CN116496403A

CN116496403A - anti-PDL 1/IGF1R bispecific antibody, preparation method and application thereof

Info

Publication number: CN116496403A
Application number: CN202210059079.XA
Authority: CN
Inventors: 顾昌玲; 黄浩旻; 朱祯平
Original assignee: Zi Da Biological Medicine Co ltd
Current assignee: Zi Da Biological Medicine Co ltd
Priority date: 2022-01-19
Filing date: 2022-01-19
Publication date: 2023-07-28
Also published as: WO2023138620A1

Abstract

The invention belongs to the field of tumor treatment and biotechnology, and discloses an anti-PDL 1/IGF1R bispecific antibody, a preparation method and application thereof, wherein the anti-PDL 1/IGF1R bispecific antibody comprises the following components: a first antigen binding domain that specifically binds PDL1, and a second antigen binding domain that specifically binds IGF 1R. The bispecific antibody can better simultaneously maintain the activities of anti-PDL 1 and anti-IGF 1R monoclonal antibodies, can inhibit the proliferation of tumor cells expressing IGF1R, can block PD1/PDL1 signal paths, and has unique effect of mediating endocytosis of cell surface receptors to lysosome degradation; the bispecific antibody has the activity of killing tumor cells obviously superior to that of monoclonal antibody, and has the potential of preparing medicines for preventing and treating tumors.

Description

anti-PDL 1/IGF1R bispecific antibody, preparation method and application thereof

Technical Field

The invention belongs to the field of tumor treatment and biotechnology, and relates to a bispecific antibody molecule for resisting PDL1 and IGF1R, and a preparation method and application thereof.

Background

The transmembrane receptor PD-1 (programmed cell death-1) is one of the members of the CD28 gene family and is expressed in activated T cells, B cells and myeloid cells. The ligands PDL1 (Programmed cell death ligand 1, also known as PD-L1) and PDL2 (Programmed cell death ligand 2, also known as PD-L2) of PD-1 belong to the B7 superfamily, wherein PDL1 is expressed in a variety of cells, including T cells, B cells, and endothelial and epithelial cells, and PDL2 is expressed only in antigen presenting cells such as dendritic cells and macrophages.

The PD-1/PDL1 signaling pathway plays an important role in regulating immune tolerance, microbial infection and tumor immune escape. PD-1 expression is mainly in immune cells such as T cells, while PDL1 ligand of PD-1 is mainly expressed in many human tumor tissues. Blocking the PD-1/PDL1 signal pathway activates the inhibited T cells, thereby attacking the cancer cells. Blocking PD-1/PDL1 signals can promote proliferation of tumor antigen specific T cells, play a role in killing tumor cells, and further inhibit local tumor growth.

Insulin-like growth factor 1 (IGF-1) and its receptor IGF-1R (also called IGF1R or IGF-I R) are effective biological transmitters capable of regulating cell growth and differentiation, and have important roles in proliferation and differentiation of cells, tissue regeneration and the like. IGF-1 regulates a range of physiological activities in cells by activating the downstream signaling pathway of its receptor IGF-1R, and is also involved in the development and progression of a variety of diseases.

IGF-1R is a cell surface transmembrane receptor protein, and consists of two alpha subunits and two beta subunits, wherein the alpha subunits are positioned outside a membrane and have binding sites for IGF-1 peptide, and the beta subunits are positioned inside the membrane to complete transmembrane signal transduction. IGF-1R is highly expressed on the surfaces of various tumor cells, and is involved in various biological effects such as proliferation differentiation, invasion, metastasis, anti-apoptosis and the like of the tumor cells. Antibodies targeting IGF-1R can block IGF1R signaling pathways, inhibiting tumor cell proliferation.

At present, the anti-PDL 1 and anti-IGF 1R antibodies have the problems of low anti-tumor activity and the like, and many tumor patients still cannot control the diseases for a long time after receiving antibody treatment, so that the survival rate is low. Therefore, research on antibodies capable of targeting both proteins at the same time, development of antibodies with high affinity, lower toxicity, more effective therapeutic means or combined dosing regimen is of great clinical importance.

According to the invention, tumor cell surface related antigen (TAA) IGF1R and immune target PDL1 are combined to prepare the TAA-PDL1 double antibody, and when the anti-IGF 1R antibody targets tumor cells, immune cells in tumor microenvironment can be activated, and the two can play a synergistic effect to kill tumors, and can play an anti-tumor effect obviously superior to that of monoclonal antibodies.

Disclosure of Invention

Through intensive research and creative labor, the inventor correspondingly modifies the structures of an anti-PD-L1 antibody and an anti-IGF 1R antibody, and constructs a bifunctional antibody capable of simultaneously targeting PD-L1 and IGF1R and blocking PD-L1 and IGF1R signaling pathways. The invention also provides isolated polynucleotides, constructs, expression systems, host cells, or the like encoding the bispecific antibodies. The invention also provides a preparation method of the bispecific antibody. The bispecific antibody can be used for preventing and/or treating tumors and is used for developing related detection kits. The invention also provides methods of preventing and/or treating cancer using the bispecific antibodies or the pharmaceutical compositions.

In a first aspect of the present invention, there is provided a bispecific antibody, characterized in that the bispecific antibody comprises:

a first antigen binding domain, and

a second antigen binding domain;

wherein the first antigen binding domain is an antibody or antigen binding fragment thereof that specifically binds PDL 1; and/or

The second antigen binding domain is an antibody or antigen binding fragment thereof that specifically binds IGF 1R;

in another preferred embodiment, the antigen binding fragment has a structure selected from the group consisting of: (i) Fab fragments; (ii) a F (ab') 2 fragment; (iii) Fv fragments; or (iv) a single chain Fv (scFv);

in another preferred embodiment, the antibody that specifically binds PDL1 comprises: single chain antibodies, diabodies, nanobodies, monoclonal antibodies, chimeric antibodies, murine antibodies, humanized antibodies, and bispecific antibodies.

In another preferred embodiment, the antibody that specifically binds IGF1R comprises: single chain antibodies, diabodies, nanobodies, monoclonal antibodies, chimeric antibodies, murine antibodies, humanized antibodies, and bispecific antibodies.

In another preferred embodiment, the first antigen binding domain and/or the second antigen binding domain is an IgG antibody.

In another preferred embodiment, the IgG antibody is an IgG1, igG2, igG3 or IgG4 antibody.

In another preferred embodiment, the first antigen binding domain and/or the second antigen binding domain is an scFv.

In another preferred embodiment, the first antigen binding domain is an scFv against PDL 1.

In another preferred embodiment, the anti-PDL 1scFv comprises a variable region VH and a variable region VL, and VH is linked to VL either directly or through a peptide linker.

In another preferred embodiment, the first antigen-binding domain is an anti-PDL 1scFv 1, the VH and VL of which are linked by a peptide linker L1, having the structure VL-L1-VH from the N-terminus to the C-terminus, wherein "-" represents a peptide bond.

In another preferred embodiment, the first antigen binding domain is an anti-PDL 1scFv 2, the VH and VL of which are linked by a peptide linker L1, having the structure VH-L1-VL from the N-terminus to the C-terminus, wherein "-" represents a peptide bond.

In another preferred embodiment, the peptide linker L1 has the sequence (G4S) n, preferably n is 1-4.

In another preferred embodiment, the second antigen binding domain is an IgG antibody directed against IGF 1R.

In another preferred embodiment, the first antigen binding domain comprises one, two, three, or more scFv against PDL 1.

In another preferred embodiment, the second antigen binding domain comprises one, two, three, or more IgG antibodies directed against IGF 1R.

In another preferred embodiment, the first antigen binding domain is linked to a region of the anti-IGF 1R antibody 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, the first antigen binding domain is an scFv against PDL 1; and the second antigen binding domain is an IgG antibody against IGF1R, wherein the first antigen binding domain is linked at the end of the heavy chain variable region of the second antigen binding domain or the first antigen binding domain is linked at the end of the heavy chain constant region of the second antigen binding domain.

In another preferred embodiment, the bispecific antibody comprises an IgG antibody against IGF1R and two scFv against PDL1, wherein each scFv comprises a variable region VH and a variable region VL, VH and VL being linked by a peptide linker L1, scFv having a structure of VL-L1-VH or VH-L1-VL from N-terminus to C-terminus, each scFv against PDL1 being in tandem with an immunoglobulin IgG against IGF1R by a peptide linker L2.

In another preferred embodiment, the VH or VL of the anti-PDL 1 scFv is linked to the heavy chain variable region, heavy chain constant region, light chain variable region, or light chain constant region of an immunoglobulin antibody IgG to IGF1R via a peptide linker L2.

In another preferred embodiment, the first antigen binding domain is an IgG antibody against PDL 1.

