TWI833227B - Specific binding protein targeting pd-l1 and cd73 and application thereof - Google Patents

Abstract

The present invention relates to specific binding proteins targeting PD-L1 and CD73, which specific binding proteins can specifically bind to PD-L1 and/or its fragments, and CD73 and/or its fragments. The present invention also relates to the use of the specific binding protein in the treatment, prevention and/or diagnosis of diseases, such as immune diseases, acute and chronic inflammatory diseases, and tumor diseases.

Description

Specific binding proteins targeting PD-L1 and CD73 and their applications

The present invention relates to the field of biomedicine, and more specifically to the field of antibody therapy.

Programmed cell death 1 ligand 1 (PD-L1) is one of the two cell surface glycoprotein ligands of the programmed death receptor 1 (PD-1) (the other is PD-L2) , which downregulates T cell priming and cytokine secretion when binding to PD-1. PD-L1, also known as cluster of differentiation 274 (CD274) or B7 homolog 1 (B7-H1), is a 40kDa type I transmembrane protein. PD-L1 contains IgV (immunoglobulin variable region)-like region, IgC (Immunoglobulin constant region)-like region, transmembrane region and cytoplasmic tail region. The cytoplasmic tail region is related to intracellular signal transduction; the IgV region and IgC region are involved in intercellular signal transduction. When PD-L1 binds to PD-1, it recruits Src homology region 2 protein tyrosine phosphatase (SHP-2), thereby attenuating lymphocyte-specific protein tyrosine kinase (LCK)-induced ζ chain-related protein ( ZAP70) phosphorylates and inhibits the RAS-MEK-ERK/PI3K-Akt-mTOR pathway, thereby effectively inhibiting the transcription of T cells, ultimately inhibiting the immune function of T cells, and plays an important role in the negative regulation of immune responses. Therefore, it is generally believed that blocking the PD-1/PD-L1 signaling pathway can upregulate T cell activation and initiate endogenous anti-tumor immune responses, thus exerting a therapeutic effect on tumors.

The first generation of immune checkpoint inhibitors, represented by PD-1/PD-L1 and CTLA-4 monoclonal antibodies, have gradually become representatives of tumor immunotherapy, especially in adoptive cell transfer therapy and immune checkpoint blockade (ICB). play an important role. Tumor immunotherapy targeting PD-1/PD-L1 has been approved for the treatment of many different solid tumors and won the Nobel Prize in Physiology or Medicine in 2018. However, clinical data on anti-PD-1/PD-L1 therapy show limited response rates. Some patients develop primary drug resistance and do not respond to PD-1/PD-L1 treatment, and some responders also develop acquired drug resistance after the initial response.

The search for a new generation of tumor immune targets that are safe, effective, and can produce synergistic effects with PD-1/PD-L1 is the focus of tumor immunotherapy at this stage. From this, LAG3, TIGIT, TIM3, CD47, etc. have emerged. OX40, 4-1BB and a series of immunosuppressive or agonistic targets.

Leukocyte differentiation antigen 73 (CD73), also known as 5'-nucleotidase, is an enzyme encoded by the NT5E gene in humans. CD73 is a dimer composed of two identical 70-KD subunits linked to the outer surface of the plasma membrane by a glycosylphospholipid inositol. CD73 is known to catalyze the dephosphorylation of extracellular monophosphate nucleosides to nucleosides, such as adenosine. The accumulation of extracellular adenosine in cancer tissues constitutes an important mechanism for tumor immune escape. Among other effects, tumor-derived adenosine largely inhibits infiltrating effector T cells via A2A receptors primed by adenylate cyclase (Ohta et al. (2006) Proc Natl Acad Sci USA 103:13132-13137) . CD73 has been reported to be expressed in a variety of tumor cells, including leukemia, bladder cancer, glioma, glioblastoma, ovarian cancer, melanoma, prostate cancer, thyroid cancer, esophageal cancer, and breast cancer (Jin et al., Cancer Res 2010 ;70:2245-55 and Stagg et al., PNAS 2010;107:1547-52).

For the treatment of solid tumors, an important aspect to overcome drug resistance and improve efficacy is to relieve the inhibitory effect of the tumor microenvironment (TME) on immune effector cells. The TME is a very complex system, consisting of a variety of cells, interstitium, enzymes, cytokines, metabolites, etc. It has significant characteristics of hypoxia, low pH, and high pressure, which is very different from normal tissues. An important immunosuppressive mechanism is mediated by the CD73-adenosine metabolism signaling pathway. Adenosine can inhibit the immune killing effect of T cells through the adenosine receptor (A2AR), allowing tumors to achieve immune escape, and CD73 is the key enzyme that catalyzes the production of adenosine.

Immune checkpoint blockade of PD-1/PD-L1 has become a milestone in immunotherapy. However, although anti-PD-1/PD-L1 therapies have shown impressive results in the treatment of solid tumors, durable responses only occur in a small proportion of patients, and some patients who initially respond to treatment eventually develop Acquired resistance, therefore, there is an urgent need for PD-1/PD-L1 inhibitors with better efficacy and tumor immune targets that can produce synergistic effects with PD-1/PD-L1.

The present invention provides antigen-binding proteins that can specifically bind to PD-L1 or fragments thereof, and can block the binding of PD-1 to PD-L1. The present invention also provides multispecific binding proteins that can bind to one or more antigens with high affinity and high specificity, and the antigens are PD-L1 or fragments thereof, and/or CD73 or its fragments. fragment. The invention also provides nucleic acid molecules encoding the antigen-binding protein and the multi-specific binding protein, expression vectors for producing the antigen-binding protein and the multi-specific binding protein, host cells and for producing the antigen-binding protein and the multi-specific binding protein. Methods of preparing said antigen-binding proteins, and said specific binding proteins. The invention also relates to the use of said antigen-binding proteins, and multispecific binding proteins, in the treatment, prevention and/or diagnosis of diseases, such as immune diseases, acute and chronic inflammatory diseases, and tumor diseases.

In a first aspect, the present invention provides an isolated antigen-binding protein that specifically binds PD-L1 or a fragment thereof.

The isolated antigen-binding protein that specifically binds to PD-L1 or its fragment has one or more of the following properties: (a) capable of binding to human PD-L1 and/or cynomolgus monkey PD-L1 with high affinity; (b) It has an inhibitory effect on the PD-1 signaling pathway; (c) it can enhance the activation of T lymphocytes; and (d) it shows tumor suppressive activity in vivo.

In some embodiments, the isolated antigen binding protein that specifically binds PD-L1 and/or fragments thereof comprises an antibody heavy chain variable region VH comprising the following complementarity determining region or a mutant thereof: SEQ ID HCDR1 represented by the amino acid sequence of NO: 9; HCDR2 represented by the amino acid sequence of SEQ ID NO: 23; and/or HCDR3 represented by the amino acid sequence of SEQ ID NO: 36.

The mutants have insertion, deletion or substitution of 1, 2, 3 or 4 amino acids respectively based on the amino acid sequences of HCDR1, HCDR2 and HCDR3 of the VH. Those skilled in the art know that one, two or three CDRs in HCDR1, HCDR2 and HCDR3 can be subjected to insertion, deletion or substitution mutations of 1, 2 or 3 amino acids respectively. For example, HCDR1 can be subjected to 1 amino acid Mutation, no amino acid mutation is performed on HCDR2 and HCDR3, or one amino acid mutation is performed on HCDR1 and HCDR2 respectively, and no amino acid mutation is performed on HCDR3.

In some embodiments, the amino acid sequence of HCDR1 is GFX ₁ FSX ₂ Y, the amino acid sequence of HCDR2 is X ₃ YX ₄ GX ₅ X ₆ , and the amino acid sequence of HCDR3 is NRAX ₇ FGVX ₈ PDX ₉ SDI, where X ₁ , X ₂ , X ₃ , X ₄ , X ₅ , X ₆ , X ₇ , _{X 8} or , any of E, I, A, V and L. In some embodiments, X ₁ is T, D, or N; X _{2 is N or S; X 3 is W or R; X 4 is D or} T; E; X ₇ is I or L; X ₈ is V or I; and/or, X ₉ is A or D.

In some embodiments, the amino acid sequence of HCDR1 is GF(N/T/D)FS(N/S)Y. In some embodiments, the amino acid sequence of HCDR2 is (W/R)Y(D/T)G(T/S)(K/R/E). In some embodiments, the amino acid sequence of HCDR3 is NRA(I/L)FGV(V/I)PD(A/D)SDI.

In some embodiments, a mutant of HCDR1 has the amino acid sequence set forth in any one of SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 13, and SEQ ID NO: 14.

In some embodiments, the mutant of HCDR2 has the amino acid sequence set forth in SEQ ID NO: 24 or SEQ ID NO: 27.

In some embodiments, a mutant of HCDR3 has the amino acid sequence set forth in any one of SEQ ID NO: 39, SEQ ID NO: 40, and SEQ ID NO: 41.

Preferably, the isolated antigen-binding protein that specifically binds PD-L1 and/or its fragments includes an antibody heavy chain variable region VH, and the VH includes the following complementarity determining region or a mutant thereof: SEQ ID NO: HCDR1 represented by the amino acid sequence of any one of 9-11 and SEQ ID NO: 13-14; HCDR1 represented by the amino acid sequence of any one of SEQ ID NO: 23-24 and SEQ ID NO: 27 HCDR2; and/or the amino acid sequence shown in any one of SEQ ID NO: 36 and SEQ ID NO: 39-41 HCDR3.

In some embodiments, the isolated antigen binding protein that specifically binds PD-L1 and/or fragments thereof comprises an antibody heavy chain variable region VH, the VH comprising HCDR1, HCDR2 and HCDR3, the amino acid sequences of which are respectively It is shown as SEQ ID NO:9, SEQ ID NO:23 and SEQ ID NO:36.

In some embodiments, the isolated antigen binding protein that specifically binds PD-L1 and/or fragments thereof comprises an antibody heavy chain variable region VH, the VH comprising HCDR1, HCDR2 and HCDR3, the amino acid sequences of which are respectively As shown in SEQ ID NO: 10, SEQ ID NO: 23 and SEQ ID NO: 36.

In some embodiments, the isolated antigen binding protein that specifically binds PD-L1 and/or fragments thereof comprises an antibody heavy chain variable region VH, the VH comprising HCDR1, HCDR2 and HCDR3, the amino acid sequences of which are respectively As shown in SEQ ID NO: 11, SEQ ID NO: 24 and SEQ ID NO: 36.

In some embodiments, the isolated antigen binding protein that specifically binds PD-L1 and/or fragments thereof comprises an antibody heavy chain variable region VH, the VH comprising HCDR1, HCDR2 and HCDR3, the amino acid sequences of which are respectively It is shown as SEQ ID NO: 11, SEQ ID NO: 23 and SEQ ID NO: 36.

In some embodiments, the isolated antigen binding protein that specifically binds PD-L1 and/or fragments thereof comprises an antibody heavy chain variable region VH, the VH comprising HCDR1, HCDR2 and HCDR3, the amino acid sequences of which are respectively It is shown as SEQ ID NO: 13, SEQ ID NO: 23 and SEQ ID NO: 36.

In some embodiments, the isolated antigen binding protein that specifically binds PD-L1 and/or fragments thereof comprises an antibody heavy chain variable region VH, the VH comprising HCDR1, HCDR2 and HCDR3, the amino acid sequences of which are respectively As shown in SEQ ID NO: 10, SEQ ID NO: 23 and SEQ ID NO: 39.

In some embodiments, the isolated antigen binding protein that specifically binds PD-L1 and/or fragments thereof comprises an antibody heavy chain variable region VH, the VH comprising HCDR1, HCDR2 and HCDR3, the amino acid sequences of which are respectively As shown in SEQ ID NO: 10, SEQ ID NO: 23 and SEQ ID NO: 40.

In some embodiments, the isolated antigen binding protein that specifically binds PD-L1 and/or fragments thereof comprises an antibody heavy chain variable region VH, the VH comprising HCDR1, HCDR2 and HCDR3, the amino acid sequences of which are respectively As shown in SEQ ID NO: 10, SEQ ID NO: 23 and SEQ ID NO: 41.

In some embodiments, the isolated antigen binding protein that specifically binds PD-L1 and/or fragments thereof comprises an antibody heavy chain variable region VH, the VH comprising HCDR1, HCDR2 and HCDR3, the amino acid sequences of which are respectively As shown in SEQ ID NO: 10, SEQ ID NO: 27 and SEQ ID NO: 36.

In some embodiments, the isolated antigen binding protein that specifically binds PD-L1 and/or fragments thereof comprises an antibody heavy chain variable region VH, the VH comprising HCDR1, HCDR2 and HCDR3, the amino acid sequences of which are respectively As shown in SEQ ID NO: 10, SEQ ID NO: 27 and SEQ ID NO: 39.

In some embodiments, the isolated antigen binding protein that specifically binds PD-L1 and/or fragments thereof comprises an antibody heavy chain variable region VH, the VH comprising HCDR1, HCDR2 and HCDR3, the amino acid sequences of which are respectively As shown in SEQ ID NO: 10, SEQ ID NO: 27 and SEQ ID NO: 40.

In some embodiments, the isolated antigen binding protein that specifically binds PD-L1 and/or fragments thereof comprises an antibody heavy chain variable region VH, the VH comprising HCDR1, HCDR2 and HCDR3, the amino acid sequences of which are respectively As shown in SEQ ID NO: 10, SEQ ID NO: 27 and SEQ ID NO: 41.

In some embodiments, the isolated antigen binding protein that specifically binds PD-L1 and/or fragments thereof comprises an antibody heavy chain variable region VH, the VH comprising HCDR1, HCDR2 and HCDR3, the amino acid sequences of which are respectively As shown in SEQ ID NO: 14, SEQ ID NO: 27 and SEQ ID NO: 36.

In some embodiments, the isolated antigen binding protein that specifically binds PD-L1 and/or fragments thereof comprises an antibody heavy chain variable region VH, the VH comprising HCDR1, HCDR2 and HCDR3, the amino acid sequences of which are respectively As shown in SEQ ID NO: 14, SEQ ID NO: 27 and SEQ ID NO: 39.

In some embodiments, the isolated antigen binding protein that specifically binds PD-L1 and/or fragments thereof comprises an antibody heavy chain variable region VH, the VH comprising HCDR1, HCDR2 and HCDR3, the amino acid sequences of which are respectively As shown in SEQ ID NO: 14, SEQ ID NO: 27 and SEQ ID NO: 40.

In some embodiments, the isolated antigen binding protein that specifically binds PD-L1 and/or fragments thereof comprises an antibody heavy chain variable region VH, the VH comprising HCDR1, HCDR2 and HCDR3, the amino acid sequences of which are respectively As shown in SEQ ID NO: 14, SEQ ID NO: 27 and SEQ ID NO: 41.

Preferably, the VH also includes a heavy chain variable region framework region (VH FWR), and the VH FWR can contain VH FWR1, VH FWR2, VH FWR3 and VH FWR4.

In some embodiments, the VH FWR1 has the amino acid sequence set forth in any one of SEQ ID NOs: 3-5.

In some embodiments, the VH FWR2 has the amino acid sequence set forth in SEQ ID NO: 17 or SEQ ID NO: 20.

In some embodiments, the VH FWR3 has the amino acid sequence set forth in SEQ ID NO: 30 or SEQ ID NO: 31.

In some embodiments, the VH FWR4 has the amino acid sequence set forth in SEQ ID NO: 43.

Preferably, the VH comprises the amino acid sequence shown in any one of SEQ ID NO: 74-77, SEQ ID NO: 79-80 and SEQ ID NO: 82-92, or has at least 80%, An amino acid sequence that is 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99% or 100% identical.

In some embodiments, the isolated antigen-binding protein that specifically binds PD-L1 and/or fragments thereof further comprises an Fc region, or a region equivalent to the Fc region of an immunoglobulin. Preferably, the Fc region Fc for people. More preferably, the human Fc is human IgG1 Fc.

Preferably, the Fc contains mutations L234A, L235A and P329G, or mutations L234A and L235A, or one, two or three mutations of S298A, E333A and K334A, more preferably simultaneously containing three mutations of S298A, E333A and K334A. a mutation.

In some embodiments, the isolated antigen binding protein that specifically binds PD-L1 or a fragment thereof comprises SEQ ID NO: 99, 100, 101, 102, 104, 105, 107, 108, 109, 110, 111 , 112, 113, 114, 115, 116 or 117 having an amino acid sequence of at least 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99% or 100% identity of heavy chains. In some embodiments, the isolated antigen binding protein that specifically binds PD-L1 or a fragment thereof comprises any of SEQ ID NOs: 99-102, SEQ ID NOs: 104-105, and SEQ ID NOs: 107-117 The amino acid sequence of the heavy chain shown in the item.

In some embodiments, the antigen-binding protein may also be an antigen-binding fragment, for example, in the form of a Fab, Fab', F(ab') ₂ , Fv, scFv, VH, or HCAb. The number of Fab, Fab', F(ab') ₂ , Fv, scFv or VH is preferably one or more.

In a second aspect, the invention provides an isolated nucleic acid encoding an isolated antigen-binding protein according to the first aspect of the invention.

In a third aspect, the present invention provides an expression vector comprising the isolated nucleic acid described in the second aspect of the present invention and capable of expressing it as the isolated antigen-binding protein of the first aspect.

The expression vector may be a eukaryotic cell expression vector and/or a prokaryotic cell expression vector, such as a plasmid.

In a fourth aspect, the invention provides a host cell comprising the isolated nucleic acid of the second aspect, or the expression vector of the third aspect.

In a fifth aspect, the present invention provides an antibody-drug conjugate, which includes: the isolated antigen-binding protein described in the first aspect of the present invention; and a drug covalently linked to the antigen-binding protein. In some embodiments, the drug is selected from the group consisting of chemotherapeutic agents, radiotherapeutic agents, immunosuppressive agents, and cytotoxic drugs.

In a sixth aspect, the present invention provides a chimeric antigen receptor, which includes an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain, wherein the extracellular antigen-binding domain includes the first aspect of the present invention. The isolated antigen-binding protein.

In a seventh aspect, the present invention provides modified immune cells comprising the chimeric antigen receptor according to the sixth aspect of the present invention.

In an eighth aspect, the present invention provides a multispecific antibody comprising two or more antigen-binding domains, wherein one antigen-binding domain comprises the isolated antigen-binding protein described in the first aspect of the present invention.

In a ninth aspect, the present invention provides a method for preparing the isolated antigen-binding protein of the first aspect.

In some embodiments, the isolated antigen-binding protein of the first aspect is prepared using hybridoma technology or other conventional techniques in the art, such as humanization technology.

In some embodiments, the preparation method includes the step of culturing the host cell of the fourth aspect.

In some embodiments, Harbor HCAb transgenic mice (hereinafter referred to as HCAb transgenic mice) are used to prepare the isolated antigen-binding protein of the first aspect.

The HCAb transgenic mouse is a transgenic mouse carrying a human immunoglobulin immune repertoire and is able to produce a new "heavy chain" only antibody that is only half the size of traditional IgG antibodies. The antibodies it produces have only the human antibody "heavy chain" variable domain and the mouse Fc constant domain.

Preferably, the method of using HCAb transgenic mice to prepare the isolated antigen-binding protein of the first aspect includes the following steps: (a) using human PD-L1 antigen to immunize HCAb transgenic mice, more preferably, the human PD -L1 antigen is recombinant human PD-L1-mFc. Specifically, the antigen is a recombinant fusion protein composed of human PD-L1 connected to Fc; (b) RNA reverse transcription of splenic B cells of transgenic mice using HCAb after immunization cDNA, and specific primers to amplify the human VH gene; (c) constructing the amplified VH gene into a mammalian cell expression vector encoding the human IgG antibody heavy chain Fc domain sequence; and (d) purification step (c) ) fully human PD-L1 monoclonal antibody.

Preferably, the IgG antibody is an IgG1 antibody or an IgG4 antibody.

In some embodiments, the method further includes the step of affinity engineering the fully human PD-L1 monoclonal antibody by mutating the CDR region.

Preferably, the fully human PD-L1 monoclonal antibody is optimized through germline backmutation and/or post-translational modification (PTM) removal.

In a tenth aspect, the present invention provides a pharmaceutical composition comprising: The isolated antigen-binding protein described in the first aspect, the antibody drug conjugate described in the fifth aspect, the chimeric antigen receptor described in the sixth aspect, the modified immune cell described in the seventh aspect or the eighth aspect The multispecific antibody; and, a pharmaceutically acceptable carrier.

The pharmaceutical composition may also include other therapeutic agents, including, but not limited to, chemotherapeutic agents, radiotherapeutic agents, immunosuppressive agents, cytotoxic drugs, and the like. In some embodiments, the chemotherapeutic agent, radiotherapeutic agent, immunosuppressive agent or cytotoxic drug in the pharmaceutical composition is the same as or different from the drug in the antibody drug conjugate described in the fifth aspect.

