CN117186235A

CN117186235A - Bispecific antibody of anti-4-1 BB/anti-EGFRvIII, preparation method and application thereof

Info

Publication number: CN117186235A
Application number: CN202210599157.5A
Authority: CN
Inventors: 戴朝辉; 陈利锋; 韩烨; 闵晨雨; 杨光; 展胜; 周嘉怡
Original assignee: Shanghai Huaiyue Biotechnology Co ltd
Current assignee: Shanghai Huaiyue Biotechnology Co ltd
Priority date: 2022-05-30
Filing date: 2022-05-30
Publication date: 2023-12-08

Abstract

The invention discloses an anti-4-1 BB/anti-EGFRvIII bispecific antibody, a preparation method and application thereof. In particular, the invention discloses a novel bispecific antibody which simultaneously targets human 4-1BB and EGFRvIII. The invention also discloses a method for preparing the bispecific antibody. The bispecific antibody of the present invention is capable of binding human 4-1BB and EGFRvIII antigens with high specificity, which has significant anti-tumor activity.

Description

Bispecific antibody resisting 4-1 BB/EGFRvIII as well as preparation method and application thereof

Technical Field

The invention belongs to the field of antibodies, and particularly relates to an anti-4-1 BB/anti-EGFRvIII bispecific antibody, and a preparation method and application thereof.

Background

4-1BB (CD 137, tumor necrosis factor receptor superfamily 9) is a costimulatory molecule widely studied and expressed on immune cells such as activated T cells and Natural Killer (NK) cells. 4-1BB mediated T cells trigger a signaling cascade that induces up-regulation of anti-apoptotic molecules and secretion of cytokines, further enhancing effector functions of immune cells. In dysfunctional T cells with reduced cytotoxic capacity, 4-1BB may mediate restoring T cell effector functions. On the other hand, on NK cells, 4-1BB signaling can increase antibody-dependent cell-mediated cytotoxicity.

The combination of 4-1BB and its ligand 4-1BBL can activate downstream NF-kappa B, JNK/SAPK, p38 MAPK and other channels, and further generate co-stimulation signals to induce the activity of CD4+ and CD8+ T cells and promote the proliferation of T cells. In addition, the binding of 4-1BB to 4-1BBL can also stimulate macrophages to produce various inflammatory cytokines, such as IL-6, TNF-alpha, etc., to further enhance anti-tumor immunity. Maintaining effective immune surveillance without eliciting an autoimmune response requires the accuracy of effector T cell responses.

4-1BB is widely used as a co-stimulatory factor domain (co-stimulatory domains, CD) for CAR-T (chimeric antigen receptor T) cell therapy. CAR-T cell therapy, with directional specificity for cancer cells, is a promising approach to treat cancer. As an intracellular co-stimulatory domain of CAR-T cells, 4-1BB can promote survival and prolonged retention of CAR-T in vivo.

Human epidermal growth factor receptor (EGFR, human epidermal growth factor receptor, also known as her-1 or Erb-B1) is a 170kDa transmembrane protein receptor that regulates a variety of cellular physiological processes, including mainly cell proliferation and differentiation, cell survival and apoptosis, angiogenesis, and cell mitosis and cell metastasis, via tyrosine kinase-mediated signal transduction pathways. EGFRvIII is one of the mutant proteins, also called de2-7EGFR, delta EGFR, mature EGFRvIIImRNA lacks 801 nucleotides from exon 2-7, the corresponding EGFRvIII protein lacks 267 amino acids (6-273) and a glycine residue is inserted, forming a unique linker peptide. Egfrvlll is specifically expressed in tumor tissue and not in normal tissue and is therefore a highly specific target in antibody therapy. The mechanism of action of egfrvlll in gliomas is not completely understood, but egfrvlll can reduce apoptosis of glioma cells and slightly enhance proliferation of glioma cells according to the existing reports.

The 4-1BB monoclonal antibody and the diabody are still in early clinical or preclinical stage at present, and the indication comprises advanced solid tumor, melanoma, non-Hodgkin lymphoma and the like. Results of a part of early clinical studies showed that 4-1BB monoclonal and diabodies have acceptable safety, tolerability and anti-tumor efficacy to some extent. However, the early 4-1BB strong agonists are toxic to the liver and if agonists have limited anti-tumor efficacy.

Given the numerous deficiencies in antigen binding activity, safety of the 4-1BB protein and EGFRvIII protein diabodies of the art, the development of novel bispecific antibodies against 4-1 BB/EGFRvIII is an urgent need for the treatment of cancer as well as autoimmune diseases.

Disclosure of Invention

The invention aims to provide a bispecific antibody for resisting 4-1 BB/EGFRvIII, and a preparation method and application thereof.

The object of the present invention is to provide an anti-4-1 BB/anti-EGFRvIII bispecific antibody having a high binding capacity to both the 4-1BB protein and the EGFRvIII protein. The bispecific antibody can be combined with EGFRvIII molecules on the surface of tumor cells and simultaneously combined with 4-1BB activated receptor molecules on the surface of immune cells, so that the immune cells are gathered around the tumor cells and stimulate the anti-tumor activity of the immune cells, the effect of cooperatively killing the tumor cells is achieved, and compared with a 4-IBB monoclonal antibody, the hepatotoxicity caused by excessive activation can be effectively reduced by using the bispecific antibody.

In order to achieve the above purpose, the invention provides an anti-4-1 BB/anti-EGFRvIII bispecific antibody, and a preparation method and application thereof. The anti-4-1 BB/anti-EGFRvIII bispecific antibody comprises: (1) An antibody against 4-1BB or an antigen-binding fragment thereof, which is a targeting molecule for 4-1BB, is capable of specifically recognizing 4-1BB protein; and (2) an anti-EGFRvIII antibody or antigen binding fragment thereof, capable of specifically recognizing an EGFRvIII protein as a targeting molecule for EGFRvIII.

In a first aspect of the invention, there is provided a bispecific antibody comprising:

a first antigen binding domain (D1); and

a second antigen binding domain (D2);

wherein the first antigen binding domain specifically binds to a target molecule 4-1BB protein;

the second antigen binding domain specifically binds to the target molecule egfrvlll protein;

the D1 is an antibody or antigen binding fragment thereof which specifically binds to 4-1BB protein; and

the D2 is an antibody or antigen-binding fragment thereof that specifically binds to egfrvlll protein.

In another preferred embodiment, the anti-4-1 BB antibody comprises a heavy chain variable region and a light chain variable region, said heavy chain variable region comprising the following three complementarity determining region CDRs:

VH-CDR1 shown in SEQ ID NO. 1,

VH-CDR2 shown in SEQ ID NO. 2, and

VH-CDR3 shown in SEQ ID No. 3;

the light chain variable region includes the following three complementarity determining region CDRs:

VL-CDR1 shown in SEQ ID No. 7,

VL-CDR2 shown in SEQ ID NO. 8, and

VL-CDR3 shown in SEQ ID No. 9.

Wherein any one of the above amino acid sequences further comprises a derivative sequence which is optionally added, deleted, modified and/or substituted with 1 or more amino acids and which is capable of retaining 4-1BB binding affinity.

In another preferred embodiment, the heavy chain variable region and/or the light chain variable region further comprises an FR region of human origin or an FR region of murine origin.

In another preferred embodiment, the heavy chain variable region sequence of the anti-4-1 BB antibody is shown in SEQ ID NO. 13; and/or the light chain variable region sequence of the anti-4-1 BB antibody is shown as SEQ ID NO. 14.

In another preferred embodiment, the anti-egfrviii antibody comprises a heavy chain variable region and a light chain variable region, said heavy chain variable region comprising the following three complementarity determining region CDRs:

VH-CDR1 shown in SEQ ID NO. 4,

VH-CDR2 shown in SEQ ID NO. 5, and

VH-CDR3 shown in SEQ ID No. 6;

VL-CDR1 shown in SEQ ID No. 10,

VL-CDR2 shown in SEQ ID NO. 11, and

VL-CDR3 shown in SEQ ID NO. 12.

Wherein any one of the above amino acid sequences further comprises a derivative sequence which is optionally added, deleted, modified and/or substituted with 1 or more amino acids and which is capable of retaining the binding affinity of egfrvlll.

In another preferred embodiment, the heavy chain variable region sequence of the anti-EGFRvIII antibody is shown in SEQ ID NO. 15; and/or the light chain variable region sequence of the anti-EGFRvIII antibody is shown in SEQ ID NO. 16.

In another preferred embodiment, the EGFRvIII protein is a human EGF deletion mutant receptor type III (EGFRvIII) protein.

In another preferred embodiment, the antibody comprises a heavy chain and a light chain, wherein the heavy chain further comprises a heavy chain constant region; the light chain further comprises a light chain constant region.

In another preferred embodiment, the heavy chain constant region and the light chain constant region are each independently of human or murine origin.

In another preferred embodiment, the antibody comprises: an animal-derived antibody (e.g., a murine antibody), a chimeric antibody, a humanized antibody, a fully human antibody, or a combination thereof.

In another preferred embodiment, the antibody comprises a single chain antibody or a double chain antibody.

In another preferred embodiment, the antibody is a monoclonal antibody.

In another preferred embodiment, the antibody is a partially or fully humanized monoclonal antibody.

In another preferred embodiment, the structure of the antigen binding fragment is selected from the group consisting of: (i) Fab fragments; (ii) F (ab') ₂ Fragments; (iii) Fd fragment; (iv) Fv fragments; (v) a single chain Fv (scFv) molecule; or (vi) a dAb fragment.

In another preferred embodiment, the antigen binding fragment is a nanobody.

In another preferred embodiment, the antigen binding fragment comprises a heavy chain variable region and a light chain variable region.

In another preferred embodiment, the antigen binding fragment comprises a single chain variable region fragment (scFv), a double chain variable region fragment (dcFv).