In another preferred embodiment, the second antigen binding domain is an scFv against IGF 1R.

In another preferred embodiment, the anti-IGF 1RscFv comprises a variable region VH and a variable region VL, and VH is linked directly to VL or via a peptide linker.

In another preferred embodiment, the second antigen binding domain is an anti-IGF 1R scFv1, the VH and VL of which are linked by a peptide linker L1, having the structure VL-L1-VH from N-terminus to C-terminus, wherein "-" represents a peptide bond.

In another preferred embodiment, the second antigen binding domain is an anti-IGF 1R scFv2, the VH and VL of which are linked by a peptide linker L1, having the structure VH-L1-VL from the N-terminus to the C-terminus, wherein "-" represents a peptide bond.

In another preferred embodiment, the first antigen binding domain comprises one, two, three, or more IgG antibodies against PDL 1.

In another preferred embodiment, the second antigen binding domain comprises one, two, three, or more scFv against IGF 1R.

In another preferred embodiment, the second antigen binding domain is linked to a region of the anti-PDL 1 antibody 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, the first antigen binding domain is an IgG antibody against PDL 1; and the second antigen-binding domain is an scFv against IGF1R, wherein the second antigen-binding domain is linked at the end of the heavy chain variable region of the first antigen-binding domain or the second antigen-binding domain is linked at the end of the heavy chain constant region of the first antigen-binding domain.

In another preferred embodiment, the first antigen binding domain and the second antigen binding domain are linked directly or via a peptide linker L2.

In another preferred embodiment, the peptide linker L2 has the sequence (G4S) n, preferably n is 1-4.

In another preferred embodiment, the bispecific antibody is a homo-or heterodimer, preferably a homodimer.

In another preferred embodiment, the bispecific antibody comprises an anti-PDL 1 IgG antibody and two anti-IGF 1R scFv, wherein each scFv comprises a variable region VH and a variable region VL, VH and VL being linked by a peptide linker L1, scFv having a VL-L1-VH or VH-L1-VL structure from N-terminus to C-terminus, each anti-IGF 1R scFv being in tandem with an anti-PDL 1 immunoglobulin IgG by a peptide linker L2.

In another preferred embodiment, the VH or VL in the anti-IGF 1R scFv is linked to the heavy chain variable region, heavy chain constant region, light chain variable region, or light chain constant region of an immunoglobulin antibody IgG to PDL1 via a peptide linker L2.

In another preferred embodiment, the bispecific antibody is a homodimer having the structure from N-terminus to C-terminus of formula Ia, ib, IIa, IIb, iiia, iiib, iva or ivb:

wherein,,

VL _A representing the light chain variable region of an anti-PDL 1 antibody;

VH _A represents the heavy chain variable region of an anti-PDL 1 antibody;

VL _B representing the light chain variable region of an anti-IGF 1R antibody;

VH _B represents the heavy chain variable region of an anti-IGF 1R antibody;

CH represents a heavy chain constant region;

CL represents the light chain constant region;

l1, L2 are each independently a bond or a peptide linker;

"-" represents disulfide or covalent bonds;

"-" represents a peptide bond;

wherein the bispecific antibody has the activity of simultaneously binding PDL1 and binding IGF 1R.

In another preferred embodiment, the heavy chain constant region is selected from the group consisting of the heavy chain constant regions of human IgG1, igG2, igG3, or IgG 4.

In another preferred embodiment, the bispecific antibody blocks the PD1/PDL1 signal pathway.

In another preferred embodiment, the bispecific antibody degrades PDL1 protein.

In another preferred embodiment, the peptide linker L1 has the sequence shown in SEQ ID NO. 1.

In another preferred embodiment, the sequence of the peptide linker L2 is shown in SEQ ID NO. 2.

In another preferred embodiment, the anti-PDL 1 antibody comprises the following three complementarity determining regions HCDR:

HCDR1 shown in SEQ ID NO. 14;

HCDR2 shown in SEQ ID NO. 15; and

HCDR3 shown in SEQ ID NO. 16; and

the anti-PDL 1 antibody comprises the following three complementarity determining regions LCDR:

LCDR1 shown in SEQ ID NO. 18;

LCDR2 as shown in SEQ ID NO. 19; and

LCDR3 as shown in SEQ ID NO. 20.

In another preferred embodiment, the anti-PDL 1 antibody comprises the heavy chain variable region shown in SEQ ID NO. 13.

In another preferred embodiment, the anti-PDL 1 antibody comprises the light chain variable region shown in SEQ ID NO. 17.

In another preferred embodiment, the heavy chain sequence of the anti-PDL 1 antibody is shown in SEQ ID NO. 21.

In another preferred embodiment, the light chain sequence of the anti-PDL 1 antibody is shown in SEQ ID NO. 22.

In another preferred embodiment, the anti-PDL 1 scFv1 sequence is as shown in SEQ ID NO. 23.

In another preferred embodiment, the anti-PDL 1 scFv2 sequence is as shown in SEQ ID NO. 24.

In another preferred embodiment, the anti-IGF 1R antibody comprises the following three complementarity determining regions HCDR:

HCDR1 shown in SEQ ID NO. 4;

HCDR2 shown in SEQ ID NO. 5; and

HCDR3 shown in SEQ ID NO. 6; and

the anti-IGF 1R antibody comprises the following three complementarity determining regions LCDR:

LCDR1 shown in SEQ ID NO. 8;

LCDR2 as shown in SEQ ID NO. 9; and

LCDR3 as shown in SEQ ID NO. 10.

In another preferred embodiment, the anti-IGF 1R antibody comprises the heavy chain variable region set forth in SEQ ID NO. 3.

In another preferred embodiment, the anti-IGF 1R antibody comprises the light chain variable region set forth in SEQ ID NO. 7.

In another preferred embodiment, the heavy chain sequence of the anti-IGF 1R antibody is set forth in SEQ ID NO. 11.

In another preferred embodiment, the light chain sequence of the anti-IGF 1R antibody is set forth in SEQ ID NO. 12.

In another preferred embodiment, the anti-IGF 1RscFv1 sequence is set forth in SEQ ID NO. 25.

In another preferred embodiment, the anti-IGF 1R scFv2 sequence is set forth in SEQ ID NO. 26.

In another preferred embodiment, the bispecific antibody is selected from the group consisting of:

(1) The heavy chain amino acid sequence of the bispecific antibody is shown as SEQ ID NO. 27, and the light chain amino acid sequence of the bispecific antibody is shown as SEQ ID NO. 12;

(2) The heavy chain amino acid sequence of the bispecific antibody is shown as SEQ ID NO. 28, and the light chain amino acid sequence of the bispecific antibody is shown as SEQ ID NO. 12;

(3) The heavy chain amino acid sequence of the bispecific antibody is shown as SEQ ID NO. 29, and the light chain amino acid sequence of the bispecific antibody is shown as SEQ ID NO. 12;

(4) The heavy chain amino acid sequence of the bispecific antibody is shown as SEQ ID NO. 30, and the light chain amino acid sequence of the bispecific antibody is shown as SEQ ID NO. 12;

(5) The heavy chain amino acid sequence of the bispecific antibody is shown as SEQ ID NO. 31, and the light chain amino acid sequence of the bispecific antibody is shown as SEQ ID NO. 22;

(6) The heavy chain amino acid sequence of the bispecific antibody is shown as SEQ ID NO. 32, and the light chain amino acid sequence of the bispecific antibody is shown as SEQ ID NO. 22;

(7) The heavy chain amino acid sequence of the bispecific antibody is shown as SEQ ID NO. 33, and the light chain amino acid sequence of the bispecific antibody is shown as SEQ ID NO. 22;

(8) The heavy chain amino acid sequence of the bispecific antibody is shown as SEQ ID NO. 34, and the light chain amino acid sequence of the bispecific antibody is shown as SEQ ID NO. 22; or (b)

(9) A polypeptide derived from any one of (1) to (8) which is formed by substitution, deletion or addition of one or more amino acid residues to the amino acid sequence of any one of (1) to (8) and has both anti-PDL 1 activity and anti-IGF 1R activity.

In another preferred embodiment, the bispecific antibody 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-PDL 1 activity and 70-100% of the anti-IGF 1R 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 embodiment, the substitution is a conservative substitution.

In another preferred embodiment, the bispecific antibody, wherein the immunoglobulin of the first antigen binding domain or antigen binding fragment thereof is in an EC of 0.05-0.40nM, e.g., 0.05, 0.08, 0.24, 0.29, 0.30, 0.40nM ₅₀ Binding to PDL1 protein. Preferably, the EC ₅₀ As measured by enzyme-linked immunosorbent assay (ELISA).