In an eleventh aspect, the present invention provides the isolated antigen-binding protein described in the first aspect, the isolated nucleic acid described in the second aspect, the antibody-drug conjugate described in the fifth aspect, and the embedded antigen-binding protein described in the sixth aspect. Combined antigen receptors, the modified immune cells described in the seventh aspect or the multispecific antibodies described in the eighth aspect or the pharmaceutical compositions of the tenth aspect are prepared for preventing, treating and/or diagnosing immune diseases, acute and applications in drugs for chronic inflammatory diseases, as well as tumor diseases.

The tumors may be breast cancer, renal cell carcinoma, melanoma, colon cancer, as well as B-cell lymphoma, melanoma, head and neck cancer, bladder cancer, gastric cancer, ovarian cancer, malignant sarcoma, urothelial cancer, liver cancer, esophageal cancer , gastroesophageal junction cancer, nasopharyngeal cancer, small cell lung cancer, cervical cancer, endometrial cancer, pancreatic cancer, prostate cancer, glioma, non-small cell lung cancer, acute myeloid leukemia, Hodgkin lymphoma, cutaneous squamous cell carcinoma Cell carcinoma, locally advanced or metastatic malignant tumors, etc.

The inflammatory disease may be atopic dermatitis, ulcerative colitis, etc.

The immune disease may be graft versus host disease, rheumatoid arthritis, systemic lupus erythematosus, asthma, etc.

In a twelfth aspect, the present invention provides a method for detecting PD-L1 in a sample, the method comprising the step of detecting PD-L1 in the sample using the isolated antigen-binding protein of the first aspect.

The sample may be a biological sample, for example, whole blood, red blood cell concentrate, platelet concentrate, leukocyte concentrate, tissue, bone marrow aspirate, plasma, serum, cerebrospinal fluid, feces, urine, cultured cells, saliva, Oral secretions, nasal secretions and other biological samples.

In some embodiments, the method of detecting PD-L1 in a sample may be for non-diagnostic purposes.

In a thirteenth aspect, the present invention provides a multispecific binding protein, said specific binding protein comprising at least two structural domains, capable of binding to PD-L1 or a fragment thereof, and/or CD73 or a fragment thereof.

In some embodiments, the specific binding protein comprises a first domain that binds CD73 or a fragment thereof and a second domain that binds PD-L1 or a fragment thereof. The first domain and the second domain are linked to form a bispecific binding protein.

In some embodiments, the first domain is a CD73 antibody or antigen-binding fragment thereof.

In some embodiments, the second domain is a PD-L1 antibody or antigen-binding fragment thereof.

In some embodiments, the first domain and/or the second domain is in the form of IgG, Fab, Fab', F(ab') ₂ , Fv, scFv, VH, or HCAb.

The number of Fab, Fab', F(ab') ₂ , Fv, scFv, and VH is one or more.

In some embodiments, the first domain is in the form of IgG. Preferably, the heavy chain constant region of the IgG is a human heavy chain constant region, more preferably a human IgG1, human IgG2, human IgG3 or human IgG4 heavy chain constant region; wherein human IgG preferably includes L234A, L235A and P329G. One, two or three mutations, more preferably the mutations of L234A and L235A, or one, two or three mutations of S298A, E333A and K334A, more preferably the mutations of S298A, E333A and K334A.

Preferably, the Fc of IgG in the first domain is the Fc of human IgG1. More preferably, the Fc includes L234A, L235A and P329G mutations, or L234A and L235A mutations, or includes S298A, E333A and K334A. One, two or three mutations, more preferably including S298A, E333A and K334A mutations.

In some embodiments, the first domain is in the form of a Fab, and more preferably, the first domain includes 2 Fabs.

In some embodiments, the second domain includes one or more VHs.

Preferably, the VH is human VH. More preferably, the second domain has two VHs.

In some embodiments, the second domain also includes an Fc, a region equivalent to the Fc region of an immunoglobulin. Preferably, the Fc is the Fc of human IgG1. More preferably, the Fc includes L234A, L235A and P329G mutations, or L234A and L235A mutations, or comprise one, two or three mutations of S298A, E333A and K334A, more preferably comprise S298A, E333A and K334A mutations.

In some embodiments, the first domain and the second domain are linked directly or via linking peptide L to form a bispecific binding protein.

In some embodiments, the second domain is linked to the C-terminus or N-terminus of the first domain.

In some embodiments, the bispecific binding protein comprises short and long chains.

Preferably, the short chain has the structure shown by N'-VL ₁ -CL ₁ -C'; the long chain has the structure shown by N'-VH ₁ -CH1-h-CH2-CH3-L-VH ₂ -C ' has the structure shown; alternatively, the short chain has the structure shown as N'-VL ₁ -CL ₁ -C'; the long chain has N'-VH ₂ -L-VH ₁ -CH1-h-CH2 -CH3-C'; or, the short chain has the structure N'-VH ₁ -CH-C'; the long chain has N'-VL ₁ -CL ₁ -L-VH ₂ The structure shown by -CH2-CH3-C', wherein the VL ₁ and VH ₁ are the VL and VH of the first domain respectively, the VH ₂ is the VH of the second domain, and the h is the hinge region , the L is a connecting peptide, and the CL ₁ is the CL of the first domain. Among them, the hinge region is a common hinge region in the field of immunoglobulin, usually contains a large amount of proline, is flexible, and forms 2-5 disulfide bonds.

Preferably, the L is a peptide with a length of 0-30 amino acids. In some embodiments, the amino acid sequence of L is as shown in any one of SEQ ID NO: 136-157.

In some embodiments, the long chain of CH3 is directly linked to _VH2 , i.e., the length of L is 0.

In some embodiments, the long chain of CH3 is linked to _VH2 via linker peptide L. In some embodiments, the linker peptide L is a peptide ranging from 0 to 30 amino acids in length. In some embodiments, the linking peptide L may be an amino acid sequence as shown in any one of SEQ ID NOs. 136-157.

In some embodiments, the long chain of VH ₁ and VH ₂ are directly linked, ie, the length of L is zero.

In some embodiments, the long chain VH ₁ is linked to VH ₂ via a linker peptide L. In some embodiments, the linker peptide L is a peptide ranging from 0 to 30 amino acids in length. In some embodiments, the linking peptide L may be an amino acid sequence as shown in any one of SEQ ID NOs. 136-157.

In some embodiments, the long chain of _VH2 is directly linked to _CL1 , i.e., the length of L is zero.

In some embodiments, the long chain _VH2 is linked to _CL1 via linker peptide L. In some embodiments, the linker peptide L is a peptide ranging from 0 to 30 amino acids in length. In some embodiments, the linking peptide L may be an amino acid sequence as shown in any one of SEQ ID NOs. 136-157.

In some embodiments, the first domain comprises a light chain variable region (VL) and a heavy chain variable region (VH), the VL comprising a complementarity determining region selected from: SEQ ID NO: 51, SEQ LCDR1 shown as the amino acid sequence of any one of ID NO: 52 and SEQ ID NO: 53; the amino acid sequence of any one of SEQ ID NO: 57, SEQ ID NO: 58 and SEQ ID NO: 59 LCDR2 shown; and/or LCDR3 shown by the amino acid sequence of any one of SEQ ID NO: 65, SEQ ID NO: 66 and SEQ ID NO: 67; the VH comprises a complementarity determining region selected from the following : HCDR1 represented by the amino acid sequence of any one of SEQ ID NO: 8, SEQ ID NO: 12 and SEQ ID NO: 10; SEQ ID NO: 22, SEQ ID NO: 25 and SEQ ID NO: 26 The amino acid sequence of any HCDR2 shown in the column; and/or HCDR3 shown in the amino acid sequence of any one of SEQ ID NO: 35, SEQ ID NO: 37 and SEQ ID NO: 38.

Preferably, the first domain includes a light chain variable region (VL) and a heavy chain variable region (VH), and the VL and VH include a complementarity determining region selected from the group consisting of SEQ ID NO: 51, respectively. , LCDR1, LCDR2 and LCDR3 as set forth in SEQ ID NO:57 and SEQ ID NO:65; and, HCDR1, HCDR2 and HCDR3 as set forth in SEQ ID NO:8, SEQ ID NO:22 and SEQ ID NO:35 respectively ; LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 52, SEQ ID NO: 58 and SEQ ID NO: 66 respectively; and, respectively as SEQ ID NO: 12, SEQ ID NO: 25 and SEQ ID NO: 37 HCDR1, HCDR2 and HCDR3 as shown; LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO:53, SEQ ID NO:59 and SEQ ID NO:67, respectively; and, respectively, as SEQ ID NO:10, SEQ ID NO :26 and HCDR1, HCDR2 and HCDR3 shown in SEQ ID NO:38.

In some embodiments, the first domain comprises VL and VH, wherein the amino acid sequence of VL has at least 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99% or 100% identity; the amino acid sequence of the VH is the same as SEQ ID NO: 73, SEQ ID NO: 78 or SEQ ID NO: 81 is at least 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99% or 100% identical.

Preferably, the first domain includes VL and VH, wherein the VL has the amino acid sequence shown in any one of SEQ ID NO: 94, SEQ ID NO: 95 and SEQ ID NO: 96; The VH has the amino acid sequence shown in any one of SEQ ID NO: 73, SEQ ID NO: 78 and SEQ ID NO: 81.

Preferably, the first domain includes VL and VH, and the amino acid sequences of VL and VH are as shown in SEQ ID NO: 94 and SEQ ID NO: 73 respectively; or as shown in SEQ ID NO: 95 and SEQ ID NO: 73 respectively. SEQ ID NO:78; or SEQ ID NO:96 and SEQ ID NO:81 respectively.

In some embodiments, the first domain comprises an amino acid sequence identical to SEQ ID NO: 119, SEQ ID NO: 120, or SEQ ID NO: 121 by at least 80%, 85%, 88%, 90%, 92 %, 95%, 97%, 98%, 99% or 100% identity of the light chain; and the amino acid sequence has at least 80%, Heavy chains of 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99% or 100% identity.

Preferably, the first domain comprises the light chain shown in any one of SEQ ID NO: 119, SEQ ID NO: 120 and SEQ ID NO: 121; and SEQ ID NO: 98, SEQ ID NO: 103 and the heavy chain shown in any one of SEQ ID NO: 106.

Preferably, the first structural domain includes the light chain shown in SEQ ID NO: 119 and the heavy chain shown in SEQ ID NO: 98.

Preferably, the first structural domain includes the light chain shown in SEQ ID NO: 120 and the heavy chain shown in SEQ ID NO: 103.

Preferably, the first structural domain includes the light chain shown in SEQ ID NO: 121 and the heavy chain shown in SEQ ID NO: 106.

In some embodiments, the second domain comprises an antibody heavy chain variable region VH, which VH comprises the following complementarity determining region or a mutant thereof: HCDR1 represented by the amino acid sequence of SEQ ID NO: 9; HCDR2 represented by the amino acid sequence of SEQ ID NO: 23; and/or HCDR3 represented by the amino acid sequence of SEQ ID NO: 36.

The mutants have the insertion, deletion or substitution of 1, 2 or 3 amino acids respectively based on the amino acid sequences of HCDR1, HCDR2 and HCDR3 of the VH. Those skilled in the art know that one, two or three CDRs in HCDR1, HCDR2 and HCDR3 can be subjected to insertion, deletion or substitution mutations of 1, 2 or 3 amino acids respectively. For example, one amino acid mutation can be performed on HCDR1, and no amino acid mutations can be performed on HCDR2 and HCDR3, or one amino acid mutation can be performed on HCDR1 and HCDR2 respectively. Amino acid mutation, no amino acid mutation is performed on HCDR3.

In some embodiments, the amino acid sequence of HCDR1 is GFX ₁ FSX ₂ Y, the amino acid sequence of HCDR2 is X ₃ YX ₄ GX ₅ X ₆ , and the amino acid sequence of HCDR3 is NRAX ₇ FGVX ₈ PDX ₉ SDI, where X ₁ , X ₂ , X ₃ , X ₄ , X ₅ , X ₆ , X ₇ , _{X 8} or , any of E, I, A, V and L. In some embodiments, X ₁ is T, D, or N; X _{2 is N or S; X 3 is W or R; X 4 is D or} T; E; X ₇ is I or L; X ₈ is V or I; and/or, X ₉ is A or D.

In some embodiments, the amino acid sequence of HCDR1 is GF(N/T/D)FS(N/S)Y. In some embodiments, the amino acid sequence of HCDR2 is (W/R)Y(D/T)G(T/S)(K/R/E). In some embodiments, the amino acid sequence of HCDR3 is NRA(I/L)FGV(V/I)PD(A/D)SDI.

In some embodiments, a mutant of HCDR1 has the amino acid sequence set forth in any one of SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 13, and SEQ ID NO: 14.

In some embodiments, the mutant of HCDR2 has the amino acid sequence set forth in SEQ ID NO: 24 or SEQ ID NO: 27.

In some embodiments, the mutant of HCDR3 has the amino acid sequence set forth in any one of SEQ ID NO:39, SEQ ID NO:40 and SEQ ID NO:41.

Preferably, the second domain includes the antibody heavy chain variable region VH, and the VH includes the following complementarity determining regions or mutants thereof: any of SEQ ID NO: 9-11 and SEQ ID NO: 13-14 HCDR1 represented by the amino acid sequence of any one of SEQ ID NO: 23-24 and SEQ ID NO: 27; HCDR2 represented by the amino acid sequence of any one of SEQ ID NO: 23-24 and SEQ ID NO: 27; and/or SEQ ID NO: 36 and SEQ ID HCDR3 represented by the amino acid sequence of any one of NO: 39-41.

In some embodiments, the second domain comprises the antibody heavy chain variable region VH, so The VH includes HCDR1, HCDR2 and HCDR3, whose amino acid sequences are shown in SEQ ID NO: 9, SEQ ID NO: 23 and SEQ ID NO: 36 respectively.

In some embodiments, the second domain comprises an antibody heavy chain variable region VH, the VH comprising HCDR1, HCDR2 and HCDR3, the amino acid sequences of which are SEQ ID NO: 10, SEQ ID NO: 23 and HCDR3, respectively. SEQ ID NO:36.

In some embodiments, the second domain comprises an antibody heavy chain variable region VH, the VH comprising HCDR1, HCDR2 and HCDR3, the amino acid sequences of which are SEQ ID NO: 11, SEQ ID NO: 24 and HCDR3, respectively. SEQ ID NO:36.

In some embodiments, the second domain comprises an antibody heavy chain variable region VH, the VH comprising HCDR1, HCDR2 and HCDR3, the amino acid sequences of which are SEQ ID NO: 11, SEQ ID NO: 23 and HCDR3, respectively. SEQ ID NO:36.

In some embodiments, the second domain comprises an antibody heavy chain variable region VH, the VH comprising HCDR1, HCDR2 and HCDR3, the amino acid sequences of which are SEQ ID NO: 13, SEQ ID NO: 23 and HCDR3, respectively. SEQ ID NO:36.

In some embodiments, the second domain comprises an antibody heavy chain variable region VH, the VH comprising HCDR1, HCDR2 and HCDR3, the amino acid sequences of which are SEQ ID NO: 10, SEQ ID NO: 23 and HCDR3, respectively. SEQ ID NO:39.

In some embodiments, the second domain comprises an antibody heavy chain variable region VH, the VH comprising HCDR1, HCDR2 and HCDR3, the amino acid sequences of which are SEQ ID NO: 10, SEQ ID NO: 23 and HCDR3, respectively. SEQ ID NO:40 is shown.

In some embodiments, the second domain comprises an antibody heavy chain variable region VH, the VH comprising HCDR1, HCDR2 and HCDR3, the amino acid sequences of which are SEQ ID NO: 10, SEQ ID NO: 23 and HCDR3, respectively. SEQ ID NO:41 is shown.

In some embodiments, the second domain comprises an antibody heavy chain variable region VH, the VH comprising HCDR1, HCDR2 and HCDR3, the amino acid sequences of which are SEQ ID NO: 10, respectively. SEQ ID NO:27 and SEQ ID NO:36 are shown.

In some embodiments, the second domain comprises an antibody heavy chain variable region VH, the VH comprising HCDR1, HCDR2 and HCDR3, the amino acid sequences of which are SEQ ID NO: 10, SEQ ID NO: 27 and HCDR3, respectively. SEQ ID NO:39.

In some embodiments, the second domain comprises an antibody heavy chain variable region VH, the VH comprising HCDR1, HCDR2 and HCDR3, the amino acid sequences of which are SEQ ID NO: 10, SEQ ID NO: 27 and HCDR3, respectively. SEQ ID NO:40 is shown.

In some embodiments, the second domain comprises an antibody heavy chain variable region VH, the VH comprising HCDR1, HCDR2 and HCDR3, the amino acid sequences of which are SEQ ID NO: 10, SEQ ID NO: 27 and HCDR3, respectively. SEQ ID NO:41.

In some embodiments, the second domain comprises an antibody heavy chain variable region VH, the VH comprising HCDR1, HCDR2 and HCDR3, the amino acid sequences of which are SEQ ID NO: 14, SEQ ID NO: 27 and HCDR3, respectively. SEQ ID NO:36.

In some embodiments, the second domain comprises an antibody heavy chain variable region VH, the VH comprising HCDR1, HCDR2 and HCDR3, the amino acid sequences of which are SEQ ID NO: 14, SEQ ID NO: 27 and HCDR3, respectively. SEQ ID NO:39.

In some embodiments, the second domain comprises an antibody heavy chain variable region VH, the VH comprising HCDR1, HCDR2 and HCDR3, the amino acid sequences of which are SEQ ID NO: 14, SEQ ID NO: 27 and HCDR3, respectively. SEQ ID NO:40 is shown.

In some embodiments, the second domain comprises an antibody heavy chain variable region VH, the VH comprising HCDR1, HCDR2 and HCDR3, the amino acid sequences of which are SEQ ID NO: 14, SEQ ID NO: 27 and HCDR3, respectively. SEQ ID NO:41 is shown.

Preferably, the VH also includes a heavy chain variable region framework region (VH FWR), and the VH FWR may include VH FWR1, VH FWR2, VH FWR3 and VH FWR4.

In some embodiments, the VH FWR1 has any one of SEQ ID NOs: 3-5 The amino acid sequence shown.

In some embodiments, the VH FWR2 has the amino acid sequence set forth in SEQ ID NO: 17 or SEQ ID NO: 20.

In some embodiments, the VH FWR3 has the amino acid sequence set forth in SEQ ID NO: 30 or SEQ ID NO: 31.

In some embodiments, the VH FWR4 has the amino acid sequence set forth in SEQ ID NO: 43.

Preferably, the VH comprises the amino acid sequence shown in any one of SEQ ID NO: 74-77, SEQ ID NO: 79-80 and SEQ ID NO: 82-92, or has at least 80%, An amino acid sequence that is 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99% or 100% identical.

In some embodiments, the second domain further comprises an Fc region, or a region equivalent to the Fc region of an immunoglobulin. Preferably, the Fc region is a human Fc. More preferably, the human Fc is human IgG1 Fc.

Preferably, the Fc includes L234A, L235A and P329G mutations, or L234A and L235A mutations, or one, two or three mutations of S298A, E333A and K334A, more preferably includes S298A, E333A and K334A mutations.

In some embodiments, the second domain comprises SEQ ID NO: 99, 100, 101, 102, 104, 105, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116 or 117 A heavy chain having an amino acid sequence that is at least 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99% or 100% identical. In some embodiments, the second domain comprises the amino acid sequence of the heavy chain set forth in any one of SEQ ID NOs: 99-102, SEQ ID NOs: 104-105, and SEQ ID NOs: 107-117.

In some embodiments, the long chain of the specific binding protein comprises at least 80%, 85%, An amino acid sequence that is 88%, 90%, 92%, 95%, 97%, 98%, 99% or 100% identical; and the short chain of the specific binding protein contains the same amino acid sequence as SEQ ID NO: 119, 120 , 121 or 131 have to Amino acid sequences that are less than 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99% or 100% identical. In some embodiments, the long chain of the specific binding protein has an amino acid sequence selected from any one of SEQ ID NO: 122-130 and SEQ ID NO: 132-133; and the specificity The short chain of the binding protein has an amino acid sequence selected from any one of SEQ ID NO: 119-121 and SEQ ID NO: 131.

In some embodiments, the specific binding protein has a long chain set forth in SEQ ID NO: 122 and a short chain set forth in SEQ ID NO: 119.

In some embodiments, the specific binding protein has a long chain set forth in SEQ ID NO: 123 and a short chain set forth in SEQ ID NO: 120.

In some embodiments, the specific binding protein has a long chain set forth in SEQ ID NO: 124 and a short chain set forth in SEQ ID NO: 121.

