaceutical preparation composition so as to exert a more stable therapeutic effect. These preparations can ensure that the bispecific antibody disclosed in the present invention The conformational integrity of the amino acid core sequence of the sex antibody, while also protecting the multifunctional groups of the protein from its degradation (including but not limited to aggregation, deamination or oxidation). The pharmaceutical composition of the present invention contains a safe and effective amount (such as 0.001-99 wt%, preferably 0.01-90 wt%, more preferably 0.1-80 wt%) of the above-mentioned bispecific antibody (or conjugate thereof) of the present invention, and A pharmaceutically acceptable carrier or excipient. Such carriers include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical preparation should match the mode of administration. The pharmaceutical composition of the present invention can be prepared in the form of injection, for example, with physiological saline or an aqueous solution containing glucose and other adjuvants for preparation by conventional methods. Pharmaceutical compositions such as injections and solutions should be manufactured under aseptic conditions. The dosage of the active ingredient is a therapeutically effective amount, for example, about 10 micrograms/kg body weight to about 50 mg/kg body weight per day. In addition, the bispecific antibodies of the present invention can also be used with other therapeutic agents.

When using the pharmaceutical composition, a safe and effective amount of the bispecific antibody or its immunoconjugate is administered to the mammal, wherein the safe and effective amount is usually at least about 10 micrograms per kilogram of body weight, and in most cases no more than about 50 mg/kg body weight, preferably the dose is about 10 micrograms/kg body weight to about 10 mg/kg body weight. Of course, the specific dosage should also consider factors such as the route of administration, the patient's health status, etc., which are all within the skill range of a skilled physician.

The experimental materials used in the following examples are described as follows:

pcDNA ^TM 3.4

vector: purchased from Thermo fisher company, article number A14697;

CHO cells: purchased from Thermofisher, catalog number A29133;

293E cells: from NRC Biotechnology Research Institute;

Human gastric cancer cell line NCI-N87: purchased from the American Type Culture Collection (ATCC);

PD-1/PD-L1 Blockade Bioassay, Propagation model: purchased from Promega, item number J1252;

VEGF Bioassay, Propagation Model: purchased from Promega, the article number is GA1082.

The experimental reagents used in the following examples are described as follows:

Anti-PD-L1 monoclonal antibody: prepared according to the sequence in PCT/CN2020/090442;

Anti-KDR monoclonal antibody: CDR region sequence comes from Dan Lu et al. Tailoring in Vitro Selection for a Picomolar Affinity Human Antibody Directed against Vascular Endothelial Growth Factor Receptor 2 The for Enhancement Biological of 2003. : 43496-43507.) in the clone number 3A10 sequence, other framework regions were obtained after mutation by the company;

HRP-labeled goat anti-human Fc antibody: purchased from sigma, catalog number A0170;

FITC-labeled goat anti-human Fc antibody: purchased from sigma, item number F9512;

HRP-labeled mouse anti-human Fab antibody: purchased from sigma, catalog number A0293;

HRP-labeled anti-6×His antibody: purchased from abcam, catalog number ab178563;

Goat anti-human IgG-FITC: purchased from sigma, item number F4143;

PBS: purchased from Shenggong Biological Engineering (Shanghai) Co., Ltd., catalog number B548117;

PBST: PBS+0.05% Tween 20;

BSA: purchased from Shenggong Biological Engineering (Shanghai) Co., Ltd., catalog number A60332;

FBS: purchased from Gibco, item number 10099;

TMB: purchased from BD company, article number 555214;

Bio-Glo Luciferase Assay System: purchased from Promega, item number G7940;

Solution: purchased from sigma, item number A6964-100mL.

The experimental instruments used in the following examples are described as follows:

PCR instrument: purchased from BioRad, article number C1000 Touch Thermal Cycler;

HiTrap MabSelectSuRe column: purchased from GE, item number 11-0034-95;

Beckman Coulter CytoFLEX flow cytometer: purchased from Beckman;

SpectraMax i3x microplate reader: purchased from Molecular Devices.