In another preferred embodiment, the bispecific antibody, wherein the immunoglobulin of the second antigen binding domain or antigen binding fragment thereof is in an EC of 0.05-0.31nM, e.g., 0.05, 0.06, 0.07, 0.08, 0.09, 0.25, 0.31nM ₅₀ Binds IGF1R protein. Preferably, the EC ₅₀ As measured by enzyme-linked immunosorbent assay (ELISA).

In a second aspect of the invention there is provided an isolated nucleotide molecule (polynucleotide) encoding a bispecific antibody according to the first aspect of the invention, or a first antigen binding domain thereof, or a second antigen binding domain thereof, or a fragment thereof. Those skilled in the art will appreciate that nucleic acid molecules encoding the amino acid sequences of the above bispecific antibodies may be appropriately introduced into substitutions, deletions, alterations, insertions, or additions to provide a homolog of the nucleotide molecule.

In a third aspect of the invention there is provided a vector comprising an isolated nucleotide molecule of the invention. Vectors are well known to those skilled in the art and include, but are not limited to: plasmids, phagemids, cosmids, artificial chromosomes, phages, animal viruses, and the like. A vector may contain a variety of elements that control expression, including, but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. In addition, the vector may also contain a replication origin. Suitable methods of providing the above constructs should be known to those skilled in the art. For example, such constructs may be constructed by inserting the isolated polynucleotides into a suitable vector.

In another preferred embodiment, the expression vector is a virus or plasmid, preferably a phage or phagemid.

In another preferred embodiment, the expression vector is selected from the group consisting of: pcDNA3.4, pDR1, pcDNA3.1 (+), pcDNA3.1/ZEO (+), pDHFR, pTT5, pDHFF, pGM-CSF or pCHO 1.0, preferably pcDNA3.4.

In a fourth aspect of the invention, there is provided an expression system comprising a construct or a polynucleotide of the invention integrated into the genome as provided by the invention, whereby the bispecific antibody can be expressed. The expression system may typically be a host cell. The host cell includes, but is not limited to, bacterial, fungal, mammalian cells, and the like. In another preferred embodiment, the host cell is selected from the group consisting of: CSO, CHO, 293E, expi293F, NS0, sf9, sf21, DH5 a, BL21 (DE 3) or TG1, more preferably e.coli TG1, BL21 (DE 3) cells (expressing single chain antibodies or Fab antibodies) or CHO-K1 cells (expressing full length IgG antibodies).

In another preferred embodiment, the host cell is a eukaryotic cell, preferably a CHO cell or 293F cell.

In a fifth aspect of the invention, there is provided a method of preparing a bispecific antibody as described in the first aspect of the invention, the method comprising the steps of:

(a) Culturing a host cell according to the fourth aspect of the invention under expression conditions, thereby expressing the bispecific antibody;

(b) Isolating and purifying the bispecific antibody of step (a).

In a sixth aspect of the invention there is provided a conjugate comprising an antibody or antigen binding fragment thereof and a conjugate moiety, wherein the antibody or antigen binding fragment thereof is a bispecific antibody of the first aspect of the invention and the conjugate moiety is a detectable label or a drug molecule; preferably, the coupling moiety is a radioisotope, a fluorescent substance, a luminescent substance, a coloured substance or an enzyme. Preferably, the drug molecule is selected from the group consisting of vinca alkaloids, substituted ureas, methyl hydrazine derivatives, adrenocortical inhibitors, hormone antagonists, antimetabolites, anthracyclines, camptothecins, tubulin inhibitors, maytansinoids, spinosads, auristatins, nitrogen mustards, pyrimidine analogs, purine analogs, antibiotics, enzyme inhibitors, epipodophyllotoxins, platinum coordination complexes, alkylating agents, ethyleneimine derivatives, alkyl sulfonates, nitrosoureas, triazenes, folic acid analogs, taxanes, antimitotics, antiangiogenic agents, tyrosine kinase inhibitors, mTOR inhibitors, heat shock protein inhibitors, proteasome inhibitors, HDAC inhibitors, pro-apoptotic agents, and combinations thereof. For example, specific drugs that are useful are selected from the group consisting of: 5-fluorouracil, afatinib, april, azalide, anastrozole, anthracycline, axitinib, bendamustine, bleomycin, bortezomib, bosutinib, busulfan, spinosyns, camptothecine, carboplatin, 10-hydroxycamptothecine, carmustine, celecoxib, chlorambucil, cisplatin, carboplatin, cladribine, camptothecine, crizotinib, cyclophosphamide, cytarabine, dacarbazine, dasatinib, desiccharide, docetaxel, dactinomycin, daunorubicin, cyano-morpholino doxorubicin, epirubicin, estramustine, epipodophyllotoxin, erlotinib, enoxolone, lenient, lenalitinib, nilotinib, nitrosourea, olmesalamine, procalcitonin, prazidine, pravastatin, and the like.

In a seventh aspect of the invention, there is provided a kit comprising a bispecific antibody according to the first aspect of the invention, or comprising a conjugate according to the invention.

Preferably, the kit further comprises a second antibody that specifically recognizes the antibody or antigen-binding fragment thereof; optionally, the secondary antibody further comprises a detectable label, such as a fluorescent substance, luminescent substance, radioisotope, colored substance, enzyme, or the like.

In an eighth aspect of the invention there is provided the use of a bispecific antibody according to the first aspect of the invention or a conjugate according to the sixth aspect of the invention in the preparation of a kit for detecting the presence or level of PD-L1 and/or IGF1R in a sample.

In a ninth aspect of the invention, there is provided a pharmaceutical composition comprising a bispecific antibody according to the first aspect of the invention or a conjugate according to the sixth aspect of the invention; optionally, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier, medium. Such acceptable carriers, vehicles, e.g., sterile or normal saline, stabilizers, excipients, antioxidants (ascorbic acid, etc.), buffers (phosphoric acid, citric acid, other organic acids, etc.), preservatives, surfactants (PEG, tween, etc.), chelating agents (EDTA, etc.), binders, and the like. Furthermore, other low molecular weight polypeptides may be included; proteins such as serum albumin, gelatin, and immunoglobulins; amino acids such as glycine, glutamine, asparagine, arginine and lysine; saccharides or carbohydrates such as polysaccharides and monosaccharides; sugar alcohols such as mannitol and sorbitol. When preparing an aqueous solution for injection, for example, physiological saline, isotonic solution containing glucose or other auxiliary drugs, such as D-sorbitol, D-mannose, D-mannitol, sodium chloride, and the like, an appropriate solubilizing agent such as alcohol (ethanol or the like), polyol (propylene glycol, PEG or the like), nonionic surfactant (Tween 80, HCO-50) or the like may be used in combination.

The amount of active ingredient in the pharmaceutical composition of the present invention is usually a safe and effective amount which should be adjustable to those skilled in the art, for example, the amount of the active ingredient applied to the bispecific antibody and pharmaceutical composition is usually dependent on the body weight of the patient, the type of application, the condition and severity of the disease, for example, the amount of the bispecific antibody applied as an active ingredient may be usually 1 to 1000mg/kg/day, 20 to 200mg/kg/day, 1 to 3mg/kg/day, 3 to 5mg/kg/day, 5 to 10mg/kg/day, 10 to 20mg/kg/day, 20 to 30mg/kg/day, 30 to 40mg/kg/day, 40 to 60mg/kg/day, 60 to 80mg/kg/day, 80 to 100mg/kg/day, 100 to 150mg/kg/day, 150 to 200mg/kg/day, 200 to 300mg/kg/day, 300 mg/day, 500 mg/day or 500 mg/day.

The bispecific antibodies provided by the present invention may be adapted for any form of administration, be it oral or parenteral, for example, be it pulmonary, nasal, rectal and/or intravenous, more particularly intradermal, subcutaneous, intramuscular, intra-articular, intraperitoneal, pulmonary, buccal, sublingual, nasal, transdermal, vaginal, oral or parenteral. The skilled artisan can select a suitable formulation depending on the mode of administration, for example, a formulation suitable for oral administration may be a formulation including, but not limited to, a pill, tablet, chew, capsule, granule, drop, or syrup, etc., and for further example, a formulation suitable for parenteral administration may be a formulation including, but not limited to, a solution, suspension, reconstitutable dry preparation, spray, etc., and for further example, a suppository may be generally suitable for rectal administration.

In the pharmaceutical composition provided by the invention, the bispecific antibody can be a single active ingredient or can be combined with other active ingredients to form a combined preparation. The other active component can be other various medicines for treating tumor, myeloma or solid tumor and cancer. In some embodiments of the invention, the additional active ingredient is a tumor chemotherapeutic including, but not limited to, tyrosine kinase inhibitor, an Luoti ni or a pharmaceutically acceptable salt thereof, or lenvatinib or a pharmaceutically acceptable salt thereof. In other embodiments, the bispecific antibody is used in combination with PBMC, having significantly better activity to kill tumor cells than monoclonal antibodies.

