CN113754773A

CN113754773A - Bispecific antibody for resisting PD1 XPDL 1

Info

Publication number: CN113754773A
Application number: CN202010487619.5A
Authority: CN
Inventors: 朱祯平; 黄浩旻; 顾昌玲; 祝海霞
Original assignee: Sunshine Guojian Pharmaceutical Shanghai Co Ltd
Current assignee: Sunshine Guojian Pharmaceutical Shanghai Co Ltd
Priority date: 2020-06-02
Filing date: 2020-06-02
Publication date: 2021-12-07
Also published as: TW202146460A; WO2021244392A1; CN115667315A

Abstract

The invention provides a bispecific antibody of anti-PD1 XPDL 1, and experimental results show that the bispecific antibody can better keep the activity of each monoclonal antibody, can specifically combine two targets of PD-1 and PD-L1 at the same time, and has good physicochemical properties.

Description

Bispecific antibody for resisting PD1 XPDL 1

Technical Field

The invention relates to the field of antibodies, and particularly discloses a bispecific antibody against PD1 × PDL 1.

Background

Human programmed cell death receptor-1 (PD-1) is a 288 amino acid type I membrane protein and is one of the known major Immune checkpoints (Immune Checkpoint) (Blank et al,2005, Cancer Immunotherapy,54: 307-) -314. PD-1 is expressed in activated T lymphocytes, and binding of the ligands PD-L1 (programmed cell death receptor-Ligand 1) and PD-L2 (programmed cell death receptor-Ligand 2) can inhibit the activity of T lymphocytes and the related in vivo cellular immune response. 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. Numerous studies have shown that the interaction of PD-1 and PD-L1 is not only necessary to maintain immune system balance in vivo, but also a major mechanism and cause PD-L1 expressing positive tumor cells to circumvent immune surveillance. By blocking the negative regulation and control of cancer cells to PD-1/PD-L1 signal channels, the immune system is activated, and the tumor specific cellular immune response related to T cells can be promoted, thereby opening the door of a new tumor treatment method, namely tumor immunotherapy.

PD-1 (encoded by gene Pdcd 1) is an immunoglobulin superfamily member that is associated with CD28 and CTLA-4. The results of the study show that PD-1 negatively regulates antigen receptor signaling when bound to its ligand (PD-L1 and/or PD-L2). The murine PD-1 structure and the cocrystal structure of murine 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 that contain an Ig variable (V-type) domain responsible for ligand binding and a cytoplasmic tail responsible for binding to a signaling molecule. The PD-1 cytoplasmic tail contains two tyrosine-based signaling motifs, the ITIM (immunoreceptor tyrosine inhibition motif) and the ITSM (immunoreceptor tyrosine transduction motif).

PD-1 plays an important role in the immune evasion mechanism of tumors. Tumor immunotherapy, namely, cancer resistance by using the immune system of the human body, is a breakthrough tumor treatment method, but the tumor microenvironment can protect tumor cells from effective immune destruction, so how to break the tumor microenvironment becomes the key point of anti-tumor research. The role of PD-1 in the tumor microenvironment has been determined by prior work: PD-L1 is expressed in a number of mouse and human tumors (and can be induced by IFN-. gamma. in most PD-L1 negative tumor cell lines) and is presumed to be an important target for mediating tumor immune evasion (Iwai Y. et al, Proc. Natl. Acad. Sci. U.S.A.99: 12293-12297 (2002); Strome S.E. et al, Cancer Res., 63: 6501-6505 (2003)). Biopsy evaluation by immunohistochemistry has revealed expression of PD-1 (on tumor infiltrating lymphocytes) and/or PD-L1 on tumor cells in many primary tumors in humans. Such tissues include lung cancer, liver cancer, ovarian cancer, cervical cancer, skin cancer, colon cancer, glioma, bladder cancer, breast cancer, kidney cancer, esophageal cancer, stomach cancer, oral squamous cell carcinoma, urothelial cell carcinoma, and pancreatic cancer, as well as head and neck tumors, among others. Therefore, the blocking of the interaction of PD-1/PD-L1 can improve the immunocompetence of tumor specific T cells and is beneficial to the immune system to eliminate tumor cells, so that PD-1 and PD-L1 become hot targets for developing tumor immunotherapy drugs.

Bispecific antibodies refer to antibody molecules that specifically bind to two antigens or two epitopes simultaneously. Bispecific antibodies can be divided into structurally symmetric and asymmetric molecules according to symmetry. Bispecific antibodies can be classified into bivalent, trivalent, tetravalent, and multivalent molecules, depending on the number of binding sites. Bispecific antibodies are evolving into a new class of therapeutic antibodies that can be used to treat a variety of inflammatory, cancer, and other diseases.

Disclosure of Invention

The invention provides a bispecific antibody against PD1 XPLA 1.

Accordingly, it is a first object of the present invention to provide a bispecific antibody against PD1 × PDL 1.

It is a second object of the present invention to provide an isolated nucleic acid encoding said bispecific antibody.

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

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

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

It is a sixth object of the present invention to provide a pharmaceutical composition comprising said bispecific antibody.

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

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

In order to achieve the purpose, the invention provides the following technical scheme:

a first aspect of the invention provides a bispecific antibody against PD1 × PDL1, comprising:

(a) two polypeptide chains comprising, from N-terminus to C-terminus, VH-PDL1-CH1-CH2-CH3-linker2-VL-PD1-linker1-VH-PD1, wherein VH-PDL1 is a heavy chain variable region binding to PD-L1, CH1-CH2-CH3 is a heavy chain constant region, linker2 is 3 GGGGGGS, VL-PD1 is a light chain variable region binding to PD-1, linker1 is 4 GGGGS, VH-PD1 is a heavy chain variable region binding to PD-1, and

(b) two light chains comprising, from N-terminus to C-terminus, VL-PDL1-CL, wherein VL-PDL1 is a light chain variable region that binds PD-L1 and CL is a light chain constant region,

wherein said VH-PDL1 forms an antigen binding site with said VL-PDL1 that specifically binds PD-L1, and said VL-PD1 forms an antigen binding site with said VH-PD1 that specifically binds PD-1.