In another preferred embodiment, said D1 and said D2 are linked by a linker peptide

In another preferred embodiment, the linker peptide comprises an antibody constant region sequence or an antibody variable region sequence.

In another preferred embodiment, the connecting peptide is (G4S) _n Preferably, (G4S) _n G, where n is a positive integer (e.g., 1, 2, 3, 4, 5, or 6), preferably n=3.

In another preferred embodiment, D1 is an anti-4-1 BB antibody and D2 is linked to a region of D1 selected from the group consisting of: heavy chain variable regions, heavy chain constant regions, light chain variable regions, light chain constant regions, or combinations thereof.

In another preferred embodiment, D1 is an anti-4-1 BB antibody and D2 is an anti-EGFRvIII antibody scFv; and D2 is linked to the heavy chain constant region end of D1 by a linker peptide.

In another preferred embodiment, D2 is an anti-egfrvlll antibody and D1 is linked to a region of D2 selected from the group consisting of: heavy chain variable regions, heavy chain constant regions, light chain variable regions, light chain constant regions, or combinations thereof.

In another preferred embodiment, D2 is an anti-EGFRvIII antibody and D1 is an scFv of an anti-4-1 BB antibody; and D1 is linked to the heavy chain constant region end of D1 by a linking peptide.

In another preferred embodiment, the bispecific antibody has the structure of formula I from N-terminus to C-terminus:

wherein,

d are each independently no or an anti-4-1 BB antibody or antigen-binding fragment thereof, preferably scFv against the 4-1BB antibody, and at least one D is an anti-4-1 BB antibody or antigen-binding fragment thereof;

l1, L2, L3, L4 are each independently a bond or a linker;

VL represents the light chain variable region of an anti-egfrvlll antibody;

CL represents the light chain constant region of an anti-egfrvlll antibody;

VH represents the heavy chain variable region of an anti-egfrvlll antibody;

CH represents the heavy chain constant region of an anti-egfrvlll antibody;

or (b)

D are each independently no or an anti-egfrvlll antibody or antigen binding fragment thereof, preferably scFv of an anti-egfrvlll antibody, and at least one D is an anti-egfrvlll antibody or antigen binding fragment thereof;

L1, L2, L3, L4 are each independently a bond or a linker;

VL represents the light chain variable region of an anti-4-1 BB antibody;

CL represents the light chain constant region of an anti-4-1 BB antibody;

VH represents the heavy chain variable region of an anti-4-1 BB antibody;

CH represents the heavy chain constant region of an anti-4-1 BB antibody;

"-" represents disulfide or covalent bonds;

"-" represents a peptide bond;

wherein the bispecific antibody has an activity of simultaneously binding to 4-1BB and binding to EGFRVIII.

In another preferred embodiment, the bispecific antibody further comprises an active fragment and/or derivative of the bispecific antibody, wherein the active fragment and/or derivative retains 70-100% (e.g. 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100%) of the anti-4-1 BB activity and 70-100% of the anti-OX 40 activity of the bispecific antibody.

In another preferred embodiment, the derivative of the antibody has at least 85% sequence identity to the antibody of the invention.

In another preferred embodiment, the derivative of the antibody is a sequence which retains at least 85% identity after deletion, insertion and/or substitution of one or more amino acids of the antibody of the invention.

In another preferred embodiment, the derivative of the antibody has at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to an antibody of the invention.

In another preferred example, the anti-4-1 BB/anti-EGFRvIII bispecific antibody may comprise an anti-4-1 BB antibody or binding fragment thereof as a 4-1BB targeting molecule,

in another preferred embodiment, the anti-4-1 BB antibody or binding fragment thereof may specifically bind to the 4-1BB protein.

In another preferred embodiment, the anti-4-1 BB antibody or binding fragment thereof is capable of enhancing an immune response and/or treating a tumor. The anti-4-1 BB antibodies or binding fragments thereof are characterized by a cross-linking dependence that significantly reduces liver toxicity and maintains an enhanced immune response and/or anti-tumor effect as compared to pre-existing 4-1BB antibodies.

In another preferred embodiment, the cross-linking means that the antibody can be enriched by binding to other mediators, including Fc fragments that specifically bind to antibodies, including but not limited to anti-Fc antibodies, cell surface Fc receptors, via its own Fc fragment.

In another preferred embodiment, the anti-4-1 BB/anti-EGFRvIII bispecific antibody is in the form of an IgG-scFv.

Wherein the bispecific antibody against 4-1 BB/anti-EGFRvIII may comprise: an IgG component comprising an anti-4-1 BB antibody, or binding fragment thereof, having a heavy chain variable region sequence set forth in SEQ ID No. 13; and the light chain variable region sequence of the antibody is shown as SEQ ID NO. 14. An scFv component comprising an anti-egfrvlll antibody or binding fragment thereof comprising an amino acid sequence having a heavy chain variable region sequence as shown in SEQ ID No. 15 or having at least 90% sequence homology; and a light chain variable region sequence as shown in SEQ ID NO. 16 or an amino acid sequence having at least 90% sequence homology.

In a second aspect of the present invention, there is provided a recombinant protein having: (i) A bispecific antibody according to the first aspect of the invention; and (ii) optionally a tag sequence to aid expression and/or purification.

In another preferred embodiment, the recombinant protein (or polypeptide) comprises a fusion protein.

In another preferred embodiment, the recombinant protein is a monomer, dimer, or multimer

In a third aspect of the invention, there is provided an isolated polynucleotide encoding a bispecific antibody according to the first aspect of the invention or a recombinant protein according to the second aspect of the invention.

In another preferred embodiment, the nucleic acid encoding the heavy chain variable region of the anti-4-1 BB antibody is as set forth in SEQ ID NO: shown at 17; the nucleic acid encoding the light chain variable region of the anti-4-1 BB antibody is shown in SEQ ID NO: shown at 18.

In another preferred embodiment, the nucleic acid encoding the heavy chain variable region of the anti-egfrvlll antibody is as set forth in SEQ ID NO: 19; the nucleic acid for encoding the light chain variable region of the anti-EGFRvIII antibody is shown in a sequence table SEQ ID NO: shown at 20.

In a fourth aspect of the invention there is provided a vector comprising a polynucleotide according to the third aspect of the invention.

In another preferred embodiment, the vector is an expression vector.

In another preferred embodiment, the vector comprises a plasmid, phage, yeast plasmid, plant cell virus, mammalian cell virus such as adenovirus, retrovirus, or other vector.

In a fifth aspect of the invention there is provided a genetically engineered host cell, characterized in that said host cell comprises a vector or genome according to the fourth aspect of the invention having integrated therein a polynucleotide according to the third aspect of the invention.

In a sixth aspect of the present invention, there is provided a method for preparing a bispecific antibody according to the first aspect of the present invention, comprising the steps of:

(i) Culturing the host cell according to the fifth aspect of the invention under suitable conditions to obtain a mixture comprising the bispecific antibody according to the first aspect of the invention; and

(ii) Purifying and/or isolating the mixture obtained in step (i) to obtain the bispecific antibody according to the first aspect of the invention.

In another preferred embodiment, the purification may be performed by protein a affinity column purification to isolate the antibody of interest.

In another preferred embodiment, the purity of the purified and isolated target antibody is greater than 95%, greater than 96%, greater than 97%, greater than 98%, greater than 99%, preferably 100%.

In a seventh aspect of the invention there is provided the use of an active ingredient selected from the group consisting of: a bispecific antibody according to the first aspect of the invention; the recombinant protein according to the second aspect of the invention, or a combination thereof, the active ingredient being used for (a) preparing a detection reagent or kit; and/or (b) preparing a medicament for preventing and/or treating related diseases.

In another preferred embodiment, the related disorder is selected from the group consisting of: cancer, autoimmune disease, viral infection, graft versus host disease, inflammatory disease, immune disease, or a combination thereof.

In another preferred embodiment, the cancer comprises a solid tumor, a hematological cancer.

In another preferred embodiment, the solid tumor is selected from the group consisting of: bladder cancer, biliary tract cancer, brain cancer, breast cancer, colon cancer, esophageal cancer, gastric cancer, glioma, head cancer, leukemia, liver cancer, lung cancer, lymphoma, myeloma, neck cancer, ovarian cancer, melanoma, pancreatic cancer, kidney cancer, salivary cancer, gastric cancer, thymus epithelial cancer, and thyroid cancer, or a combination thereof.

In another preferred embodiment, the autoimmune disease is selected from the group consisting of: systemic lupus erythematosus, rheumatoid arthritis, ulcerative colitis, type I diabetes, psoriasis, multiple sclerosis, or a combination thereof.

In another preferred embodiment, the detection reagent or kit is used to detect 4-1BB in a sample.

In another preferred embodiment, the detection reagent or kit is used to detect egfrvlll in a sample.

In another preferred embodiment, the detection reagent or kit is used to detect 4-1BB in a sample and EGFRvIII in the sample simultaneously.

In an eighth aspect of the present invention, there is provided a pharmaceutical composition characterized in that it comprises:

(I) A bispecific antibody according to the first aspect of the invention; and

(II) a pharmaceutically acceptable carrier.

In another preferred embodiment, the pharmaceutical composition may be used for the treatment and/or prevention of cancer.

In another preferred embodiment, the pharmaceutical composition further comprises an additional antineoplastic agent.

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

In another preferred embodiment, the anti-neoplastic agent comprises paclitaxel, doxorubicin, cyclophosphamide, axitinib, lenvatinib, or pembrolizumab.

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

In another preferred embodiment, the pharmaceutical composition is a liquid formulation.

In another preferred embodiment, the pharmaceutical composition is an injection.

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

In another preferred embodiment, the parenteral administration comprises intravenous injection, intravenous drip, subcutaneous injection, topical injection, intramuscular injection, intratumoral injection, intraperitoneal injection, intracranial injection, or intracavity injection.