The following examples and experimental examples are to further illustrate the present invention, and should not be construed as limiting the present invention. The examples do not include detailed descriptions of traditional methods, such as those used to construct vectors and plasmids, methods of inserting genes encoding proteins into such vectors and plasmids, or methods of introducing plasmids into host cells. Such methods are well known to those of ordinary skill in the art, and are described in many publications, including Sambrook, J., Fritsch, EF and Maniais, T. (1989) Molecular Cloning: A Laboratory Manual , 2nd edition, Cold spring Harbor Laboratory Press. The experimental methods that do not indicate specific conditions in the following examples are usually in accordance with conventional conditions or in accordance with the conditions recommended by the manufacturer. Unless otherwise specified, percentages and parts are weight percentages and parts by weight.

Example 1 Construction of anti-PDL1×KDR double antibody molecule

In the present invention, the anti-human KDR monoclonal antibody scFv1 (VL-linker1-VH) is connected in series to the C-terminus of the heavy chain of the anti-human PD-L1 monoclonal antibody through linker2 to construct an anti-PDL1×KDR bispecific antibody named It is anti-PDL1×KDR BsAb1. The structure is shown in Figure 1A (the VL of scFv1 is connected to the C-terminus of the heavy chain of anti-human PD-L1 monoclonal antibody through linker2).

In the present invention, the anti-human KDR monoclonal antibody scFv2 (VH-linker1-VL) is connected in series to the C-terminus of the heavy chain of the anti-human PD-L1 monoclonal antibody through linker2 to construct an anti-PDL1×KDR bispecific antibody, which is named anti -PDL1×KDR BsAb2. The structure is shown in Figure 1A (the VH of scFv2 is connected to the C-terminus of the heavy chain of anti-human PD-L1 monoclonal antibody through linker2).

In the present invention, the anti-human PD-L1 monoclonal antibody scFv3 (VL-linker1-VH) is connected in series to the N-terminus of the heavy chain of the anti-human KDR monoclonal antibody through linker2 to construct an anti-PDL1×KDR bispecific antibody, which is named anti -PDL1×KDR BsAb3. The structure is shown in Figure 1B (the VH of scFv3 is connected to the N-terminus of the heavy chain of anti-human KDR monoclonal antibody through linker2).

In the present invention, the anti-human KDR monoclonal antibody scFv2 (VH-linker1-VL) is connected in series to the N-terminus of the heavy chain of the anti-human PD-L1 monoclonal antibody through linker2 to construct an anti-PDL1×KDR bispecific antibody named It is anti-PDL1×KDR Rev3 (the VL of scFv2 is connected to the N-terminus of the heavy chain of anti-human PD-L1 monoclonal antibody through linker2).

In the present invention, the anti-human KDR monoclonal antibody scFv1 (VL-linker1-VH) is connected in series to the N-terminus of the heavy chain of the anti-human PD-L1 monoclonal antibody through linker2 to construct an anti-PDL1×KDR bispecific antibody named It is anti-PDL1×KDR Rev4 (the VH of scFv1 is connected to the N-terminus of the heavy chain of anti-human PD-L1 monoclonal antibody through linker2).

The present invention uses the scFv3 (VL-linker1-VH) of the anti-human PD-L1 monoclonal antibody, which is connected in series to the C-terminus of the heavy chain of the anti-human KDR monoclonal antibody through linker2, to construct an anti-PDL1×KDR bispecific antibody, named as anti -PDL1×KDR Rev5 (the VL of scFv3 is connected to the C-terminus of the heavy chain of anti-human KDR monoclonal antibody through linker2).

The present invention uses the scFv4 (VH-linker1-VL) of the anti-human PD-L1 monoclonal antibody, which is connected in series to the C-terminus of the heavy chain of the anti-human KDR monoclonal antibody through linker2, to construct an anti-PDL1×KDR bispecific antibody, named as anti -PDL1×KDR Rev6 (the VH of scFv4 is connected to the C-terminus of the heavy chain of anti-human KDR monoclonal antibody through linker2).

In the present invention, the anti-human PD-L1 monoclonal antibody scFv4 (VH-linker1-VL) is connected in series to the N-terminus of the heavy chain of the anti-human KDR monoclonal antibody through linker2 to construct an anti-PDL1×KDR bispecific antibody, which is named anti -PDL1×KDR Rev7 (the VL of scFv4 is connected to the N-terminus of the heavy chain of anti-human KDR monoclonal antibody through linker2).