In another preferred embodiment, the unit dose of the pharmaceutical composition is 100mg to 200mg, 200mg to 300mg, 300mg to 500mg, 500mg to 800mg, 800mg to 1000mg, etc., calculated according to the mass of the bispecific antibody therein.

In a tenth aspect of the invention, there is provided a combination product comprising a first product and a second product packaged separately,

the first product comprises a bispecific antibody according to the first aspect of the invention, a conjugate according to the sixth aspect of the invention or a pharmaceutical composition according to the ninth aspect of the invention;

The second product comprises one or more tumor chemotherapeutic;

preferably, the first product and the second product further independently comprise one or more pharmaceutically acceptable excipients;

preferably, the combination further comprises a product instruction.

In another preferred embodiment, the unit dose of the first product is 100mg to 200mg, 200mg to 300mg, 300mg to 500mg, 500mg to 800mg, 800mg to 1000mg, etc., calculated on the mass of the bispecific antibody therein.

In another preferred embodiment, the unit dose of the second product is 0.1mg to 10mg, 10mg to 20mg, 20mg to 30mg, 30mg to 40mg, 40mg to 50mg or 50mg to 100mg, calculated as the mass of the active ingredient therein.

In an eleventh aspect of the invention there is provided the use of a bispecific antibody according to the first aspect of the invention, a conjugate according to the sixth aspect of the invention, a pharmaceutical composition according to the ninth aspect of the invention or a combination product according to the tenth aspect of the invention for the manufacture of a medicament for the prevention and/or treatment of a tumor, or for the manufacture of a product for the diagnosis of a tumor.

Preferably, the tumor may be a solid tumor or hematological tumor, including but not limited to one or more of bowel cancer, esophageal cancer, head and neck cancer, skin cancer, kidney cancer, hepatocellular carcinoma, ovarian serous cyst adenocarcinoma, thyroid cancer, lung adenocarcinoma, head and neck squamous cell carcinoma, glioblastoma multiforme, thymus cancer, low-grade glioma of the brain, rectal adenocarcinoma, pheochromocytoma and paraganglioma, esophageal cancer, renal clear cell carcinoma, cervical squamous carcinoma and adenocarcinoma, melanoma, renal tumor, prostate cancer, bladder cancer, colon cancer, rectal cancer, stomach cancer, liver cancer, lung cancer, ovarian cancer, leukemia, breast cancer, mesothelioma, cervical cancer, endometrial cancer, lymphoma, nasopharyngeal carcinoma, bladder urothelial cancer, renal papillary cell carcinoma, pancreatic cancer, renal chromocytocarcinoma, breast infiltration carcinoma, lung squamous carcinoma, sarcoma, acute myeloid leukemia, and the like.

Preferably, the tumor is human in situ pancreatic adenocarcinoma or breast cancer. Further preferably, the tumor cell is BxPC-3 (human in situ pancreatic adenocarcinoma cell) or MCF7 (human breast cancer cell).

In a twelfth aspect of the invention there is provided the use of a bispecific antibody according to the first aspect of the invention, a conjugate according to the sixth aspect of the invention, a pharmaceutical composition according to the ninth aspect of the invention or a combination product according to the tenth aspect of the invention for one or more of the following:

Preparing a medicament for preventing and/or treating tumors;

preparing an antibody drug conjugate;

preparing a medicament for diagnosing tumors;

preparing a drug blocking a PDL1/PD-1 pathway;

preparing a medicament that down-regulates the activity or level of PDL 1;

preparing a medicament for relieving immunosuppression of PDL1 on an organism;

preparing a drug that blocks IGF1R binding to IGF 1;

preparing a medicament that down regulates IGF1R activity or IGF1R levels;

preparing the medicine for relieving the immune inhibition of IGF1R to organisms.

In a thirteenth aspect of the present invention there is provided a method of preventing and/or treating and/or assisting in the treatment and/or diagnosis of a tumor comprising the step of administering to a subject in need thereof an effective amount of a bispecific antibody according to the first aspect of the present invention, a conjugate according to the sixth aspect of the present invention, a pharmaceutical composition according to the ninth aspect of the present invention or a combination product according to the tenth aspect of the present invention; preferably, the tumor may be a solid tumor or hematological tumor, including but not limited to one or more of bowel cancer, esophageal cancer, head and neck cancer, skin cancer, kidney cancer, hepatocellular carcinoma, ovarian serous cyst adenocarcinoma, thyroid cancer, lung adenocarcinoma, head and neck squamous cell carcinoma, glioblastoma multiforme, thymus cancer, low-grade glioma of the brain, rectal adenocarcinoma, pheochromocytoma and paraganglioma, esophageal cancer, renal clear cell carcinoma, cervical squamous carcinoma and adenocarcinoma, melanoma, renal tumor, prostate cancer, bladder cancer, colon cancer, rectal cancer, stomach cancer, liver cancer, lung cancer, ovarian cancer, leukemia, breast cancer, mesothelioma, cervical cancer, endometrial cancer, lymphoma, nasopharyngeal carcinoma, bladder urothelial cancer, renal papillary cell carcinoma, pancreatic cancer, renal chromocytocarcinoma, breast infiltration carcinoma, lung squamous carcinoma, sarcoma, acute myeloid leukemia, and the like.

In another preferred embodiment, the method of the invention, the step of administering to the subject in need thereof an effective amount of an anti-PD-L1/IGF 1R bispecific antibody is before or after surgical treatment, and/or before or after radiation treatment.

In another preferred example, the method wherein the bispecific antibody is administered in an amount of usually 1 to 1000mg/kg/day, 20 to 200mg/kg/day, 1 to 3mg/kg/day, 3 to 5mg/kg/day, 5 to 10mg/kg/day, 10 to 20mg/kg/day, 20 to 30mg/kg/day, 30 to 40mg/kg/day, 40 to 60mg/kg/day, 60 to 80mg/kg/day, 80 to 100mg/kg/day, 100 to 150mg/kg/day, 150 to 200mg/kg/day, 200 to 300mg/kg/day, 300 to 500mg/kg/day, or 500 to 1000mg/kg/day.

In the methods of the invention, the dosing interval may be once every 1 day, 2 days, 3 days, 4 days, 5 days, 8 days, 10 days, 2 weeks, or 3 weeks. The administration may be intravenous drip or intravenous injection.

Compared with the prior art, the invention has the beneficial effects that:

the bispecific antibody disclosed by the invention can be combined with PD-L1 and IGF1R with high affinity, and can better simultaneously maintain the activities of anti-IGF 1R and anti-PDL 1 monoclonal antibody. The bispecific antibody can inhibit proliferation of tumor cells expressing IGF1R, can block PD1/PDL1 signal paths, specifically relieve immunosuppression of PDL1 on an organism, and activate T lymphocytes. The bispecific antibody has unique effects of mediating endocytosis of cell surface receptor to lysosome degradation and the like. The bispecific antibody can also be used in combination with other active ingredients, has activity obviously superior to that of monoclonal antibody in killing tumor cells, has potential in preventing and/or treating diseases related to PD-L1 and IGF1R activity, and has wide application prospect especially in preparing medicines for preventing and/or treating tumors related to PD-L1 and IGF1R activity.

Drawings

Fig. 1A: d31-1/2/5/6 structure schematic diagram.

Fig. 1B: d31-3/4/7/8 structure schematic diagram.

Fig. 2A: affinity of D31-1 to D31-4 with PDL1 antigen.

Fig. 2B: affinity of D31-5 to D31-8 with PDL1 antigen.

Fig. 2C: affinity of D31-1 to D31-4 with IGF1R antigen.

Fig. 2D: affinity of D31-5 to D31-8 with IGF1R antigen.

Fig. 3: d31-1 to D31-8 bind simultaneously to the affinity of PDL1 antigen and IGF1R antigen.

Fig. 4A: d31-1 to D31-4 block the cell pathway activity of PD1/PD-L1 on the cells.

Fig. 4B: d31-5 to D31-8 block the cell pathway activity of PD1/PD-L1 on the cells.

Fig. 5A: d31-1 to D31-4 inhibit proliferation of BxPC3 cells in vitro.

Fig. 5B: d31-5 to D31-8 inhibit proliferation of BxPC3 cells in vitro.

Fig. 6A: synergistic killing by bispecific antibody D31-2.

Fig. 6B: synergistic killing by bispecific antibody D31-8.

Fig. 7: bispecific antibody mediated endocytosis of BxPC3 cell surface receptor IGF 1R.

Fig. 8: western Blot detects changes in BxPC3 cell surface receptors IGF1R and PDL 1.