In a ninth aspect of the invention, there is provided an immunoconjugate comprising:

(a) A bispecific antibody according to the first aspect of the invention; and

(b) A coupling moiety selected from the group consisting of: a detectable label, drug, toxin, cytokine, radionuclide, enzyme, or a combination thereof.

In another preferred embodiment, the conjugate moiety is selected from the group consisting of: fluorescent or luminescent labels, radioactive labels, MRI (magnetic resonance imaging) or CT (computed tomography) contrast agents, or enzymes capable of producing a detectable product, radionuclides, biotoxins, cytokines (e.g., IL-2, etc.), antibodies, antibody Fc fragments, antibody scFv fragments, gold nanoparticles/nanorods, viral particles, liposomes, nanomagnetic particles, prodrug-activating enzymes (e.g., DT-diaphorase (DTD) or biphenyl hydrolase-like proteins (BPHL)), chemotherapeutic agents (e.g., cisplatin), or any form of nanoparticle, etc.

In another preferred embodiment, the antibody moiety is coupled to the coupling moiety via a chemical bond or linker.

In a tenth aspect of the invention there is provided the use of a bispecific antibody according to the first aspect of the invention, a recombinant protein according to the second aspect of the invention, or an immunoconjugate according to the ninth aspect of the invention, for the preparation of (a) a detection reagent or kit; and/or (b) preparing a medicament for preventing and/or treating related diseases.

In another preferred embodiment, the tumor is a tumor that highly expresses 4-1BB and/or EGFRVIII.

In another preferred embodiment, the medicament or formulation is for the manufacture of a medicament or formulation for the prevention and/or treatment of a disease associated with 4-1BB and/or EGFRVIII (positive for expression).

In another preferred embodiment, the antibody is in the form of A Drug Conjugate (ADC).

In another preferred embodiment, the detection reagent or kit is used for diagnosing 4-1BB and/or EGFRVIII related diseases.

In another preferred embodiment, the detection reagent or kit is used to detect 4-1BB and/or EGFRVIII protein in a sample.

In another preferred embodiment, the detection reagent is a detection chip.

In an eleventh aspect of the invention, there is provided a composition and method for in vitro detection (including diagnostic or non-diagnostic) of 4-1BB, EGFRvIII or simultaneous detection of 4-1BB, EGFRvIII in a sample, the composition comprising an anti-4-1 BB/anti-EGFRvIII bispecific antibody; the method comprises the steps of:

(1) Contacting the sample in vitro with a bispecific antibody according to the first aspect of the invention;

(2) Detecting whether an antigen-antibody complex is formed, wherein the formation of a complex indicates the presence of 4-1BB and/or EGFRvIII in the sample.

In another preferred embodiment, the composition comprises a bispecific antibody.

In another preferred embodiment, the detection method comprises the use of a bispecific antibody for the diagnosis of a disease associated with 4-1BB, EGFRvIII, or both.

In another preferred example, the disease associated with 4-1BB, EGFRvIII or both may be a disease associated with activation of 4-1BB and/or overexpression of EGFRvIII.

In a twelfth aspect of the present invention, there is provided a method for producing a recombinant polypeptide, the method comprising:

(a) Expressing in a cell, under conditions suitable for expression, nucleotides encoding the heavy and light chains of the bispecific antibody;

(b) Isolating from the culture a recombinant polypeptide which is a bispecific antibody according to the first aspect of the invention or a recombinant protein according to the second aspect of the invention.

In a thirteenth aspect of the invention there is provided a method of treating a tumour, cancer or autoimmune disease comprising the steps of: administering to a subject in need thereof a safe and effective amount of a bispecific antibody according to the first aspect of the present invention, or a pharmaceutical composition according to the eighth aspect of the present invention, or an immunoconjugate according to the ninth aspect of the present invention, or a combination thereof.

It is understood that within the scope of the present invention, the above-described technical features of the present invention and technical features specifically described below (e.g., in the examples) may be combined with each other to constitute new or preferred technical solutions. And are limited to a space, and are not described in detail herein.

Drawings

FIG. 1 shows ELISA detection of binding of full length antibody PE0116 to h4-1BB protein.

FIG. 2 shows FACS detection of binding of scFv single chain and full length antibodies to EGFRvIII antibody 043-1 to CHO-K1-hEGFRvIII.

FIG. 3a shows the FACS detection of binding of anti-4-1 BB/anti-EGFRvIII bispecific antibody (HY 1001007) to CHO-K1-h4-1 BB. Wherein PE0116 is an anti-4-1 BB antibody.

FIG. 3b shows the FACS detection of binding of anti-4-1 BB/anti-EGFRvIII bispecific antibody (HY 1001007) to CHO-K1-blank. Wherein PE0116 is an anti-4-1 BB antibody.

FIG. 3c shows the FACS detection of binding of anti-4-1 BB/anti-EGFRvIII bispecific antibody (HY 1001007) to CHO-K1-c4-1 BB. Wherein PE0116 is an anti-4-1 BB antibody.

FIG. 4a shows FACS detection of binding of anti-4-1 BB/anti-EGFRvIII bispecific antibody (HY 1001007) to CHO-K1-hEGFRvIII. Wherein 043-1IgG4 is an anti-EGFRvIII antibody.

FIG. 4b shows the FACS detection of binding of anti-4-1 BB/anti-EGFRvIII bispecific antibody (HY 1001007) to CHO-K1-blank. Wherein 043-1IgG4 is an anti-EGFRvIII antibody.

FIG. 5 shows a reporter gene assay for activation of NF-. Kappa.B downstream promoters by anti-4-1 BB/anti-EGFRvIII bispecific antibody (HY 1001007). Wherein PE0116 is an anti-4-1 BB antibody.

FIG. 6 shows the effect of anti-4-1 BB/anti-EGFRvIII bispecific antibody (HY 1001007) on IFN-gamma secretion in a T lymphocyte stimulation assay. Wherein PE0116 is an anti-4-1 BB antibody, and PE0116 cross-linked is an anti-4-1 BB antibody after artificial cross-linking.

FIG. 7a shows the lysis rate of target cells in an anti-4-1 BB/anti-EGFRvIII bispecific antibody (HY 1001007) mediated PBMC killing CT26-EGFRvIII assay, wherein HY0101 is the 4-1BB antibody PE0116 and HY0502 is the EGFRvIII antibody 043-1IgG4.

FIG. 7b shows the concentration of IFN-. Gamma.secretion in anti-4-1 BB/anti-EGFRvIII bispecific antibody (HY 1001007) mediated PBMC killing CT26-EGFRvIII assay, wherein HY0101 is the 4-1BB antibody PE0116 and HY0502 is the EGFRvIII antibody 043-1IgG4.

FIG. 8a shows the tumor volume changes of different groups of BALB/c-hCD137 mice subcutaneously vaccinated with CT26 colon cancer model.

FIG. 8b shows the experimental results of anti-4-1 BB/anti-EGFRvIII bispecific antibody (HY 1001007) on mouse CT26 tumor growth.

Figure 8c shows tumor volumes of mice individuals of different antibody-treated groups.

FIG. 9 shows the structure of a bispecific antibody of the present invention.

Detailed Description

The present inventors have studied extensively and intensively, and have unexpectedly developed for the first time an anti-4-1 BB/anti-EGFRvIII bispecific antibody having a high binding capacity for both the 4-1BB protein and the EGFRvIII protein. The bispecific antibody can be combined with EGFRvIII molecules on the surfaces of tumor cells and simultaneously combined with 4-1BB activated receptor molecules on the surfaces of immune cells, so that the immune cells are gathered around the tumor cells and stimulate the anti-tumor activity of the immune cells, the effect of cooperatively killing the tumor cells is achieved, and compared with a 4-IBB monoclonal antibody, the hepatotoxicity caused by overactivation can be effectively reduced by using the bispecific antibody. The present invention has been completed on the basis of this finding.

Terminology

The terms "peptide," "polypeptide," "protein" each refer to a molecule formed of two or more amino acid residues joined to one another by peptide bonds. These terms include, for example, natural and artificial protein sequences, protein fragments and polypeptide analogs (e.g., mutations, variants and fusion proteins), and proteins that are post-translationally modified or otherwise covalently or non-covalently modified. The peptide, polypeptide or protein may be monomeric or polymeric.

The term "immunoglobulin" is a tetrameric molecule consisting of two identical pairs of polypeptide chains, each pair having one "light" chain (about 25 kDa) and one "heavy" chain (about 50-70 kDa). The amino-terminal portion of each chain comprises a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition; the carboxy-terminal portion of each chain is composed of a constant region responsible for effector function. Human light chains are classified as either kappa or lambda light chains. Heavy chains were classified as mu, delta, gamma, alpha or epsilon and the isotypes of antibodies were defined as IgM, igD, igG, igA and IgE, respectively.

In light and heavy chains, the variable and constant regions are joined by a "J" region of about 12 or more amino acids, and the heavy chain also includes a "D" region of about 10 or more amino acids. The variable regions of each light/heavy chain pair form an antibody binding site such that the intact immunoglobulin has two binding sites.

The variable regions of an immunoglobulin chain exhibit the same general structure, namely relatively conserved Framework Regions (FR) joined by three hypervariable regions (also known as complementarity determining regions or CDRs). From the N-terminus to the C-terminus, both the light and heavy chains comprise domains FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4.

The term "antibody" refers to an intact immunoglobulin or an antigen-binding portion thereof that competes for specific binding with the intact antibody. In one embodiment, an antibody comprises a heavy chain variable domain, a light chain constant region (CL), and heavy chain constant regions CH1, CH2, and CH3-. The antigen binding portion of an antibody may be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of the intact antibody. The antigen binding portion "antibody fragment" or "antigen binding fragment of an antibody" may be selected from the group consisting of Fab, fab ', F (ab') 2, fv fragments, linear antibodies, domain antibodies (dAbs) and Complementarity Determining Region (CDR) fragments, single chain antibodies (scFv), chimeric antibodies, diabodies, trisomy, tetrasomy, and polypeptides. They each contain at least a portion of an immunoglobulin and are sufficient to accomplish specific antigen binding.