Among them, linker1 is 4 GGGGS, and linker2 is 3 GGGGS. The CDR region sequence of anti-human KDR monoclonal antibody comes from Dan Lu et al. 2 For Enhanced Neutralizing Activity. The Journal of Biological Chemistry, 2003,278:43496-43507.) The sequence of clone number 3A10, the other framework regions are obtained after mutation of the company, and the source of the sequence of anti-human PD-L1 monoclonal antibody M8 In PCT/CN2020/090442.

The heavy chain and light chain expression vectors of each bispecific antibody and its corresponding monoclonal antibody were obtained through gene synthesis and conventional molecular cloning methods. The corresponding amino acid sequences are shown in Table 1, and the CDRs are encoded according to Kabat rules.

Table 1. Sequence information of the antibody of the present invention

Note: The polypeptide chain of the anti-PDL1×KDR double antibody in the above table refers to the heavy chain of the double antibody (formed by the scFv connected to the end of the heavy chain of the anti-human KDR monoclonal antibody through a linker). The light chain of the double antibody does not contain Inside.

Example 2 Expression and purification of anti-PDL1×KDR double antibodies

The polypeptide chain and light chain DNA fragments of the anti-PDL1×KDR double antibody were respectively subcloned into pcDN3.4 vector, and the recombinant plasmids were extracted and co-transfected into CHO cells and/or 293E cells. After 5-7 days of cell culture, the cells were cultured After the solution is filtered by high-speed centrifugation and vacuum filtration with a microporous membrane, the sample is loaded on a HiTrap MabSelect SuRe column, and the protein is eluted with an eluent containing 100 mM citric acid and pH 3.5, and the target sample is recovered and dialyzed to pH 7.4 PBS.

The purified protein was detected by HPLC. The HPLC detection pattern of the anti-PDL1×KDR double antibody is shown in Figure 2A, and the purity of the anti-PDL1×KDR BsAb1 double antibody monomer reaches more than 96%. The other two double antibodies (anti-PDL1×KDR BsAb2, BsAb3) have similar profiles, and the monomer purity is above 96%. The results of SDS-PAGE detection are shown in Figure 2B.

Lanes

1 and 2 are reduced and non-reduced SDS-PAGE of anti-PDL1×KDR BsAb1, and

lanes

3 and 4 are reduced and non-reduced SDS-PAGE of anti-PDL1 monoclonal antibody. 5 and 6 are the reduced and non-reduced SDS-PAGE of anti-PDL1×KDR BsAb2, and

lanes

7 and 8 are the reduced and non-reduced SDS-PAGE of anti-PDL1×KDR BsAb3. The theoretical molecular weight of anti-PDL1×KDR BsAb1 and anti-PDL1×KDR BsAb2 is 197KD, and the theoretical molecular weight of anti-PDL1×KDR BsAb3 is 196KD.

Example 3 Enzyme-linked immunosorbent assay (ELISA) to determine the affinity of anti-PDL1×KDR double antibodies to antigen

3.1 Detection of affinity with PD-L1 antigen

In order to detect the affinity of the anti-PDL1×KDR double antibody with the PD-L1 antigen, the PDL1-ECD-His protein (according to the sequence provided by NCBI (NCBI registration number NP_054862.1) was synthesized into PD-L1 with pH 7.4 PBS buffer The extracellular domain gene is added with a signal peptide sequence at the N-terminus and a 6×His tag at the C-terminus. It is constructed into the expression vector through the two restriction sites of EcoRI and HindIII, and transfected into HEK-293E cells for expression and purification. ) Dilute to 2000ng/ml, then add 100μl/well to the ELISA plate; incubate overnight at 4°C; wash the plate twice with PBST the next day; add PBST+1% BSA to each well for blocking, block at 37°C for 1 hour; wash the plate with PBST Twice; then add the antibody to be tested diluted with PBS + 1% BSA, and anti-PD-L1 monoclonal antibody as a positive control, with an initial concentration of 300 nM, and 12 gradients of 3-fold dilution. Incubate at 37°C for 1h; wash the plate twice with PBST, add HRP-labeled goat anti-human Fc antibody, and incubate at 37°C for another 40min; wash the plate three times with PBST and pat dry, add 100μl TMB to each well, and avoid light at room temperature (20±5°C) for 5 min; per well was added 50μl 2M H ₂ SO ₄ stop solution to stop the substrate reaction, microplate read OD at 450nm, GraphPad Prism data analysis, plotting and calculation of EC _50. _{The experimental results are shown in Figure 3A. The EC 50 of} anti-PD-L1 monoclonal antibody, anti-PDL1×KDR BsAb1, anti-PDL1×KDR BsAb2, and anti-PDL1×KDR BsAb3 are 0.13nM, 0.13nM, 0.14nM, 0.15, respectively. nM, the affinity of the three double antibodies is equivalent to that of the monoclonal antibody, and the anti-PD-L1 monoclonal antibody platform is slightly higher, which may be due to the fact that the secondary antibody is anti-Fc. It has been experimentally proved that the ELISA of this sample is different due to the secondary antibody, and the result is slightly There are different. It can be considered that the affinity of the double antibody is not weaker than that of the monoclonal antibody.