Detailed Description

Other advantages and effects of the present invention will become apparent to those skilled in the art from the following disclosure, which describes the embodiments of the present invention with reference to specific examples. The invention may be practiced or carried out in other embodiments that depart from the specific details, and the details of the present description may be modified or varied from the spirit and scope of the present invention.

Before the embodiments of the invention are explained in further detail, it is to be understood that the invention is not limited in its scope to the particular embodiments described below; it is also to be understood that the terminology used in the examples of the invention is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the invention; in the description and claims of the invention, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise.

Where numerical ranges are provided in the examples, it is understood that unless otherwise stated herein, both endpoints of each numerical range and any number between the two endpoints are significant both in the numerical range. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In addition to the specific methods, devices, materials used in the embodiments, any methods, devices, and materials of the prior art similar or equivalent to those described in the embodiments of the present invention may be used to practice the present invention according to the knowledge of one skilled in the art and the description of the present invention.

Unless otherwise indicated, the experimental methods, detection methods, and preparation methods disclosed in the present invention employ techniques conventional in the art of molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA techniques, and related arts.

In the present invention, unless otherwise indicated, scientific and technical terms used herein have the meanings commonly understood by one of ordinary skill in the art. Moreover, the cell culture, molecular genetics, nucleic acid chemistry, immunological laboratory procedures used herein are all conventional procedures widely used in the corresponding field. Meanwhile, in order to better understand the present invention, definitions and explanations of related terms are provided below.

As used herein, when referring to the amino acid sequence of a PDL1 (also known as PD-L1) protein, it includes the full length of the PDL1 protein, or an extracellular fragment of PDL 1; bispecific antibodies comprising extracellular fragments of PDL1, such as fragments fused to Fc protein fragments of mouse or human IgG, are also included. However, it is understood by those skilled in the art that mutations or variations (including but not limited to substitutions, deletions and/or additions) may be naturally occurring or artificially introduced in the amino acid sequence of the PDL1 protein without affecting its biological function. And, when describing a sequence fragment of a PDL1 protein, it also includes the corresponding sequence fragment in its natural or artificial variant.

As used herein, when referring to the amino acid sequence of an IGF1R (also known as IGF-1R) protein, it includes the full length of the IGF1R protein, or an extracellular fragment of IGF 1R; bispecific antibodies comprising extracellular fragments of IGF1R, such as fragments fused to Fc protein fragments of mouse or human IgG, are also included. However, it is understood by those skilled in the art that mutations or variations (including but not limited to substitutions, deletions and/or additions) may be naturally occurring or artificially introduced in the amino acid sequence of IGF1R protein without affecting its biological function. And, when describing a sequence fragment of IGF1R protein, it also includes the corresponding sequence fragment in its natural or artificial variant.

As used herein, the term EC ₅₀ Refers to half maximal effect concentration, refers to concentration that causes 50% maximal effect.

In this context, unless the context clearly indicates otherwise, when referring to the term "antibody" it includes not only whole antibodies, but also antigen-binding fragments of antibodies. The term "antibody", or "immunoglobulin" includes monoclonal antibodies, isolated, engineered, chemically synthesized or recombinant antibodies, polyclonal antibodies, multivalent antibodies or multispecific antibodies (e.g., bispecific antibodies), and antibody fragments so long as they exhibit the desired biological activity.

Antigen binding fragments of antibodies can be obtained from a given antibody using conventional techniques known to those skilled in the art (e.g., recombinant DNA techniques or enzymatic or chemical cleavage methods).

As used herein, the terms "mab" and "monoclonal antibody" refer to an antibody or a fragment of an antibody from a population of highly homologous antibody molecules, i.e., a population of identical antibody molecules except for natural mutations that may occur spontaneously. Monoclonal antibodies have a high specificity for a single epitope on an antigen.

As used herein, the term "humanized antibody" refers to an antibody or antibody fragment obtained by replacing all or part of the CDR regions of a human immunoglobulin (recipient antibody) with those of a non-human antibody (donor antibody), which may be a non-human (e.g., mouse, rat, or rabbit) antibody of desired specificity, affinity, or reactivity.

As used herein, the term "epitope" refers to a site on an antigen that is specifically bound by an immunoglobulin or antibody. "epitopes" are also known in the art as "antigenic determinants".

Bispecific antibodies

The bispecific antibody of the present invention (anti-PDL 1/IGF1R bispecific antibody or anti-PDL 1×igf1R bispecific antibody) is a bispecific antibody capable of specifically binding to PDL1 and IGF1R, which comprises an anti-PDL 1 antibody moiety and an anti-IGF 1R antibody moiety. Specifically, it comprises an immunoglobulin antibody IgG and two identical single chain variable region fragments scFv, wherein each single chain variable region fragment scFv comprises a variable region VH and a variable region VL, VH and VL are linked by a peptide linker L1, having a structure of VL-L1-VH or VH-L1-VL from N-terminus to C-terminus, and each single chain variable region fragment scFv is in tandem with the immunoglobulin antibody IgG by a peptide linker L2.

The "bispecific antibody" or "diabody" as used herein refers to a bispecific antibody having two different antigen binding sites capable of simultaneously binding PDL1 and IGF1R, comprising two single chain variable region fragments scFv and an immunoglobulin antibody IgG conjugated thereto, each scFv being linked to each heavy chain of the immunoglobulin IgG via a peptide linker L2 to form a heavy chain fusion protein of the bispecific antibody, wherein each scFv comprises a variable region VH and a variable region VL, VH and VL being linked by a peptide linker L1, having the structure VL-L1-VH or VH-L1-VL from the N-terminal to the C-terminal.

As described above, the anti-PDL 1 antibody moiety in the bispecific antibody of the present invention is an antibody or antigen-binding fragment thereof that specifically binds PDL1, and the anti-IGF 1R antibody moiety is an antibody or antigen-binding fragment thereof that specifically binds IGF 1R; the structure of the antigen binding fragment is selected from the group consisting of: (i) Fab fragments; (ii) a F (ab') 2 fragment; (iii) Fv fragments; or (iv) a single chain Fv (scFv);

bispecific antibodies of the invention may be dimers, trimers or multimers, preferably homo-or heterodimers. The anti-PDL 1 or anti-IGF 1R antibody moiety in the bispecific antibodies of the invention may comprise one or more antibodies or antigen binding fragments thereof, preferably 1, 2, 3, 4, 5, 6.

As a preferred embodiment, the bispecific antibody of the present invention comprises an scFv of an anti-PDL 1 antibody and an IgG antibody of an anti-IGF 1R, wherein the VH of the anti-PDL 1 antibody comprises complementarity determining regions HCDR1, HCDR2 and HCDR3, wherein the amino acid sequence of HCDR1 is shown as SEQ ID NO. 14, the amino acid sequence of HCDR2 is shown as SEQ ID NO. 15, and the amino acid sequence of HCDR3 is shown as SEQ ID NO. 16;

the VL of the anti-PDL 1 antibody comprises complementarity determining regions LCDR1, LCDR2 and LCDR3, wherein the amino acid sequence of LCDR1 is shown as SEQ ID NO. 18, the amino acid sequence of LCDR2 is shown as SEQ ID NO. 19, and the amino acid sequence of LCDR3 is shown as SEQ ID NO. 20;

the VH of the IgG antibody against IGF1R comprises complementarity determining regions HCDR1, HCDR2 and HCDR3, wherein the amino acid sequence of HCDR1 is shown as SEQ ID NO. 4, the amino acid sequence of HCDR2 is shown as SEQ ID NO. 5, and the amino acid sequence of HCDR3 is shown as SEQ ID NO. 6;

the VL of the IgG antibody against IGF1R comprises complementarity determining regions LCDR1, LCDR2 and LCDR3, wherein the amino acid sequence of LCDR1 is as set forth in SEQ ID NO:8, the amino acid sequence of LCDR2 is shown as SEQ ID NO:9, the amino acid sequence of LCDR3 is shown in SEQ ID NO: shown at 10.

As a preferred embodiment, the bispecific antibody of the present invention comprises an anti-PDL 1 IgG antibody and an anti-IGF 1R antibody scFv, wherein the anti-PDL 1 IgG antibody comprises the complementarity determining regions HCDR1, HCDR2 and HCDR3, wherein the amino acid sequence of HCDR1 is shown as SEQ ID NO. 14, the amino acid sequence of HCDR2 is shown as SEQ ID NO. 15, and the amino acid sequence of HCDR3 is shown as SEQ ID NO. 16;

The VL of the anti-PDL 1 IgG antibody comprises complementarity determining regions LCDR1, LCDR2 and LCDR3, wherein the amino acid sequence of LCDR1 is shown as SEQ ID NO. 18, the amino acid sequence of LCDR2 is shown as SEQ ID NO. 19, and the amino acid sequence of LCDR3 is shown as SEQ ID NO. 20;

the VH of the anti-IGF 1R antibody comprises complementarity determining regions HCDR1, HCDR2 and HCDR3, wherein the amino acid sequence of the HCDR1 is shown as SEQ ID NO. 4, the amino acid sequence of the HCDR2 is shown as SEQ ID NO. 5, and the amino acid sequence of the HCDR3 is shown as SEQ ID NO. 6;

the VL of the anti-IGF 1R antibody comprises complementarity determining regions LCDR1, LCDR2 and LCDR3, wherein the amino acid sequence of LCDR1 is as shown in SEQ ID NO:8, the amino acid sequence of LCDR2 is shown as SEQ ID NO:9, the amino acid sequence of LCDR3 is shown in SEQ ID NO: shown at 10.