According to a preferred embodiment of the invention, said VH-PDL1 comprises an amino acid sequence as set forth in SEQ ID NO: 1-3, and VL-PDL1 comprises an amino acid sequence set forth in SEQ ID NO: 4-6, and VH-PD1 comprising a light chain CDR having an amino acid sequence set forth in SEQ ID NO: 7-9, and the VL-PD1 comprises an amino acid sequence set forth in SEQ ID NO: 10-12, and a light chain CDR.

According to a preferred embodiment of the invention, said VH-PDL1 has the amino acid sequence as shown in SEQ ID NO: 13, and VL-PDL1 has the amino acid sequence shown in SEQ ID NO: 14, and the VH-PD1 has an amino acid sequence shown as SEQ ID NO: 15, and VL-PD1 has an amino acid sequence as shown in SEQ ID NO: 16.

According to a preferred embodiment of the invention, said polypeptide chain has the amino acid sequence as shown in SEQ ID NO: 17, and the light chain has an amino acid sequence shown as SEQ ID NO: 18, or a pharmaceutically acceptable salt thereof.

According to the invention, the heavy chain constant region comprises an IgG1, IgG2, IgG3, or IgG4 heavy chain constant region and the light chain constant region comprises a kappa or lambda light chain constant region.

In a second aspect, the invention provides an isolated nucleotide encoding said bispecific antibody.

In a third aspect, the invention provides an expression vector, wherein the expression vector contains the nucleotide as described above.

In a fourth aspect, the present invention provides a host cell comprising an expression vector as described above.

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

(a) culturing a host cell as described above under expression conditions, thereby expressing the bispecific antibody;

(b) isolating and purifying the bispecific antibody of (a).

A sixth aspect of the invention provides a pharmaceutical composition comprising a bispecific antibody as described above and a pharmaceutically acceptable carrier.

A seventh aspect of the invention provides the use of a bispecific antibody as described above or a pharmaceutical composition as described above for the manufacture of a medicament for the treatment of cancer.

According to the invention, the cancer is selected from the group consisting of: melanoma, renal cancer, lung cancer, liver cancer, gastric cancer, lymph cancer, breast cancer, colorectal cancer, leukemia, prostate cancer, bone marrow cancer and other neoplastic malignant diseases.

An eighth aspect of the invention provides a method of treating cancer comprising administering to a subject in need thereof a bispecific antibody as described above or a pharmaceutical composition as described above.

Has the advantages that: the invention provides a bispecific antibody of anti-PD1 XPDL 1, and experimental results show that the bispecific antibody can better keep the activity of each monoclonal antibody, can specifically combine two targets of PD-1 and PD-L1 at the same time, and has good physicochemical properties.

Drawings

FIG. 1 is a schematic diagram of the structure of an anti-PD1 XPDL 1 diabody molecule of the present invention.

FIG. 2 shows the HPLC detection pattern and SDS-PAGE detection result of anti-PD1 XPDL 1 double antibody, wherein FIG. 2A shows the HPLC detection pattern, and FIG. 2B shows the SDS-PAGE detection result.

FIG. 3 shows the results of ELISA detection of the anti-PD1 XPLA 1 double antibody binding to PD-1 and PD-L1 respectively, wherein FIG. 3A shows the results of binding to PD-1 and FIG. 3B shows the results of binding to PD-L1.

FIG. 4 shows the results of the dual-specificity ELISA for detecting the binding of anti-PD1 XPDL 1 dual antibody to PD-1 and PD-L1 simultaneously.

FIG. 5 shows the results of FACS detection of the binding of anti-PD1 XPL 1 double antibody to PD-1/CHO and N87-PDL1 cells, respectively, wherein FIG. 5A shows the result of binding to PD-1/CHO cells and FIG. 5B shows the result of binding to N87-PDL1 cells.

FIG. 6 shows the results of the activity of PD1/PD-L1 on anti-PD1 XPDL 1 double antibody blocking cells.

Detailed Description

In the present invention, the terms "Antibody (abbreviated Ab)" and "Immunoglobulin G (abbreviated IgG)" are heterotetrameric proteins of about 150000 daltons having the same structural features, which consist of two identical light chains (L) and two identical heavy chains (H). Each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide bonds varies between heavy chains of different immunoglobulin isotypes (isotypes). Each heavy and light chain also has regularly spaced intrachain disulfide bonds. Each heavy chain has at one end a variable region (VH) followed by a constant region consisting of three domains, CH1, CH2, and CH 3. Each light chain has a variable region (VL) at one end and a constant region at the other end, the light chain constant region comprising a domain CL; the constant region of the light chain is paired with the CH1 domain of the heavy chain constant region, and the variable region of the light chain is paired with the variable region of the heavy chain. The constant regions are not directly involved in binding of an antibody to an antigen, but they exhibit different effector functions, such as participation in antibody-dependent cell-mediated cytotoxicity (ADCC) and the like. Heavy chain constant regions include IgG1, IgG2, IgG3, IgG4 subtypes; light chain constant regions include κ (Kappa) or λ (Lambda). The heavy and light chains of an antibody are covalently linked together by disulfide bonds between the CH1 domain of the heavy chain and the CL domain of the light chain, and the two heavy chains of the antibody are covalently linked together by interpoly disulfide bonds formed between the hinge regions.