In some embodiments, the specific binding protein has a long chain set forth in SEQ ID NO: 125 and a short chain set forth in SEQ ID NO: 119.

In some embodiments, the specific binding protein has a long chain set forth in SEQ ID NO: 126 and a short chain set forth in SEQ ID NO: 119.

In some embodiments, the specific binding protein has a long chain set forth in SEQ ID NO: 127 and a short chain set forth in SEQ ID NO: 119.

In some embodiments, the specific binding protein has a long chain set forth in SEQ ID NO: 128 and a short chain set forth in SEQ ID NO: 119.

In some embodiments, the specific binding protein has a long chain set forth in SEQ ID NO: 129 and a short chain set forth in SEQ ID NO: 119.

In some embodiments, the specific binding protein has a long chain set forth in SEQ ID NO: 132 and a short chain set forth in SEQ ID NO: 131.

In some embodiments, the specific binding protein has a long chain set forth in SEQ ID NO: 130 and a short chain set forth in SEQ ID NO: 119.

In some embodiments, the specific binding protein has a long chain set forth in SEQ ID NO: 133 and a short chain set forth in SEQ ID NO: 131.

In some embodiments, the dual-specific binding protein is a tetravalent symmetric structure formed by two first polypeptide chains and two second polypeptide chains.

In a fourteenth aspect, the present invention provides an isolated nucleic acid encoding the specific binding protein or fragment thereof according to the thirteenth aspect of the present invention.

In a fifteenth aspect, the present invention provides an expression vector capable of expressing the isolated nucleic acid described in the fourteenth aspect into the multispecific binding protein of the thirteenth aspect or a fragment thereof.

The expression vector may be a eukaryotic cell expression vector and/or a prokaryotic cell expression vector, such as a plasmid.

In a sixteenth aspect, the present invention provides a host cell comprising the isolated nucleic acid of the fourteenth aspect, or the expression vector of the fifteenth aspect.

The host cell is a conventional host cell in the art, as long as the expression vector of the fifteenth aspect can stably express the carried nucleic acid into the specific binding protein of the thirteenth aspect or a fragment thereof. Preferably, the host cell is a prokaryotic cell and/or a eukaryotic cell, the prokaryotic cell is preferably E.coli cells such as TG1, BL21 (expressing single chain antibodies or Fab antibodies), and the eukaryotic cell is preferably HEK293 cells or CHO cells (expressing full-length IgG antibodies). By transforming the expression vector of the fifteenth aspect into a host cell, the host cell of the present invention can be obtained. The transformation method is a conventional transformation method in this field, preferably a chemical transformation method, a heat shock method or an electroporation method.

In a seventeenth aspect, the present invention provides an antibody drug conjugate comprising the specific binding protein of the thirteenth aspect or a fragment thereof. The antibody drug conjugate further comprises a drug covalently linked to the binding protein or fragment thereof. In some embodiments, the drug is selected from the group consisting of chemotherapeutic agents, radiotherapeutic agents, immunosuppressive agents, and cytotoxic drugs.

In an eighteenth aspect, the present invention provides a method for preparing the specific binding protein or fragment thereof of the thirteenth aspect.

In some embodiments, the specific binding protein or fragment thereof of the thirteenth aspect is prepared using hybridoma technology or other conventional techniques in the art, such as humanization technology, etc.

In some embodiments, the preparation method includes the step of culturing the host cell of the sixteenth aspect.

In some embodiments, Harbor HCAb transgenic mice (hereinafter referred to as HCAb transgenic mice) are used to prepare the second domain of the specific binding protein of the thirteenth aspect.

The HCAb transgenic mouse is a transgenic mouse carrying a human immunoglobulin immune repertoire and is able to produce a new "heavy chain" only antibody that is only half the size of traditional IgG antibodies. The antibodies it produces have only the human antibody "heavy chain" variable domain and the mouse Fc constant domain.

Preferably, the method for preparing the second domain of the specific binding protein of the thirteenth aspect using HCAb transgenic mice includes the following steps: (a) immunizing HCAb transgenic mice with human PD-L1, more preferably, The human PD-L1 antigen is recombinant human PD-L1-Fc. Specifically, the antigen is a recombinant fusion protein composed of PD-L1 connected to Fc; (b) RNA from splenic B cells of HCAb transgenic mice after immunization is used The reverse-transcribed cDNA and specific primers amplify the human VH gene; (c) construct the amplified VH gene into a mammalian cell expression vector encoding the human IgG antibody heavy chain Fc domain sequence; and (d) purify Fully human PD-L1 monoclonal antibody obtained in step (c).

In some embodiments, the method further includes the step of affinity engineering the fully human PD-L1 monoclonal antibody by mutating the CDR region.

Preferably, the fully human PD-L1 monoclonal antibody can also be optimized through germline backmutation and/or post-translational modification (PTM) removal.

In some embodiments, Harbor H2L2 transgenic mice (hereinafter referred to as H2L2 transgenic mice) are used to prepare the first domain of the specific binding protein of the thirteenth aspect.

The H2L2 transgenic mouse is a transgenic mouse carrying a human immunoglobulin immune repertoire. Mouse, which produces antibodies with complete human antibody variable domains and rat constant domains.

Preferably, the method of using H2L2 transgenic mice to prepare the first domain of the specific binding protein of the thirteenth aspect includes the following steps: (a) using human CD73 to immunize the H2L2 transgenic mice, more preferably, the The human CD73 antigen is soluble recombinant human CD73-hFc. Specifically, the antigen is a recombinant fusion protein composed of CD73 connected to Fc; (b) hybridoma cells are obtained by fusion of spleen cells of H2L2 transgenic mice after immunization and myeloma cell lines. , the isolated hybridoma expresses an antibody molecule with a complete human variable domain and a heavy chain and a light chain of a rat constant domain; (c) converting the antibody light chain variable domain sequence (VL) obtained in step (b) ) is genetically synthesized and cloned into a mammalian cell expression vector encoding the human antibody kappa light chain constant domain sequence to encode the full-length light chain of the antibody produced; (d) converting the antibody heavy chain variable obtained in step (b) The domain sequence (VH) is genetically synthesized and cloned into a mammalian cell expression vector encoding a human IgG antibody heavy chain constant domain sequence to encode a full-length heavy chain that produces an IgG antibody; (e) combining steps (c) and The expression vector in (d) is simultaneously transfected into mammalian host cells, and conventional recombinant protein expression and purification techniques can be used to obtain recombinant antibodies with correctly paired and assembled light and heavy chains.

Preferably, the antibody heavy chain variable domain sequence (VH) obtained in step (b) is gene synthesized and cloned into a mammalian cell expression vector encoding the human IgG1 antibody heavy chain constant domain sequence to encode and produce Full-length heavy chain of IgG1 antibody.

In some embodiments, the method further includes the step of affinity engineering the fully human CD73 monoclonal antibody by mutating the CDR region.

Preferably, the fully human CD73 monoclonal antibody can also be optimized through germline backmutation and/or post-translational modification (PTM) removal.

In some embodiments, the prepared second domain and the prepared first domain are combined into a bispecific binding protein. The bispecific binding protein can bind two targets at the same time. The first domain can recognize CD73 specifically expressed on the surface of tumor cells, while the second domain can bind to PD-L1 molecules on T cells. After the specific binding protein binds to the surface of tumor cells, it can recruit and start tumor cells. nearby T cells, thereby killing tumor cells.

In some embodiments, the prepared second domain and the prepared first domain are constructed into a bispecific binding protein. The bispecific binding protein can bind to two targets simultaneously, in which the first domain can recognize CD73 specifically expressed on the surface of tumor cells, and the second domain can bind to the PD-L1 molecule expressed by the cells. The bispecific binding protein can effectively bind to soluble and cell surface CD73 and PD-L1, inhibit the enzymatic activity of CD73, block the binding of PD-L1 to PD-1, and activate T cells.

Preferably, the bispecific binding protein has a tetravalent symmetrical structure.

Preferably, the bispecific binding protein has the structure and sequence described in the thirteenth aspect.

In a nineteenth aspect, the present invention provides a pharmaceutical composition comprising the specific binding protein described in the thirteenth aspect or the antibody-drug conjugate of the seventeenth aspect, and a pharmaceutically acceptable carrier. .

In some embodiments, the pharmaceutical composition further includes other ingredients as active ingredients, such as other small molecule drugs or antibodies or polypeptides as active ingredients.

In some embodiments, the pharmaceutical composition further comprises a therapeutic agent selected from the group consisting of chemotherapeutic agents, radiotherapeutic agents, immunosuppressive agents, and cytotoxic drugs. In some embodiments, the therapeutic agents of chemotherapeutic agents, radiotherapeutic agents, immunosuppressive agents and cytotoxic drugs of the pharmaceutical composition are different or the same as the drugs in the antibody-drug conjugate of the seventeenth aspect.

The pharmaceutically acceptable carrier can be a conventional carrier in the art, and the carrier can be any suitable physiological or pharmaceutically acceptable pharmaceutical excipient. The pharmaceutical excipients are conventional pharmaceutical excipients in this field, and preferably include pharmaceutically acceptable excipients, fillers or diluents, etc. More preferably, the pharmaceutical composition includes 0.01~99.99% of the specific binding protein and/or other small Molecular drugs, antibodies or polypeptides, and 0.01 to 99.99% of pharmaceutical carriers, the percentages being the quality percentage of the pharmaceutical composition.

The administration route of the pharmaceutical composition may be parenteral, injection or oral administration. The pharmaceutical composition can be prepared in a form suitable for administration, such as a solid, semi-solid or liquid form, an aqueous solution, a non-aqueous solution or a suspension, a powder, a tablet, a capsule, a granule, an injection or an infusion. form. Administration may be via intravascular, subcutaneous, intraperitoneal, intramuscular, inhalation, intranasal, airway instillation or intrathoracic instillation. The pharmaceutical composition may also be administered in the form of an aerosol or spray, for example nasally; alternatively, intrathecally, intramedullary or intraventricularly, and may also be administered transdermally, percutaneously, topically, enterally, intravaginally. , sublingual or rectal administration. The pharmaceutical composition can be made into various dosage forms as needed, and can be administered by a physician based on the patient's type, age, weight, general disease status, administration method and other factors to determine a dose that is beneficial to the patient.

In some embodiments, the first domain and the second domain of the specific binding protein can be administered simultaneously or sequentially.

In some embodiments, the specific binding protein and other active ingredients in the pharmaceutical composition can be administered simultaneously or sequentially.

In some embodiments, the specific binding protein is a bispecific binding protein.

In a twentieth aspect, the present invention provides the specific binding protein described in the thirteenth aspect, the isolated nucleic acid described in the fourteenth aspect, the antibody drug conjugate of the seventeenth aspect, or the drug combination of the nineteenth aspect The application of substances in the preparation of drugs for the prevention, treatment and/or diagnosis of immune diseases, acute and chronic inflammatory diseases, and tumor diseases.

The tumors may be breast cancer, renal cell carcinoma, melanoma, colon cancer, as well as B-cell lymphoma, melanoma, head and neck cancer, bladder cancer, gastric cancer, ovarian cancer, malignant sarcoma, urothelial cancer, liver cancer, esophageal cancer , gastroesophageal junction cancer, nasopharyngeal cancer, small cell lung cancer, cervical cancer, endometrial cancer, pancreatic cancer, prostate cancer, glioma, non-small cell lung cancer, acute myeloid leukemia, Hodgkin lymphoma, cutaneous squamous cell carcinoma Cell carcinoma, locally advanced or metastatic malignant tumors, etc.

The inflammatory disease may be atopic dermatitis, ulcerative colitis, etc.

The immune disease may be graft versus host disease, rheumatoid arthritis, systemic lupus erythematosus, asthma, etc.

In a twenty-first aspect, the present invention provides a method for detecting PD-L1 and CD73 in a sample, which method includes the step of detecting PD-L1 and CD73 in the sample using the specific binding protein of the thirteenth aspect.

The sample may be a biological sample, for example, whole blood, red blood cell concentrate, platelet concentrate, leukocyte concentrate, tissue, bone marrow aspirate, plasma, serum, cerebrospinal fluid, feces, urine, cultured cells, saliva, Oral secretions, nasal secretions and other biological samples.

In a twenty-second aspect, the present invention provides a pharmaceutical kit, which includes one or more pharmaceutical kits, including the antigen-binding protein described in the first aspect, the antibody-drug conjugate of the fifth aspect, or the tenth aspect. The pharmaceutical composition described in the aspect.

In some embodiments, the set of kits includes a first kit and a second kit, and the first kit includes the antigen-binding protein of the first aspect, the antibody-drug conjugate of the fifth aspect, or the second pharmaceutical kit. The pharmaceutical composition of the tenth aspect, and the second kit contains other therapeutic agents, which include, but are not limited to, chemotherapeutic agents, radiotherapeutic agents, immunosuppressive agents and cytotoxic drugs. In some embodiments, the chemotherapeutic agents, radiotherapeutic agents, immunosuppressive agents and cytotoxic drugs in the kit are the same as or different from the antibody drug conjugate of the fifth aspect.

In some embodiments, the first medicine box and the second medicine box can be used at the same time, or the first medicine box can be used first and then the second medicine box, or the second medicine box can be used first and then the third medicine box. A pill box can be determined according to the actual needs of the specific application.

In a twenty-third aspect, the present invention provides a pharmaceutical kit, which includes one or more pharmaceutical kits, including the specific binding protein described in the thirteenth aspect and the antibody drug conjugate of the seventeenth aspect. Or the pharmaceutical composition described in the nineteenth aspect.

In some embodiments, the kit includes a first kit that contains It includes the bispecific binding protein composed of the first structural domain and the second structural domain described in the thirteenth aspect, the antibody drug conjugate of the seventeenth aspect, or the pharmaceutical composition described in the nineteenth aspect.

In some embodiments, the kit may further include a second kit that includes other therapeutic agents including, but not limited to, chemotherapeutic agents, radiotherapeutic agents, immunosuppressive agents, agents and cytotoxic drugs. In some embodiments, the chemotherapeutic agents, radiotherapeutic agents, immunosuppressive agents and cytotoxic drugs in the kit are the same as or different from the antibody drug conjugate of the seventeenth aspect.

In some embodiments, the first medicine box and the second medicine box can be used at the same time, or the first medicine box can be used first and then the second medicine box, or the second medicine box can be used first and then the third medicine box. A pill box can be determined according to the actual needs of the specific application.

In a twenty-fourth aspect, the present invention provides a drug delivery device comprising the antigen-binding protein of the first aspect, the antibody-drug conjugate of the fifth aspect, or the pharmaceutical composition of the tenth aspect. In some embodiments, the drug delivery device is a prefilled syringe.

In a twenty-fifth aspect, the present invention provides a drug delivery device comprising the antigen-binding protein of the thirteenth aspect, the antibody-drug conjugate of the seventeenth aspect, or the pharmaceutical composition of the nineteenth aspect. In some embodiments, the drug delivery device is a prefilled syringe.

In a twenty-sixth aspect, the present invention provides methods for preventing, treating and/or diagnosing immune diseases, acute and chronic inflammatory diseases, and tumor diseases, comprising administering to a subject a therapeutically effective amount of the antigen binding agent of the first aspect. The protein, the antibody-drug conjugate of the fifth aspect, or the pharmaceutical composition of the tenth aspect.

In a twenty-seventh aspect, the present invention provides methods for preventing, treating and/or diagnosing immune diseases, acute and chronic inflammatory diseases, and tumor diseases, comprising administering to a subject a therapeutically effective amount of the antigen of the thirteenth aspect Binding protein, the antibody-drug conjugate of the seventeenth aspect, or the pharmaceutical composition of the nineteenth aspect.

The bispecific binding protein of the present invention has one or more of the following properties: (a) The bispecific binding protein can specifically bind to human PD-L1 and CD73 with high affinity; (b) can inhibit the enzymatic activity of CD73; (c) the bispecific binding protein has an inhibitory effect on the PD-1 signaling pathway; (d) The dual-specific binding protein can enhance the activation of T lymphocytes to secrete the cytokines IL-2 and IFN-γ; and (e) show tumor suppressive activity in vivo.

definition

Unless otherwise defined, all technical and scientific terms used herein have the same meanings as commonly used in the art to which this invention belongs. For the purposes of interpreting this specification, the following definitions will apply and, where appropriate, terms used in the singular will also include the plural and vice versa.

"About" and "approximately" generally mean an acceptable range of error for a measured value given the nature or precision of the measurement. Typically the margin of error is within 20% of the given value or range of values, typically within 10%, and even more typically within 5%.

The term "antigen-binding molecule" or "specific binding protein" generally refers to a molecule that specifically binds to an antigenic determinant. Antigen-binding molecules or specific binding proteins include, for example, antibodies, antibody fragments, and scaffold antigen-binding proteins.

The term "antibody" as used herein encompasses a variety of antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, monospecific and multispecific antibodies (e.g., bispecific or trispecific antibodies), single chain molecules, and Antibody fragments are sufficient as long as they exhibit the required antigen-binding activity.

The term "monoclonal antibody" as used herein refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., except for the possible presence of trace amounts of variant antibodies (e.g. containing naturally occurring mutations or generated during the production of monoclonal antibody preparations). , usually present in small amounts), the individual antibodies included in the antibody population are identical and/or bind to the same epitope. Unlike polyclonal antibody preparations, which typically include different antibodies directed against different antigenic determinants (epitopes), each monoclonal antibody in a monoclonal antibody preparation is directed against a single part of the antigen. determinants.

The term "multispecific antibody" herein is used in its broadest sense to encompass antibodies with multiple epitope specificities. These multispecific antibodies include, but are not limited to: antibodies containing a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH-VL unit has multi-epitope specificity; having two or more Antibodies with VL and VH regions, each VH-VL unit binds to different targets or different epitopes of the same target; antibodies with two or more single variable regions, each single variable region binds to Binding to different targets or different epitopes of the same target; full-length antibodies, antibody fragments, bispecific antibodies (diabodies), triabodies (triabodies), antibody fragments covalently or non-covalently linked together, etc. .

The term "bispecific binding protein" or "bispecific antibody" of the present invention refers to one that can specifically bind at least two different antigenic determinants, for example, each consisting of an antibody heavy chain variable domain (VH) and an antibody light chain. The two binding sites formed by the chain variable domain (VL) bind to different antigens or different epitopes on the same antigen. Bispecific antibodies can be in a 1+1 format, a 2+1 format (comprising two binding sites for a first antigen or epitope and one binding site for a second antigen or epitope) or a 2+2 format (comprising a second binding site for a second antigen or epitope). two binding sites for one antigen or epitope and two binding sites for a second antigen or epitope). Typically, bispecific antibodies contain two antigen-binding sites, each specific for a different antigenic determinant.

The term "valency" in the present invention means that the antigen-binding molecule is present with a specified number of binding domains. Therefore, the terms "bivalent", "tetravalent" and "hexavalent" refer to the presence of two binding domains, four binding domains and six binding domains, respectively, in the antigen-binding molecule. The bispecific antibody is at least "bivalent" and may be "trivalent", "tetravalent" or "more valent"). In some cases, the antibodies have two or more binding sites and are bispecific. That is, an antibody can be bispecific even where more than two binding sites are present (ie, the antibody is trivalent or multivalent).

The terms "full-length antibody" and "intact antibody" of the present invention are used interchangeably herein to refer to antibodies that are structurally substantially similar to native antibodies. "Native antibodies" refer to naturally occurring immunoglobulin molecules. For example, natural IgG class antibodies are heterotetrameric glycoproteins of approximately 150,000 daltons, consisting of two light and two heavy chains bonded by disulfide bonds. From N-terminus to C-terminus, each heavy chain has a variable region (VH) (also called a variable heavy chain domain or a heavy chain variable domain) and three constant domains (CH1, CH2, and CH3) ( Also called heavy chain constant region). From the N-terminus to the C-terminus, each light chain has a variable region (VL) (also called a variable light domain or a light chain variable domain) and a light chain constant domain (CL) (also called a light chain domain). chain constant region). The heavy chain of an antibody can be one of five types, alpha (IgA), delta (IgD), epsilon (IgE), gamma (IgG), or mu (IgM), and can be further divided into subtypes. Types, such as γ1 (IgG1), γ2 (IgG2), γ3 (IgG3), γ4 (IgG4), α 1 (IgA1) and α 2 (IgA2). The light chain of an antibody can be one of two types, based on the amino acid sequence of its constant domain, namely kappa light chain and lambda light chain.

Within the light and heavy chains, the variable and constant regions are connected by a "J" region of approximately 12 or more amino acids, and the heavy chain also contains a "D" region of approximately 3 or more amino acids. . Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region consists of 3 domains (CH1, CH2 and CH3). Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL). The light chain constant region consists of one domain, CL. The constant region of an antibody may mediate binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (eg, effector cells) and the first component (Clq) of the classical complement system.