The term "hybridoma" refers to a cell line that is produced by fusing a B cell with a cancer cell line (typically a myeloma cell) to extend the time that the B cell can produce a particular monoclonal antibody of clinical or other research interest.

Fab is a monovalent fragment consisting of VL, VH, CL and CH1 domains; f (ab') 2 is a bivalent fragment of two Fab linked by a disulfide bridge at the hinge region; fd is a fragment consisting of VH and CH1 domains; fv is a fragment with VL and VH domains in the light/heavy single arm. scFv are also known as single chain antibodies, in which the VL and VH regions are linked by a linker (e.g., a synthetic sequence of amino acid residues, (GGGGS) 3) to form a continuous protein chain, wherein the linker is long enough to allow the protein chain to fold upon itself and form a monovalent antigen binding site.

The terms "Fc polypeptide", "Fc constant structure", "Fc fragment" refer to a polypeptide derived from the natural or mutant form of an antibody Fc region, and the presence of a truncated form of the polypeptide comprising only a hinge region that promotes dimerization is also included within this concept. Fusion proteins containing an Fc portion (and oligomers formed therefrom) offer the possibility of being purified by affinity chromatography such as protein A or protein G columns. In one embodiment, "hFc" refers to an Fc fragment derived from a human immunoglobulin.

The term "human antibody" includes all antibodies having one or more variable and constant region domains derived from human immunoglobulins. In one embodiment, all domains of an antibody are derived from a human immunoglobulin (referred to as a "fully human antibody"). These antibodies can be prepared in a variety of ways, examples of which are described below, including by immunization with an antigen of interest from a mouse that can express antibodies derived from human heavy and/or light chain encoding genes by genetic modification.

The term "humanized antibody" sequences are derived from antibody sequences of non-human species in which one or more amino acids have been substituted, deleted and/or added. Humanized antibodies are less susceptible to inducing an immune response and/or induce a less severe immune response when administered to a human subject than antibodies of non-human species. In one embodiment, the constant domain from a human antibody is fused to a humanized variable domain in which certain amino acids are mutated.

The term "chimeric antibody" refers to an antibody that comprises one or more regions from one antibody and one or more regions from another antibody or antibodies.

Antibodies of the invention may be full length proteins (e.g., igG1, igG2a, igG2b, or IgG2 c), or may be protein fragments (e.g., fab, F (ab'), sdabs, scFv fragments) comprising an antigen-antibody binding domain or antigen-binding fragment.

Non-limiting examples of antigen binding fragments, as used herein, include: (i) Fab fragments; (ii) F (ab') ₂ Fragments; (iii) Fd fragment; (iv) Fv fragments; (v) a single chain Fv (scFv) molecule; (vi) a dAb fragment; and (vii) a minimal recognition unit consisting of amino acid residues mimicking an antibody hypervariable region (e.g., an independent Complementarity Determining Region (CDR) such as a CDR3 peptide) or a constrained FR3-CDR3-FR4 peptide. In another preferred embodiment, the antibody of the present invention or a fragment thereof The antigen binding fragment is selected from the group consisting of: camel-derived single domain antibodies, scFv dimers, bsFv, dsFv, dsFv2, dsFv-dsFv ', fv fragments, fab ', F (ab ') ₂ A ds bifunctional antibody, nanobody, domain antibody, or diabody.

As used herein, the terms "single domain antibody", "nanobody" have the same meaning, referring to the variable region of a cloned antibody heavy chain, a single domain antibody consisting of only one heavy chain variable region is constructed, which is the smallest antigen binding fragment with complete function. Typically, after an antibody is obtained which naturally lacks the light and heavy chain constant region 1 (CH 1), the variable region of the heavy chain of the antibody is cloned, and a single domain antibody consisting of only one heavy chain variable region is constructed.

As used herein, the term "framework region" (FR) refers to the amino acid sequence inserted between CDRs, i.e., refers to those portions of the light and heavy chain variable regions of immunoglobulins that are relatively conserved among different immunoglobulins in a single species. The light and heavy chains of immunoglobulins each have four FRs, designated FR1-L, FR2-L, FR3-L, FR-L and FR1-H, FR2-H, FR3-H, FR-H, respectively. Accordingly, the light chain variable domain may thus be referred to as (FR 1-L) - (CDR 1-L) - (FR 2-L) - (CDR 2-L) - (FR 3-L) - (CDR 3-L) - (FR 4-L) and the heavy chain variable domain may thus be denoted as (FR 1-H) - (CDR 1-H) - (FR 2-H) - (CDR 2-H) - (FR 3-H) - (CDR 3-H) - (FR 4-H). Preferably, the FR of the invention is a human antibody FR or a derivative thereof which is substantially identical to a naturally occurring human antibody FR, i.e. has a sequence identity of up to 85%, 90%, 95%, 96%, 97%, 98% or 99%.

As used herein, the term "affinity" is theoretically defined by equilibrium association between an intact antibody and an antigen. The affinity of the subject diabodies can be assessed or determined by KD values (dissociation constants) (or other assay means), such as by biofilm layer interference techniques (Bio-layer interferometry BLI), using a FortebioRed96 instrument.

As used herein, the term "linker" refers to one or more amino acid residues inserted into an immunoglobulin domain that provide sufficient mobility for the domains of the light and heavy chains to fold into an exchanged double variable region immunoglobulin.

Examples of suitable linkers include mono glycine (Gly), or serine (Ser) residues, the identity and sequence of the amino acid residues in the linker may vary with the type of secondary structural element that needs to be achieved in the linker.

Bispecific antibodies

Bispecific antibodies (Bispecific Antibody, bsAb) are unnatural antibodies that target two different antigens or proteins simultaneously, block two different signaling pathways, elicit specific immune responses, and their specificity and bifunctional roles in tumor immunotherapy are becoming increasingly important, as research hotspots in antibody engineering for tumor therapy in the world today. Research shows that bispecific antibodies mainly mediate the killing of immune cells to tumors in tumor immunotherapy; the double targets are combined to block the double signal paths, so that a unique or overlapped function is exerted, and drug resistance can be effectively prevented; has strong specificity, targeting property and reduced off-target toxicity; the advantages of effectively reducing the treatment cost and the like are achieved, so that the bispecific antibody medicament can reduce the escape probability of tumor cells, clear away the tumor cells and improve the curative effect.

Bispecific antibodies can be prepared by means of double hybridoma cells, chemical coupling, recombinant gene technology, and the like, wherein the recombinant gene technology has strong flexibility in terms of binding sites, yield, and the like. According to incomplete statistics, more than 60 bispecific antibodies exist at present, and according to the characteristics and structural differences, the bispecific antibody structure mainly comprises two structures of a bispecific antibody (IgG-like bispecific antibody with Fc-mediated effect function) containing an Fc fragment and a bispecific antibody (non-IgG-like bispecific antibody) without the Fc fragment, and the bispecific antibody has the advantages of small molecular weight, low immunogenicity and the like through antigen binding force.

As used herein, the terms "bispecific antibody", "bifunctional antibody", "inventive diabody", "bifunctional fusion antibody" are used interchangeably and refer to an anti-4-1 BB/egfrvlll bispecific antibody that binds both 4-1BB and egfrvlll.

In one embodiment, an anti-4-1 BB antibody, or binding fragment thereof, comprises heavy and light chain variable regions having the amino acid sequence of VH-CDR1 shown in SEQ ID NO. 1, VH-CDR2 shown in SEQ ID NO. 2, and VH-CDR3 shown in SEQ ID NO. 3; the light chain variable region has the amino acid sequence of VL-CDR1 shown in SEQ ID NO. 7, VL-CDR2 shown in SEQ ID NO. 8 and VL-CDR3 shown in SEQ ID NO. 9; wherein the anti-EGFRvIII antibody or binding fragment thereof comprises a heavy chain and a light chain variable region, said heavy chain variable region having the amino acid sequence of VH-CDR1 shown in SEQ ID NO. 4, VH-CDR2 shown in SEQ ID NO. 5, and VH-CDR3 shown in SEQ ID NO. 6; the light chain variable region has the amino acid sequence of VL-CDR1 shown in SEQ ID NO. 10, VL-CDR2 shown in SEQ ID NO. 11 and VL-CDR3 shown in SEQ ID NO. 12.

A "multispecific antibody" is an antibody that recognizes more than one epitope on one or more antigens. A subclass of such antibodies is "bispecific antibodies" which recognize two different epitopes on the same or different antigens.

In one embodiment, a bispecific antibody against 4-1 BB/anti-EGFRvIII is provided, which is in the form of an IgG-scFv. Wherein the bispecific antibody against 4-1 BB/anti-EGFRvIII may comprise: an IgG component comprising an anti-4-1 BB antibody or binding fragment thereof, characterized in that the heavy chain variable region sequence of said antibody is shown in SEQ ID No. 13; and the light chain variable region sequence of the antibody is shown as SEQ ID NO. 14. An scFv component comprising an anti-egfrvlll antibody or binding fragment thereof, characterized in that said single chain antibody has an amino acid sequence as shown in SEQ ID No. 16 or an amino acid sequence having at least 90% sequence homology.

The variant forms of the diabody include: homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants, proteins encoded by DNA which hybridizes under high or low stringency conditions with the encoding DNA of an antibody of the invention, and polypeptides or proteins obtained using antisera raised against an antibody of the invention.

In the present invention, the diabodies of the present invention also include conservative variants thereof, meaning that up to 10, preferably up to 8, more preferably up to 5, most preferably up to 3 amino acids are replaced by amino acids of similar or similar nature to the amino acid sequence of the diabodies of the present invention to form a polypeptide. These conservatively variant polypeptides are preferably generated by amino acid substitutions according to Table A.