In the present invention, the term "bispecific antibody (diabody)" refers to an antibody molecule capable of specifically binding to two antigens (targets) or two epitopes simultaneously.

In the present invention, the term "monoclonal antibody (mab)" refers to an antibody obtained from a substantially homogeneous population, i.e., the individual antibodies comprised in the population are identical, except for a few naturally occurring mutations that may be present. Monoclonal antibodies are directed against a single antigenic site with high specificity. Moreover, unlike conventional polyclonal antibody preparations (typically a mixture of different antibodies with epitopes against different antigens), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, monoclonal antibodies are also advantageous in that they can be synthesized by hybridoma culture, uncontaminated by other immunoglobulins. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method.

In the present invention, the terms "Fab" and "Fc" mean that papain cleaves an antibody into two identical Fab fragments and one Fc fragment. The Fab fragment consists of the VH and CH1 domains of the heavy chain and the VL and CL domains of the light chain of the antibody. The Fc fragment, i.e., the crystallizable fragment (Fc), consists of the CH2 and CH3 domains of the antibody. The Fc region has no antigen binding activity and is the site of antibody interaction with effector molecules or cells.

In the present invention, the term "scFv" is a single chain antibody (scFv), and is formed by connecting an antibody heavy chain variable region and an antibody light chain variable region via a short peptide (linker) of 15 to 25 amino acids.

In the present invention, the term "variable" means that certain portions of the variable regions of an antibody differ in sequence, which results in the binding and specificity of each particular antibody for its particular antigen. However, the variability is not evenly distributed throughout the antibody variable region. It is concentrated in three segments called complementarity-determining regions (CDRs) or hypervariable regions in the heavy chain variable region and the light chain variable region. The more conserved portions of the variable regions are called Framework Regions (FR). The variable regions of native heavy and light chains each comprise four FR regions, which are in a substantially β -sheet configuration, connected by three CDRs that form a connecting loop, and in some cases may form part of a β -sheet structure. The CDRs in each chain are held close together by the FR region and form the antigen binding site of the antibody with the CDRs of the other chain (see Kabat et al, NIH Publ. No.91-3242, Vol I, 647-669 (1991)).

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

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 to which an antibody binds.

In the present invention, the term "expression vector" may be pTT5, pSECtag series, pCGS3 series, pcDNA series vectors, etc., and other vectors used in mammalian expression systems, etc., and the expression vector includes a fusion DNA sequence to which appropriate transcription and translation regulatory sequences are ligated.

In the present invention, the term "host cell" refers to a cell suitable for expressing the above expression vector, and may be a eukaryotic cell, such as mammalian or insect host cell culture system, which can be used for the expression of the fusion protein of the present invention, and CHO (Chinese Hamster Ovary), HEK293, COS, BHK and derived cells of the above cell can be suitable for the present invention.

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 composition for more stable therapeutic effect, which can ensure the conformational integrity of the amino acid core sequence of the bispecific antibody disclosed in the present invention, while protecting the multifunctional group of the protein from degradation (including but not limited to aggregation, deamination or oxidation).

The experimental materials used in the following examples are illustrated below:

pcDNA^TM3.4

vector: available from Thermo fisher corporation under item number a 14697;

CHO cells: purchased from Thermo fisher corporation, cat # a 29133;

293E cells: from the NRC biotechnology Research Institute;

PD-1/PD-L1 Block Bioassay, Propagation model: available from Promega corporation under the designation J1252;

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

SD rat: purchased from Shanghai Ling Biotech, Inc.

The experimental reagents used in the following examples are illustrated below:

anti-PD-L1 mab: prepared according to the sequence in PCT/CN 2020/090442;

anti-PD-1 monoclonal antibody: prepared according to the sequence in WO 2018/137576;

HRP-labeled murine anti-human Fab antibodies: purchased from sigma, cat # a 0293;

HRP-labeled anti-6 × His antibody: purchased from abcam, cat # ab 178563;

goat anti-human IgG-FITC: purchased from sigma, cat # F4143;

PBS: purchased from bio-engineering (shanghai) gmbh, cat # B548117;

PBST：PBS+0.05％Tween 20；

BSA: purchased from bio-engineering (shanghai) gmbh, cat # a 60332;

FBS: purchased from Gibco, cat # 10099;

TMB: available from BD corporation, cat # 555214;

Bio-Glo Luciferase Assay System: purchased from Promega, cat # G7940.

The experimental equipment used in the following examples is illustrated below:

a PCR instrument: purchased from BioRad, cat # C1000 Touch Thermal Cycler;

HiTrap MabSelectSuRe column: available from GE, cat # 11-0034-95;

beckman Coulter CytoFLEX flow cytometer: from Beckman corporation;

SpectraMax i3x microplate reader: purchased from Molecular Devices, Inc.

The following examples and experimental examples are intended to further illustrate the present invention and should not be construed as limiting the present invention. The examples do not include detailed descriptions of conventional 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 having ordinary skill in the art and are described in numerous publications, including Sambrook, j., Fritsch, e.f. and maniis, T. (1989) Molecular Cloning: a Laboratory Manual, 2nd edition, Cold spring Harbor Laboratory Press.

Example 1 construction of anti-PD1 XPDL 1 diabodies

The invention adopts a mode that anti-human PD-1 monoclonal antibody mAb1-25-Hu (sequence is derived from WO2018/137576) scFv (VL-linker1-VH, linker1 is 4 GGGGGGS), and linker2 (is 3 GGGGS) is connected in series at the C-terminal of the heavy chain of anti-human PD-L1 monoclonal antibody M8 (sequence is derived from PCT/CN2020/090442) to construct an anti-PD1 x PDL1 bispecific antibody which is named anti-PD1 x PDL1 BsAb. The structure is shown in figure 1.