In the art, the binding region of an antibody typically comprises a light chain variable region and a heavy chain variable region, each variable region comprising 3 CDR domains. The CDR domains of the heavy and light chains of antibodies are referred to as HCDR and LCDR, respectively. Thus, a conventional antibody antigen binding site comprises six CDRs, including sets of CDRs from the heavy and light chain V regions, respectively.

In the present invention, the term "bispecific antibody" also includes conservative variants thereof, which means that up to 10, preferably up to 8, more preferably up to 5, and most preferably up to 3 amino acids are replaced by amino acids of similar or similar nature, as compared to the amino acid sequence of the bispecific antibody of the present invention, to form a polypeptide.

As a preferable scheme, the amino acid sequence of the peptide linker L1 is shown as SEQ ID NO. 1.

As a preferable scheme, the amino acid sequence of the peptide linker L2 is shown as SEQ ID NO. 2.

Preferably, the single chain variable fragment scFv1 has a molecular structure of VL-L1-VH, and the N-terminal of each scFv1 is connected to the C-terminal of an immunoglobulin antibody IgG heavy chain via a peptide linker L2.

Preferably, the single chain variable fragment scFv2 has a molecular structure of VH-L1-VL, and the N-terminal of each scFv2 is connected to the C-terminal of the heavy chain of the immunoglobulin antibody IgG via a peptide linker L2.

Preferably, the single chain variable fragment scFv1 has a molecular structure of VL-L1-VH, and the C-terminal end of each scFv1 is connected to the N-terminal end of the heavy chain of the immunoglobulin antibody IgG via a peptide linker L2.

Preferably, the single chain variable fragment scFv2 has a molecular structure of VH-L1-VL, and the C-terminal end of each scFv2 is connected to the N-terminal end of the heavy chain of the immunoglobulin antibody IgG via a peptide linker L2.

As a preferred embodiment, the amino acid sequence of the single-chain variable fragment anti-PDL 1 scFv1 is shown in SEQ ID NO. 23.

As a preferred embodiment, the amino acid sequence of the single-chain variable fragment anti-PDL 1 scFv2 is shown in SEQ ID NO. 24.

As a preferred embodiment, the amino acid sequence of the single-chain variable fragment anti-IGF 1R scFv1 is shown in SEQ ID NO. 25.

As a preferred embodiment, the amino acid sequence of the single-chain variable fragment anti-IGF 1R scFv2 is shown in SEQ ID NO. 26.

As a preferable scheme, the heavy chain amino acid sequence of the bispecific antibody is shown as SEQ ID NO. 27, and the light chain amino acid sequence is shown as SEQ ID NO. 12.

As a preferable scheme, the heavy chain amino acid sequence of the bispecific antibody is shown as SEQ ID NO. 28, and the light chain amino acid sequence is shown as SEQ ID NO. 12.

As a preferable scheme, the heavy chain amino acid sequence of the bispecific antibody is shown as SEQ ID NO. 29, and the light chain amino acid sequence is shown as SEQ ID NO. 12.

As a preferable scheme, the heavy chain amino acid sequence of the bispecific antibody is shown as SEQ ID NO. 30, and the light chain amino acid sequence is shown as SEQ ID NO. 12.

As a preferable scheme, the heavy chain amino acid sequence of the bispecific antibody is shown as SEQ ID NO. 31, and the light chain amino acid sequence is shown as SEQ ID NO. 22.

As a preferable scheme, the heavy chain amino acid sequence of the bispecific antibody is shown as SEQ ID NO. 32, and the light chain amino acid sequence is shown as SEQ ID NO. 22.

As a preferable scheme, the heavy chain amino acid sequence of the bispecific antibody is shown as SEQ ID NO. 33, and the light chain amino acid sequence is shown as SEQ ID NO. 22.

As a preferable scheme, the heavy chain amino acid sequence of the bispecific antibody is shown as SEQ ID NO. 34, and the light chain amino acid sequence is shown as SEQ ID NO. 22.

In constructing the bispecific antibodies of the present invention, problems associated with the chemical and physical stability of the bispecific antibodies are also solved, such as expression of physically stable molecules, increased heat and salt dependent stability, reduced aggregation, increased solubility at high concentrations, and maintenance of affinity for the two antigens PDL1 and IGF1R, respectively, and the like.

The terms "polynucleotide" and "nucleic acid" are used interchangeably herein and generally refer to polymers of any length consisting essentially of nucleotides, such as deoxyribonucleotides and/or ribonucleotides. Nucleic acids may comprise purine and/or pyrimidine bases, and/or other natural, chemically or biochemically modified (e.g., methylated), non-natural, or derivatized nucleotide bases. The backbone of the nucleic acid may comprise sugar and phosphate groups, as commonly found in RNA or DNA, and/or one or more modified or substituted (e.g., 2' -O-alkylated, e.g., 2' -O-methylated or 2' -O-ethylated; or 2' -O,4' -C-alkynylated, e.g., 2' -O,4' -C-ethylated) sugar or one or more modified or substituted phosphate groups.

The term "expression system" or "recombinant host cell" or "recombinant engineering bacterium" or "host cell" refers to those cells used for transformation, i.e. cells used for expression of a gene of interest. The recombinant engineering bacteria may be isolated cells or cell lines cultured in culture, or cells present in living tissue or organisms. In the context of the present invention, the host cell is preferably a cell capable of growing in culture. The host cell may be a bacterial, fungal or mammalian cell. The bacteria are selected from one or more of escherichia coli, bacteroides ovatus, campylobacter jejuni, staphylococcus saprophyticus, enterococcus faecalis, bacteroides thetaiotaomicron, bacteroides vulgatus, lactobacillus casei, bacteroides fragilis, acinetobacter rouxii, fusobacterium nucleatum, bacteroides johnsonii, arabidopsis thaliana, lactobacillus rhamnosus, bacteroides massiliensis, parabacteroides faecalis, fusobacterium and bifidobacterium breve. The mammalian cells are selected from one or more of CSO, CHO, NS, sf9, sf21, 293E and Expi293F cells.

As used herein, the term "isolated" or "isolated" refers to obtained from a natural state by artificial means. The term "isolated" or "separated" does not exclude the presence of substances mixed with artificial or synthetic substances, nor the presence of other impurities which do not affect the activity of the substances.

As used herein, the term "vector" refers to a nucleic acid vehicle into which a polynucleotide may be inserted. When a vector enables expression of a protein encoded by an inserted polynucleotide, the vector is referred to as an expression vector. The vector may be introduced into a host cell by transformation, transduction or transfection such that the genetic material elements carried thereby are expressed in the host cell.

As used herein, the term "specific binding" refers to a non-random binding reaction between two molecules, such as a reaction between an antibody and an antigen against which it is directed.

Sequence identity or homology, as used herein, refers to the percentage of identical residues in the sequences involved in the alignment. The sequence identity of two or more sequences may be calculated using calculation software known in the art, which is available from sources such as NCBI.

In the present invention, "individual" generally includes human, non-human primates, such as mammals, dogs, cats, horses, sheep, pigs, cattle, etc., which may benefit from treatment with the above-described drugs, compositions, formulations, kits, or combination preparations.

In the present invention, a "therapeutically effective amount" generally means an amount that, after a suitable period of administration, achieves the effect of treating the diseases as set forth above.

The dosage of the formulation of the pharmaceutical composition of the present invention and the like may vary within a wide range depending on the disease or disorder to be treated, the age and condition of the individual patient, and the like. The appropriate dosage to be administered will be ultimately determined by the physician.

In the present invention, the "first antigen binding domain", and "second antigen binding domain" are intended to refer to distinguishing or descriptive clarity and not to have a typical sequential meaning unless otherwise specified.