"Antibody fragment" includes a portion of an intact antibody. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab') _2, and Fv; diabodies, tribodies, tetrabodies, crossed Fab fragments; linear antibodies; single chain antibody molecules (e.g., scFv); Multispecific antibodies formed from antibody fragments and single domain antibodies (single domain antibodies).

The term "antigen-binding domain" or "antigen-binding site" of the present invention refers to the portion of an antigen-binding molecule that specifically binds to an antigenic determinant. More specifically, the term "antigen-binding domain" refers to that portion of an antibody that contains a region that specifically binds and is complementary to part or all of the antigen. Where the antigen molecule is large, the antigen-binding molecule can bind only to a specific part of the antigen, called an epitope. The antigen binding domain may be provided, for example, by one or more variable domains (also called variable regions). Preferably, the antigen-binding domain includes an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH). In one aspect, the antigen binding domain is capable of binding its antigen and blocking or partially blocking the function of said antigen.

The term "antigenic determinant" of the present invention is synonymous with "antigen" and "epitope" and refers to a site on a polypeptide macromolecule (such as a stretch of continuous amino acids or different regions composed of non-continuous amino acids). Conformational configuration), the antigen-binding moiety binds to the site, thereby forming an antigen-binding moiety-antigen complex. Antigenic determinants may be present, for example, on the surface of tumor cells, on the surface of microbial-infected cells, on the surface of other diseased cells, on the surface of immune cells, in free substances in serum and/or in the extracellular matrix (ECM). Unless otherwise stated, the protein used as an antigen in the present invention may be any native form of the protein from any vertebrate source, including animals such as primates (e.g., humans) and rodents (e.g., mice and rats) and other mammals. The antigen may also be a human protein, or the antigen may be a "full-length," unprocessed protein, any form of protein resulting from intracellular processing, or a naturally occurring protein variant, such as a splice variant or an allelic variant. body.

1μM、

100nM、

10nM、

1nM、

0.1nM、

0.01nM或

0.001nM(例如10^-7M或更低，例如10^-7M至10^-13M，例如10^-9M至10^-13M)。 "Specifically binds" means that binding is selective for an antigen and can be distinguished from undesired or non-specific binding. The ability of an antigen-binding molecule to bind to a specific antigen can be measured by enzyme-linked immunosorbent assay (ELISA) or other techniques familiar to those skilled in the art, such as surface plasmon resonance (SPR) technology as well as traditional binding assays. In one embodiment in which the antigen-binding molecule binds to the unrelated protein to a degree that is less than about 10% of the degree of binding of the antigen-binding molecule to the antigen, such as as measured by SPR. In certain embodiments, dissociation of the molecule bound to the antigen The constant (Kd) is

1μM,

100nM,

10nM,

1nM,

0.1nM,

0.01nM or

0.001 nM (eg 10 ^-7 M or less, eg 10 ^-7 M to 10 ^-13 M, eg 10 ^-9 M to 10 ^-13 M).

"Affinity" or "binding affinity" refers to the strength of the non-covalent interaction between a single binding site of a molecule (eg, an antibody) and its binding partner (eg, an antigen). Binding affinity can often be expressed in terms of the dissociation constant (Kd), which is the ratio of the dissociation rate constant to the association rate constant (K _off and K _on , respectively). Therefore, equivalent affinities can include different rate constants as long as the ratio of the rate constants remains the same. Affinity can be measured by conventional methods known in the art, such as surface plasmon resonance (SPR).

The term "high affinity" in the present invention means that the Kd of the antibody to the target antigen is ^10-9 M or lower, or even ^10-10 M or lower. The term "low affinity" of the present invention refers to an antibody with a Kd of 10 ^-8 M or higher.

An "affinity matured" antibody refers to an antibody that has one or more modifications in one or more hypervariable regions (HVRs) that results in a greater affinity for the antibody compared to the parent antibody without such modifications. Improvement of antigen affinity.

The terms "single domain antibody" and "nanobody" of the present invention have the same meaning and refer to having only the variable region of the heavy chain of the antibody. The construction of a single domain antibody consisting of only one heavy chain variable region has a complete The smallest functional antigen-binding fragment.

The term "heavy chain antibody" of the present invention, also known as HCAb antibody, refers to an antibody that lacks the antibody light chain and only contains a heavy chain relative to a diabody (immunoglobulin), specifically a heavy chain variable structure. domain and Fc constant domain.

The specific binding protein includes a first domain that binds CD73 or a fragment thereof and a second domain that binds PD-L1 or a fragment thereof.

The terms "bispecific antibody comprising a first domain that specifically binds CD73 and a second domain that specifically binds PD-L1" "bispecific antibody that specifically binds CD73 and PD-L1" "specific for CD73 and Bispecific antigen-binding molecules for PD-L1" or "anti-CD73/anti-PD-L1 antibodies" are used interchangeably herein and refer to the ability of the antibody to bind CD73 and PD-L1 with sufficient affinity such that the antibody can be used as Bispecific antibodies for diagnostic and/or therapeutic agents targeting CD73 and PD-L1.

The term "T effector cells" of the present invention refers to T cells with cytolytic activity (for example, CD4+ and CD8+ T cells) and T helper (Th) cells. T effector cells secrete cytokines and activate and guide other immune cells. But it does not include regulatory T cells (Treg cells).

The term "regulatory T cells" or "Treg cells" in the present invention refers to a special type of CD4+ T cells that can block the responses of other T cells. Treg cells are characterized by expression of CD4, the alpha subunit of the IL-2 receptor (CD25), and the transcription factor FOXP3, and play a crucial role in the induction and maintenance of peripheral autologous tolerance to tumor-expressed antigens. .

The term "CD73", cluster of differentiation 73, also known as 5'-exonucleotidase, Usually refers to the enzyme (nucleotidase) that can convert extracellular nucleoside 5' monophosphate into nucleosides, that is, adenosine monophosphate (AMP) into adenosine. CD73 is a cell surface enzyme linked by glycosylphosphoinositol (GPI). CD73 is widely expressed on the surface of human endothelial cells, lymphocytes, such as Treg, DC, MDSC, NK and other cells. CD73 can also be expressed on cancer cells, including colon cancer, lung cancer, pancreatic cancer, ovarian cancer, bladder cancer, leukemia, glioma, glioblastoma, melanoma, thyroid cancer, esophageal cancer, prostate cancer, and breast cancer. High CD73 expression has been reported to be associated with poor prognosis in multiple cancer indications (eg, lung cancer, melanoma, breast cancer, squamous head and neck cancer, and colorectal cancer). The main function of CD73 is to convert extracellular nucleotides (such as 5'-AMP) into the highly immunosuppressive molecule adenosine. The term "CD73" as used herein includes any variant or isoform of CD73 naturally expressed by a cell. CD73, or any variants and isoforms thereof, can be isolated from the cells or tissues in which they are naturally expressed, or can be produced recombinantly using techniques well known in the art and/or described herein. Accordingly, the antigen-binding proteins described herein may cross-react with species other than humans (eg, cynomolgus CD73). Alternatively, the CD73-directed antigen-binding proteins, antibodies or domains of the invention may be specific for human CD73 and may not exhibit any cross-reactivity with other species.

1μM，

100nM，

10nM，

1nM，

0.1nM，

0.01nM，或

0.001nM(例如10^-8M或更低，例如10^-13M到10^-8M，例如10^-13M到10^-9M)的解離常數(KD)。 The term "CD73 antibody" or "isolated antigen-binding protein that specifically binds CD73 and/or fragments thereof" of the present invention is capable of binding to CD73 with sufficient affinity such that the antibody can be used as a diagnostic and/or CD73-targeting agent. or therapeutic agents. Typically, CD73 antibodies have less ability to bind to unrelated CD73 proteins than to CD73, as determined by radioimmunoassay (RIA) or flow cytometry (FACS) or by surface plasmon resonance using a biosensor system. about 10% of the binding capacity. In certain embodiments, an antibody that binds CD73 has

1μM,

100nM,

10nM,

1nM,

0.1nM,

0.01nM, or

A dissociation constant (KD) of 0.001 nM (eg 10 ^-8 M or less, eg 10 ^-13 M to 10 ^-8 M, eg 10 ^-13 M to 10 ^-9 M).

The term programmed cell death 1 ligand 1 (PD-L1), also known as cluster of differentiation CD274 or B7 homolog 1 (B7-H1), is a member of the B7 family that regulates activation or inhibition of the PD-1 receptor. a member of. The open reading frame of PD-L1 encodes a 290-amino acid type I transmembrane protein, which includes an extracellular Ig domain. (N-terminal V-like domain and IgC-like domain), a hydrophobic transmembrane domain and a 30-amino acid cytoplasmic tail. The 30-amino acid intracellular (cytoplasmic) domain contains no obvious signaling but does have potential sites for protein kinase C phosphorylation. Studies have shown that the expression of PD-L1 on immune cells is enhanced under the stimulation of interferon gamma. PD-L1 is also expressed on non-immune cells, including pancreatic islets, Kupffer cells of the liver, vascular endothelium, and selected epithelia, such as tracheal epithelium and renal tubular epithelium, where its expression is enhanced during inflammatory episodes. Increased levels of PD-L1 expression have also been found on a variety of tumors, including but not limited to breast, ovarian, cervical, colon, colorectal, lung including non-small cell lung cancer, including renal cell carcinoma Cancers include kidney cancer, stomach cancer, esophageal cancer, bladder cancer, hepatocellular carcinoma, squamous cell carcinoma of the head and neck (SCCHN) and pancreatic cancer, melanoma and uveal melanoma.

1μM、

100nM、

10nM、

1nM、

0.1nM、

0.01nM或

0.001nM(例如10^-8M或更低，例如10^-13M至10^-8M，例如10^-13M至10^-9M)。在一些實施方案中，在表面電漿共振測定中，使用人PD-L1，尤其是帶有組胺酸標籤的人PD-L1測定結合親和力的相應KD值，以獲得PD-L1結合親和力。PD-1/PD-L1抗體的治療策略是幾種轉移性腫瘤中的標準治療策略，並已顯示出它們在早期疾病階段和輔助治療中的作用，尤其是黑色素瘤和非小細胞肺癌。 The term "PD-L1 antibody" or "isolated antigen-binding protein that specifically binds PD-L1 and/or fragments thereof" of the present invention is capable of binding PD-L1, especially PD-L1 polypeptide expressed on the cell surface, and Have sufficient affinity such that the antibody can be used as a diagnostic and/or therapeutic agent targeting PD-L1. Typically, PD-L1 antibodies have less than About 10% of the antibody's binding ability to PD-L1. In certain embodiments, the KD value of the antigen-binding protein that binds human PD-L1

1μM,

100nM,

10nM,

1nM,

0.1nM,

0.01nM or

0.001 nM (eg 10 ^-8 M or less, eg 10 ^-13 M to 10 ^-8 M, eg 10 ^-13 M to 10 ^-9 M). In some embodiments, the corresponding KD value of the binding affinity is determined using human PD-L1, especially human PD-L1 with a histidine tag, in a surface plasmon resonance assay to obtain the PD-L1 binding affinity. Therapeutic strategies with PD-1/PD-L1 antibodies are standard treatment strategies in several metastatic tumors and have shown their role in early disease stages and in adjuvant therapy, particularly in melanoma and non-small cell lung cancer.

The term "H2L2 transgenic mouse" or "Harbour H2L2 mouse" in the present invention is a transgenic mouse carrying a human immunoglobulin immune repertoire that produces a fully human variable region consisting of two heavy chains and A traditional tetrameric antibody composed of two light chains (H2L2), with a complete human antibody variable domain and a rat constant domain. The antibodies produced by the transgenic mice are affinity matured and the variable regions Fully humanized and has excellent solubility.

The term "Harbour HCAb mouse" (WO2002/085945A2) in the present invention is a transgenic mouse carrying a human immunoglobulin immune repertoire that is capable of producing a new "heavy chain" only antibody that is only the size of a traditional IgG antibody. half. The antibodies produced have only the human antibody "heavy chain" variable domain and the mouse Fc constant domain. Due to the fact that it does not contain a light chain, the antibody almost solves the problems of light chain mismatching and heterodimerization, allowing this technology platform to develop products that are difficult to achieve with traditional antibody platforms.

The term "variable region" or "variable domain" of the present invention refers to the domain of an antibody heavy chain or light chain that participates in the binding of an antigen-binding molecule to an antigen. The variable domains of the heavy and light chains of natural antibodies (VH and VL, respectively) generally have similar structures, with each domain containing four conserved framework regions (FR) and three hypervariable regions (HVR). A single VH or VL domain may be sufficient to confer antigen binding specificity.

The term "variable" as used herein means that certain segments of the variable domains generally differ in sequence between antibodies. The V domain mediates antigen binding and defines the specificity of a particular antibody for its particular antigen. However, variability is not evenly distributed throughout the variability domain. Instead, it is concentrated in three segments called hypervariable regions (HVR) within the light and heavy chain variable domains. The more highly conserved portion of the variable domain is called the framework region (FR). The variable domains of native heavy and light chains each contain four FR regions, mostly in a β-sheet configuration, connected by three HVRs, which form loop connections and in some cases form part of the β-sheet structure. The HVRs in each chain are held tightly together by the FR region and, together with the HVRs of other chains, contribute to the formation of the antibody's antigen-binding site (see Kabat et al., Sequences of Immunological Interest, 5th ed., National Institute of Health, Bethesda , MD (1991)). The constant domain is not directly involved in the binding of the antibody to the antigen and has other effector functions, such as participating in the antibody-dependent cellular cytotoxicity of the antibody.

The term "hypervariable region" or "HVR" of the present invention refers to a region within an antibody variable domain region that is hypervariable in sequence and/or forms a structurally defined loop ("hypervariable loop"). Typically, natural four-chain antibodies contain six HVRs: three in VH (H1, H2, H3) and three in VL (L1, L2, L3). HVRs typically contain amino acid residues from hypervariable loops and/or from "complementarity determining regions (CDRs)", derived from "interactions" The amino acid residues of the "complementary determining region (CDR)" have the highest sequence variability and/or are involved in antigen recognition. Exemplary CDRs (CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2 and CDR-H3) occurs at amino acid residues 26-32(L1), 50-52(L2), 91-96(L3), 26-32(H1), 53-55(H2) and 96-101( H3) (Chothia et al., J. Mol. Biol. 196:901-917 (1987). Exemplary CDRs (CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3 ) occurs at amino acid residues 24-34 (L1), amino acid residues 50-56 (L2), amino acid residues 89-97 (L3), and amino acid residues 31-35 (H1) , amino acid residues 50-65 (H2), and amino acid residues 95-102 (H3) (Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health , Bethesda, MD (1991). For comparison, the corresponding amino acid residues containing the CDRs defined in the references cited above are listed in Table 1 below. In the present invention, the amine groups of the CDRs listed above The acid sequences are all shown according to the Chothia definition rules (the claims of the present invention are also the sequences shown according to the Chothia definition rules). However, it is well known to those in the art that antibodies can be defined in a variety of ways in the art CDR, for example, Kabat definition rules based on sequence variability and Chothia definition rules based on the position of the structural loop region (see J Mol Biol 273:927-48, 1997). In the present invention, it is also possible to use the Kabat definition and Chothia definition The Combined definition rule determines the amino acid residues in the variable domain sequence. The Combined definition rule combines the ranges defined by Kabat and Chothia, and takes a larger range based on this. Those skilled in the art should understand It is understood that, unless otherwise specified, the terms "CDR" and "complementarity determining region" of a given antibody or region thereof (e.g., variable region) shall be understood to encompass any of the above-described known schemes as described by the present invention. Defined complementarity determining region. Although the scope of protection claimed in the present invention is the sequence shown based on the Chothia definition rules, the corresponding amino acid sequences according to other CDR definition rules should also fall within the protection scope of the present invention.

"Framework" or "FR" refers to the variable domain residues other than the hypervariable region (HVR) residues. The FR of the variable domain usually consists of the following four FR domains: FR1, FR2, FR3 and FR4. Therefore, HVR and FR sequences usually appear in VH (or VL) as the following sequence: FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

The "class" of an antibody refers to the type of constant domain or constant region that the heavy chain of the antibody has. There are five classes of antibodies: IgA, IgD, IgE, IgG and IgM, and several of these classes can be further divided into subclasses (isotypes), such as IgGl, IgG2, IgG3, IgG4, IgA1 and IgA2. The heavy chain constant domains corresponding to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively.

"Humanized antibodies" comprise amino acid residues from a non-human HVR and amino acid residues from a human FR. In certain embodiments, a humanized antibody comprises at least one, and typically two, variable domains, wherein all or substantially all of the HVRs (e.g., CDRs) correspond to the HVRs of the non-human antibody, and all or substantially all of the FRs Corresponds to the FR of human antibodies. A humanized antibody optionally can comprise at least a portion of an antibody constant region derived from a human antibody. A "humanized form" of an antibody, such as a non-human antibody, is an antibody that has undergone humanization.

A "human antibody" has an amino acid sequence that corresponds to the amino acid sequence of an antibody produced by humans or human cells or derived from non-human sources utilizing human antibody libraries or other human antibody coding sequences. This definition of human antibodies specifically excludes humanized antibodies containing non-human antigen-binding residues.

The term "Fc domain" or "Fc region" of the present invention is used to define the C-terminal region of an antibody heavy chain containing at least a portion of the constant region. The term includes native sequence Fc regions and variant Fc regions. The IgG Fc region contains the IgG CH2 domain and the IgG CH3 domain. The CH2 domains herein may be native sequence CH2 domains or variant CH2 domains. The CH3 region herein may be a native sequence CH3 domain or a variant CH3 domain. The CH2 domain may comprise one or more mutations that reduce or eliminate binding of the CH2 domain to one or more Fcγ receptors (eg, FcγRI, FcγRIIa, FcγRIIb, FcγRIII) and/or complement. It is hypothesized that reducing or eliminating binding to Fc receptor gamma will reduce or eliminate ADCC mediated by antibody molecules. Similarly, reducing or eliminating complement binding would be expected to reduce or eliminate CDC mediated by antibody molecules. Reduce or eliminate the CH2 domain Mutations that bind one or more Fcγ receptors and/or complement are known in the art (Wang et al., 2018). These mutations include the so-called "LALA mutations" which involve the replacement of leucine residues at positions 1.3 and 1.2 of the IMGT of the CH2 domain with alanine (L1.3A and L1.2A). Alternatively, by changing the CH2 structure The asparagine (N) at position 84.4 of the IMGT domain in the domain is mutated to alanine, glycine, or glutamine (N84.4A, N84.4G, or N84.4Q), thus converting the conserved N-chain sugar Mutation of the sylation site to generate α-glycosyl antibodies to reduce IgG1 effector function is also known (Wang et al., 2018). As an alternative, it is known that complement initiation (C1q binding) and ADCC can be induced by combining the CH2 domain The IMGT position 114 is reduced by mutating proline to alanine or glycine (P114A or P114G) (Idusogie et al., 2000; Klein et al., 2016). These mutations can be combined to produce products with further reduction or no ADCC or CDC-active antibody molecules.

"Region equivalent to the Fc region of an immunoglobulin" includes variants of naturally occurring alleles of the Fc region of an immunoglobulin, as well as variants that produce substitutions, additions, or deletions that do not substantially reduce the immunoglobulin-mediated effector function (such as antibody-dependent cytotoxicity). For example, one or more amino acids can be deleted from the N-terminus or C-terminus of the Fc region of an immunoglobulin without substantial loss of biological function. Such variants may be selected so as to have minimal impact on activity according to general rules known in the art (see, eg, Bowie, J.U. et al., Science 247:1306-10 (1990)).

The term "effector function" of the present invention is attributable to the Fc region of an antibody and is a biological activity that changes with the isotype of the antibody. Examples of antibody effector functions include: C1q binding and complement-dependent cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), cytokines Secretion, immune complex-mediated antigen uptake by antigen-presenting cells, downregulation of cell surface receptors (such as B cell receptors), and B cell priming.

The term "peptide linker" or "linking peptide" of the present invention refers to a peptide containing one or more amino acids, usually about 2 to 20 amino acids. Peptide linkers are known in the art or described herein.

The term "fused to", "fusion linked" or "linked to" in the present invention means that the segments (eg antigen binding domain and FC domain) are linked either directly or via one or more peptide linkers by peptide bonds.

The invention also relates to amino acid sequence variants of the bispecific antibodies of the invention. bispecific Amino acid sequence variants of antibodies can be prepared by introducing appropriate modifications into the nucleotide sequence of the encoding molecule or by peptide synthesis. Such modifications include, for example, deletions, insertions and/or substitutions of residues in the antibody amino acid sequence. Any combination of deletions, insertions and substitutions can be made to obtain a final construct having the desired properties, such as antigen-binding activity. Sites used for substitution typically include HVR and framework (FR). See table 2 below for possible substitutions of amino acids.