The bispecific antibody and the corresponding heavy chain and light chain expression vector of the monoclonal antibody are obtained by gene synthesis and conventional molecular cloning methods, the corresponding amino acid sequences are shown in table 1, wherein the CDRs are encoded according to the Kabat rules.

TABLE 1 sequence information of antibodies of the invention

SEQ ID NO：	Sequence name
		1	Amino acid sequence of heavy chain complementarity determining region H-CDR1 of anti-PD-L1 monoclonal antibody
2	Amino acid sequence of heavy chain complementarity determining region H-CDR2 of anti-PD-L1 monoclonal antibody
		3	Amino acid sequence of heavy chain complementarity determining region H-CDR3 of anti-PD-L1 monoclonal antibody
4	Amino acid sequence of light chain complementarity determining region L-CDR1 of anti-PD-L1 monoclonal antibody
		5	Amino acid sequence of light chain complementarity determining region L-CDR2 of anti-PD-L1 monoclonal antibody
6	Amino acid sequence of light chain complementarity determining region L-CDR3 of anti-PD-L1 monoclonal antibody
		7	Amino acid sequence of heavy chain complementarity determining region H-CDR1 of anti-PD-1 monoclonal antibody
8	Amino acid sequence of heavy chain complementarity determining region H-CDR2 of anti-PD-1 monoclonal antibody
		9	Amino acid sequence of heavy chain complementarity determining region H-CDR3 of anti-PD-1 monoclonal antibody
10	Amino acid sequence of light chain complementarity determining region L-CDR1 of anti-PD-1 monoclonal antibody
		11	Amino acid sequence of light chain complementarity determining region L-CDR2 of anti-PD-1 monoclonal antibody
12	Amino acid sequence of light chain complementarity determining region L-CDR3 of anti-PD-1 monoclonal antibody
		13	Amino acid sequence of heavy chain variable region of anti-PD-L1 monoclonal antibody
14	Amino acid sequence of light chain variable region of anti-PD-L1 monoclonal antibody
		15	Amino acid sequence of heavy chain variable region of anti-PD-1 monoclonal antibody
16	Amino acid sequence of light chain variable region of anti-PD-1 monoclonal antibody
		17	amino acid sequence of polypeptide chain of anti-PD1 XPDL 1 BsAb
18	Amino acid sequence of light chain of anti-PD-L1 monoclonal antibody
		19	Amino acid sequence of heavy chain of anti-PD-L1 monoclonal antibody
20	Amino acid sequence of heavy chain of anti-PD-1 monoclonal antibody
		21	Amino acid sequence of light chain of anti-PD-1 monoclonal antibody

Example 2 expression and purification of anti-PD1 XPDL 1 diabody

DNA fragments of heavy and light chains of anti-PD1 XPDL 1 double antibody are respectively subcloned into pcDN3.4 vector, recombinant plasmid is extracted to co-transfect CHO cells and/or 293E cells, after the cells are cultured for 5-7 days, culture solution is loaded to HiTrap MabSelectSuRe column after being filtered by high speed centrifugation and millipore filter, protein is eluted by eluent containing 100mM citric acid and pH3.5, and the solution is dialyzed to PBS of pH7.4.

The purified protein is detected by HPLC, and the HPLC-SEC detection pattern of the anti-PD1 XPDL 1 double antibody is shown in figure 2A, and the purity of the double antibody monomer reaches over 96 percent. The SDS-PAGE results are shown in FIG. 2B,

lanes

1 and 2 are reduced and non-reduced SDS-PAGE of anti-PD1 × PDL1 double antibody, and

lanes

3 and 4 are reduced and non-reduced SDS-PAGE of anti-PD-L1 monoclonal antibody. The molecular weight of double antibody is 197 KD.

Example 3 detection of affinity of anti-PD1 XPDL 1 double antibodies to antigen by enzyme-linked immunosorbent assay (ELISA)

Example 3.1 affinity detection with PD-1 antigen

In order to detect the affinity of the anti-PD1 XPLA 1 diabody with PD-1 antigen, PD1-ECD-hFc protein (extracellular domain gene synthesized according to the sequence provided by UniProt (sequence number Q15116) and added with a signal peptide sequence at the N end, hFc at the C end, constructed into an expression vector through two enzyme cutting sites of EcoRI and HindIII respectively, transfected into HEK-293E cells for expression and purified) is diluted to 200ng/ml by PBS buffer solution with pH7.4, and then 100 ul/hole is added into an ELISA plate; incubating overnight at 4 ℃; washing the plate twice the next day with PBST; adding PBST + 1% BSA into each hole for blocking, and blocking for 1h at 37 ℃; wash the plate twice with PBST; then adding the antibody to be detected diluted by PBS + 1% BSA gradient, taking the anti-PD-1 monoclonal antibody as a positive control, and diluting the antibody to be detected by 12 gradients by 3 times step by step with the initial concentration of 100 nM. Incubating at 37 ℃ for 1 h; PBST washing twice, adding a secondary antibody HRP-labeled mouse anti-human Fab, and incubating for 40min at 37 ℃; PBST washing plate three times and pat dry, each hole add 100 u l TMB, room temperature (20 + -5 ℃) dark place 5 minutes; add 50. mu.l of 2M H per well₂SO₄Stopping the substrate reaction by the stop solution, reading OD value at 450nm of an enzyme labeling instrument, performing data analysis by GraphPad Prism, mapping and calculating EC₅₀。

The results of the experiments are shown in FIG. 3A, and the EC of the anti-PD-1 monoclonal antibody and the anti-PD1 XPDL 1 double antibody combined with the PD-1 antigen₅₀0.29nM and 0.30nM, respectively, with comparable affinities.