The experimental materials and reagents used in the following examples are described below:

pcDNA ^TM 3.4vector: purchased from Thermo fisher company under the product number a14697;

293E cells: from NRC biotechnology Research Institute;

PD-1/PD-L1 Blockade Bioassay, propagation model: purchased from Promega company under the product number J1252;

BxPC3 cells: purchased from ATCCCRL-1687;

MCF7 cells: purchased from ATCCHTB-22;

SD rats: purchased from Shanghai Ling Biotechnology Co., ltd;

anti-PD-L1 mAb (anti-PDL 1 mAb): preparation according to the anti-PD-L1 mAb sequence in PCT/CN 2020/090442;

anti-IGF1R mab (anti-IGF 1 RmAb): prepared according to the A12 sequence in PCT/US 2004/013865;

IGF1R antigen: prepared according to UniprotKB sequence P08069;

PD-L1 antigen: prepared according to UniprotKB sequence Q9NZQ 7;

HRP-labeled goat anti-human Fc antibody: purchased from sigma, cat No. a0170;

HRP-labeled murine anti-human Fab antibody: purchased from sigma, cat No. a0293;

HRP-labeled anti-6 xhis antibody: purchased from abcam, cat No. ab178563;

FITC-labeled goat anti-human IgG: purchased from sigma, cat No. F4143;

PBS: purchased from the division of bioengineering (Shanghai), cat No. B548117;

PBST：PBS+0.05％Tween 20；

BSA: purchased from the division of bioengineering (Shanghai) under the product number A60332;

FBS: purchased from Gibco under the accession number 10099;

TMB: purchased from BD company, cat number 555214;

RPMI 1640 medium: purchased from gibco under accession number 22400-089;

Bio-Glo Luciferase Assay System: purchased from Promega, cat No. G7940;

cell Counting Kit-8: japanese same-core chemical institute (Dojindo), cat# CK04;

MEM medium: purchased from gibco under the accession number 11095-080;

red DND-99: purchased from Thermo Fisher, cat No. L7528;

Alexa Fluor ^TM 488NHS Ester (Succinimidyl Ester): purchased from Thermo Fisher, cat No. a2000;

IGF-I (N-Met), human: jinsri, cat No. Z0398-1;

western and IP cell lysate: green cloud, cat No. P0013;

PMSF (100 mM): biyun Tian, ST506;

BCA protein concentration assay kit (enhanced): biyundian, goods number P0010 (500 times)

SDS-PAGE protein loading buffer (5X): biyun day, goods number P0015L;

BeyoColor ^TM color pre-dye protein molecular weight standard (6.5-270 kD): biyundian, P0071;

PVDF film: doctor, goods number AR0136-04;

IGF-IR beta anti-body (D-11): santa Cruz biotechnology, cat# sc-398250;

Anti-PD-L1 antibody [ EPR19759] (Rabbit antibody): abcam, cat No. ab213524;

beta Actin Antibody: wuhan Sanying, cat# 66009-1-Ig;

TBS rinse buffer (dry powder): doctor, item AR0144;

HRP-labeled goat anti-mouse antibody: jackson, cat No. 115-035-003;

HRP-labeled goat anti-rabbit IgG (h+l): boolon, cat No. BF03008;

BeyoECL Star (ultra-hypersensitive ECL chemiluminescent kit): biyun day, cat No. P0018AS. The method comprises the steps of carrying out a first treatment on the surface of the

The experimental apparatus used in the following examples is illustrated as follows:

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

beckman Coulter CytoFLEX flow cytometer: purchased from Beckman corporation;

SpectraMax i3x microplate reader: purchased from Molecular Devices company;

SpectraMaxM5 microplate reader: purchased from Molecular Devices company;

high content analysis system: purchased from Perkinelmer.

EXAMPLE 1 construction of anti-PDL1×IGF1R bispecific antibody molecules

The anti-PD-L1 monoclonal antibody sequence adopts anti-human PD-L1 monoclonal antibody (the sequence is derived from PCT/CN 2020/090442); anti-IGF 1R mab was used with mab A12 (sequence derived from PCT/US 2004/013865).

An anti-PDL 1×igf1r bispecific antibody was constructed and designated D31. The molecular structure of the double antibody is shown in figures 1A-1B, wherein figure 1A is a structural schematic diagram of D31-1/2/5/6; FIG. 1B is a schematic diagram of the structure of D31-3/4/7/8. The sequences are shown in Table 5.

The bispecific antibodies D31-1 to D31-4 are constructed by adopting a mode that IgG1 of the anti-IGF 1R antibody A12 is connected in series with scFv of the anti-PDL 1 antibody; bispecific antibodies D31-5 to D31-8 were constructed by tandem connection of IgG1 of the anti-PDL 1 antibody with the scFv of the anti-IGF 1R antibody A12. The sequences of the antibodies are shown in Table 5.

Wherein, the connecting sequence of VH and VL of the scFv is 4 GGGGS, which is named L1 (SEQ ID NO: 1); the scFv was linked to IgG1 by 3 GGGGS, designated L2 (SEQ ID NO: 2).

The scFv1 (SEQ ID NO: 23) ligation sequence of the anti-PDL 1 antibody is VL-L1-VH;

the scFv2 (SEQ ID NO: 24) linkage sequence of the anti-PDL 1 antibody is VH-L1-VL;

the scFv1 (SEQ ID NO: 25) of anti-IGF 1R antibody A12 has a ligation sequence of VL-L1-VH;

The scFv2 (SEQ ID NO: 26) linkage sequence of anti-IGF1R antibody A12 is VH-L1-VL.

The light chain of D31-1 to D31-4 is the light chain of A12 (SEQ ID NO: 12);

the connection sequence of the heavy chain of D31-1 is anti-IGF1R-IgG1-L2-scFv1 (SEQ ID NO: 23), and the full-length sequence is shown as SEQ ID NO: 27;

the connection sequence of the heavy chain of D31-2 is anti-IGF1R-IgG1-L2-scFv2 (SEQ ID NO: 24), and the full-length sequence is shown as SEQ ID NO: 28;

the connection sequence of the heavy chain of D31-3 is scFv2 (SEQ ID NO: 24) -L2-anti-IGF1R-IgG1, and the full-length sequence is shown as SEQ ID NO: 29;

the connection sequence of the heavy chain of D31-4 is scFv1 (SEQ ID NO: 23) -L2-anti-IGF1R-IgG1, and the full-length sequence is shown as SEQ ID NO: 30.

The light chain of D31-5 to D31-8 is the light chain of an anti-PDL1 antibody (SEQ ID NO: 22);

the connection sequence of the heavy chain of D31-5 is anti-PDL1-IgG1-L2-scFv1 (SEQ ID NO: 25), and the full-length sequence is shown as SEQ ID NO: 31;

the connection sequence of the heavy chain of D31-6 is anti-PDL1-IgG1-L2-scFv2 (SEQ ID NO: 26), and the full-length sequence is shown as SEQ ID NO: 32;

the connection sequence of the heavy chain of D31-7 is scFv2 (SEQ ID NO: 26) -L2-anti-PDL1-IgG1, and the full-length sequence is shown as SEQ ID NO: 33;

the connection sequence of the heavy chain D31-8 is scFv1 (SEQ ID NO: 25) -L2-anti-PDL1-IgG1, and the full-length sequence is shown as SEQ ID NO: 34.

EXAMPLE 2 expression and purification of anti-PDL1×IGF1R bispecific antibodies

The DNA fragments of the heavy chain and the light chain of the anti-PDL1xIGF 1R bispecific antibody are respectively connected to a pcDNA3.4 vector, the recombinant plasmid is extracted to co-transfect 293E cells, after the cells are cultured for 5 to 7 days, the culture solution is filtered through high-speed centrifugation and vacuum filtration by a microporous filter membrane, and then is loaded to a ProteinA affinity chromatography column, protein is eluted by eluent containing 100mM citric acid and having the pH value of 3.5, and the protein is dialyzed to PBS having the pH value of 7.4. Filtering with 0.22 mu M filter membrane, measuring concentration, and placing in a refrigerator at 4deg.C for use.

Example 3 detection of affinity of anti-PDL1×IGF1R bispecific antibodies for antigen

The affinity of the antibodies for the antigen was detected by enzyme-linked immunosorbent assay (ELISA).