Table A

Vector, plasmid and expression vector

"vector" refers to a nucleic acid sequence used to introduce a nucleic acid fragment linked thereto into a cell. "plasmid" is one type of which refers to a linear or circular double stranded DNA molecule into which additional nucleic acid fragments may be ligated. Another type of vector is a viral vector (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses), in which additional DNA segments can likewise be introduced. Certain vectors are capable of autonomous replication upon introduction into a host cell (e.g., bacterial vectors comprising a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. An "expression vector" is a vector that can be used for protein expression after it has been introduced into a cell.

The term "purification" refers to the isolation of a protein (e.g., an antibody) that is substantially free of other cellular material and/or chemicals. In one embodiment, the antibody is isolated from other proteins in the cell expression supernatant of the same species and is made substantially free of other proteins.

The term "4-1BB" refers to CD137 or TNFRSF9 (TNF receptor 25 superfamily member 9), which is expressed after immune cells (innate and adaptive immune cells) are activated. In one embodiment, the 4-1BB may be derived from a protein sequence such as mammalian Chinesemedicine (NCBI accession number NP-001552). In another embodiment, 4-1BB may be derived from the monkey source 4-1BB amino acid sequence (Uniprot database accession number A9YYE 7).

The term "EGFRvIII" is a mutant protein of the HER (EGFR, human epidermal growth factor receptor, also known as her-1 or Erb-B1), also known as de2-7EGFR, ΔEGFR, deleted 801 nucleotides of exon 2-7 from the mature EGFRvIII mRNA, and the corresponding EGFRvIII protein deleted 267 amino acids (6-273) with a glycine residue inserted, forming a unique linker peptide. In the examples, the amino acid sequence of the extracellular domain of the human EGFRvIII protein is derived from (NCBI: NP-005219.2).

The term "T lymphocyte" refers to one of the important cell types that make up the immune system, playing a central role in the adaptive immune response. T cells can be distinguished from other lymphocytes by the presence of T Cell Receptors (TCRs) on their cell surfaces. Immune activated T cells secrete and release "INF- γ", regulate T cell differentiation, and enhance their tumor killing efficacy.

Detection uses refer to the provision of a bispecific antibody that can be used in detection applications, e.g. for detecting a sample, thereby providing diagnostic information.

Samples (samples) employed in the examples include cell, tissue samples and biopsy specimens. The term "biopsy" as used herein shall include all kinds of biopsies known to a person skilled in the art. Thus biopsies used in the present invention may include tissue samples prepared, for example, by endoscopic methods or by puncture or needle biopsy of an organ.

The sample used includes a fixed or preserved cell or tissue sample.

Kits means that a kit is provided that comprises an antibody (or fragment thereof) of the invention.

In embodiments, the kit further comprises a container, instructions for use, buffers, and the like.

In another embodiment, the antibodies of the invention may be immobilized to a detection plate.

In another embodiment, there is also provided a method of detecting cells overexpressing 4-1BB protein and/or EGFRvIII, comprising the steps of: the protein contacts with the sample to be detected in vitro, and the combination of the protein and the sample to be detected is detected.

The meaning of overexpression is conventional in the art and refers to overexpression of the 4-1BB protein in the sample to be tested (due to increased transcription, post-transcriptional processing, translation, post-translational processing and altered protein degradation), as well as to local overexpression and increased functional activity due to altered protein transport patterns (increased nuclear localization), as in the case of increased enzymatic hydrolysis of the substrate.

The detection means for binding is conventional in the art, preferably FACS detection.

In another embodiment, a composition for detecting cells overexpressing 4-1BB and/or EGFRvIII proteins is provided, comprising as an active ingredient the above-described bispecific antibody. Preferably, it further comprises a compound composed of a functional fragment of the above protein as an active ingredient.

The use of the active ingredient refers to therapeutic and diagnostic uses.

In one embodiment, a bispecific antibody against 4-1 BB/anti-EGFRvIII is provided that is capable of blocking EGFR pathway while activating anti-tumor activity of immune cells such as T cells, and thus exhibits improved efficacy in cancer treatment.

In another embodiment, a pharmaceutical composition comprising the bispecific antibody described above is provided. The pharmaceutical composition may also include a pharmaceutically acceptable carrier. The pharmaceutical composition can be used to enhance immune response and treat or prevent diseases associated with 4-1BB, EGFRvIII, or both.

The disease associated with 4-1BB, EGFRvIII, or both, may be selected from cancer, infectious diseases, autoimmune reactions, neurological disorders, and the like.

The cancer may be a solid tumor or a hematological tumor.

In another embodiment, there is provided the use of a bispecific antibody or pharmaceutical composition against 4-1 BB/anti-EGFRvIII in the treatment and/or prevention of cancer. The disease associated with 4-1BB, EGFRvIII, or both may be a disease associated with activation of 4-1BB and/or overexpression of EGFRvIII.

Pharmaceutical composition

The invention also provides a composition. Preferably, the composition is a pharmaceutical composition comprising the bispecific antibody or active fragment thereof or fusion protein thereof of the present invention as described above, and a pharmaceutically acceptable carrier. Typically, these materials are formulated in a nontoxic, inert and pharmaceutically acceptable aqueous carrier medium, wherein the pH is typically about 5 to 8, preferably about 6 to 8, although the pH may vary depending on the nature of the material being formulated and the condition being treated. The formulated pharmaceutical compositions may be administered by conventional routes including, but not limited to: intravenous injection, intravenous drip, subcutaneous injection, local injection, intramuscular injection, intratumoral injection, intraperitoneal injection (e.g., intraperitoneal), intracranial injection, or intracavity injection.

The pharmaceutical composition of the invention can be directly used for binding to PD-L1 protein molecules or PD-L1, and thus can be used for treating tumors. In addition, other therapeutic agents may also be used simultaneously.

The pharmaceutical compositions of the invention contain a safe and effective amount (e.g., 0.001-99wt%, preferably 0.01-90wt%, more preferably 0.1-80 wt%) of the nanobody (or conjugate thereof) of the invention as described above, and a pharmaceutically acceptable carrier or excipient. Such vectors include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical formulation should be compatible with the mode of administration. The pharmaceutical compositions of the invention may be formulated as injectables, e.g. by conventional means using physiological saline or aqueous solutions containing glucose and other adjuvants. The pharmaceutical compositions, such as injections, solutions are preferably manufactured under sterile conditions. The amount of active ingredient administered is a therapeutically effective amount, for example, from about 10 micrograms per kilogram of body weight to about 50 milligrams per kilogram of body weight per day. In addition, the polypeptides of the invention may also be used with other therapeutic agents.

In the present invention, bispecific antibodies alone can be used to obtain the best target response by adjusting the dosing regimen. For example, a single administration, or multiple administrations over a period of time, or the dosage may be proportionally reduced or increased as the degree of urgency of the treatment situation.

When a pharmaceutical composition is used, a safe and effective amount of the immunoconjugate is administered to the mammal, wherein the safe and effective amount is typically at least about 10 micrograms per kilogram of body weight, and in most cases no more than about 50 milligrams per kilogram of body weight, preferably the dose is from about 10 micrograms per kilogram of body weight to about 10 milligrams per kilogram of body weight. Of course, the particular dosage should also take into account factors such as the route of administration, the health of the patient, etc., which are within the skill of the skilled practitioner.

The main advantages of the invention include

(1) The anti-4-1 BB/anti-EGFRvIII bispecific antibody can enable immune cells to gather around tumor cells and stimulate the anti-tumor activity of the immune cells, achieves the effect of cooperatively killing the tumor cells, and can effectively reduce hepatotoxicity caused by overactivation.

(2) The bispecific antibody of the invention for resisting 4-1 BB/EGFRvIII enhances the activity of T cells, promotes the release of cytokine INF-gamma, and is further beneficial to killing tumor cells.

(3) The specificity of the 4-1BB antibody in an in-vivo activation region is increased, the targeting of EGFRvIII is utilized to increase the targeting of the whole double antibody to tumors, and meanwhile, the activation intensity of 4-1BB is further improved depending on EGFRvIII.

The invention is further illustrated below in conjunction with specific embodiments. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. The experimental procedure, in which the detailed conditions are not noted in the following examples, is generally followed by routine conditions such as Sambrook et al, molecular cloning: conditions described in the laboratory Manual (New York: cold Spring Harbor Laboratory Press, 1989) or as recommended by the manufacturer. Percentages and parts are by weight unless otherwise indicated.

Example 1 4-1 preparation of BB fully human antibody

(one) obtaining of 4-1BB fully human antibody

Cloning the extracellular region amino acid sequence of human 4-1BB protein onto pCpCpC carrier with human IgG Fc fragment, preparing plasmid transfected HEK293 cell by standard molecular biological method, and purifying with protein A protein purifying column to obtain 4-1BB extracellular region protein, i.e. immunogen A. Cloning human 4-1BB full-field amino acid sequence into pIRES vector, preparing plasmid transfected HEK293 and CHO-K1 cell line, subcloning and amplifying in 96-well plate by limiting dilution method, and flow screening amplified clone with known 4-1BB antibody to obtain monoclonal with better growth condition and high fluorescence intensity, i.e. immunogen B. Immunogen a and immunogen B were immunized with 6-8 week old, respectively, transgenic mice (purchased from beiveltui rituximab) and splenocytes were collected after sacrifice. According to 5:1, mixing the mouse myeloma cells SP2/0, culturing and screening to obtain hybridoma cells. Total RNA from hybridoma cells was isolated, reverse transcribed, PCR amplified to obtain heavy and light chain variable region genes and sequenced, and the antibody was designated PE0116.

Wherein the amino acid sequence of the heavy chain variable region is shown in a sequence table SEQ ID NO. 13, and the amino acid sequence of the light chain variable region is shown in a sequence table SEQ ID NO. 14.