The term "polynucleotide" or "nucleic acid" or "nucleotide sequence" of the present invention refers to an isolated nucleic acid molecule or construct, such as messenger RNA (mRNA), virus-derived RNA or plasmid DNA (pDNA). Polynucleotides may contain conventional phosphodiester linkages or unconventional linkages (eg, amide linkages, such as found in peptide nucleic acids (PNA)). The term "nucleic acid molecule" refers to any one or more nucleic acid segments present in a polynucleotide, such as a DNA or RNA segment.

The term "isolated" nucleic acid molecule or polynucleotide as used herein refers to a nucleic acid molecule, DNA or RNA, that has been separated from its natural environment. In the present invention, the heavy protein encoding the polypeptide contained in the vector Groups of polynucleotides are also isolated. Other examples of isolated polynucleotides include recombinant polynucleotides in heterologous host cells or polynucleotides purified in solution. Isolated polynucleotides include polynucleotide molecules that are normally present in the cell containing the polynucleotide molecule, but which are present extrachromosomally or in a chromosomal location that is different from its native chromosomal location. Isolated RNA molecules include in vivo or in vitro RNA transcripts of the present invention, in positive and negative stranded forms, and in double-stranded form. The isolated polynucleotides or nucleic acids of the invention further include synthetically produced such molecules. Additionally, a polynucleotide or nucleic acid may be or may include regulatory elements, such as a promoter, a ribosome binding site, or a transcription terminator.

The term "expression cassette" as used herein refers to a recombinantly or synthetically produced polynucleotide having a series of nucleic acid elements that permit the transcription of a specific nucleic acid in a target cell. Recombinant expression cassettes can be introduced into plasmids, chromosomes, mitochondrial DNA, plastid DNA, viruses or nucleic acid fragments. Typically, the recombinant expression cassette portion of the expression vector includes, among other sequences, the nucleic acid sequence to be transcribed and a promoter. In certain embodiments, expression cassettes of the invention comprise polynucleotide sequences encoding bispecific antigen-binding molecules of the invention, or fragments thereof.

The terms "vector" or "expression vector" and "expression construct" of the present invention are used interchangeably, and are DNA molecules to which a specific gene operably linked is introduced into a target cell and directs expression. Such vectors include vectors that are self-replicating nucleic acid structures as well as vectors that are incorporated into the genome of the host cell into which they have been introduced. The expression vector of the present invention contains an expression cassette. Expression vectors enable the transcription of large amounts of stable mRNA. Once the expression vector is within the target cell, the ribonucleic acid molecule or protein encoded by the gene is produced by the cellular transcription and/or translation machinery. In one embodiment, the expression vector of the invention includes an expression cassette containing a polynucleotide sequence encoding a bispecific antigen-binding molecule of the invention or a fragment thereof.

The terms "host cell", "host cell line" and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, and also include the progeny of such cells. Host cells include "transformants/transformants" and "transformed cells", including primary transformed cells and progeny derived therefrom. The nucleic acid of the offspring may not be completely identical to that of the parent cell and may contain mutations. A host cell is any type of cell that can be used to produce the bispecific antigen-binding molecules of the invention. Host cells include cultured cells, e.g. Cultured mammalian cells, such as CHO cells, HEK293 cells, BHK cells, NS0 cells, SP2/0 cells, YO myeloma cells, P3X63 mouse myeloma cells, PER cells, PER.C6 cells or hybridoma cells, yeast cells , insect cells and plant cells, also include cells contained within transgenic animals, transgenic plants or cultured plant or animal tissues.

Germline backmutation refers to the process of backmutating the supersomatic mutation of an antibody to the corresponding germline sequence. The heavy chain variable domain sequence of the antibody is derived from events such as gene rearrangements and somatic high-frequency mutations of the germline gene V, D, and J gene segments of the heavy chain gene group on the chromosome; the light chain variable domain sequence is derived from Events such as gene rearrangement of germline gene V and J gene segments of the light chain gene group and somatic high-frequency mutations. Gene rearrangements and high-frequency somatic mutations are major factors that increase antibody diversity. Antibodies derived from the same germline V gene fragment may also produce different sequences, but overall the similarity is high. Use some algorithms, such as IMGT/DomainGapAlign (http://imgt.org/3Dstructure-DB/cgi/DomainGapAlign.cgi) or NCBI/IgBLAST (https://www.ncbi.nlm.nih.gov/igblast/) , the possible germline gene fragments when gene rearrangement occurs can be inferred from the variable domain sequence of the antibody.

The term "post-translational modification (PTM)" refers to the process of introducing chemical modifications into protein or polypeptide amino acid chains after they are translated and synthesized in cells. For antibodies, some PTM sites are very conserved. For example, the conserved amino acid asparagine Asn at position 297 (EU numbering) of the constant domain of human IgG1 antibodies usually undergoes glycosylation. Modifications form glycans whose structure is critical to antibody structure and associated effector functions. However, if there are PTMs in the variable domain of the antibody, especially the antigen-binding region (such as CDR), then the presence of these PTMs may have a greater impact on the binding of the antigen, and may also affect the physical and chemical properties of the antibody. to change. For example, glycosylation, deamidation, isomerization, oxidation, etc. may increase the instability or heterogeneity of antibody molecules, thereby increasing the difficulty and risk of antibody development. Therefore, the process of removing PTMs to reduce sequence instability and thereby avoid some potential PTMs is very important for the development of therapeutic antibodies. With the accumulation of experience, people have discovered that some PTMs are highly correlated with the composition of amino acid sequences, especially with the "pattern" of adjacent amino acid compositions. This allows prediction of potential PTMs from the primary amino acid sequence of a protein. For example, the sequence pattern N-x-S/T (the first position is asparagine, the second position is any amino acid except proline, and the third position is serine or threonine) predicts N-linked sugars base site. The amino acid sequence pattern that causes PTM may originate from germline gene sequences. For example, the human germline gene fragment IGHV3-33 naturally has a glycosylation pattern NST in the FR3 region; it may also originate from somatic high-frequency mutations. The amino acid sequence pattern of PTMs can be disrupted through amino acid mutation, thereby reducing or eliminating the formation of specific PTMs. Depending on the antibody sequence and PTM sequence pattern, there are different mutation design methods. One approach is to replace "hotspot" amino acids (such as N or S in the NS pattern) with amino acids with similar physical and chemical properties (such as mutating N to Q). If the PTM sequence pattern is derived from somatic high-frequency mutations and does not exist in the germline gene sequence, then another approach can be to replace the sequence pattern with the corresponding germline gene sequence. In actual operation, multiple mutation design methods may be used for the same PTM sequence pattern.

The term "antibody drug conjugate" or "ADC" refers to a binding protein (such as an antibody or antigen-binding fragment thereof) chemically linked to one or more chemical drugs. In preferred embodiments, the ADC includes a binding protein, a drug, and a linker connecting the binding protein to the drug.

The term "chimeric antigen receptor" or "CAR" refers to a receptor with a desired antigen specificity and signaling domains to propagate an intracellular signal upon antigen binding. For example, T lymphocytes recognize specific antigens via the interaction of the T cell receptor (TCR) with short peptides presented by class I or class II major histocompatibility complex (MHC) molecules. For initial activation and selective expansion, naïve T cells rely on antigen-presenting cells (APCs) that provide additional costimulatory signals. In some embodiments, monocytes and macrophages can be engineered, for example, to express chimeric antigen receptors (CARs). Modified cells can be recruited to the tumor microenvironment where they serve as potent immune effectors by infiltrating tumors and killing target cancer cells. A CAR may include an antigen-binding domain, a transmembrane domain, and an intracellular domain. The antigen binding domain binds to the antigen on the target cell. Examples of cell surface markers that can be used as antigens for binding to the antigen-binding domain of a CAR include those associated with viruses, bacteria, parasitic infections, autoimmune diseases, and cancer cells (eg, tumor antigens).

The term "modified immune cells" refers to immune cells that have been genetically modified to express a CAR. In some embodiments, the immune cells are T cells, or cells derived therefrom. In some embodiments, the immune cells are natural killer (NK) cells, or cells derived therefrom. In some embodiments, the immune cell is a B cell, or a cell derived therefrom. In some embodiments, the immune cell is a B cell, or a cell derived therefrom. In some embodiments, the immune cells are monocytes or macrophages, or cells derived therefrom.

The term "kit" refers to a combination of one or more active ingredients present in more than one unit. When multiple active ingredients are present in the kit, each active ingredient can be administered simultaneously or sequentially.

The term "delivery device" refers to any means used to administer medication to a subject.

The term "prefilled syringe" refers to a syringe that has been loaded with medication prior to access or use by the operator of the syringe. Prefilled syringes can be of any material (eg, glass, plastic, or metal). In some embodiments, the prefilled syringe is a glass syringe.

An "effective amount" of a drug is that amount necessary to produce physiological changes in the cells or tissues to which it is administered. An "effective amount" includes an amount sufficient to ameliorate or prevent symptoms or conditions of a medical disease. An effective amount also means an amount sufficient to allow or facilitate diagnosis. The effective amount for a particular patient or veterinary subject may vary depending on factors such as, for example, the condition being treated, the general health of the patient, the method, route and dosage of administration, and the severity of the side effects. An effective amount may be the maximum dosage or dosage regimen that avoids significant side effects or toxic effects.

The bispecific antibodies according to the invention have synergistic effects. "Synergistic effect" means that the combined effect of two drugs is greater than the sum of their individual effects and is statistically different from the control and the single drugs. Additive effect in the present invention means that the combined effect of two drugs is the sum of their individual effects and is statistically different from the control and/or single drug.

A "therapeutically effective amount" of a drug (eg, a pharmaceutical composition) is that amount, in dosage and administration interval and time, that is effective in achieving the desired therapeutic or preventive effect. For example, a therapeutically effective amount of a drug eliminates, alleviates/reduces, delays, minimizes, or prevents the adverse effects of a disease.

The term "individual" or "subject" refers to a mammal. Mammals include, but are not limited to, domesticated animals (e.g., cattle, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates, such as monkeys), rabbits, and rodents (e.g., mice and rats). In particular, the individual or subject is a human being.

The term "pharmaceutical composition" refers to a mixture containing one or more antibodies or antigen-binding fragments thereof of the present disclosure together with other chemical components, such as physiologically/pharmaceutically acceptable carriers or excipients. The purpose of pharmaceutical compositions is to facilitate administration to living organisms and facilitate the absorption of active ingredients to exert biological activity.

The term "pharmaceutically acceptable carrier" refers to a component of a pharmaceutical composition, other than the active ingredient, that is non-toxic to the subject. Pharmaceutically acceptable excipients include, but are not limited to, buffers, stabilizers and/or preservatives.

The term "cancer" is used to describe a disorder in mammals characterized by unregulated cell growth. Examples of cancers include, but are not limited to, tumors, lymphomas, blastomas, sarcomas, and leukemias or lymphoid malignancies. More specific examples of cancer include, but are not limited to, squamous cell carcinoma (eg, epithelial squamous cell carcinoma), lung cancer (including small cell lung cancer, non-small cell lung cancer, adenocarcinoma, and squamous cell carcinoma of the lung), peritoneal cancer, hepatocellular carcinoma, Gastric cancer (including gastrointestinal cancer and gastrointestinal stromal cancer), bone cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, urethra cancer, breast cancer, colon cancer, rectal cancer, colorectal cancer, Endometrial or cervical cancer, salivary gland cancer, kidney or ureter cancer, prostate cancer, vaginal cancer, vulvar cancer, thyroid cancer, anal cancer, penile cancer, melanoma, cholangiocarcinoma, central nervous system (CNS) tumors , spinal axial tumors, brainstem glioma, glioblastoma multiforme, astrocytoma, schwannoma, ependymoma, medulloblastoma, meningioma, squamous cell carcinoma, pituitary adenoma and Ewing sarcoma, superficial spreading melanoma, lentigo maligna melanoma, acral melanoma, nodular melanoma, multiple myeloma and B-cell lymphoma, chronic lymphocytic leukemia (CLL), acute Lymphoblastic leukemia (ALL), hairy cell leukemia, chronic myeloblastic leukemia and post-transplant lymphoproliferative disorder (PTLD), as well as phakomatoses, edema (such as those associated with brain tumors) and Abnormal blood vessel proliferation, brain tumors and cancers associated with Meigs syndrome, and head or neck cancers and related metastases.

The term "treatment" refers to the administration of an internal or external therapeutic agent, such as a composition comprising any of the antibodies or antigen-binding fragments thereof of the present disclosure or a nucleic acid molecule encoding an antibody or antigen-binding fragment thereof, to a patient who has one or more Diseases or symptoms for which the therapeutic agent has a therapeutic effect. Generally, a therapeutic agent is administered to a subject or population in an amount effective to alleviate one or more diseases or symptoms, to induce regression of such symptoms or to inhibit the progression of such symptoms to any clinically measurable extent.

The term "preventing cancer" refers to delaying, inhibiting or preventing the onset of cancer in a mammal in which the initiation of carcinogenesis or tumorigenesis has not been proven, but has been identified, for example by genetic screening or other methods. Have cancer susceptibility. The term also includes treatment of a mammal with a precancerous condition to terminate the progression of the precancerous condition to malignancy or to cause regression thereof.

The sequence number (SEQ ID NO:) of the antibody of the present invention is shown in Table 3 below.

The sequence number (SEQ ID NO:) of the bisantibody of the present invention is shown in Table 4 below.

Figure 1 shows the results of the antigen-binding protein of the present invention binding to CHO-K1 cells overexpressing human PD-L1. Among them, A and B show the results of the PD-L1 antigen-binding protein of the present invention binding to CHO-K1 cells overexpressing human PD-L1; C shows the PR000960 post-translational modification (PTM) variant of the present invention binding to overexpressing human PD. The results of -L1 CHO-K1 cells; D, E, and F show the results of binding the affinity mature variant of PR002082 of the present invention to CHO-K1 cells overexpressing human PD-L1.

Figure 2 shows the results of the antigen-binding protein of the present invention binding to CHO-K1 cells overexpressing cynomolgus monkey PD-L1. Among them, A shows the results of the PD-L1 antigen-binding protein of the present invention binding to CHO-K1 cells overexpressing cynomolgus monkey PD-L1; B shows the binding of the PR000960 post-translational modification (PTM) variant of the present invention to overexpressing cynomolgus monkeys. The results of CHO-K1 cells expressing monkey PD-L1; C shows the results of binding the affinity mature variant of PR002082 of the present invention to CHO-K1 cells overexpressing monkey PD-L1.

Figure 3 shows the inhibitory effect of the antigen-binding protein of the present invention on the PD-1 signaling pathway. Among them, A shows the inhibitory effect of the PD-L1 antigen-binding protein of the present invention on the PD-1 signaling pathway; B shows the inhibitory effect of the PR000960 post-translational modification (PTM) variant of the present invention on the PD-1 signaling pathway; C, D shows the inhibitory effect of the affinity mature variant of PR002082 of the present invention on the PD-1 signaling pathway.

Figure 4 shows the effect of the antigen-binding protein of the present invention on cytokine secretion by T lymphocytes in the MLR experiment of donor pair #1. Among them, A shows the result that the PD-L1 antigen-binding protein of the present invention enhances the activation of T lymphocytes to secrete the cytokine IL-2; B shows that the PD-L1 antigen-binding protein of the present invention enhances the activation of T lymphocytes to secrete the cytokine IFN-γ. result.

Figure 5 shows the effect of the antigen-binding protein of the present invention on cytokine secretion by T lymphocytes in the MLR experiment of donor pair #2. Among them, A shows the result that the PD-L1 antigen-binding protein of the present invention enhances the activation of T lymphocytes to secrete the cytokine IL-2; B shows that the PD-L1 antigen-binding protein of the present invention enhances the activation of T lymphocytes to secrete the cytokine IFN-γ. result.

Figure 6 shows the effect of the antigen-binding protein of the present invention on cytokine secretion by T lymphocytes in the MLR experiment of donor pair #3. Among them, A shows the result that the PR000960 post-translational modification (PTM) variant of the present invention enhances the activation of T lymphocytes to secrete the cytokine IL-2; B shows that the PR000960 post-translational modification (PTM) variant of the present invention enhances the activation of T lymphocytes to secrete. Results for the cytokine IFN-γ.

Figure 7 shows the effect of the antigen-binding protein of the present invention on cytokine secretion by T lymphocytes in the MLR experiment of donor pair #4. Among them, A shows the result that the affinity mature variant of PR002082 of the present invention enhances the secretion of cytokine IL-2 by T lymphocytes; B shows the result that the affinity mature variant of PR002082 of the present invention enhances the secretion of cytokine IFN-γ by T lymphocytes.

Figure 8 shows the effect of the antigen-binding protein of the present invention on cytokine secretion by T lymphocytes in the MLR experiment of donor pair #5. Among them, A shows the result that the affinity mature variant of PR002082 of the present invention enhances the secretion of cytokine IL-2 by T lymphocytes; B shows the result that the affinity mature variant of PR002082 of the present invention enhances the secretion of cytokine IFN-γ by T lymphocytes.

Figure 9 shows the effect of the antigen-binding protein of the present invention on cytokine secretion by T lymphocytes in the MLR experiment of donor pair #6. Among them, A shows the result that the affinity mature variant of PR002082 of the present invention enhances the secretion of cytokine IL-2 by T lymphocytes; B shows the result that the affinity mature variant of PR002082 of the present invention enhances the secretion of cytokine IFN-γ by T lymphocytes.

Figure 10 shows the effect of the antigen-binding protein of the present invention on cytokine secretion by T lymphocytes in the MLR experiment of donor pair #7. Among them, A shows the result that the affinity mature variant of PR002082 of the present invention enhances the secretion of cytokine IL-2 by T lymphocytes; B shows the result that the affinity mature variant of PR002082 of the present invention enhances the secretion of cytokine IFN-γ by T lymphocytes.

Figure 11 shows the effect of the antigen-binding proteins of the invention on T lymphocytes in MLR experiments on donor pair #8. The role of cytokines secreted by B cells. Among them, A shows the result that the affinity mature variant of PR002082 of the present invention enhances the secretion of cytokine IL-2 by T lymphocytes; B shows the result that the affinity mature variant of PR002082 of the present invention enhances the secretion of cytokine IFN-γ by T lymphocytes.

Figure 12 shows the effect of the antigen-binding protein of the present invention on cytokine secretion by T lymphocytes in the MLR experiment of donor pair #9. Among them, A shows the result that the affinity mature variant of PR002082 of the present invention enhances the secretion of cytokine IL-2 by T lymphocytes; B shows the result that the affinity mature variant of PR002082 of the present invention enhances the secretion of cytokine IFN-γ by T lymphocytes.

Figure 13 shows the tumor inhibitory effect of the antigen-binding protein of the present invention in the A375 tumor model of NCG mice reconstructing the human PBMC immune system. Among them, A shows the anti-tumor effect in vivo; B shows the results of body weight changes of animals in each group during the experiment.

Figure 14 shows a schematic structural diagram of the bispecific molecule of the present invention.

Figure 15 shows antibody binding to CHO-K1 cells stably expressing human CD73 and human PD-L1.

Figure 16 shows the experimental results of antibodies inhibiting CD73 enzyme activity.

Figure 17 shows the use of reporter gene cell lines to detect the inhibitory effect of antibodies on the PD-1/PD-L1 signaling pathway.

Figure 18 shows the MLR method for in vitro detection of the priming effect of fusion protein on T cells.

The examples shown below are intended to illustrate specific embodiments of the invention and are not intended to limit the scope of the specification or claims in any way. The examples do not include a detailed description of traditional methods, such as those used to construct vectors and plasmids, methods of inserting protein-encoding genes 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 numerous publications, including Sambrook, J., Fritsch, E. F. and Maniais, T. (1989) MolecuLar Cloning: A Laboratory Manual, 2nd edition, Cold spring Harbor Laboratory Press.

Example

Example 1. Preparation of PD-L1 antigen binding protein

In order to obtain antibody molecules that specifically bind to PD-L1, PD-L1 antigen can usually be used to immunize experimental animals. The experimental animals can be mice, rats, rabbits, sheep, camels, etc., but the antibody molecules obtained are not of human origin. After obtaining non-human antibodies, these molecules need to be humanized using antibody engineering technology to reduce immunogenicity and improve druggability. However, the process of humanizing antibodies has its technical complexities, and humanized molecules often have reduced affinity for the antigen. On the other hand, advances in transgenic technology have made it possible to create genetically engineered mice carrying a human immunoglobulin immune repertoire and deleting the endogenous murine immune repertoire. The antibodies produced by this transgenic mouse have fully human sequences, so there is no need for further humanization, which greatly improves the efficiency of therapeutic antibody development.