Example 3.2 affinity detection with PD-L1 antigen

To examine the affinity of the anti-PD1 XPDL 1 diabody with the PD-L1 antigen, PDL1-ECD-His protein (according to the sequence provided by NCBI (NCBI accession number NP-054862.1)) was synthesized into the PD-L1 ectodomain gene using PBS buffer at pH7.4 and added with a signal peptide sequence at its N-terminus, 6 XHis tag at its C-terminus, and digested with EcoRI and HindIIIThe spots are respectively constructed into an expression vector, transfected into HEK-293E cells for expression and purified) to be diluted to 1000ng/ml, and then 100 mu l/hole is added into an ELISA plate; incubating overnight at 4 ℃; washing the plate twice the next day with PBST; adding PBST + 1% BSA into each hole for blocking, and blocking for 1h at 37 ℃; wash the plate twice with PBST; then, the antibody to be detected diluted with a PBS + 1% BSA gradient, and the anti-PD-L1 monoclonal antibody as a positive control were added at an initial concentration of 100nM, and the 12 gradients were diluted 3-fold step by step. Incubating at 37 ℃ for 1 h; PBST washing twice, adding a secondary antibody HRP-labeled mouse anti-human Fab, and incubating for 40min at 37 ℃; PBST washing plate three times and pat dry, each hole add 100 u l TMB, room temperature (20 + -5 ℃) dark place 5 minutes; add 50. mu.l of 2M H per well₂SO₄Stopping the substrate reaction by the stop solution, reading OD value at 450nm of an enzyme labeling instrument, performing data analysis by GraphPad Prism, mapping and calculating EC₅₀。

The experimental results are shown in FIG. 3B, and the anti-PD-L1 monoclonal antibody and anti-PD1 XPDL 1 double antibody bind to the EC of PD-L1 antigen₅₀0.27nM and 0.29nM, which are comparable in affinity.

Example 4 bispecific ELISA to detect the ability of anti-PD1 × PDL1 diabodies to bind to two antigens simultaneously

To test the ability of the anti-PD1 XPLA 1 double antibody to bind both PD-1 antigen and PD-L1 antigen, the PD1-ECD-hFc protein was diluted to 200ng/ml with PBS buffer at pH7.4 and then added to an ELISA plate at 100. mu.l/well; incubating overnight at 4 ℃; washing the plate twice the next day with PBST; adding PBST + 1% BSA into each hole for blocking, and blocking for 1h at 37 ℃; wash the plate twice with PBST; the antibody to be detected was then added in a gradient diluted with PBS + 1% BSA at an initial concentration of 100nM, and diluted 3-fold over 12 gradients. Incubating at 37 ℃ for 1 h; the plates were washed twice with PBST, and 1000ng/ml PDL1-ECD-His diluted in PBS at pH7.4 was added to 100. mu.l/well to the ELISA plates. Incubating at 37 ℃ for 1 h; PBST washing plate twice, adding secondary antibody HRP-anti-His, and incubating for 40min at 37 ℃; PBST washing plate three times and pat dry, each hole add 100 u l TMB, room temperature (20 + -5 ℃) dark place 5 minutes; add 50. mu.l of 2M H per well₂SO₄Stopping the substrate reaction by the stop solution, reading OD value at 450nm of an enzyme labeling instrument, performing data analysis by GraphPad Prism, mapping and calculating EC₅₀。

The results of the experiment are shown in FIG. 4EC against PD1 XPDL 1 double antibody₅₀0.36nM, whereas anti-PD-1 mAb and anti-PD-L1 mAb did not have the ability to bind both antigens simultaneously.

Example 5 detection of binding affinity of anti-PD1 XPDL 1 diabodies to target cells

CHO stably transfected cells expressing PD-1 on the cell surface were used as target cells, and the binding affinity of the anti-PD1 XPDL 1 double antibody to the cells was determined by flow cytometry. The cells were washed three times with PBS containing 0.5% BSA, centrifuged at 300g each for 5 minutes, and the supernatant was discarded. 0.5% BSA in PBS resuspended cells at a cell density of 1X 10⁶cells/mL, 100. mu.L/well were added to 96-well plates. Starting with 200nM of anti-PD1 XPDL 1 double antibody and positive control anti-PD-1 monoclonal antibody, gradually diluting 11 gradients, adding 100. mu.L/well into 96-well plate, and incubating for 1h at 4 ℃. The cells were washed twice with PBS to remove unbound test antibody. Mu.l of goat anti-human IgG-FITC was added thereto, and the mixture was incubated at 4 ℃ for 30 minutes. The cells were centrifuged at 300g for 5 minutes and washed twice with PBS to remove unbound secondary antibody. Finally, the cells were resuspended in 200. mu.l PBS and the binding affinity of the diabodies to the cells was determined by Beckman Coulter Cytoflex flow cytometer. The data obtained were analyzed by GraphPad Prism software fitting. The results are shown in FIG. 5A, and the EC of anti-PD-1 monoclonal antibody and anti-PD1 XPLA 1 double antibody₅₀0.64nM and 1.43nM, respectively, with comparable affinities.

N87-PDL1 is a stable transfectant cell strain constructed by transfecting PD-L1 to NCI-N87 by a lentivirus transfection method in the laboratory. N87-PDL1 in the logarithmic growth phase was digested with trypsin, washed three times with PBS containing 0.5% BSA, centrifuged at 300g for 5 minutes each, and the supernatant was discarded. 0.5% BSA in PBS resuspended cells at a cell density of 1X 10⁶cells/mL, 100. mu.L/well were added to 96-well plates. The anti-PD1 XPDL 1 double antibody and the positive control anti-PD-L1 monoclonal antibody are diluted to 120nM, and are diluted by 11 gradients step by step, 100 mu L of monoclonal antibody is added into a 96-well plate per well, and is uniformly mixed with N87-PDL1 cells. The rest of the method is the same as above. The results are shown in FIG. 5B, EC against PD-L1 mAb₅₀0.11nM, EC against PD1 × PDL1 double antibody₅₀The affinity of the two was 0.15 nM.