3.2 Affinity test with KDR antigen

In order to detect the affinity of the anti-PDL1×KDR double antibody with the KDR antigen, the KDR-ECD-His protein (according to the sequence provided by UniProt (SEQ ID P35968)) was synthesized into the extracellular domain gene with pH 7.4 PBS buffer and added at its N-terminus. The upper signal peptide sequence, the 6×His tag is added to the C-terminus, the two restriction sites of EcoRI and HindIII are respectively constructed into the expression vector, and the HEK-293E cell is transfected to express and purified.) Dilute to 2000ng/ml, then 100μl /Well add to ELISA plate; incubate overnight at 4°C; wash the plate twice with PBST the next day; add PBST+1% BSA to each well for blocking, block at 37°C for 1h; wash the plate twice with PBST; then add PBS+1 The antibody to be tested is diluted in %BSA, and anti-KDR monoclonal antibody is used as a positive control. The initial concentration is 300nM, and 12 gradients are gradually diluted 3 times. Incubate at 37°C for 1h; wash the plate twice with PBST, add HRP-anti-Fab antibody, and incubate at 37°C for another 40min; wash the plate three times with PBST and pat dry, add 100μl TMB to each well, and place in the dark at room temperature (20±5°C) 5 min; per well was added 50μl 2M H ₂ SO ₄ stop solution to stop the substrate reaction, microplate read OD at 450nm, GraphPad Prism data analysis, plotting and calculation of EC _50. _{The experimental results are shown in Figure 3B. The EC 50 of} anti-KDR monoclonal antibody, anti-PDL1×KDR BsAb1, anti-PDL1×KDR BsAb2, and anti-PDL1×KDR BsAb3 are 0.26nM, 0.23nM, 0.23nM, 0.41nM, respectively. The affinity of anti-PDL1×KDR BsAb1 and anti-PDL1×KDR BsAb2 is slightly stronger than that of monoclonal antibodies. This may be due to the fact that the secondary antibody is anti-Fab. Experiments have shown that the ELISA of this sample is slightly different due to different secondary antibodies. . It can be considered that the affinity of the double antibody is not weaker than that of the monoclonal antibody.

Example 4 Double specific ELISA to detect the ability of anti-PDL1×KDR double antibodies to simultaneously bind to two antigens

In order to test the ability of the anti-PDL1×KDR double antibody to simultaneously bind to the KDR antigen and the PD-L1 antigen, the PDL1-ECD-hFc protein was replaced with a pH 7.4 PBS buffer (the C-terminus of the PDL1-ECD-His protein was replaced with the hFc tag) Dilute to 1μg/ml, then add 100μl/well to the ELISA plate; incubate overnight at 4°C; wash the plate twice with PBST the next day; add PBST+1% BSA to each well for blocking, and block at 37°C for 1 hour; wash the plate twice with PBST Times; then add the antibody to be detected that is diluted with PBS+1% BSA, the initial concentration is 12nM, and 8 gradients of 3-fold dilution are gradually added. Incubate at 37°C for 1h; wash the plate twice with PBST, then add 1μg/ml KDR-ECD-His antigen diluted with pH7.4 PBS, and add 100μl/well to the ELISA plate. Incubate at 37°C for 1h; wash the plate twice with PBST, add the secondary antibody HRP-anti-His, and incubate at 37°C for another 40min; wash the plate three times with PBST and pat dry, add 100μl TMB to each well, and store at room temperature (20±5°C) away from light 5 minutes; per well was added 50μl 2M H ₂ SO ₄ stop solution to stop the substrate reaction, microplate read OD at 450nm, GraphPad Prism data analysis, plotting and calculation of EC _50. The experimental results are shown in Figure 4. The EC50 of anti-PDL1×KDR BsAb1, anti-PDL1×KDR BsAb2, and anti-PDL1×KDR BsAb3 are 0.13nM, 0.14nM, 0.20nM, respectively. Among them, anti-PDL1×KDR BsAb3 is slightly weaker than the other two double antibodies, and the monoclonal antibody does not have the ability to bind these two antigens at the same time.