Harbor HCAb mouse (Harbour Antibodies BV, WO 2002/085945 A3) is a transgenic mouse carrying a human immunoglobulin immune repertoire, capable of producing a new "heavy chain" only antibody that is half the size of traditional IgG antibodies . The antibodies it produces have only the human antibody "heavy chain" variable domain and the mouse Fc constant domain. Due to the fact that it does not contain a light chain, the antibody almost solves the problems of light chain mismatching and heterodimerization, allowing this technology platform to develop products that are difficult to achieve with traditional antibody platforms.

1.1 PD-L1 immunized HCAb mice

Multiple rounds of immunization were performed on Harbor HCAb human antibody transgenic mice aged 6 to 8 weeks with soluble recombinant human PD-L1-mFc fusion protein (Novo Protein Inc. #CM06, lot number 0330837). The total injection dose received by each mouse per immunization was 100 μL by subcutaneous inguinal injection or by intraperitoneal injection. In the first round of immunization, each mouse received immunization with an immunogenic reagent prepared by mixing 50 μg of antigen protein and complete Freund's adjuvant (Sigma, #F5881) at a volume ratio of 1:1. In each subsequent round of boosted immunization, each mouse received immunization with an immunogenic reagent mixed with 25 μg of antigenic protein and Ribi adjuvant (Sigma Adjuvant System, #S6322). The interval between each round of immune boosting is at least two weeks, usually no more than Enhance immunity after five rounds. The immunization time is day 0, day 14, day 28, day 42, day 56, and day 70; and on day 49 and day 77, the mouse serum antibody titer is detected. Five days before isolation of splenic B cells from HCAb mice, a final booster immunization was performed at a dose of 25 μg of antigen protein per mouse.

1.2 Obtain the HCAb antibody sequence against PD-L1

When the PD-L1-specific antibody titer in the mouse serum reaches a certain level, the spleen cells of the mice are removed to isolate B cells, and CD138-positive plasma cells and human PD-L1 antigen are sorted using BD FACS AriaII Cell Sorter. Positive B cell population. RNA from B cells was extracted, cDNA was reverse transcribed (SuperScript IV First-Strand synthesis system, Invitrogen, #18091200), and then specific primers were used to PCR amplify the human VH gene. PCR primers 5'-GGTGTCCA GTGTSAGGTGCAGCTG-3' (SEQ ID NO: 134), 5'-AATCCCTGGGCACTGAAGAGACGG TGACC-3' (SEQ ID NO: 135). The amplified VH gene fragment was constructed into the mammalian cell expression plasmid pCAG vector encoding the human IgG1 antibody heavy chain Fc domain sequence.

The constructed plasmid is transfected into mammalian host cells (such as human embryonic kidney cells HEK293) to express the obtained HCAb antibody. Detect the binding of the HCAb-expressing supernatant to the CHO-K1/hPD-L1 (GenScript, #M00543) stable cell line that overexpresses human PD-L1, and at the same time use the positive antibody PR000151 (Atezolizumab analogue, which the applicant synthesized and expressed based on the Atezolizumab sequence ) as a positive control, Miroball fluorescence flow instrument (Sptlabtech) screening was performed.

The specific steps are: wash CHO-K1/hPD-L1 cells with serum-free F12K medium (Thermofisher, #21127022), and resuspend them in serum-free medium to 1×10 ⁶ /mL. Add Draq5 fluorescent probe (CTS, #4048L) (1 μL Draq5 to 1 mL CHO-K1/hPD-L1 cells, 1:1000 dilution) and incubate in a dark place for 30 minutes. After centrifuging the cells, wash the cells with culture medium and adjust the cell density to 1.0×10 ⁵ cells/mL. Then add Alexa Fluor® 488 AffiniPure Goat Anti-Human IgG, Fcγ Fragment Specific secondary antibody (Jackson ImmunoResearch, #109-545-098) diluted 1:1000, and add 30 μL of this mixture from each well to a 384-well plate (Greiner, #781091). Then add 10 μL of positive control or HCAb-expressing supernatant to the 384-well plate and culture for 2 hours. Fluorescence values were read on a Miroball fluorescence flow instrument. Positive plant antibodies were further tested by FACS for binding to CHO-K1/hPD-L1 cells, and ELISA for cynomolgus monkey PD-L1-his protein (Acrobiosystems, #PD-1-C52H4) to verify binding and cross-binding activity. Use conventional sequencing methods to sequence the positive antibody to obtain the nucleotide sequence encoding the variable domain of the antibody molecule and the corresponding amino acid sequence.

1.3 Preparation of recombinant fully human antibodies

After obtaining the heavy chain variable domain sequence encoding the antibody molecule in the above Example 1.2, conventional recombinant DNA technology can be used to fuse and express the heavy chain variable domain sequence and the corresponding human antibody heavy chain constant domain sequence. , to obtain recombinant antibody molecules.

The plasmid encoding the antibody heavy chain is transfected into mammalian host cells (such as human embryonic kidney cells HEK293), and conventional recombinant protein expression and purification techniques are used to obtain recombinant antibodies with HCAb heavy chains.

Specifically, HEK293 cells were expanded in FreeStyle ^™ F17 Expression Medium (Thermo, #A1383504). Before starting transient transfection, adjust the cell concentration to (6~8) × 10 ⁵ cells/mL, and culture it in a shaker at 37°C and 8% CO ₂ for 24 hours. The cell concentration is 1.2 × 10 ⁶ cells/mL. Prepare 30 mL of cultured cells. The above heavy chain plasmid encoding HCAb was dissolved in 1.5 mL of Opti-MEM serum-reduced medium (Thermo, #31985088), and sterilized by filtration with a 0.22 μm filter. Then take 1.5mL Opti-MEM and dissolve 120μL of 1mg/mL PEI (Polysciences Inc, #23966-2), and let it stand for 5 minutes. Slowly add PEI to the plasmid, incubate at room temperature for 10 minutes, slowly drop in the plasmid PEI mixed solution while shaking the culture bottle, and culture in a 37°C 8% _CO2 shaker for 5 days. The cell viability was observed after 5 days. Collect the culture, centrifuge at 3300g for 10 minutes and take the supernatant; then centrifuge the supernatant at high speed to remove impurities. Equilibrate a gravity column (Bio-Rad, #7311550) containing MabSelect ^™ (GE Healthcare Life Science, #71-5020-91 AE) with PBS (pH 7.4) and rinse with 2-5 column volumes. Pass the supernatant sample through the column. Rinse the column with 5-10 column volumes of PBS. Then use 0.1M glycine at pH 3.5 to elute the target protein, and then use Tris-HCl at pH 8.0 to adjust to neutrality. Finally, use an ultrafiltration tube (Millipore, #UFC901024) to concentrate and change the medium into PBS buffer to obtain purification. of antibody solution. Then use NanoDrop (Thermo Scientific ^TM NanoDrop ^TM One) to measure the concentration, aliquot and store for later use.

1.4 Sequence of fully human recombinant PD-L1 antibody

Table 5 lists the amino acid sequence of the heavy chain variable domain of the PD-L1 antibody in this example, the full-length amino acid sequence of the heavy chain, and the amino acid sequence of the CDR defined according to the Chothia definition rules. At the same time, the corresponding amino acid sequence of the anti-PD-L1 positive control antibody Atezolizumab analog of the present invention is derived from the IMGT database, the antibody heavy chain sequence is SEQ ID NO: 97, and the antibody light chain sequence is SEQ ID NO: 118.

1.5 Analysis of protein purity and polymers using HPLC-SEC

Protein samples are analyzed for purity and aggregate form using analytical size exclusion chromatography (SEC). Connect the analytical chromatography column TSKgel G3000SWxl (Tosoh Bioscience, #08541, 5 μm, 7.8 mm × 30 cm) to a high-pressure liquid chromatograph (HPLC) (model: Agilent Technologies, Agilent 1260 Infinity II), and use PBS buffer at room temperature. Equilibrate for at least 1 hour. An appropriate amount of protein sample (at least 10 μg, sample concentration adjusted to 1 mg/mL) was filtered with a 0.22 μm filter and injected into the system, and the HPLC program was set: use PBS (pH 7.4) buffer to flow the sample at a flow rate of 1.0 mL/min. Chromatographic column, the maximum time is 25 minutes; the detection wavelength is 280nm. After collection, use ChemStation software to integrate the chromatogram and calculate relevant data, generate an analysis report, and report the retention times of different molecular size components in the sample.

The yield and purity analysis results of PD-L1 antibodies are shown in Table 6.

Example 2 Optimization of antibody sequences

2.1 Removal of PTM sites

The antibody sequence in Example 1 was analyzed. The germline gene V gene fragment of its heavy chain variable domain (VH) and the predicted PTM of the variable domain VH are listed in Table 7.

Table 8 lists new antibody molecules (called PTM variants) obtained by amino acid mutation of the sequence of the PR000960 antibody with potential PTM sites from Example 1. Table 9 lists the amino acid sequences of the CDRs, VH and HC of the PTM variants of PR000960, and the amino acid sequences of the CDRs defined according to the Chothia definition rules. Purified recombinant antibodies were obtained from all designed PTM variants according to the method described in Example 1.3, and were further verified in subsequent functional experiments. The yield and purity analysis results of the PTM variants of PR000960 are shown in Table 10.

2.2 Optimize HCAb antibody sequence to improve binding affinity to PD-L1

A yeast display antibody mutation library was established by randomly mutating the CDR region, and the PR002082 molecule was modified for affinity using the BD FACS AriaIII sorting platform. This affinity maturation approach is divided into three rounds.

First, mutations were randomly introduced into the three CDRs of the parent molecule PR002082 to establish a yeast display mutation library of 3 CDRs (CDR1, CDR2, CDR3); then the MACS sorting method was used for enrichment; and then flow FACS was used for multiple rounds of sorting. Enrich high-affinity mutant molecules. In this example, the CDR sequences of antibody variable domains were analyzed by Chothia rules.

In the first round, 0.1 nM biotinylated PD-L1-his was used to sort out yeast cells with higher binding capacity; then culture induction was continued.

In the second round, 0.01 nM biotinylated PD-L1-his was used to further sort out yeast cells with higher binding capacity; then culture induction was continued.

In the third round, continue to reduce the concentration of the antigen during sorting, and use 0.003nM biotinylated PD-L1-his for the final round of sorting.

Finally, the molecules sorted in the previous three rounds were sequenced, the mutation sites were found and randomly combined to establish a combined mutation library. FACS is then used to continue sorting higher affinity mutant molecules at lower antigen concentrations. In this example, the antibody heavy chain variable domain sequence (VH) obtained through affinity maturation was genetically synthesized and cloned into a mammalian cell expression plasmid vector encoding Flag and 6xHis tags to encode and produce recombinant HCAb single Domain antibody molecules.

The sequences of the PR002082 variants (all single domain antibodies) were obtained as shown in Table 11 below.

Table 11: Sequence number list of PR002082 variants (SEQ ID NO:)

2.3 Production and Purification of PR002082 Affinity Variants

After obtaining the sequence encoding the PR002082 affinity variant variable domain in Example 2.2 above, conventional recombinant DNA technology can be used to fuse and express the heavy chain variable domain sequence and the corresponding purification tag to obtain a recombinant HCAb single domain antibody. molecular.

The plasmid encoding the recombinant HCAb single domain antibody is transfected into mammalian host cells (such as Chinese hamster ovary cells CHO), and the corresponding purified recombinant antibody can be obtained using conventional recombinant protein expression and purification techniques.

Specifically, ExpiCHO-S ^™ cells (Gibco, #A29127) were expanded in ExpiCHO ^™ Expression Medium (Gibco, #A2910001). Before starting transient transfection, adjust the cell concentration to (3~4)×10 ⁶ cells/mL, culture it in a 37°C 8% CO ₂ shaker for 24 hours, and adjust the cell concentration to (7~10)×10 ⁶ cells/mL. mL, then dilute to 6×10 ⁶ cells/mL, and prepare 10 mL of cultured cells. Dissolve 8 μg of the above plasmid encoding HCAb single domain antibody (the ratio of plasmid to cells is 0.8 μg: 1 mL) in 0.4 mL OptiPRO ^™ SFM medium (Gibco, #12309019), and filter and sterilize with a 0.22 μm filter. Then add 32 μL of ExpiFectamine ^TM CHO reagent (Gibco, #A29129) to 0.37 mL of OptiPRO ^TM SFM medium (Gibco, #12309019). Immediately add the ExpiFectamine ^TM CHO reagent solution slowly to the plasmid solution, mix by inverting, and slowly drip in the mixed solution of plasmid and transfection reagent while shaking the culture bottle, and culture in a 37°C 8% _CO2 shaker for 8-9 days. The cell viability was observed after 8 days. Collect the culture, centrifuge at 3300g for 10 minutes, take the supernatant, and then filter the supernatant with a 0.22 μm filter to remove impurities. Equilibrate a gravity column (Bio-Rad, #7311550) containing Ni Sepharose excel (GE Healthcare Life Science, #17531802) with PBS (pH 7.4) and rinse with 2-5 column volumes. Pass the supernatant sample through the column. Wash the column with 5-10 times column volume of PBS and 5-10 times column volume of 20mM imidazole solution (pH7.4). Then use 500mM imidazole solution (pH7.4) to elute the target protein, and finally use an ultrafiltration tube (Millipore, #UFC901024) to concentrate and replace the solution with PBS buffer to obtain a purified antibody solution. Then use NanoDrop (Thermo Scientific ^TM NanoDrop ^TM One) to measure the concentration, aliquot and store for later use. Preparation of PR002082 affinity matured variants is shown in Table 12.

Example 3 Binding of antigen-binding proteins to cells overexpressing human/cynomolgus PD-L1 (FACS)

In this example, in order to study the activity of PD-L1 antigen-binding protein in binding to human/cynomolgus PD-L1 in vitro, the CHO-K1 cell line overexpressing human PD-L1 (CHO-K1/hPD-L1, Nanjing Gens Cynomolgus, M00543) or the CHO-K1 cell line overexpressing cynomolgus PD-L1 (CHO-K1/cynoPD-L1, GenScript, Nanjing, M00573) was used for binding experiments at the cellular level. Briefly, PD-L1 cells were digested and resuspended in F-12K complete medium, and the cell density was adjusted to 1 × ¹⁰ cells/mL. Inoculate 100 μL cells/well in a 96-well V-bottom plate (Corning, 3894), and then add 100 μL/well of the antigen-binding protein to be tested at a gradient dilution of 2 times the final concentration and 5 times the final concentration, and mix evenly. The antigen-binding protein has the highest final concentration. The concentration is 100nM, with a total of 8 concentrations, and the isotype antibody hIgG1 iso is used as a control. Place the cells at 4°C and incubate in the dark for 1 hour. Afterwards, add 100 μL/well pre-cooled PBS to rinse the cells twice, centrifuge at 500 g for 5 minutes at 4°C, and discard the supernatant. Then add 100 μL/well fluorescent secondary antibody (goat anti-human IgG (H+L) secondary antibody, Alexa Fluor 488 conjugate, Invitrogen, A11013, 1:1000 dilution), and incubate at 4°C in the dark for 30 minutes. Wash the cells twice with 200 μL/well pre-cooled PBS, centrifuge at 500 g at 4°C for 5 minutes, and discard the supernatant. Finally, resuspend cells in 200 μL/well pre-chilled PBS. Use BD FACS CANTOII to read the fluorescence luminescence signal value. Use the flow cytometer BD FACS CANTOII or FACS ACEA to read the fluorescence signal value, and use the software FlowJo v10 (FlowJo, LLC) to process and analyze the data. The software GraphPad Prism 8 was used for data processing and graphing analysis, and parameters such as the binding curve and EC ₅₀ value were obtained through four-parameter nonlinear fitting.

Figure 1 (A, B) results show that the PD-L1 antigen-binding protein of the present invention can bind CHO-K1 cells overexpressing human PD-L1.

The results in Figure 1(C) show that all PR000960 post-translational modification (PTM) variants of the present invention can bind to CHO-K1 cells overexpressing human PD-L1.

The results in Figure 1 (D, E, F) show that the affinity mature variants of PR002082 of the present invention can all bind to CHO-K1 cells overexpressing human PD-L1, and the effects are stronger than the parent sequence antibody PR002082.

The results in Figure 2(A) show that the PD-L1 antigen-binding proteins of the present invention can bind to CHO-K1 cells overexpressing cynomolgus monkey PD-L1.

The results in Figure 2(B) show that all PR000960 post-translational modification (PTM) variants of the present invention can bind to CHO-K1 cells overexpressing cynomolgus monkey PD-L1.

The results in Figure 2(C) show that the affinity mature variants of PR002082 described in the present invention can all bind to CHO-K1 cells overexpressing monkey PD-L1, and their effects are stronger than those of the parent sequence antibody PR002082.

Example 4. Determination of affinity between antigen-binding proteins and human PD-L1 using BLI method

In this example, the Octet Red96e instrument was used to measure the affinity of the PR002082 affinity mature mutant. The specific method is that the human PD-L1 protein with histamine tag was purchased from the manufacturer ACROBiosystems (# PD-1-H5258), and the test buffer was 1× kinetic buffer (from 10× kinetic buffer (ForteBio, # 18-1105) dilution), used for affinity testing and dilution of antigens and antibodies. Biofilm interference (BLI) technology is used to use the Octet molecular interaction analyzer (ForteBio, model Octet Red96e) to perform the binding between antigens and antibodies. Kinetic analysis.

When measuring the affinity of antigen and antibody, the sensor rotation speed is 1000 rpm. First, equilibrate the two HIS1K sensors placed in a row in the test buffer for 10 minutes, then use the HIS1K sensor to capture His PD-L1 with a capture height of 0.9-1.1nm, and then place the HIS1K sensor in the test buffer. Equilibrate for 2 minutes, then bind the HIS1K sensor to the antigen-binding protein (60 nM and 0 nM) for 3 minutes, and finally dissociate for 15 minutes. The HIS1K sensor was regenerated by immersing it in a 10mM glycine (pH 1.5) solution to elute the protein bound to the sensor.

When using Octet Data Analysis software (Fortebio, version 11.0) for data analysis, select the single subtraction mode (reference well) to subtract the reference signal, select the "1:1 Local/full fitting" method for data fitting, and calculate the antigen-antibody binding Kinetic parameters, K _on (1/Ms) value, K _dis (1/s) value and KD (M) value were obtained.

The results are shown in Table 13. The affinity mature variants of PR02082 can all bind to PD-L1, and the affinity of most variants is significantly higher than that of PR002082.

Example 5. Using reporter gene cell lines to detect the inhibitory effect of antigen-binding proteins on the PD-1 signaling pathway

293T (eBioscience) cells expressing PD-L1 and OS8 (CD3 single-chain antibody transmembrane protein) were spread on a 96-well plate, the cell volume was 1.25×10 ⁴ /well, 100 μL/well. Incubate overnight at 37°C in a 5% _CO2 environment. Remove the supernatant and add 50 μL/well of the antigen-binding protein dilution to be tested. The starting concentration is 100 nM, diluted 5 times, and hlgG1 is the control group. Add 5×10 ⁴ /well Jurkat reporter cells (UPharm) that can sustainably express PD-1 and NFAT-luciferase reporter genes, 50 μL/well. Incubate at 37°C for 6 hours in a 5% _CO2 environment. Add ONE-GloTM luciferase reagent (Promega, #E6110), incubate at room temperature for 5 minutes, and detect the luminescence value with a microplate reader. The results are shown in Figure 3. The results show that the HCAb antibody of the present invention has an inhibitory effect on the PD-1 signaling pathway.

The results in Figure 3(A) show that the PD-L1 antigen-binding proteins of the present invention all have inhibitory effects on the PD-1 signaling pathway.

The results in Figure 3(B) show that the PR000960 post-translational modification (PTM) variants of the present invention all have inhibitory effects on the PD-1 signaling pathway.

The results of Figure 3 (C, D) show that the affinity mature variants of PR002082 of the present invention all have inhibitory effects on the PD-1 signaling pathway, and the inhibitory effects of most variants are significantly improved compared to PR002082.

Example 6. Antigen-binding proteins stimulate cytokine secretion in mixed lymphocyte reaction (MLR)

Purchase AllCells PBMC cells, isolate monocytes, and add recombinant human interleukin 4 (IL-4) (R&D, #204-GMP) and human GM-CSF (R&D, #215-GM/CF) for induction for 6 days. Immature human CD14+ dendritic cells (iDC cells) were obtained. Add 1 μg/mL lipopolysaccharide (LPS; Sigma, #L2630) and induce for 24 hours to obtain mature dendritic cells (mDC cells). T lymphocytes were isolated from the second donor PBMC cells using a T cell isolation kit (StemCell, #17951). Inoculate 1×10 ⁵ /well T lymphocytes and 1×10 ⁴ /well mDC cells into a 96-well plate at a ratio of 10:1, and add 10 times or corresponding dilutions of each antigen-binding protein and negative and positive control antibodies. . Culture for 5 days in a 37°C 5% _CO2 incubator. Collect the supernatants on the third and fifth days, and use ELISA kits to detect the secretion of IL-2 (ThermoFisher, #88-7025-88) and IFN-γ (ThermoFisher, #88-7316-88). .