Example 6 anti-PD1 XPDL 1 double antibody blocks the cellular level of activity of PD1/PD-L1

The experiment used Promega's PD-1/PD-L1 Block Bioassay, Propagation model and method.

Taking PD-L1 aAPC/CHO-K1 growing in logarithmic phase, pancreatin digesting into single cells, transferring to a white background 96-well plate, 100 mu L/well and 40000 cells/well, placing at 37 ℃ and 5% CO₂And incubated overnight. And (3) diluting the anti-PD1 XPDL 1 double antibody, the anti-PD-L1 monoclonal antibody and the anti-PD-1 monoclonal antibody into 2 Xworking solution with the initial concentration of 100nM by 3-fold gradient step by step. Taking the density of 1.4-2 × 10⁶PD1 effector cells with cell viability above 95% per mL, and trypsinized to 1.25 × 10⁶Cells/ml single cell suspension. Taking PD-L1 aAPC/CHO-K1 cells paved on the previous day, discarding supernatant, and adding 40 mu L of working solution of the antibody to be detected in gradient dilution; an additional volume of PD1 effector cells was added. Standing at 37 deg.C for 5% CO₂And incubated for 6 hours. Add 80. mu.l of detection reagent Bio-Glo per well. After incubation at room temperature for 10 minutes, the luminescences were read with spectramax i 3. Data analysis with GraphPad Prism, plotting and calculating IC₅₀。

The experimental results are shown in FIG. 6, IC of anti-PD1 XPDL 1 BsAb, anti-PD-L1 monoclonal antibody and anti-PD-1 monoclonal antibody₅₀Respectively at 0.24nM, 0.42nM, 1.60nM, IC of double antibody and anti-PD-L1 monoclonal antibody₅₀IC of near, anti-PD-1 monoclonal antibody₅₀Larger, but with a slightly higher plateau.

Example 7 pharmacokinetic Studies of anti-PD1 × PDL1 double antibody

4 SD rats per group, weighing about 200g, were injected with 2mg of antibody per rat via the tail vein. The post-dose bleeding time points were: 0h, 3h, 24h, 48h, 96h, 168h, 336h and 504 h. Blood is collected from the orbit, and serum is obtained by centrifugation at 8000rpm/min after the blood is naturally coagulated.

The drug concentration in the serum of the anti-PD1 XPL 1 double antibody is detected by the following method:

1) protein A was coated on ELISA plates and antibody Fab fragments were detected. Coating with protein A, wherein the coating amount is 100 ng/hole, and the temperature is 4 ℃ overnight; the next day the plates were washed twice with PBST and then blocked with PBS + 2% BSA at 37 ℃ for 2 hours. The PBST plates were washed twice. The standard against PD1 XPL 1 double antibody was diluted two-fold stepwise in 12 gradients starting at 1000 ng/mL. Rat serum sample1000-. Adding the two groups of samples into the sealed ELISA plate, and incubating for 1 hour; after PBST washing the plate twice, adding a mouse anti-human Fab antibody marked by HRP, and standing at 37 ℃ for 30 minutes; after PBST washing for 3 times, the residual liquid drops are patted dry on absorbent paper, 100 mu l of TMB is added into each hole, and the plate is placed for 5 minutes in a dark place at room temperature (20 +/-5 ℃); add 50. mu.l of 2M H per well₂SO₄The stop solution stops the substrate reaction, and the OD value is read at 450nm of the microplate reader. The calculated half-life was 275.6 hours.

2) PD1-ECD-hFc coated ELISA plate, 20 ng/well. The rest of the method is the same as above. The calculated half-life was 303.5 hours.

3) ELISA plates were coated with PDL1-ECD-hFc at 100 ng/well. The rest of the method is the same as above. The calculated half-life was 307 hours.

As shown in tables 2-4, the half-life results calculated by the above three groups of ELISA results are similar, and are all about 300 hours, which indicates that the analysis data is reliable.

TABLE 2 protein A plate ELISA results

Experimental group	Half-life (hours)	Maximum concentration (μ g/mL)
			1	247.50433	170
2	316.48482	166
			3	273.29592	150
4	265.20196	164

Table 3, PD1-ECD-hFc plate ELISA results

Experimental group	Half-life (hours)	Maximum concentration (μ g/mL)
			1	299.46777	196
2	348.73796	202
			3	295.3837	170
4	270.42874	174

TABLE 4 PDL1-ECD-hFc plate ELISA results

Experimental group	Half-life (hours)	Maximum concentration (μ g/mL)
			1	280.15463	166
2	336	152
			3	320.97912	146
4	291.3996	166