TABLE 1 heavy chain amino acid sequence of anti-4-1 BB antibody (PE 0116)

Heavy chain	SEQ ID NO:
		PE0116VH	13
H-CDR1	1
		H-CDR2	2
H-CDR3	3

TABLE 2 light chain amino acid sequence of anti-4-1 BB antibody (PE 0116)

Light chain	SEQ ID NO:
		PE0116VL	14
L-CDR1	7
		L-CDR2	8
L-CDR3	9

Preparation of (II) fully human antibody IgG and Single chain antibody

(1) The fully human scFv single chain antibody is produced according to the antibody sequence obtained by the hybridoma technology, the synthesized humanized VH and VL genes synthesize scFv sequences in the structure of VH-GGGGS-VL, the scFv sequences are cloned into a pFUSE vector with an hFc tag, and the recombinant plasmid is subjected to sequencing verification. The IgG full-length antibody is synchronously constructed, the synthesized humanized VH and VL genes are respectively cloned into a vector containing a human Fc constant domain, and the recombinant plasmid is subjected to sequencing verification. High purity plasmids were extracted from endotoxin using a plasmid extraction kit (available from TIANGEN) for transfection.

(2) The 293Fectin transfection reagent and the recombinant expression plasmid are mixed according to the volume-mass ratio of 30 Mul: 30. Mu.g of the cells were mixed, 30ml of 293Freestyle suspension cells were added, and the mixture was subjected to shaking culture at 37℃for 72 to 96 hours at a rotation speed of 125rpm, and the supernatant was collected after centrifugation and filtered with a 0.22 μm filter for antibody purification.

(3) The antibody was purified using endotoxin free chromatography columns and Protein A packing (from GE), the packed chromatography columns were first washed with 5 column volumes of 0.5M NaOH, washed to neutrality with endotoxin free deionized water and equilibrated with 5-10 column volumes of PBS. Loading the filtered cell culture supernatant onto a column, washing the hetero protein by using PBS (phosphate buffer solution) with 5-10 times of column volume after loading, eluting the protein by using Glycine-HCL with 0.1M of pH3.0 and 5 times of column volume, and collecting eluent and neutralizing with 1M of Tris with pH8.5 to pH 7.0-7.5. The protein eluate was transferred to an ultrafiltration tube (from Milipore) for concentration and antibody concentration was detected with reference to the instructions of BCA method protein quantification kit (from Thermo Fisher).

Detection of binding Capacity of (tri) anti-4-1 BB antibody to human 4-1BB antigen

And (3) carrying out a binding reaction on the purified fully human 4-1BB antibody obtained in the step (II) and the human 4-1BB-hFc protein.

Human 4-1BB-hFc protein was diluted with PBS to a final concentration of 1.0. Mu.g/mL and then added to a 96-well ELISA plate at 100. Mu.l per well. Incubation was performed overnight at 4℃with plastic film, and the next day was performed with plate wash [ PBS containing 0.01% (v/v) Tween20 ] 2 times, and blocking solution [ PBS containing 0.01% (v/v) Tween20 and 1% (v/v) BSA ] was added thereto for 2 hours at room temperature. The blocking solution was removed, and 100. Mu.l of the purified 4-1BB antibody obtained in (II) was added to each well. After incubation for 2 hours at 37℃the plates were washed 3 times with plate wash [ PBS containing 0.01% (v/v) Tween20 ]. HRP (horseradish peroxidase) -labeled secondary antibody (purchased from Sigma) was added and after incubation for 2 hours at 37 ℃ the plates were washed 3 times with plate wash [ PBS containing 0.01% (v/v) Tween20 ]. 100. Mu.l of TMB substrate per well was added and after incubation for 30 minutes at room temperature, 100. Mu.l of stop solution (1.0N HCl) per well was added. The A450nm values were read with ELISA plate reader (SpectraMax 384plus,Molecular Device) and the results are shown in FIG. 1, where the purified antibodies bind specifically to the 4-1BB recombinant protein at ELISA level. Wherein the negative control is human IgG.

EXAMPLE 2 preparation of EGFR fully human antibodies

(one) obtaining of Anti-EGFRvIII fully human antibody

The nucleotide sequence containing the amino acid sequence encoding the extracellular region of the human EGFRvIII protein (NCBI: NP-005219.2) was cloned into pCpC vector (available from Invitrogen, V044-50) with a human IgG Fc fragment (hFc) and plasmids were prepared according to established standard molecular biology methods, HEK293 cells were transfected, and 4-1BB extracellular fragment protein, i.e., immunogen A, was obtained after expression via a protein A protein purification column. The full-length amino acid sequence encoding human EGFRvIII was cloned into pIRES vector (purchased from Clontech) and plasmids were prepared and transfected into HEK293 cell line to obtain immunogen B. Immunogen a and immunogen B were immunized with 6-8 week old, respectively, transgenic mice (purchased from beiveltui rituximab) and splenocytes were collected after sacrifice. According to 5:1, mixing the mouse myeloma cells SP2/0, culturing and screening to obtain hybridoma cells. The total RNA of the hybridoma cells is isolated, and the heavy chain and light chain variable region genes are obtained through reverse transcription and PCR amplification. Selecting a human Germline antibody heavy chain and light chain variable region template which is matched with a non-CDR region of the chimeric antibody in a Germline database, transplanting chimeric antibody heavy chain and light chain CDR into the selected human Germline template respectively, and replacing the CDR region of the human Germline template to obtain the humanized antibody which is named 043-1.

TABLE 3 heavy chain amino acid sequence of anti-EGFRvIII antibody (043-1)

Heavy chain	SEQ ID NO:
		043-1VH	15
H-CDR1	4
		H-CDR2	5
H-CDR3	6

TABLE 4 light chain amino acid sequence of anti-EGFRvIII antibody (043-1)

Preparation of (II) fully human antibody IgG and Single chain antibody

The fully human scFv single chain antibody is produced according to the antibody sequence obtained by the hybridoma technology, the synthesized humanized VH and VL genes synthesize scFv sequences in the structure of VL-GGGGS-VH, the scFv sequences are cloned into a pFUSE vector with an hFc tag, and the recombinant plasmids are subjected to sequencing verification. The IgG full-length antibody is synchronously constructed, the synthesized humanized VH and VL genes are respectively cloned into a vector containing a human Fc constant domain, and the recombinant plasmid is subjected to sequencing verification. High purity plasmids were extracted from endotoxin using a plasmid extraction kit (available from TIANGEN) for transfection. After transfection of 293 cells, single-chain and full-length IgG antibodies were obtained by protein a protein purification.

Detection of the ability of fully human antibody IgG and Single chain antibody to bind human EGFRvIII

CHO-K1-egfrvlll cells were expanded to 90% confluency in T-75 cell culture flasks, medium was drained, washed 2 times with HBSS buffer (Hanks Balanced Salt Solution) (from Invitrogen), then treated with enzyme-free cell dissociation solution (verine solution: from Life technology company) and the cells were collected. The cells were washed 2 times with HBSS buffer, and after cell counting, the cells were diluted with HBSS buffer to 2×10 ⁶ Cells per ml, 1% goat serum blocking solution was added, the percentages are mass percentages, incubated on ice for 30 minutes, and then washed 2 times with HBSS buffer by centrifugation. The collected cells were suspended to 2X 10 with FACS buffer (HBSS+1% BSA, the percentages are mass percent) ⁶ cells/mL were added to a 96-well FACS reaction plate at 100 μl per well, and the resulting purified egfrvlll antibody test sample was added (two) and incubated on ice for 2 hours at 100 μl per well. The cells were washed 2 times with FACS buffer, and 100 μl of fluorescent (Alexa 488) -labeled secondary antibody (available from Invitrogen) per well was added and incubated on ice for 1 hour. Centrifuge wash 3 times with FACS buffer, add 100. Mu.l fixative [4% (v/v) paraformaldehyde per well]Cells were suspended and after 10 min washed 2 times with FACS buffer. Cells were suspended with 100 μl FACS buffer and the mean fluorescence density (MFI) of the cells was measured. Analysis results As shown in FIG. 2, both the single-chain and full-length forms of anti-EGFRvIII antibody 043-1 specifically recognized EGFRvIII protein on the cell surface.

Example 3 preparation of anti-4-1 BB/anti-EGFRvIII bispecific antibody

(1) Constructing a plasmid: different forms of 4-1BB/EGFRvIII bispecific antibodies were constructed based on the scFv and IgG structures of the anti-4-1BB and anti-EGFRvIII antibodies of examples 1 and 2, PE0116 was used as IgG, scFv in the form of 043-1VL-VH was modified at the C-terminus of the heavy chain to construct a bispecific antibody CH-scFv (043-1 VLVH) -IgG4 (PE 0116), designated HY1001007. Wherein the amino acid sequence is (GGGGS) ₃ The encoded linker links the variable regions VH and VL among all scFv versions, as well as the linkage between IgG-scFv. The structure of which is shown in fig. 9.

The constructed anti-EGFRvIII scFv (043-1 VLVH) modified anti-4-1 BB antibody heavy chain (PE 0116VH-CH-043-1 VLVH) was homologously recombined onto a heavy chain expression vector of human IgG4 (expression vector was purchased from Invitrogen), and anti-4-1 BB antibody light chain (PE 0116 VL) was homologously recombined onto a light chain expression vector of human IgG4 to obtain recombinant plasmid and verified by sequencing. High purity plasmids were extracted using a deiotonic plasmid extraction kit (purchased from the root organism) and sterilized with a 0.22 μm filter for cell transfection.

(2) Antibody production: the expression plasmid prepared in (1) was transiently transfected into 293F cells, the plasmid and the Lipofectamine 2000 transfection reagent (purchased from Invitrogen) were diluted with Opti-MEM, respectively, the plasmid and the transfection reagent were mixed within 5 minutes, left to stand at room temperature for 15 minutes, added to 293F cell solution, cultured for 48-72 hours, and the cell supernatant was collected and filtered through a 0.22 μm filter.