Example 5 FACS detection of the binding affinity of anti-PDL1×KDR double antibodies to target cells

N87-PDL1 used the lentiviral transfection method in our laboratory to transfect NCI-N87 with a stable cell line constructed by PD-L1. After taking the N87-PDL1 in the logarithmic growth phase and digesting it with trypsin, it was washed three times with PBS containing 0.5% BSA, and centrifuged at 300 g for 5 minutes each time, and the supernatant was discarded. Resuspend the cells in 0.5% BSA in PBS at a cell density of 1×10 ⁶ cells/mL, and add 100 μL/well to a 96-well plate. Dilute the anti-PDL1×KDR double antibody and the positive control anti-PD-L1 monoclonal antibody to 120nM, dilute 11 gradients step by step, add 100μL/well to a 96-well plate, and mix well with N87-PDL1 cells. Incubate at 4°C for 1h. Wash the cells twice with PBS to remove unbound antibody to be tested. Then add 100 μL/well of FITC-labeled goat anti-human Fc antibody, and incubate at 4°C for 30 minutes. Centrifuge at 300g for 5 minutes, and wash the cells twice with PBS to remove unbound secondary antibodies. Finally, the cells were resuspended in 200μl PBS, and the binding affinity of the double antibody to the cells was determined by the Beckman Coulter CytoFLEX flow cytometer. The data obtained was fitted and analyzed by GraphPad Prism software.

The experimental results are shown in Figure 5. The EC ₅₀ of anti-PD-L1 monoclonal antibody is 0.13nM, the EC 50 of anti-PDL1×KDR BsAb1 _{is 0.15nM, the EC 50} of anti-PDL1×KDR BsAb2 is 0.15nM, and _{the EC 50 of anti-PDL1} _{The EC 50} of ×KDR BsAb3 is 0.21 nM, and the affinity of the three double antibodies is equivalent to that of the positive control anti-PD-L1 monoclonal antibody.

Example 6 Anti-PDL1×KDR double antibody blocks the activity of PD1/PD-L1 at the cellular level

This experiment uses Promega's PD-1/PD-L1 Blockade Bioassay, Propagation model and method.

Take the PD-L1 aAPC/CHO-K1 grown in logarithmic phase, trypsinize into single cells, transfer to a white bottom permeable 96-well plate, 100μL/well, 40,000 cells/well, place at 37℃, 5% CO ₂ , Incubate overnight. Take anti-PDL1×KDR double antibody and anti-PD-L1 monoclonal antibody, and dilute to 2× working solution concentration, starting concentration is 66nM, step by step 3 times gradient. Take the PD1 effector cells with a density of 1.4-2×10 ⁶ cells/mL and a cell viability above 95%, and trypsinize them into a single cell suspension of ^{1.25×10 6 cells/ml.} Take the PD-L1aAPC/CHO-K1 cells that were plated the day before, discard the supernatant, and add 40 μl of serially diluted double antibody/PD-L1 monoclonal antibody working solution; then add an equal volume of PD1 effector cells. Place at 37°C, 5% CO ₂ , and incubate for 6 hours. Add 80μl of Bio-Glo detection reagent to each well. After incubating for 10 minutes at room temperature, read the luminescence with spectramax i3. Seal the bottom of the plate with an opaque film before reading the plate. Data analysis was performed using GraphPad Prism, and plotting calculated IC _50.