In this example, PBMC from 18 donors were divided into 9 donor pairs for mixed lymphocyte reaction (MLR).

The results of Figures 4 and 5 show that in two independent MLR experiments, the PD-L1 antigen-binding protein of the present invention can enhance the secretion of cytokines IL-2 and IFN-γ by activated T lymphocytes.

The results of Figure 6 show that the PR000960 post-translational modification (PTM) variants of the present invention can enhance the secretion of cytokines IL-2 and IFN-γ by primed T lymphocytes.

Figure 7, Figure 8, Figure 9, Figure 10, Figure 11, and Figure 12 show that in 6 independent MLR In experiments, the affinity matured variants of PR002082 described in the present invention can all enhance the secretion of cytokines IL-2 and IFN-γ by activated T lymphocytes.

Example 7. In vivo tumor inhibitory activity of antigen-binding proteins

The in vivo anti-tumor effect of the A375 tumor model using NCG mice to reconstruct the human PBMC immune system is shown in Figure 13A. Specifically, the average tumor volume of mice in the vehicle control group was 1336 mm ³ on the 23rd day after administration. The average tumor volume of the test drug Atezolizumab (10 mg/kg) treatment group was 343 mm ³ on the 23rd day after vaccination, which was significantly different from the vehicle control group (p value = 0.002), and the tumor inhibition rate TGI (%) was 74.33%. The average tumor volume of the test drug PR002079 (5mg/kg) treatment group was 728mm ³ on the 23rd day after vaccination, which was significantly different from the vehicle control group (p value = 0.021), and the tumor inhibition rate TGI (%) was 45.51%. The average tumor volume of the test drug PR002082 (5mg/kg) treatment group was 891mm ³ on the 23rd day after vaccination. There was no significant difference compared with the vehicle control group (p value = 0.139), and the tumor inhibition rate TGI (%) was 33.31%.

The results of Figure 13 B show that during the experiment, the weight of animals in each group increased, indicating that the animals tolerated the test product well. It has no obvious toxic effects on animals and has good safety.

Example 8. Obtaining CD73 antibodies

For the acquisition of CD73 antibodies, please refer to the descriptions in Example 1, Example 2 and Example 3 in the specification of published application WO2021032173A1.

Specifically, Harbor H2L2 mice were used, and CD73 protein was used as an immunogen to generate anti-CD73 antibodies. Each mouse was given a first boost of 50 μg of protein and subsequent boosts of 25 μg of protein by subcutaneous and intraperitoneal injection with adjuvant (Sigma, S6322). This immunization is given every two weeks for a total of 6 times. Final immunization was performed by intraperitoneal injection of the immunogen diluted in PBS. Serum titers against human CD73 were tested using ELISA and FACS. At indicated time points, mouse sera were sampled and titrated for ELISA and FACS analysis to test binding to CD73 protein or CD73 stable cell lines. Good serum titers were observed in the protein immunization cohort.

Mouse spleen cells isolated from immunized mice were fused with the mouse myeloma cell line SP2/0 (ATCC, CRL-1581) via an electric field-based electroporator using a cell fusion generator (BEX-LF301), to obtain hybridomas. Human anti-CD73 antibodies were screened in individual wells 9-14 days after fusion. After hybridoma sub-selection, a single strain 38H6 that showed good binding activity to CD73 was selected for sequencing. Identification and acquisition of heavy chain variable regions and light chain variable regions. Then the heavy chain variable region and the light chain variable region were synthesized and cloned into plasmids encoding the human IgG1 constant region and the plasmid encoding the human Igκ region respectively.

Recombinant plasmids encoding the antibodies of interest were transiently co-transfected into HEK293-6E or HEK293-F cell cultures using PEI (Polyscience, 24885). 30 μg plasmid was mixed with 120 μL PEI and incubated for 15 min at room temperature. Next, the mixture was added dropwise to 293 cells suspended in Opti-MEM at a concentration of 1×106 cells/ml. After transfection, the cells were cultured at 37°C, 5% CO2, and shaken at 120 rpm. Cell culture supernatants collected on days 6-7 were used for purification.

The supernatant containing the target antibody is harvested 6-7 days after transfection by centrifugation and filtration. Monoclonal antibodies were purified by passing rProtein A ( GE , 17-1279-02) prepacked columns (Bio-Rad, 7311550). Prepare a Protein A column by packing 0.2 mL of rProtein A resin into a column and then flushing with 10 column volumes of ddH2O and 10 column volumes of PBS. The cell culture supernatant was passed through the column, and then washed with 10 times the column volume of PBS. The protein was then eluted with 8 column volumes of elution buffer (Thermo, 21004) and mixed with 640 μL of neutralization buffer (1 M Tris-HCl, pH 9.0; Teknova, T1090) immediately after elution. In a concentrator (Millipore, UFC903024), the buffer was exchanged more than 500 times with DPBS by centrifugation at 3800 rpm (Eppendorf, 5810R) at 4°C, and finally concentrated to an appropriate volume. The concentration of purified anti-CD73 antibody was determined by UV absorbance at 280 nm (NanoDrop). Antibody purity was determined by SEC-HPLC and SDS-PAGE. Recombinant antibody PR000506 was successfully expressed and purified for characterization.

The VH and VL sequences of the anti-CD73 antibody were further optimized through germline backmutation and PTM removal operations: In the germline program, the antibody VH or VL sequence was first analyzed through, for example, NCBI/ The Ig-BLAST algorithm aligns to the closest human germline sequence and then inverts residues in the framework region that are different from the germline sequence to the corresponding residues in the germline sequence. Germlined antibodies composed of sequence variants are then recombinantly produced through mature molecular biology techniques.

Post-translational modifications (PTMs) are widely observed in proteins expressed in mammalian cells. In addition to conserved PTM sites in antibodies (e.g., the conserved N-glycosylation site on the CH2 domain of IgG1 antibodies), other PTM sites occurring within the antibody antigen-binding site (i.e., the CDR region) may degrade the antigen Binding activity or reduced chemical stability. For example, deamidation or isomerization can render the molecule unstable and heterogeneous. To reduce sequence instability, the PTM motif can be removed through mutation. Look for PTM motifs of VH or VL sequences, such as isomerization motifs (e.g. DG). "Hotspot" residues (e.g., D or G in the DG motif) are then mutated to corresponding residues in the germline sequence or other residues with similar biophysical properties. Then, antibodies composed of sequence variants after PTM removal are produced recombinantly through mature molecular biology techniques.

The following anti-CD73 antibodies of Table 14 were obtained:

Example 9 Preparation of CD73/PD-L1 bispecific antibodies

This example utilizes the antigen-binding domain Fab of anti-CD73 IgG antibodies PR000846, PR001408, and PR003836, and the antigen-binding structures of anti-PD-L1 HCAb antibody PR002082 and affinity mature variants PR005263, PR005872, PR005875, PR005878, PR006246, and PR006248. domain VH to construct CD73×PD-L1 bispecific antibody molecules.

The sequence number of the anti-CD73 H2L2 antibody is shown in Table 15, and the sequence number of the anti-PD-L1 HCAb antibody is shown in Table 16.

Figure 14 lists the molecular structures of dual-specific binding proteins encompassed by the invention, each structure being further described below. In this example and other parts of the present application, when referring to the number of polypeptide chains contained in a molecular structure, it usually refers to the number of "different polypeptide chains"; for example, a conventional IgG antibody has two different polypeptide chains, namely Heavy chain and light chain, although the IgG antibody molecule itself is a tetrapeptide chain protein molecule containing two identical heavy chains and two identical light chains, its two different polypeptide chains are specifically referred to when describing its structural characteristics. The binding valence refers to the number of antigen-binding sites in the molecular structure. For example, a conventional IgG antibody can bind two identical antigen molecules at the same time, and its binding valence is two.

Table 17 lists the connecting peptide sequences that may be used in the structural design of the present invention.

9.1 Construction of bispecific antibodies with IgG_HC-VH quadrivalent symmetric structure

A bispecific antibody with an IgG-VH tetravalent symmetric structure was constructed using anti-CD73 H2L2 antibody and anti-PD-L1 HCAb antibody. The binding protein of the IgG_HC-VH tetravalent symmetry structure (shown in Figure 14A) contains two polypeptide chains: polypeptide chain 1, also called a short chain, from the amino end to the carboxyl end, which includes VL_A-CL; polypeptide chain 2. Also called long chain, from the amine end to the carboxyl end, it contains VH_A-CH1-h-CH2-CH3-L-VH_B. h is the hinge region or derived sequence of an IgG antibody. In one embodiment, CH3 of polypeptide chain 2 is directly fused to VH_B, that is, the length of L is 0. In another embodiment, CH3 of polypeptide chain 2 is linked to VH_B via a linker peptide L; L can be the sequence listed in Table 17.

9.2 Construction of bispecific antibodies with VH-IgG_HC quadrivalent symmetric structure

A bispecific antibody with an IgG-VH tetravalent symmetric structure was constructed using anti-CD73 H2L2 antibody and anti-PD-L1 HCAb antibody. The binding protein of the VH-IgG_HC tetravalent symmetric structure (shown in Figure 14, B) contains two polypeptide chains: polypeptide chain 1, also called a short chain, from the amino end to the carboxyl end, which includes VL_A-CL; polypeptide chain 2. Also called long chain, from the amine end to the carboxyl end, it contains VH_B-L-VH_A-CH1-h-CH2-CH3. h is the hinge region or derived sequence of an IgG antibody. VH_A of polypeptide chain 2 is linked to VH_B via a linker peptide L, which may be the sequence listed in Table 17. In one embodiment, VH_A and VH_B of polypeptide chain 2 are directly fused and linked, that is, the length of L is 0.

9.3 Construction of bispecific antibody molecules with Fab(CL)-VH-Fc structure

A bispecific antibody with an IgG-VH tetravalent symmetric structure was constructed using anti-CD73 H2L2 antibody and anti-PD-L1 HCAb antibody. The binding protein of the Fab(CL)-VH-Fc symmetric structure (shown in Figure 14, C) contains two polypeptide chains, polypeptide chain 1, also known as short chain, from the amino terminus to the carboxyl terminus, which contains VH_A-CH1 ;Polypeptide chain 2, also called long chain, from the amino end to the carboxyl end, it contains VL_A-CL-L1-VH_B-L2-CH2-CH3. The connecting peptides L1 and L2 of polypeptide chain 2 can be the sequences listed in Table 17. In one embodiment, CL of polypeptide chain 2 is directly fused to VH_B, that is, the length of L1 is 0.

The CD73×PD-L1 bispecific antibody is an IgG1 with Fc mutations L234A and L235A or L234A and L235A and G237A (according to EU index numbering).

The CD73×PD-L1 bispecific antibodies prepared by the present invention are summarized in Table 18; the sequence number of the amino acid sequence of the polypeptide chain of the obtained biantibody molecule is shown in Table 19. The sequence numbers of the CDRs of the antigen-binding domain of the obtained bisantibody molecule are shown in Table 20. The physical and chemical properties analysis of the prepared CD73×PD-L1 double anti-antibody molecular samples are summarized in Table 21.

Table 20: Sequence of the CDRs of the antigen-binding domain of the CD73×PD-L1 bis-antibody molecule

Example 10. Binding of CD73×PD-L1 bispecific antibodies to cells overexpressing human PD-L1 or human CD73 (FACS)

In order to study the activity of CD73×PD-L1 bispecific antibodies binding to human CD73 and PD-L1, CHO-K1 cell lines overexpressing human CD73 or PD-L1 (CHO-K1-hCD73, CHO-K1-hPD- L1) Conduct binding experiments at the cellular level. Briefly, CHO-K1-hCD73 and CHO-K1-hPD-L1 cells were digested and resuspended in F-12K complete medium, and the cell density was adjusted to 1 × ¹⁰ cells/mL. Seed 100 μL cells/well in a 96-well V-bottom plate (Corning, #3894), then add 100 μL/well, centrifuge at 500 g at 4°C for 5 minutes, and discard the supernatant. Dilute the antibody to a maximum final concentration of 50 nM, 3-fold dilution, for a total of 8 concentrations. Add 100 μL of diluted antibody to the cells, place the cells at 4°C, and incubate in the dark for 1 hour. Afterwards, add 100 μL/well pre-cooled PBS to rinse the cells twice, centrifuge at 500 g and 4°C for 5 minutes, and discard the supernatant. Then add 100 μL/well fluorescent secondary antibody (goat anti-human IgG (H+L) secondary antibody, AlexaFluor® 488 conjugate, Invitrogen, #A11013, 1:1000), and incubate at 4°C in the dark for 30 minutes. Wash the cells twice with 200 μL/well pre-cooled PBS, centrifuge at 500 g and 4°C for 5 minutes, and discard the supernatant. Finally, resuspend the cells in 200 μL/well pre-cooled PBS, and use BD FACS CANTOII to read the fluorescence signal value.

The results of Figure 15 show that the anti-CD73 antibodies PR000846 and PR001408 of the present invention can specifically bind to CHO-K1 cells overexpressing human CD73 (A in Figure 15); the CD73×PD-L1 bispecific antibodies of the present invention (PR003569 and PR003739 ) is able to bind to CHO-K1 cells overexpressing human CD73 (A of Figure 15); the anti-CD73 antibody PR003836 of the present invention can specifically bind to CHO-K1 cells overexpressing human CD73 (B of Figure 15); the CD73×PD-L1 bispecific antibody PR004295 of the present invention can specifically bind to CHO-K1 cells expressing human CD73 (B in Figure 15); The anti-CD73 antibody PR000846 of the present invention can specifically bind to CHO-K1 cells overexpressing human CD73 (Figure 15C, Figure 15D); the CD73×PD-L1 bispecific antibodies of the present invention (PR006268, PR006269, PR006270 , PR006271, PR006661, PR006662, PR006663, PR006664, PR006667, PR006668) can bind to CHO-K1 cells overexpressing human CD73 (Figure 15C, Figure 15D); the anti-PD-L1 antibody PR002082 of the present invention can bind to CHO-K1 cells expressing human PD-L1 (E in Figure 15); CD73×PD-L1 bispecific antibodies PR003569, PR003739 and PR004295 of the present invention can bind to CHO-K1 cells overexpressing human PD-L1 (Figure 15 E, Figure 15 F).

The EC50 values of the anti-CD73 antibody, anti-PD-L1 antibody and CD73×PD-L1 bispecific antibody of the present invention are in the range of 0.3418nM-2.929nM. The starting concentration in C of Figure 15 is 50 nM, 3-fold gradient dilution, a total of eight concentrations; the starting concentration in D of Figure 15 is 100 nM, 3-fold gradient dilution, a total of eight concentrations.

Example 11. CD73×PD-L1 bispecific antibody inhibits the enzymatic activity of CD73

The malachite green method was used to determine the enzyme activity of antibodies against soluble recombinant CD73. First, 12.5 μL of 1 nM recombinant CD73 protein and 12.5 μL of 1 nM antibody were added to a 384-well plate (Corning, #3799) (the experimental buffer was 25 mM Tris pH 7.5, 5 mM MgCl ₂ , 0.005% Tween-20), incubate at room temperature for 1 hour. Add 25 μL AMP (maximum concentration 200 μM, diluted 2-fold to 8 concentrations with experimental buffer) and incubate at room temperature for 15 minutes. The concentration of inorganic phosphate in each well was measured according to the supplier's instructions. After the measurement, the 620nm light absorption value was recorded using a Molecular Devices (SPECTRAMax plus384) plate reader. The experimental results were analyzed and graphed using GraphPad Prism 8.0.

Figure 16 results show that the CD73xPD-L1 bispecific antibodies PR003569, PR003739, PR006268, PR006269, PR006270, PR006271, PR006663, PR006664, PR006667 and PR006668 can non-competitively inhibit the activity of CD73 protease, which is equivalent to the activity of the parent monoclonal antibody PR000846.

Example 12. Using reporter gene cell lines to detect the inhibitory effect of CD73×PD-L1 bispecific antibodies on the PD-1 signaling pathway

HEK293T (eBioscience) cells expressing PD-L1 and OS8 (CD3 single-chain antibody transmembrane protein) were spread on a 96-well plate, the cell volume was 1.25×10 ⁴ /well, 100 μL/well. Incubate overnight at 37°C in a 5% _CO2 environment. Add 50 μL/well of the antigen-binding protein dilution to be tested, with a starting concentration of 100 nM and a 5-fold dilution, and hlgG1 is the control group. Add 5×10 ⁴ /well Jurkat reporter cells (UPharm) that can sustainably express PD-1 and NFAT-luciferase reporter genes, 50 μL/well. Incubate for 6 hours at 37°C in a 5% _CO2 environment. Add ONE-GloT luciferase reagent (Promega, #E6110), incubate at room temperature for 5 minutes, and detect the luminescence value with a microplate reader.

The results of Figure 17 show that the CD73×PD-L1 bispecific antibodies PR003569, PR003739 and PR004295 of the present invention have inhibitory effects on the PD-1 signaling pathway using reporter gene cell lines.

Example 13. CD73×PD-L1 bispecific antibody primes T cells in mixed lymphocyte reaction (MLR) assay

Purchase AllCells PBMC cells, isolate CD14+ monocytes, and add recombinant human interleukin 4 (IL-4) (R&D, #204-GMP) and human GM-CSF (R&D, #215-GM/CF) for induction for 6 days. , to obtain immature human dendritic cells (iDC cells). Continue to add 1 μg/mL lipopolysaccharide (LPS; Sigma, #L2630) and induce for 24 hours to obtain mature dendritic cells (mDC cells). T lymphocytes were isolated from the second donor PBMC cells using Pan-T cell isolation kit (Miltenyibiotec, #130-096-535). Inoculate 1×10 ⁵ /well T lymphocytes and 1×10 ⁴ /well mDC cells into a 96-well plate at a ratio of 10:1, and add diluted antigen-binding protein and negative and positive control antibodies. Culture in a 37°C 5% _CO2 incubator for 5 days. Collect the supernatants on the third and fifth days, and use ELISA kits to detect the secretion of IL-2 (ThermoFisher, #88-7025-88) and IFN-γ (ThermoFisher, #88-7316-88). .

The results of Figure 18 show that the CD73xPD-L1 bispecific antibodies PR003569, PR003739 and PR004295 of the present invention have a priming effect on T cells detected in vitro by MLR method. Anti-PD-L1 mAb PR002082 alone compared with human isotype IgG, and compared with anti-CD73 mAb PR000846, PR001408 and PR003836 alone, and anti-PD-L1 mAb PR002082 compared with anti-CD73 mAb CD73xPD-L1 bispecific antibodies PR003569, PR003739 and PR004295 were effective in priming T cell secretion at a concentration of 10 nM compared with the combination of anti-PR000846 and the combination of anti-PD-L1 mAb PR002082 with anti-CD73 mAb PR003836. IL-2 was more significantly effective. Furthermore, the CD73xPD-L1 bispecific antibodies PR003569, PR003739 and PR004295 were compared with the anti-PD-L1 mAb PR002082 in combination with the anti-CD73 mAb PR000846, and the anti-PD-L1 mAb PR002082 with the anti-CD73 mAb PR003836. Compared with the more significant effect of the combination, it can be considered that CD73xPD-L1 bispecific antibodies PR003569, PR003739 and PR004295 have a synergistic effect.

Similarly, compared with human isotype IgG, compared with the anti-PD-L1 mAb PR002082 alone, and compared with the anti-CD73 mAb PR000846, PR001408, and PR003836 alone, and compared with the anti-PD-L1 mAb PR002082 The CD73xPD-L1 bispecific antibodies PR003569, PR003739 and PR004295 at a concentration of 10 nM were Priming T cells to secrete IFN-γ was significantly more effective. Furthermore, the CD73xPD-L1 bispecific antibodies PR003569, PR003739 and PR004295 were compared with the anti-PD-L1 mAb PR002082 in combination with the anti-CD73 mAb PR000846, and the anti-PD-L1 mAb PR002082 with the anti-CD73 mAb PR003836. Compared with the more significant effect of the combination, it can be considered that CD73xPD-L1 bispecific antibodies PR003569, PR003739 and PR004295 have a synergistic effect.