Protein A (from GE) was packed into endotoxin-free chromatography columns, washed with 5 column volumes of 0.1M NaOH, and then washed with ultrapure water until the effluent was neutral. After equilibration with 5-10 column volumes of PBS (pH approximately 7.2), the filtered cell culture supernatant was passed to the column and the effluent was collected as necessary. After the completion of the column, the mixed proteins were removed by washing with 10 column volumes of PBS. Eluting with 5 times of 0.1M Glycine-HCl with pH 3.0, collecting eluate, neutralizing the eluate with Tris-HCl with pH8.5 to pH about 7.0-7.5, and concentrating the eluate with 30KD ultrafiltration column (commercially available from Milpore) to obtain purified bispecific antibody. Purified antibody concentrations were further determined by BCA protein concentration detection kit (available from Thermo Fisher).

EXAMPLE 4 detection of binding Activity of bispecific antibodies

(1) Construction of human 4-1BB stably transformed cell line

Cloning of the full-length amino acid sequence of human 4-1BB into pIRES vector (from Clontech) and preparation of plasmid, transfection of CHO-K1 cell line (from Invitrogen), cultivation in selective Medium containing 0.5. Mu.g/ml puromycin for two weeks, subcloning in 96 well plates by limiting dilution and placing at 37℃5% CO ₂ Is cultured in an incubator of (a). After two weeks, the monoclonal is picked up and amplified to a 6-hole plate, and a commercial 4-1BB antibody (purchased from BD) is used for carrying out monoclonal screening through a flow, and a monoclonal cell line with better growth vigor and higher fluorescence intensity is selected to continue to be subjected to expansion culture and liquid nitrogen freezing storage, so that a CHO-K1 cell strain capable of stably expressing the human 4-1BB is obtained.

(2) Construction of overexpressing monkey 4-1BB stably transfected cell line

Cloning the monkey 4-1BB amino acid sequence (Uniprot database accession number A9YYE 7) into a pIRES vector, and obtaining a CHO-K1 cell strain for stably expressing cynomolgus 4-1BB, wherein the monoclonal screening process is the same as that of the step (1).

(3) Construction of overexpression human EGFRvIII stably transformed cell line

The full-length amino acid sequence (NCBI: NP-005219.2, deletion of amino acids 30-297) encoding human EGFRvIII was cloned into the pIRES vector and the monoclonal screening procedure was the same as in step (1). The amplified clone was screened with a commercial EGFRvIII antibody (purchased from Absolute antibody, # Ab 00184-1.1) by flow cytometry, and a monoclonal cell line with better growth vigor and higher fluorescence intensity was selected to continue the expansion culture and liquid nitrogen freezing storage, so as to obtain a CHO-K1 cell line stably expressing human EGFRvIII.

(4) Flow cytometry (FACS) detection of bispecific antibody binding to 4-1BB/EGFRvIII overexpressing cells

Culturing the stable transgenic cell strain constructed in the steps (1), 2 and 3) until the density is more than 90%, wherein the activity rate is more than 95%. Cells were dispensed into 96-well plates after pancreatin digestion, 2×10 per well ⁵ Individual cells were centrifuged at 1000rpm at 4℃for 5 minutes. 150 μl FACS Buffer was washed twice and centrifuged at 300g for 5min at 4deg.C. Diluting the antibody to be tested: a total of 7 spots were diluted 5-fold at an initial concentration of 30. Mu.l/ml, and the diluted antibodies were incubated with cells at 4℃for 1 hour in a volume of 100. Mu.l per well. The FACS Buffer was washed twice with 150. Mu.l, and the supernatant was discarded after each wash. FITC anti-human IgG Fc secondary antibody (available from BioLegend) and 100. Mu.l/well FACS Buffer resuspended cells were added at 1.5. Mu.l/well and incubated for 1h at 4 ℃. The cells were washed twice with 200. Mu.l FACS Buffer, centrifuged at 300g for 5min at 4℃and the supernatant was discarded after each washing. Cells were resuspended in 100. Mu.l per well using PBS cold at 4℃and then run on the machine.

As shown in FIG. 3, the bispecific antibody HY1001007 can specifically recognize human 4-1BB and monkey 4-1BB on the surfaces of CHO-K1 cells, and does not specifically bind to CHO-K1. As shown in FIG. 4, bispecific antibody HY1001007 can specifically recognize human EGFRvIII on the surface of CHO-K1 cells.

Example 5 test for the Activity of bispecific antibodies against NF-. Kappa.B Signal pathway

(1) Construction of NF- κB signaling pathway activation reporter cell lines: based on NF- κB signaling pathway, a lentiviral vector was constructed using a luciferase reporter vector from promega, a monoclonal cell line was obtained by infecting HEK293 cells with lentivirus, and positive control stimulation validation was performed using TNF- α. In TNF-alpha stimulation experiments, the response value and the TNF-alpha concentration show a dose-dependent effect, and the signal to noise ratio can reach more than 100 times, which indicates that the establishment of the cell line is successful.

(2) Construction of NF- κB signaling pathway reporter cell lines stably expressing human 4-1 BB: the full length amino acid sequence of human 4-1BB was cloned into pIRES vector (purchased from Clontech) and plasmids were prepared and transfected into HEK293 NF-. Kappa.B signaling cell lines. The subcloning was performed in 96-well plates by limiting dilution and placed in an incubator at 37℃with 5% CO2, after two weeks in selective medium containing 0.5. Mu.g/ml puromycin. After two weeks, the monoclonal is picked up and amplified to a 6-hole plate, and a commercial 4-1BB antibody (purchased from BD) is used for carrying out monoclonal screening through a flow, and a monoclonal cell line with better growth vigor and higher fluorescence intensity is selected to continue to be subjected to expansion culture and liquid nitrogen freezing storage, so that an NF- κB signal channel reporting cell line for stably expressing the human 4-1BB is obtained.

(3) Report cell activation detection assay: preparing CHO-K1 cell strain stably expressing human EGFRvIII and human 4-1BB/NF- κB signal pathway activation report cell strain constructed above, mixing at a ratio of 1:1 after cell count, and mixing at a ratio of 2×10 ⁴ The cells are mixed uniformly and then added with purified 4-1BB antibody PE0116, anti-4-1 BB/anti-EGFRvIII bispecific antibody HY1001007 and control antibody IgG4 isotype with different concentrations and equal volume ratio. After 24 hours incubation of cells with antibodies, luminescence values were detected using a luciferase substrate detection kit.

The results are shown in FIG. 5, where the 4-1BB antibody HY1001007 targeting EGFRvIII activated NF-. Kappa.B signaling pathway to a greater extent and at a lower half-effective antibody concentration than the 4-1BB antibody PE0016 alone.

Example 6T lymphocyte stimulation experiments

(1) Construction of a CHOK1-OS8-EGFRvIII cell line: the ScFv sequence of anti-CD3 (OKT 3) was ligated with the C-terminal 113-220 amino acid sequence of mouse CD8a (NCBI Accession No:NP-001074579.1) to construct plasmid pIRES-OS8, which was transfected into CHO-K1 cells, and stably passaged cell line CHOK1-OS8 was constructed as described in example 4. The full-length amino acid sequence (NCBI: NP-005219.2, deletion of amino acids 30-297) encoding human EGFRvIII was further cloned into pIRES vector and CHOK1-OS8 cells were transfected to prepare a stable passaged cell line CHOK1-OS8-EGFRvIII.

(2) Cell and antibody preparation: cells were resuspended to 1X 10 ⁶ cell/mL, and 10ug/mL Mitomycin C was added at 37℃in 5% CO ₂ The cells were cultured in an incubator for 2 hours to inhibit division. Treating the treated CHOK1-OS8-EGFRvIII at 2.5X10 ⁴ cells/well were added to 96-well plates. Preparing purified 4-1BB antibody PE0116 and anti-4-1 BB/anti-EGFRvIII bispecific antibody diluted in equal volume ratio, and anti-human Fc F (ab') ₂ A cross-linked PE0116 antibody.

(3) T lymphocyte stimulation experiments: human CD3 positive T cells were purified from human peripheral blood mononuclear lymphocytes (available from ORiCELLS) using a T cell purification kit (available from Stemcell) according to the method provided by the kit. The CD3 positive T cells were added to a 96-well cell culture plate, 1X 10 ⁵ Cells per well, eventually each reaction well has a final volume of 200 μl. At 37℃with 5% CO ₂ The culture was carried out in an incubator for 72 hours, and the supernatant was collected.

(4) Detection of INF-gamma in cell supernatants: cytokine IFN-gamma ELISA assays (double antibody sandwich ELISA kits from R & D systems) were performed using cell supernatants obtained in T lymphocyte stimulation experiments. The experiment is according to the instruction of the kit, all detection reagents are provided by the kit: and coating the IFN-gamma polyclonal antibody on an ELISA micro-pore plate, taking the obtained cell supernatant as a sample to be detected, adding a standard substance and the sample to be detected, and incubating for 2 hours at room temperature. 300 μl of wash solution was added to each well and the plate was washed repeatedly 3 times; then adding an anti-human IFN-gamma biotin labeled antibody, incubating for 2 hours at room temperature, and cleaning micropores; adding horseradish peroxidase-labeled streptavidin, incubating at room temperature for 20 minutes, forming an immune complex with IFN-gamma or IL-2 on a microplate, adding a substrate for color development, keeping away from light at room temperature for 30 minutes, adding a stop solution, and measuring the absorbance of A450nm by using an enzyme-labeled instrument.