The experimental results are shown in Figure 6, the IC ₅₀ of the anti-PD-L1 monoclonal antibody is 0.23 nM. _{The IC 50 of} anti-PDL1×KDR BsAb1, anti-PDL1×KDR BsAb2, anti-PDL1×KDR BsAb3 are 0.27nM, 0.22nM, and 0.30nM, respectively, which are equivalent to monoclonal antibodies.

anti-PDL1×KDR Rev3, Rev4, Rev5, Rev6, and Rev7 block the activity of PD1/PD-L1 signaling pathway on cells. The results are shown in Figure 8. Anti-PDL1×KDR rev5 is significantly more single than the positive control anti-PD-L1 Anti-poor; anti-PDL1×KDR rev6 is slightly worse than the positive control anti-PD-L1 monoclonal antibody; anti-PDL1×KDR rev3, rev4, rev7 has no significant difference with the positive control anti-PD-L1 monoclonal antibody.

Example 7 Anti-PDL1×KDR double antibody blocks the activity of KDR and VEGF binding on cells

This experiment uses Promega's VEGF Bioassay, Propagation Model and method.

KDR/NFAT-RE HEK293 cells express KDR on the surface. When VEGF binds to the KDR on the cell surface, the signal is transmitted to the cell, and the fluorescent reporter gene is expressed, and the biological fluorescent signal can be detected. When the anti-KDR antibody is added to block the binding of VEGF to KDR on the cell surface, the fluorescence signal is weakened, and there is a dose-effect relationship with the concentration of KDR antibody within a certain range.

Take a bottle of T75 cultured KDR/NFAT-RE HEK293 cells in the logarithmic growth phase, wash it with D-PBS, and then add 3 mL

The solution was digested at 37°C for about 2 minutes, then an equal volume of assay buffer (DMED+10% FBS) was added to neutralize, and the cells were dispersed by pipetting the cells. Centrifuge at 200g for 5 min. Count the trypan blue, then adjust the cell density to 1.6×10 ⁶ cells/mL, and pave a 96-well white translucent bottom plate with 25 μL/well. VEGF was diluted with assay buffer (DMED+10% FBS) to a 3x dilution, 60ng/mL, working concentration 20ng/mL, 25μL/well was added to 96-well white transparent bottom plate. Dilute the antibody to be tested and the positive control with assay buffer (DMED+10% FBS) to 1000 nM 3× dilution, and then dilute it four-fold step by step, and add 25 μL/well to 96-well white translucent bottom plate. Continue to incubate for 6 hours in a 37°C, 5% CO ₂ incubator, take out the 96-well plate, add 75 μL/well of Bio-Glo detection reagent, incubate at room temperature for 10 minutes, and read the luminescence with spectramax i3. Seal the bottom of the plate with an opaque film before reading the plate. Data analysis was performed using GraphPad Prism, and plotting calculated IC _50.

The experimental results are shown in Figure 7, the IC ₅₀ of the anti-KDR monoclonal antibody is 3.10 nM. _{The IC 50 of} anti-PDL1×KDR BsAb1, anti-PDL1×KDR BsAb2, anti-PDL1×KDR BsAb3 are 13.47nM, 6.09nM, 4.35nM, respectively, of which anti-PDL1×KDR BsAb1 is slightly weaker than anti- PDL1×KDR BsAb2 and anti-PDL1×KDR BsAb3. There is little difference in the activity of the three groups of double antibodies and monoclonal antibodies.

anti-PDL1×KDR Rev3, Rev4, Rev5, Rev6, Rev7 block the activity of KDR and VEGF binding on cells, the results are shown in Figure 9A, 9B, 9C, anti-PDL1×KDR Rev3, rev4, rev5, rev6, rev7 The activity of the anti-KDR monoclonal antibody is not significantly different from that of the positive control.

All documents mentioned in the present invention are cited as references in this application, as if each document was individually cited as a reference. In addition, it should be understood that after reading the above teaching content of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application.