Sequence listing

<110> Sansheng Guojian pharmaceutical industry (Shanghai) GmbH

<120> a bispecific antibody against PD1 XPLA 1

<160> 21

<170> SIPOSequenceListing 1.0

<210> 1

<211> 5

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 1

Ser Tyr Gly Val His

1 5

<210> 2

<211> 16

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 2

Leu Ile Trp Ser Gly Gly Gly Thr Asp Tyr Asn Pro Ser Leu Lys Ser

1 5 10 15

<210> 3

<211> 9

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 3

Gln Leu Gly Leu Arg Ala Met Asp Tyr

1 5

<210> 4

<211> 11

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 4

Arg Ala Ser Gln Ser Ile Gly Thr Thr Ile His

1 5 10

<210> 5

<211> 7

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 5

Tyr Ala Ser Gln Ser Phe Ser

1 5

<210> 6

<211> 9

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 6

Gln Gln Ser Asn Ser Trp Pro Leu Thr

1 5

<210> 7

<211> 5

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 7

Ser Tyr Asp Met Ser

1 5

<210> 8

<211> 17

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 8

Thr Ile Ser Gly Gly Gly Arg Tyr Thr Tyr Tyr Pro Asp Thr Val Lys

1 5 10 15

Gly

<210> 9

<211> 8

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 9

Pro Tyr Gly Gly Tyr Phe Asp Val

1 5

<210> 10

<211> 11

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 10

Arg Ala Ser Gln Ser Ile Ser Asn Phe Leu His

1 5 10

<210> 11

<211> 7

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 11

Tyr Ala Ser Gln Ser Ile Ser

1 5

<210> 12

<211> 9

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 12

Gln Gln Ser Asn Ser Trp Pro His Thr

1 5

<210> 13

<211> 117

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 13

Gln Val Gln Leu Gln Gln Ser Gly Gly Gly Leu Val Lys Pro Ser Gln

1 5 10 15

Ser Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Ser Tyr

20 25 30

Gly Val His Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Leu Ile Trp Ser Gly Gly Gly Thr Asp Tyr Asn Pro Ser Leu Lys

50 55 60

Ser Arg Leu Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Val Ser Phe

65 70 75 80

Lys Ile Ser Ser Leu Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Gln Leu Gly Leu Arg Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser

100 105 110

Val Thr Val Ser Ser

115

<210> 14

<211> 107

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 14

Glu Ile Val Leu Thr Gln Ser Pro Asp Phe Leu Ser Val Thr Pro Lys

1 5 10 15

Glu Lys Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Gly Thr Thr

20 25 30

Ile His Trp Tyr Gln Gln Lys Pro Asp Gln Ser Pro Lys Leu Leu Ile

35 40 45

Lys Tyr Ala Ser Gln Ser Phe Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Asn Ser Val Glu Ala

65 70 75 80

Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Ser Asn Ser Trp Pro Leu

85 90 95

Thr Phe Gly Ala Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 15

<211> 117

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 15

Glu Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ala Phe Ser Ser Tyr

20 25 30

Asp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Arg Leu Glu Trp Val

35 40 45

Ala Thr Ile Ser Gly Gly Gly Arg Tyr Thr Tyr Tyr Pro Asp Thr Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser His Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Phe Cys

85 90 95

Ala Ser Pro Tyr Gly Gly Tyr Phe Asp Val Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser

115

<210> 16

<211> 107

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 16

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Ile Ser Asn Phe

20 25 30

Leu His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

35 40 45

Lys Tyr Ala Ser Gln Ser Ile Ser Gly Ile Pro Ala Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro

65 70 75 80

Glu Asp Phe Ala Val Tyr Phe Cys Gln Gln Ser Asn Ser Trp Pro His

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

<210> 17

<211> 706

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 17

Gln Val Gln Leu Gln Gln Ser Gly Gly Gly Leu Val Lys Pro Ser Gln

1 5 10 15

Ser Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Ser Tyr

20 25 30

Gly Val His Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Leu Ile Trp Ser Gly Gly Gly Thr Asp Tyr Asn Pro Ser Leu Lys

50 55 60

Ser Arg Leu Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Val Ser Phe

65 70 75 80

Lys Ile Ser Ser Leu Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Gln Leu Gly Leu Arg Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser

100 105 110

Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu

115 120 125

Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys

130 135 140

Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser

145 150 155 160

Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser

165 170 175

Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser

180 185 190

Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn

195 200 205

Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His

210 215 220

Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val

225 230 235 240

Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr

245 250 255

Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu

260 265 270

Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys

275 280 285

Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser

290 295 300

Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys

305 310 315 320

Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile

325 330 335

Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro

340 345 350

Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu

355 360 365

Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn

370 375 380

Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser

385 390 395 400

Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg

405 410 415

Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu

420 425 430

His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Gly

435 440 445

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Ile

450 455 460

Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly Glu Arg

465 470 475 480

Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Ile Ser Asn Phe Leu His

485 490 495

Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile Lys Tyr

500 505 510

Ala Ser Gln Ser Ile Ser Gly Ile Pro Ala Arg Phe Ser Gly Ser Gly

515 520 525

Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro Glu Asp

530 535 540

Phe Ala Val Tyr Phe Cys Gln Gln Ser Asn Ser Trp Pro His Thr Phe

545 550 555 560

Gly Gln Gly Thr Lys Val Glu Ile Lys Gly Gly Gly Gly Ser Gly Gly

565 570 575

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Lys

580 585 590

Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg

595 600 605

Leu Ser Cys Ala Ala Ser Gly Phe Ala Phe Ser Ser Tyr Asp Met Ser

610 615 620

Trp Val Arg Gln Ala Pro Gly Lys Arg Leu Glu Trp Val Ala Thr Ile

625 630 635 640

Ser Gly Gly Gly Arg Tyr Thr Tyr Tyr Pro Asp Thr Val Lys Gly Arg

645 650 655

Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser His Tyr Leu Gln Met

660 665 670

Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Phe Cys Ala Ser Pro

675 680 685

Tyr Gly Gly Tyr Phe Asp Val Trp Gly Gln Gly Thr Leu Val Thr Val

690 695 700

Ser Ser

705

<210> 18

<211> 214

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 18

Glu Ile Val Leu Thr Gln Ser Pro Asp Phe Leu Ser Val Thr Pro Lys

1 5 10 15

Glu Lys Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Gly Thr Thr

20 25 30

Ile His Trp Tyr Gln Gln Lys Pro Asp Gln Ser Pro Lys Leu Leu Ile

35 40 45

Lys Tyr Ala Ser Gln Ser Phe Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Asn Ser Val Glu Ala