The results of a portion of the T cell stimulation experiments are shown in FIG. 6, in which bispecific antibodies both enhance T cell activity and promote release of cytokine INF-gamma. Which is a kind ofIn the method, the bispecific antibody HY1001007 can greatly promote the release of the cytokine INF-gamma at 10nM, and the content exceeds 3×10 ⁴ pg/ml。

EXAMPLE 7 peripheral blood mononuclear lymphocyte killing experiments

(1) CT26-EGFRvIII stable transgenic cell strain construction: the full-length amino acid sequence encoding human EGFRvIII (NCBI: NP-005219.2, deletion of amino acids 30-297) was cloned into the pIRES vector and CT26 cells were transfected and the monoclonal screening procedure was the same as in example 4. The amplified clone is screened by a commercial EGFRvIII antibody (purchased from Absolute antibody, # Ab 00184-1.1) through a flow cytometry method, a monoclonal cell line with better growth vigor and higher fluorescence intensity is selected to be subjected to expansion culture and liquid nitrogen freezing storage, a stable passage cell strain CT26-EGFRvIII is prepared, and monoclonal 2B5 with the highest EGFRvIII expression level is selected as a target cell of a killing experiment. CT26-EGFRvIII cells were added to 96-well plates. Preparing purified 4-1BB antibody and 4-1BB and EGFRVIII double anti-IgG 4 antibody with equal volume ratio dilution, wherein anti-human Fc F (ab') is prepared for 4-1BB monoclonal antibody simultaneously ₂ And the antibody to be detected is crosslinked well or is not crosslinked. The sample to be tested was added to a 96-well plate at 100. Mu.L per reaction well volume at 37℃with 5% CO ₂ Incubators were incubated for 30 minutes. Human peripheral blood mononuclear lymphocytes (from ORiCELLS) were resuspended in medium to a suitable target specific concentration and 50 μl per well was added to 96-well plates. A final concentration of 5ug/mL of humanized OKT3 CD3 antibody was added to each well, and finally each reaction well had a final volume of 200. Mu.L. At 37℃with 5% CO ₂ The incubator was co-cultured for 72 hours.

(2) The killing effect was detected using the lactate dehydrogenase method (kit from promega) and the amount of cytokine IFN-gamma released was detected (double antibody sandwich ELISA kit from R)&D systems). The experimental operation strictly meets the requirements of the specification of the kit, and all detection reagents are provided by the kit. The specific experiments are briefly described as follows: before the co-incubation of peripheral blood mononuclear lymphocytes with CT26-EGFRVIII-1A8 is completed, a cell lysate is added into a control well with maximum lactate dehydrogenase release of target cells, and the mixture is subjected to the treatment of 5% CO at 37 DEG C ₂ Incubator co-incubate for 45 minutes. Centrifugal sedimentation in 96-well platesCells, supernatant was taken for the experiment. Taking a supernatant to be detected in the lactate dehydrogenase method detection, adding a reaction substrate provided by a kit into a reaction plate, and incubating for 30 minutes at room temperature in a dark place; the reaction termination solution was added, and the absorbance at A490nm was measured by an enzyme-labeled instrument. The results of the partial kill test experiments are shown in the figures. The amount of cytokine IFN-gamma released was measured as described in example 5, step (2).

As shown in FIG. 7a and FIG. 7b, the bispecific antibody mediated killing effect is stronger than that of the monoclonal antibody, and the monoclonal antibody can better enhance the release of the T cell factor INF-gamma.

EXAMPLE 8 pharmacodynamic evaluation in a BALB/c-hCD137 mouse subcutaneously transplanted CT26 colon cancer model

(1) Study of BALB/c-hCD137 mice subcutaneously inoculated with colon cancer cell CT26 model

Recovering and subculturing colon cancer cells of the mice, recovering and replacing times to be Pn+0, collecting CT26 cells in a logarithmic growth phase (replacing times to be Pn+5), removing culture solution, washing twice with PBS, and inoculating (cell survival rates before and after tumor loading are 96.8% and 96.7%, respectively), wherein the inoculation amount is as follows: g1 group 1×10 ⁶ cells/100. Mu.L/alone (without matrigel addition), group G2, 2X10 ⁶ cells/100. Mu.L/alone (without matrigel addition). Tumor size was observed after inoculation and mice were weighed. The tumor volume calculation mode is as follows: tumor volume (mm) ³ ) =0.5×tumor long diameter×tumor short diameter ² 。

The results are shown in FIG. 8a, which shows that the BALB/c-hCD137 mice were analyzed at 2X10 according to tumor volume data ⁶ The cell/100 mu L subcutaneous inoculation CT26 cell tumor grows faster and has better uniformity; at 1x10 ⁶ cell/100. Mu.L subcutaneous inoculation of CT26 cells resulted in slower tumor formation and regression of some mouse tumors.

(2) In vivo pharmacodynamic evaluation of bispecific antibodies

Resuscitates and subcultures colon cancer cells of the mice, wherein the resuscitating time is Pn+4, the vaccinating time is Pn+7, and the cell survival rates before and after tumor loading are respectively as follows: 98.0% and 95.9% with an inoculum size of 2X 10 ⁶ cells/100. Mu.L/alone (without matrigel addition), average tumor volume reached 84.44mm on day 6 post inoculation ³ When the left and right sides are used, the left and right sides,30 mice were selected and randomly divided into 5 groups of 6 mice each based on tumor volume. Wherein, the G1 group is Isotype control group, the G2 group is commercial 4-1BB antibody Urerlumab (1 mpk) group, the G3 group is bispecific antibody HY1001007 (0.3 mpk) group, the G4 group is bispecific antibody HY1001007 (1 mpk), the G5 group is EGFRvIII antibody 043-1 (1 mpk) group, and the antibody is in IgG4 form.

The day of the grouping was defined as D0 and dosing was started on day D0 of the grouping, twice weekly for 3 weeks. Experimental results are expressed as Mean ± standard error (Mean ± SEM). Comparison between the two groups of samples used independent sample T-Test (T-Test), data were analyzed using SPSS, P <0.05 was significantly different. The mapping software is Graphpad prism.

As shown in fig. 8b, 8c, statistical analysis of tumor volume data at D20 days compared to control group G1: group G2 Urelumab (1 mpk, TGI) _TV =100.00%), G3 group HY1001007 (0.3 mpk, tgi _TV =77.71%), G4 group HY1001007 (1 mpk, tgi _TV =100.00%) has a significant inhibition effect on tumor growth, and group G5 043-1 (1 mpk, tgi _TV =36.11%) has a certain inhibition effect on tumor growth, but no significant statistical difference (P>0.05)。

Discussion of the application

Compared to the bispecific antibody in the reference WO2021020845A1 application:

different targets are aimed at by the double antibody, namely EGFRvIII and 4-1BB, and comparison files are EGFR and 4-1BB. In addition, the diabodies of the application significantly promote the release of the cytokine INF-gamma, which is on the order of 10 ⁴ pg/ml. Whereas bispecific antibodies in the control document promote release of the cytokine INF-gamma on the order of 10 ² pg/ml (example 7.1 in the control document) is far lower than the bispecific antibody in the present application.

All documents mentioned in this disclosure are incorporated by reference in this disclosure as if each were individually incorporated by reference. Further, it will be appreciated that various changes and modifications may be made by those skilled in the art after reading the above teachings, and such equivalents are intended to fall within the scope of the application as defined in the appended claims.

Sequence listing

<110> Shanghai Bow Biotech Co., ltd

<120> bispecific antibody against 4-1 BB/EGFRvIII, and preparation method and application thereof

<130> P2022-0527

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Claims

1. A bispecific antibody, characterized in that the bispecific antibody comprises:

a first antigen binding domain (D1); and

a second antigen binding domain (D2);

the D2 is an antibody or an antigen binding fragment thereof which specifically binds to EGFRvIII protein;

the anti-4-1 BB antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the following three complementarity determining regions CDR:

VH-CDR1 shown in SEQ ID NO. 1,

VH-CDR2 shown in SEQ ID NO. 2, and

VH-CDR3 shown in SEQ ID No. 3; and

VL-CDR1 shown in SEQ ID No. 7,

VL-CDR2 shown in SEQ ID NO. 8, and

VL-CDR3 shown in SEQ ID No. 9.

2. The bispecific antibody of claim 1, wherein said anti-egfrviii antibody comprises a heavy chain variable region and a light chain variable region, said heavy chain variable region comprising the following three complementarity determining region CDRs:

VH-CDR1 shown in SEQ ID NO. 4,

VH-CDR2 shown in SEQ ID NO. 5, and

VH-CDR3 shown in SEQ ID No. 6; and

VL-CDR1 shown in SEQ ID No. 10,

VL-CDR2 shown in SEQ ID NO. 11, and

VL-CDR3 shown in SEQ ID NO. 12.

3. A recombinant protein, said recombinant protein comprising: (i) the bispecific antibody of claim 1; and (ii) optionally a tag sequence to aid expression and/or purification.

4. An isolated polynucleotide encoding the bispecific antibody of claim 1 or the recombinant protein of claim 3.

5. A vector comprising the polynucleotide of claim 4.

6. A genetically engineered host cell comprising the vector or genome of claim 5 having incorporated therein the polynucleotide of claim 4.

7. A pharmaceutical composition, comprising:

(I) The bispecific antibody of claim 1; and

(II) a pharmaceutically acceptable carrier.

8. An immunoconjugate, the immunoconjugate comprising:

(a) The bispecific antibody of claim 1; and

9. Use of the bispecific antibody of claim 1, the recombinant protein of claim 2, or the immunoconjugate of claim 8 for the preparation of (a) a detection reagent or kit; and/or (b) preparing a medicament for preventing and/or treating related diseases.

10. Compositions and methods for in vitro detection (including diagnostic or non-diagnostic) of 4-1BB, EGFRvIII or simultaneous detection of 4-1BB, EGFRvIII in a sample, the compositions comprising anti-4-1 BB/anti-EGFRvIII bispecific antibodies; characterized in that the method comprises the steps of:

(1) Contacting the sample with the bispecific antibody of claim 1 in vitro;

(2) Detecting whether an antigen-antibody complex is formed, wherein the formation of a complex indicates the presence of 4-1BB and/or EGFRv III in the sample.