Claims

The anti-PDL1×KDR bispecific antibody is characterized by comprising two polypeptide chains and two light chains selected from the group consisting of:

(a) The polypeptide chain from N-terminus to C-terminus contains VH-PDL1-CH1-CH2-CH3-linker2-VL-KDR-linker1-VH-KDR or VH-PDL1-CH1-CH2-CH3-linker2-VH -KDR-linker1-VL-KDR, the light chain includes VL-PDL1-CL from N-terminus to C-terminus; or

(b) The polypeptide chain includes VL-PDL1-linker1-VH-PDL1-linker2-VH-KDR-CH1-CH2-CH3 from N-terminus to C-terminus, and the light chain includes VL-PDL1-linker1-VH-PDL1-linker2-VH-KDR-CH1-CH2-CH3 from N-terminus to C-terminus. -KDR-CL; or

(c) The polypeptide chain includes VH-KDR-linker1-VL-KDR-linker2-VH-PDL1-CH1-CH2-CH3 or VL-KDR-linker1-VH-KDR-linker2-VH from N-terminus to C-terminus -PDL1-CH1-CH2-CH3, the light chain includes VL-PDL1-CL from N-terminus to C-terminus; or

(d) The polypeptide chain includes VH-KDR-CH1-CH2-CH3-linker2-VL-PDL1-linker1-VH-PDL1 or VH-KDR-CH1-CH2-CH3-linker2-VH from N-terminus to C-terminus -PDL1-linker1-VL-PDL1, the light chain includes VL-KDR-CL from N-terminus to C-terminus; or

(e) The polypeptide chain includes VH-PDL1-linker1-VL-PDL1-linker2-VH-KDR-CH1-CH2-CH3 from N-terminus to C-terminus, and the light chain includes VL from N-terminus to C-terminus -KDR-CL;

Wherein, the VH-PDL1 is a heavy chain variable region that binds PD-L1,

The VL-PDL1 is the light chain variable region that binds to PD-L1,

The VH-KDR is a heavy chain variable region that binds to KDR,

The VL-KDR is the light chain variable region that binds to KDR,

The linker1 and linker2 are each independently a flexible peptide linker,

The CH1-CH2-CH3 is the heavy chain constant region, the CL is the light chain constant region, and the VH-PDL1 and the VL-PDL1 form an antigen binding site that specifically binds to PD-L1, The VH-KDR and the VL-KDR form an antigen binding site that specifically binds to the KDR.
The anti-PDL1×KDR bispecific antibody according to claim 1, characterized in that it comprises two polypeptide chains and two light chains, wherein: (a) the polypeptide chain comprises VH from N-terminus to C-terminus -PDL1-CH1-CH2-CH3-linker2-VL-KDR-linker1-VH-KDR or VH-PDL1-CH1-CH2-CH3-linker2-VH-KDR-linker1-VL-KDR, the light chain is from N The end to the C-terminus contains VL-PDL1-CL; or

(b) The polypeptide chain includes VL-PDL1-linker1-VH-PDL1-linker2-VH-KDR-CH1-CH2-CH3 from N-terminus to C-terminus, and the light chain includes VL-PDL1-linker1-VH-PDL1-linker2-VH-KDR-CH1-CH2-CH3 from N-terminus to C-terminus. -KDR-CL;

Wherein, the VH-PDL1 is a heavy chain variable region that binds PD-L1,

The VL-PDL1 is the light chain variable region that binds to PD-L1,

The VH-KDR is a heavy chain variable region that binds to KDR,

The VL-KDR is the light chain variable region that binds to KDR,

The linker1 and linker2 are each independently a flexible peptide linker; the CH1-CH2-CH3 is the heavy chain constant region, the CL is the light chain constant region, the VH-PDL1 and the VL- PDL1 forms an antigen binding site that specifically binds to PD-L1, and said VH-KDR and said VL-KDR form an antigen binding site that specifically binds to KDR.
The anti-PDL1×KDR bispecific antibody according to claim 1, characterized in that it comprises two polypeptide chains and two light chains, wherein: (a) the polypeptide chain comprises VH from N-terminus to C-terminus -PDL1-CH1-CH2-CH3-linker2-VL-KDR-linker1-VH-KDR or VH-PDL1-CH1-CH2-CH3-linker2-VH-KDR-linker1-VL-KDR, the light chain is from N The end to the C-terminus contains VL-PDL1-CL; or

(b) The polypeptide chain includes VL-PDL1-linker1-VH-PDL1-linker2-VH-KDR-CH1-CH2-CH3 from N-terminus to C-terminus, and the light chain includes VL-PDL1-linker1-VH-PDL1-linker2-VH-KDR-CH1-CH2-CH3 from N-terminus to C-terminus. -KDR-CL;