65 70 75 80

Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Ser Asn Ser Trp Pro Leu

85 90 95

Thr Phe Gly Ala Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 19

<211> 447

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 19

Gln Val Gln Leu Gln Gln Ser Gly Gly Gly Leu Val Lys Pro Ser Gln

1 5 10 15

Ser Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Ser Tyr

20 25 30

Gly Val His Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Leu Ile Trp Ser Gly Gly Gly Thr Asp Tyr Asn Pro Ser Leu Lys

50 55 60

Ser Arg Leu Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Val Ser Phe

65 70 75 80

Lys Ile Ser Ser Leu Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Gln Leu Gly Leu Arg Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser

100 105 110

Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu

115 120 125

Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys

130 135 140

Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser

145 150 155 160

Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser

165 170 175

Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser

180 185 190

Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn

195 200 205

Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His

210 215 220

Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val

225 230 235 240

Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr

245 250 255

Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu

260 265 270

Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys

275 280 285

Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser

290 295 300

Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys

305 310 315 320

Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile

325 330 335

Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro

340 345 350

Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu

355 360 365

Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn

370 375 380

Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser

385 390 395 400

Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg

405 410 415

Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu

420 425 430

His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

435 440 445

<210> 20

<211> 444

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 20

Glu Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ala Phe Ser Ser Tyr

20 25 30

Asp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Arg Leu Glu Trp Val

35 40 45

Ala Thr Ile Ser Gly Gly Gly Arg Tyr Thr Tyr Tyr Pro Asp Thr Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser His Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Phe Cys

85 90 95

Ala Ser Pro Tyr Gly Gly Tyr Phe Asp Val Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu

115 120 125

Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys

130 135 140

Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser

145 150 155 160

Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser

165 170 175

Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser

180 185 190

Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn

195 200 205

Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro

210 215 220

Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe

225 230 235 240

Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val

245 250 255

Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe

260 265 270

Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro

275 280 285

Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr

290 295 300

Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val

305 310 315 320

Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala

325 330 335

Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln

340 345 350

Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly

355 360 365

Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro

370 375 380

Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser

385 390 395 400

Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu

405 410 415

Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His

420 425 430

Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys

435 440

<210> 21

<211> 214

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 21

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Ile Ser Asn Phe

20 25 30

Leu His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

35 40 45

Lys Tyr Ala Ser Gln Ser Ile Ser Gly Ile Pro Ala Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro

65 70 75 80

Glu Asp Phe Ala Val Tyr Phe Cys Gln Gln Ser Asn Ser Trp Pro His

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

Claims

1. A bispecific antibody against PD1 × PDL1, comprising:

(a) two polypeptide chains comprising, from N-terminus to C-terminus, VH-PDL1-CH1-CH2-CH3-linker2-VL-PD1-linker1-VH-PD1, wherein VH-PDL1 is a heavy chain variable region that binds PD-L1, CH1-CH2-CH3 is a heavy chain constant region, and linker2 is 3G₄S, the VL-PD1 is a light chain variable region combined with PD-1, and the linker1 is 4G₄S, said VH-PD1 is a heavy chain variable region that binds PD-1, and

2. The bispecific antibody of claim 1, wherein said VH-PDL1 comprises an amino acid sequence set forth in SEQ ID NO: 1-3, and VL-PDL1 comprises an amino acid sequence set forth in SEQ ID NO: 4-6, and VH-PD1 comprising a light chain CDR having an amino acid sequence set forth in SEQ ID NO: 7-9, and the VL-PD1 comprises an amino acid sequence set forth in SEQ ID NO: 10-12, and a light chain CDR.

3. The bispecific antibody of claim 2, wherein said VH-PDL1 has the amino acid sequence as set forth in SEQ ID NO: 13, and VL-PDL1 has the amino acid sequence shown in SEQ ID NO: 14, and the VH-PD1 has an amino acid sequence shown as SEQ ID NO: 15, and VL-PD1 has an amino acid sequence as shown in SEQ ID NO: 16.

4. The bispecific antibody of claim 3, wherein said polypeptide chain has an amino acid sequence as set forth in SEQ ID NO: 17, and the light chain has an amino acid sequence shown as SEQ ID NO: 18, or a pharmaceutically acceptable salt thereof.

5. The bispecific antibody of claim 1, wherein the heavy chain constant region comprises an IgG1, IgG2, IgG3, IgG4 heavy chain constant region and the light chain constant region comprises a kappa or lambda light chain constant region.

6. An isolated nucleotide encoding the bispecific antibody of any one of claims 1 to 5.

7. An expression vector comprising the nucleotide of claim 6.

8. A host cell comprising the expression vector of claim 7.

9. The method of making a bispecific antibody of any one of claims 1-5, comprising the steps of:

(a) culturing the host cell of claim 8 under expression conditions, thereby expressing the bispecific antibody;

(b) isolating and purifying the bispecific antibody of (a).

10. A pharmaceutical composition comprising the bispecific antibody of any one of claims 1-5 and a pharmaceutically acceptable carrier.

11. Use of the bispecific antibody of any one of claims 1-5 or the pharmaceutical composition of claim 10 in the manufacture of a medicament for the treatment of cancer.

12. The use of claim 11, wherein the cancer is selected from the group consisting of: melanoma, renal cancer, lung cancer, liver cancer, gastric cancer, lymphoma, breast cancer, colorectal cancer, leukemia, prostate cancer, bone marrow cancer and other neoplastic malignant diseases.

13. A method of treating cancer, comprising administering to a subject in need thereof the bispecific antibody of any one of claims 1-5 or the pharmaceutical composition of claim 10.

14. The method of claim 13, wherein the cancer is selected from the group consisting of: melanoma, renal cancer, lung cancer, liver cancer, gastric cancer, lymphoma, breast cancer, colorectal cancer, leukemia, prostate cancer, bone marrow cancer and other neoplastic malignant diseases.