<110> HARBOUR BIOMED(SHANGHAI)CO.,LTD

<120> Specific binding proteins targeting PD-L1 and CD73 and their applications

<130> 111P000707TW

<140> 111119822

<141> 2022-05-27

<150> CN 202110594756.3

<151> 2021-05-28

<150> CN 202210579248.2

<151> 2022-05-16

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<400> 50

<210> 51

<211> 11

<212> PRT

<213> Artificial Sequence

<400> 51

<210> 52

<211> 11

<212> PRT

<213> Artificial Sequence

<400> 52

<210> 53

<211> 11

<212> PRT

<213> Artificial Sequence

<400> 53

<210> 54

<211> 15

<212> PRT

<213> Artificial Sequence

<400> 54

<210> 55

<211> 15

<212> PRT

<213> Artificial Sequence

<400> 55

<210> 56

<211> 7

<212> PRT

<213> Artificial Sequence

<400> 56

<210> 57

<211> 7

<212> PRT

<213> Artificial Sequence

<400> 57

<210> 58

<211> 7

<212> PRT

<213> Artificial Sequence

<400> 58

<210> 59

<211> 7

<212> PRT

<213> Artificial Sequence

<400> 59

<210> 60

<211> 32

<212> PRT

<213> Artificial Sequence

<400> 60

<210> 61

<211> 32

<212> PRT

<213> Artificial Sequence

<400> 61

<210> 62

<211> 32

<212> PRT

<213> Artificial Sequence

<400> 62

<210> 63

<211> 32

<212> PRT

<213> Artificial Sequence

<400> 63

<210> 64

<211> 9

<212> PRT

<213> Artificial Sequence

<400> 64

<210> 65

<211> 9

<212> PRT

<213> Artificial Sequence

<400> 65

<210> 66

<211> 9

<212> PRT

<213> Artificial Sequence

<400> 66

<210> 67

<211> 9

<212> PRT

<213> Artificial Sequence

<400> 67

<210> 68

<211> 10

<212> PRT

<213> Artificial Sequence

<400> 68

<210> 69

<211> 10

<212> PRT

<213> Artificial Sequence

<400> 69

<210> 70

<211> 10

<212> PRT

<213> Artificial Sequence

<400> 70

<210> 71

<211> 10

<212> PRT

<213> Artificial Sequence

<400> 71

<210> 72

<211> 118

<212> PRT

<213> Artificial Sequence

<400> 72

<210> 73

<211> 119

<212> PRT

<213> Artificial Sequence

<400> 73

<210> 74

<211> 123

<212> PRT

<213> Artificial Sequence

<400> 74

<210> 75

<211> 123

<212> PRT

<213> Artificial Sequence

<400> 75

<210> 76

<211> 123

<212> PRT

<213> Artificial Sequence

<400> 76

<210> 77

<211> 123

<212> PRT

<213> Artificial Sequence

<400> 77

<210> 78

<211> 123

<212> PRT

<213> Artificial Sequence

<400> 78

<210> 79

<211> 123

<212> PRT

<213> Artificial Sequence

<400> 79

<210> 80

<211> 123

<212> PRT

<213> Artificial Sequence

<400> 80

<210> 81

<211> 122

<212> PRT

<213> Artificial Sequence

<400> 81

<210> 82

<211> 123

<212> PRT

<213> Artificial Sequence

<400> 82

<210> 83

<211> 123

<212> PRT

<213> Artificial Sequence

<400> 83

<210> 84

<211> 123

<212> PRT

<213> Artificial Sequence

<400> 84

<210> 85

<211> 123

<212> PRT

<213> Artificial Sequence

<400> 85

<210> 86

<211> 123

<212> PRT

<213> Artificial Sequence

<400> 86

<210> 87

<211> 123

<212> PRT

<213> Artificial Sequence

<400> 87

<210> 88

<211> 123

<212> PRT

<213> Artificial Sequence

<400> 88

<210> 89

<211> 123

<212> PRT

<213> Artificial Sequence

<400> 89

<210> 90

<211> 123

<212> PRT

<213> Artificial Sequence

<400> 90

<210> 91

<211> 123

<212> PRT

<213> Artificial Sequence

<400> 91

<210> 92

<211> 123

<212> PRT

<213> Artificial Sequence

<400> 92

<210> 93

<211> 107

<212> PRT

<213> Artificial Sequence

<400> 93

<210> 94

<211> 107

<212> PRT

<213> Artificial Sequence

<400> 94

<210> 95

<211> 107

<212> PRT

<213> Artificial Sequence

<400> 95

<210> 96

<211> 107

<212> PRT

<213> Artificial Sequence

<400> 96

<210> 97

<211> 448

<212> PRT

<213> Artificial Sequence

<400> 97

<210> 98

<211> 449

<212> PRT

<213> Artificial Sequence

<400> 98

<210> 99

<211> 355

<212> PRT

<213> Artificial Sequence

<400> 99

<210> 100

<211> 355

<212> PRT

<213> Artificial Sequence

<400> 100

<210> 101

<211> 355

<212> PRT

<213> Artificial Sequence

<400> 101

<210> 102

<211> 355

<212> PRT

<213> Artificial Sequence

<400> 102

<210> 103

<211> 453

<212> PRT

<213> Artificial Sequence

<400> 103

<210> 104

<211> 355

<212> PRT

<213> Artificial Sequence

<400> 104

<210> 105

<211> 355

<212> PRT

<213> Artificial Sequence

<400> 105

<210> 106

<211> 452

<212> PRT

<213> Artificial Sequence

<400> 106

<210> 107

<211> 355

<212> PRT

<213> Artificial Sequence

<400> 107

<210> 108

<211> 355

<212> PRT

<213> Artificial Sequence

<400> 108

<210> 109

<211> 355

<212> PRT

<213> Artificial Sequence

<400> 109

<210> 110

<211> 355

<212> PRT

<213> Artificial Sequence

<400> 110

<210> 111

<211> 355

<212> PRT

<213> Artificial Sequence

<400> 111

<210> 112

<211> 355

<212> PRT

<213> Artificial Sequence

<400> 112

<210> 113

<211> 355

<212> PRT

<213> Artificial Sequence

<400> 113

<210> 114

<211> 355

<212> PRT

<213> Artificial Sequence

<400> 114

<210> 115

<211> 355

<212> PRT

<213> Artificial Sequence

<400> 115

<210> 116

<211> 355

<212> PRT

<213> Artificial Sequence

<400> 116

<210> 117

<211> 355

<212> PRT

<213> Artificial Sequence

<400> 117

<210> 118

<211> 214

<212> PRT

<213> Artificial Sequence

<400> 118

<210> 119

<211> 214

<212> PRT

<213> Artificial Sequence

<400> 119

<210> 120

<211> 214

<212> PRT

<213> Artificial Sequence

<400> 120

<210> 121

<211> 214

<212> PRT

<213> Artificial Sequence

<400> 121

<210> 122

<211> 587

<212> PRT

<213> Artificial Sequence

<400> 122

<210> 123

<211> 591

<212> PRT

<213> Artificial Sequence

<400> 123

<210> 124

<211> 590

<212> PRT

<213> Artificial Sequence

<400> 124

<210> 125

<211> 587

<212> PRT

<213> Artificial Sequence

<400> 125

<210> 126

<211> 587

<212> PRT

<213> Artificial Sequence

<400> 126

<210> 127

<211> 587

<212> PRT

<213> Artificial Sequence

<400> 127

<210> 128

<211> 587

<212> PRT

<213> Artificial Sequence

<400> 128

<210> 129

<211> 577

<212> PRT

<213> Artificial Sequence

<400> 129

<210> 130

<211> 577

<212> PRT

<213> Artificial Sequence

<400> 130

<210> 131

<211> 222

<212> PRT

<213> Artificial Sequence

<400> 131

<210> 132

<211> 569

<212> PRT

<213> Artificial Sequence

<400> 132

<210> 133

<211> 569

<212> PRT

<213> Artificial Sequence

<400> 133

<210> 134

<211> 24

<212> DNA

<213> Artificial Sequence

<400> 134

<210> 135

<211> 29

<212> DNA

<213> Artificial Sequence

<400> 135

<210> 136

<211> 4

<212> PRT

<213> Artificial Sequence

<400> 136

<210> 137

<211> 5

<212> PRT

<213> Artificial Sequence

<400> 137

<210> 138

<211> 6

<212> PRT

<213> Artificial Sequence

<400> 138

<210> 139

<211> 7

<212> PRT

<213> Artificial Sequence

<400> 139

<210> 140

<211> 15

<212> PRT

<213> Artificial Sequence

<400> 140

<210> 141

<211> 20

<212> PRT

<213> Artificial Sequence

<400> 141

<210> 142

<211> 25

<212> PRT

<213> Artificial Sequence

<400> 142

<210> 143

<211> 15

<212> PRT

<213> Artificial Sequence

<400> 143

<210> 144

<211> 15

<212> PRT

<213> Artificial Sequence

<400> 144

<210> 145

<211> 15

<212> PRT

<213> Artificial Sequence

<400> 145

<210> 146

<211> 15

<212> PRT

<213> Artificial Sequence

<400> 146

<210> 147

<211> 17

<212> PRT

<213> Artificial Sequence

<400> 147

<210> 148

<211> 18

<212> PRT

<213> Artificial Sequence

<400> 148

<210> 149

<211> 18

<212> PRT

<213> Artificial Sequence

<400> 149

<210> 150

<211> 19

<212> PRT

<213> Artificial Sequence

<400> 150

<210> 151

<211> 19

<212> PRT

<213> Artificial Sequence

<400> 151

<210> 152

<211> 9

<212> PRT

<213> Artificial Sequence

<400> 152

<210> 153

<211> 19

<212> PRT

<213> Artificial Sequence

<400> 153

<210> 154

<211> 29

<212> PRT

<213> Artificial Sequence

<400> 154

<210> 155

<211> 6

<212> PRT

<213> Artificial Sequence

<400> 155

<210> 156

<211> 17

<212> PRT

<213> Artificial Sequence

<400> 156

<210> 157

<211> 2

<212> PRT

<213> Artificial Sequence

<400> 157

Claims (24)

An isolated antigen-binding protein that specifically binds to PD-L1 and/or fragments thereof, comprising an antibody heavy chain variable region VH, the VH comprising the following complementarity determining regions: SEQ ID NO: 9, SEQ ID NO: respectively. 23 and HCDR1, HCDR2 and HCDR3 as set forth in SEQ ID NO: 36; or HCDR1, HCDR2 and HCDR3 as set forth in SEQ ID NO: 10, SEQ ID NO: 23 and SEQ ID NO: 36 respectively; or as set forth in SEQ ID NO: 10, SEQ ID NO: 23 and HCDR3 respectively; NO: 13, HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 23 and SEQ ID NO: 36; or HCDR1, HCDR2 as shown in SEQ ID NO: 10, SEQ ID NO: 23 and SEQ ID NO: 39 respectively and HCDR3; or HCDR1, HCDR2 and HCDR3 as set forth in SEQ ID NO: 10, SEQ ID NO: 23 and SEQ ID NO: 40 respectively; or as set forth in SEQ ID NO: 10, SEQ ID NO: 23 and SEQ ID NO respectively : HCDR1, HCDR2 and HCDR3 shown in SEQ ID NO: 41; or HCDR1, HCDR2 and HCDR3 shown in SEQ ID NO: 10, SEQ ID NO: 27 and SEQ ID NO: 36 respectively; or respectively SEQ ID NO: 10, SEQ HCDR1, HCDR2 and HCDR3 as set forth in ID NO:27 and SEQ ID NO:39; or HCDR1, HCDR2 and HCDR3 as set forth in SEQ ID NO:10, SEQ ID NO:27 and SEQ ID NO:40 respectively; or respectively HCDR1, HCDR2 and HCDR3 as set forth in SEQ ID NO: 10, SEQ ID NO: 27 and SEQ ID NO: 41; or as set forth in SEQ ID NO: 14, SEQ ID NO: 27 and SEQ ID NO: 36 respectively. HCDR1, HCDR2 and HCDR3; or HCDR1, HCDR2 and HCDR3 as set forth in SEQ ID NO: 14, SEQ ID NO: 27 and SEQ ID NO: 39 respectively; or as set forth in SEQ ID NO: 14, SEQ ID NO: 27 and SEQ ID NO: 39 respectively. HCDR1, HCDR2 and HCDR3 as shown in NO:40; or HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO:14, SEQ ID NO:27 and SEQ ID NO:41 respectively. The isolated antigen-binding protein as described in claim 1, wherein the VH further includes a heavy chain variable region framework region VH FWR, the VH FWR includes VH FWR1, VH FWR2, VH FWR3 and VH FWR4, and the VH FWR1 has the amino acid sequence shown in any one of SEQ ID NO: 3-5, the VH FWR2 has the amino acid sequence shown in SEQ ID NO: 17 or SEQ ID NO: 20, and the VH FWR3 has The amino acid sequence shown in SEQ ID NO: 30 or SEQ ID NO: 31, and the VH FWR4 has the amino acid sequence shown in SEQ ID NO: 43. The isolated antigen-binding protein of claim 1 or 2, wherein the VH comprises the amine shown in any one of SEQ ID NO: 74, SEQ ID NO: 79-80 and SEQ ID NO: 82-92 amino acid sequence. The isolated antigen-binding protein as described in claim 1 or 2, further comprising an Fc region, and the Fc region is human Fc. The isolated antigen-binding protein as described in claim 1 or 2, comprising SEQ ID NO: 99, 104, 105, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116 or 117 The heavy chain showing the amino acid sequence. The isolated antigen-binding protein as described in claim 1 or 2, which is an antigen-binding fragment in the form of HCAb or nanobody. A chimeric antigen receptor, which includes an extracellular antigen-binding domain, a transmembrane domain and an intracellular signaling domain, wherein the extracellular antigen-binding domain includes as claimed in claims 1 to 6 The isolated antigen-binding protein of any one of the above. A modified immune cell comprising the chimeric antigen receptor as described in claim 7. A multispecific antibody comprising two or more antigen-binding domains, wherein one antigen-binding domain comprises the isolated antigen-binding protein as described in any one of claims 1 to 6. A specific binding protein comprising a first structural domain and a second structural domain, the first structural domain binds CD73 or a fragment thereof, and the second structural domain binds PD-L1 or a fragment thereof, the first structural domain The domain is connected to the second structural domain to form a bispecific binding protein; the second structural domain includes the isolated antigen-binding protein as described in any one of claims 1 to 6; the first structural domain includes a light Chain variable region VL and heavy chain variable region VH, said VL and said VH comprising complementarity determining regions selected from the group consisting of SEQ ID NO: 51, SEQ ID NO: 57 and SEQ ID NO: 65, respectively. LCDR1, LCDR2, and LCDR3; and, HCDR1, HCDR2, and HCDR3 as set forth in SEQ ID NO:8, SEQ ID NO:22, and SEQ ID NO:35, respectively; or, respectively, SEQ ID NO:52, SEQ ID NO: 58 and LCDR1, LCDR2 and LCDR3 as set forth in SEQ ID NO: 66; and, HCDR1, HCDR2 and HCDR3 as set forth in SEQ ID NO: 12, SEQ ID NO: 25 and SEQ ID NO: 37 respectively; or respectively as SEQ ID NO: 12, SEQ ID NO: 25 and HCDR3 LCDR1, LCDR2 and LCDR3 as shown in ID NO: 53, SEQ ID NO: 59 and SEQ ID NO: 67; and, HCDR1 as shown in SEQ ID NO: 10, SEQ ID NO: 26 and SEQ ID NO: 38 respectively , HCDR2 and HCDR3. The specific binding protein of claim 10, wherein the first structural domain includes two Fabs, the second structural domain has two VHs, and the first structural domain and the second structural domain are directly The second domain is connected to the C-terminus or N-terminus of the first domain by connecting or connecting via the linking peptide L to form a bispecific binding protein. The specific binding protein of claim 11, wherein the bispecific binding protein includes a short chain and a long chain, and the short chain has a structure represented by N'-VL ₁ -CL ₁ -C'; The long chain has the structure shown by N'-VH ₁ -CH ₁ -h-CH ₂ -CH ₃ -L-VH ₂ -C'; or, the short chain has N'-VL ₁ -CL ₁ -C ' has the structure shown; the long chain has the structure shown as N'-VH ₂ -L-VH ₁ -CH ₁ -h-CH ₂ -CH ₃ -C'; or the short chain has N'- The structure represented by VH ₁ -CH ₁ -C'; the long chain has the structure represented by N'-VL ₁ -CL ₁ -L-VH ₂ -CH ₂ -CH ₃ -C', wherein the VL ₁ and VH ₁ are the VL and VH of the first domain respectively, the VH ₂ is the VH of the second domain, the h is the hinge region, the L is the connecting peptide, and the CL ₁ is the first domain CL. The specific binding protein of claim 10, wherein the specific binding protein has: the long chain shown in SEQ ID NO: 122 and the short chain shown in SEQ ID NO: 119; or SEQ ID NO: 123 The long chain shown and the short chain shown in SEQ ID NO: 120; or the long chain shown in SEQ ID NO: 124 and the short chain shown in SEQ ID NO: 121; or the long chain shown in SEQ ID NO: 125 chain and the short chain shown in SEQ ID NO: 119; or the long chain shown in SEQ ID NO: 126 and the short chain shown in SEQ ID NO: 119; or the long chain shown in SEQ ID NO: 127 and SEQ ID The short chain shown in NO: 119; or the long chain shown in SEQ ID NO: 128 and the short chain shown in SEQ ID NO: 119; or the long chain shown in SEQ ID NO: 129 and the short chain shown in SEQ ID NO: 119 or the long chain shown in SEQ ID NO: 132 and the short chain shown in SEQ ID NO: 131; or the long chain shown in SEQ ID NO: 130 and the short chain shown in SEQ ID NO: 119 ; Or the long chain shown in SEQ ID NO: 133 and the short chain shown in SEQ ID NO: 131. The specific binding protein of claim 10, wherein the bispecific binding protein includes two first polypeptide chains and two second polypeptide chains to form a tetravalent symmetrical structure. An isolated nucleic acid encoding the isolation described in any one of claims 1 to 6 The antigen-binding protein or the specific binding protein or fragment thereof as described in any one of claims 10 to 14. An expression vector comprising the isolated nucleic acid of claim 15. A host cell comprising the isolated nucleic acid as described in claim 15, or the expression vector as described in claim 16. An antibody drug conjugate, which includes: the isolated antigen-binding protein as described in any one of claims 1 to 6 or the specific binding protein as described in any one of claims 10 to 14; and The isolated antigen-binding protein or the drug covalently linked to the specific binding protein. The antibody drug conjugate of claim 18, wherein the drug is selected from the group consisting of chemotherapeutic agents, radiotherapeutic agents, immunosuppressants and cytotoxic drugs. A pharmaceutical composition comprising: the isolated antigen-binding protein as described in any one of claims 1 to 6, the chimeric antigen receptor as described in claim 7, and the modified immune system as described in claim 8 Cells or multispecific antibodies as described in claim 9, specific binding proteins as described in any one of claims 10 to 14, or antibody drug conjugates as described in claim 18 or 19; and pharmaceuticals acceptable carrier. The pharmaceutical composition according to claim 20, further comprising a therapeutic agent selected from the group consisting of chemotherapeutic agents, radiotherapeutic agents, immunosuppressive agents and cytotoxic drugs. An isolated antigen-binding protein as described in any one of claims 1 to 6, a chimeric antigen receptor as described in claim 7, a modified immune cell as described in claim 8 or as claimed in claim 9 The multispecific antibody, the specific binding protein as described in any one of claims 10 to 14, the isolated nucleic acid as described in claim 15, the antibody drug conjugate as described in claim 18 or 19 Or the application of the pharmaceutical composition as described in claim 20 or 21 in the preparation of medicaments for preventing, treating and/or diagnosing immune diseases, acute and chronic inflammatory diseases, and tumor diseases. The application as described in claim 22, wherein the tumor is breast cancer, renal cell carcinoma, melanoma, colon cancer, B-cell lymphoma, melanoma, head and neck cancer, bladder cancer, gastric cancer, ovarian cancer, malignant sarcoma, Urothelial cancer, liver cancer, esophageal cancer, gastroesophageal junction cancer, nasopharyngeal cancer, small cell carcinoma Cellular lung cancer, cervical cancer, endometrial cancer, pancreatic cancer, prostate cancer, glioma, non-small cell lung cancer, acute myeloid leukemia, Hodgkin lymphoma, cutaneous squamous cell carcinoma, locally advanced and metastatic malignancies One or more of the following; the inflammatory disease is one or more of atopic dermatitis or ulcerative colitis; the immune disease is graft versus host disease, rheumatoid arthritis, systemic lupus erythematosus or One or more types of asthma. A set of pharmaceutical kits, comprising one or more pharmaceutical kits, said pharmaceutical kits comprising the isolated antigen-binding protein as described in any one of claims 1 to 6, as described in any one of claims 10 to 14 The specific binding protein is an antibody-drug conjugate as described in claim 18 or 19, or a pharmaceutical composition as described in claim 20 or 21.