Wherein, the VH-PDL1 is a heavy chain variable region that binds PD-L1,

The VL-PDL1 is the light chain variable region that binds to PD-L1,

The VH-KDR is a heavy chain variable region that binds to KDR,

The VL-KDR is the light chain variable region that binds to KDR,

The linker1 is 4 G4S, the linker2 is 3 G4S, the CH1-CH2-CH3 is the heavy chain constant region, the CL is the light chain constant region, and the VH-PDL1 is the same as the The VL-PDL1 forms an antigen binding site that specifically binds to PD-L1, and the VH-KDR and the VL-KDR form an antigen binding site that specifically binds to KDR.
The bispecific antibody of claim 3, wherein the VH-PDL1 comprises an amino acid sequence such as the heavy chain CDR shown in SEQ ID NO: 1-3, and the VL-PDL1 comprises an amino acid sequence such as The light chain CDR shown in SEQ ID NO: 4-6, the VH-KDR includes the heavy chain CDR shown in SEQ ID NO: 7-9, and the VL-KDR includes the amino acid sequence shown in SEQ ID NO: light chain CDR shown by 10-12.
The bispecific antibody of claim 4, wherein the VH-PDL1 has an amino acid sequence as shown in SEQ ID NO: 13, and the VL-PDL1 has an amino acid sequence as shown in SEQ ID NO: 14 The VH-KDR has the amino acid sequence shown in SEQ ID NO: 15, and the VL-KDR has the amino acid sequence shown in SEQ ID NO: 16.
The bispecific antibody according to any one of claims 1-5, wherein the polypeptide chain has SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 24 or SEQ ID NO: 25, the light chain has the amino acid sequence shown in SEQ ID NO: 19; or the polypeptide chain has the amino acid sequence shown in SEQ ID NO: 20, SEQ ID NO: 26, SEQ ID NO: 27 Or the amino acid sequence shown in SEQ ID NO: 28, and the light chain has the amino acid sequence shown in SEQ ID NO: 21.
The bispecific antibody of any one of claims 1-6, wherein the heavy chain constant region comprises an IgG1, IgG2, IgG3 or IgG4 heavy chain constant region, and the light chain constant region comprises Constant region of kappa or lambda light chain.
An isolated nucleotide, characterized in that the nucleotide encodes the bispecific antibody according to any one of claims 1-7.
An expression vector, characterized in that the expression vector contains the nucleotide according to claim 8.
A host cell, characterized in that the host cell contains the expression vector according to claim 9.
The method for preparing a bispecific antibody according to any one of claims 1-7, wherein the method comprises the following steps:

(a) Culturing the host cell according to claim 10 under expression conditions, thereby expressing the bispecific antibody;

(b) Isolation and purification of the bispecific antibody described in (a).
A pharmaceutical composition, characterized in that it contains the bispecific antibody according to any one of claims 1-7 and a pharmaceutically acceptable carrier.
Use of the bispecific antibody according to any one of claims 1-7 or the pharmaceutical composition according to claim 11 in the preparation of a medicament for the treatment of cancer.
The use according to claim 13, wherein the cancer is selected from the group consisting of colorectal cancer, non-small cell lung cancer, gastric cancer, gastroesophageal junction adenocarcinoma, melanoma, lung cancer, liver cancer, lymphoma , Leukemia, prostate cancer, bone marrow cancer and other neoplastic malignant diseases.
A method for treating cancer, characterized in that it comprises administering the bispecific antibody according to any one of claims 1-7, or an immunoconjugate thereof, or according to claim 12 to a subject in need The pharmaceutical composition.
The method of claim 15, wherein the cancer is selected from the group consisting of colorectal cancer, non-small cell lung cancer, gastric cancer, gastroesophageal junction adenocarcinoma, melanoma, lung cancer, liver cancer, lymphoma , Leukemia, prostate cancer, bone marrow cancer and other neoplastic malignant diseases.
An immunoconjugate, characterized in that, the immunoconjugate comprises:

(a) The bispecific antibody of any one of claims 1-7; and

(b) A coupling moiety selected from the group consisting of detectable markers, drugs, toxins, cytokines, radionuclides, or enzymes.
The use of the immunoconjugate according to claim 17, characterized in that it is used to prepare a pharmaceutical composition for treating tumors.