3.1 affinity detection with PD-L1 antigen

For detection of anti-PDL1XIGF 1R bispecific antibodies and PD-Affinity of L1 antigen, PDL1-ECD-His protein (synthesized PD-L1 ectodomain gene according to the sequence Q9NZQ of UniprotKB, added with signal peptide sequence at its N end, added with 6 XHis tag at its C end, respectively constructed into expression vector through EcoRI and HindIII cleavage sites, transfected HEK-293E cell expressed and purified) was diluted to 1000ng/mL with PBS buffer of pH7.4, and then 100. Mu.L/well was added into 96-well plate; incubating overnight at 4 ℃; the next day the plates were washed twice with PBST; blocking by adding PBST+1% BSA, blocking for 1 hour at 37 ℃; washing the plate twice with PBST; then, the antibody to be detected diluted with PBS+1% BSA gradient was added, and anti-PDL1 mAb (anti-PDL 1 mAb) was used as a positive control, the initial concentration was 12nM, and the 12 gradients were diluted step by step 3 times. Incubation at 37 ℃ for 1 hour; PBST washing the plate twice, adding a secondary anti-HRP-labeled goat anti-human Fc antibody, and incubating for 40 minutes at 37 ℃; PBST plates were washed three times, 100. Mu.L/well TMB was added, and left at room temperature (20.+ -. 5 ℃ C.) in the dark for 5 minutes; add 50. Mu.L/well 2M H ₂ SO ₄ Stopping substrate reaction by using a stopping solution, reading an OD value at 450nm of an enzyme-labeled instrument, performing data analysis by using a GraphPad Prism, drawing and calculating EC ₅₀ 。

As shown in FIGS. 2A-2B, the affinities of D31-2 to D31-4 and D31-5 to D31-8 were not greatly different from those of the positive control, but the affinities of D31-1 were slightly worse. EC (EC) ₅₀ The values are shown in tables 1A-1B.

TABLE 1 affinity of D31-1 to D31-4 with PDL1 antigen

Sample	D31-1	D31-2	D31-3	D31-4	anti-PDL1 mAb
						EC ₅₀ (nM)	0.3914	0.2957	0.3008	0.2494	0.1455

TABLE 1 affinity of D31-5-D31-8 for PDL1 antigen

Sample	D31-5	D31-6	D31-7	D31-8	anti-PDL1mAb
						EC ₅₀ (nM)	0.08583	0.07903	0.07491	0.0572	0.0836

3.2 affinity detection with IGF1R antigen

To examine the affinity of anti-PDL 1×igf1r bispecific antibody to IGF1R antigen, IGF1R-ECD-His protein (sequence provided by UniProt (sequence number P08069) was synthesized with PBS buffer ph7.4 and the ectodomain gene was added with a signal peptide sequence at its N-terminus, his-tag was added at the C-terminus, and two cleavage sites, ecoRI and HindIII, were respectively constructed into expression vectors, transfected HEK-293E cells expressed and purified), and after dilution, coated in ELISA plates (first plate coating concentration 100 ng/well, second plate coating concentration 20 ng/well); incubating overnight at 4 ℃; the next day the plates were washed twice with PBST; PBST+1% BSA is added to each hole for blocking, and blocking is carried out for 1 hour at 37 ℃; washing the plate twice with PBST; then, the antibody to be detected diluted with PBS+1% BSA gradient was added, and the anti-IGF 1R monoclonal antibody A12 was used as a positive control, the initial concentration was 12nM, and the mixture was diluted step by step 3 times for 12 gradients. Incubation at 37 ℃ for 1 hour; PBST washing the plate twice, adding a secondary anti-HRP-anti-hFc antibody, and incubating for 40 minutes at 37 ℃; PBST plates were washed three times and patted dry, 100. Mu.L TMB was added to each well and left in the dark at room temperature (20.+ -. 5 ℃ C.) for 5 minutes; 50 mu L of 2M H are added to each well ₂ SO ₄ Stopping substrate reaction by using a stopping solution, reading an OD value at 450nm of an enzyme-labeled instrument, performing data analysis by using GraphPad Prism9, drawing and calculating EC ₅₀ 。

The experimental results are shown in FIGS. 2C-2D, where the affinity of the D31-1 to D31-8 bispecific antibodies is not significantly different from that of the positive control A12. EC (EC) ₅₀ The values are shown in tables 1C-1D.

Table 1C.D31-1 to D31-4 affinity for IGF1R antigen

Sample	D31-1	D31-2	D31-3	D31-4	A12
						EC ₅₀ (nM)	0.1933	0.1851	0.3154	0.2492	0.1574

TABLE 1 affinity of D31-5-D31-8 for IGF1R antigen

Sample	D31-5	D31-6	D31-7	D31-8	A12
						EC ₅₀ (nM)	0.137	0.0961	0.06483	0.04653	0.06757

EXAMPLE 4ELISA detection of the ability of anti-PDL1×IGF1R bispecific antibodies to simultaneously bind PDL1 antigen and IGF1R antigen

To test the ability of anti-PDL1 xigf 1R bispecific antibodies to bind both IGF1R antigen and PDL1 antigen, PDL1-ECD-hFc protein (C-terminal of PDL1-ECD-His protein replaced with hFc tag) was diluted to 1 μg/mL with PBS buffer ph7.4, and then 100 μl/well was added to ELISA plates; incubating overnight at 4 ℃; the next day the plates were washed twice with PBST; PBST+1% BSA is added to each hole for blocking, and blocking is carried out for 1 hour at 37 ℃; washing the plate twice with PBST; then, the antibody to be detected diluted with PBS+1% BSA gradient, anti-PDL1 mAb and A12 as controls, the initial concentration was 30nM, and the mixture was diluted step by 3 times for 12 gradients. Incubation at 37 ℃ for 1 hour; PBST plates were washed twice, and 1. Mu.g/mL IGF1R-ECD-His antigen diluted in PBS pH7.4 was added, 100. Mu.L/well to ELISA plates. Incubation at 37 ℃ for 1 hour; PBST washing the plate twice, adding secondary antibody HRP-anti-His, and incubating for 40 minutes at 37 ℃; PBST plates were washed three times, 100. Mu.L/well TMB was added, and left at room temperature (20.+ -. 5 ℃ C.) in the dark for 5 minutes; add 50. Mu.L/well 2M H ₂ SO ₄ Stopping substrate reaction by using a stopping solution, reading an OD value at 450nm of an enzyme-labeled instrument, performing data analysis by using a GraphPad Prism, drawing and calculating EC ₅₀ 。

As shown in FIG. 3, D31-1, D31-2, D31-5, and D31-8 have the highest affinity for binding two antigens simultaneously, D31-3, D31-4, D31-6, and D31-7 times, while anti-PDL 1 mab and anti-IGF 1R mab do not have the ability to bind both antigens simultaneously. Its EC (EC) ₅₀ And Top values are shown in Table 2.

TABLE 2 affinity of D31-1 to D31-8 for simultaneous binding of PDL1 antigen and IGF1R antigen

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Example 5 anti-PDL1XIGF 1R bispecific antibody blocks cell pathway Activity of PD1/PD-L1 on cells

The experiment uses Promega PD-1/PD-L1 Blockade Bioassay, propagation model and methods.

Taking PD-L1 aAPC/CHO-K1 cells growing in logarithmic phase, transferring to a white bottom transparent 96-well plate after pancreatin digestion into single cells, 100 mu L/well, 2×10 ⁴ Individual cells/wells were incubated at 37℃with 5% CO ₂ Incubate overnight. The antibody to be detected, anti-PDL 1 monoclonal antibody, is taken as a positive control, diluted into 2X working solution concentration, the initial concentration is 33nM, and the concentration is diluted step by 3 times for 8 gradients. The density is 1.4-2 x 10 ⁶ PD1 effector cells with cell viability of more than 95% per cell/mL, and pancreatin digestion into 1.25X10 ⁶ Single cell suspension of individual cells/mL. Taking PD-L1 aAPC/CHO-K1 cells paved the day before, discarding the supernatant, and adding 40 mu L of gradient diluted antibody to be detected or anti-PDL 1 monoclonal antibody working solution; an equal volume of PD1 effector cells was then added. Placed at 37 ℃ and 5% CO ₂ Incubation was performed for 6 hours. mu.L of detection reagent Bio-Glo was added to each well, and after incubation for 10 minutes at room temperature, luminencement was read with spectramax i 3. The bottom of the plate is sealed by an opaque film before reading the plate. Data analysis was performed using GraphPad Prism, mapping and IC calculation ₅₀ 。

As shown in FIGS. 4A-4B, the PD1/PD-L1 cell pathway blocking activities of the bispecific antibodies D31-2 to D31-8 were not significantly different from those of the positive control anti-PDL 1 mab, except that the activity of D31-1 was poor. EC (EC) ₅₀ The values are shown in tables 3A-3B.

TABLE 3A.D31-1 to D31-4 blocking the cell pathway Activity of PD1/PD-L1 on cells

Sample	D31-1	D31-2	D31-3	D31-4	anti-PDL1mAb
						EC ₅₀ (nM)	2.98	1.75	1.826	1.313	0.901

TABLE 3 B.D31-5-D31-8 blocking the cell pathway Activity of PD1/PD-L1 on cells

Sample	D31-5	D31-6	D31-7	D31-8	anti-PDL1mAb
						EC ₅₀ (nM)	0.4012	0.3357	0.4957	0.3905	0.3635

EXAMPLE 6 inhibition of in vitro proliferation of BxPC3 cells by anti-PDL1×IGF1R bispecific antibody

The experimental procedure was as follows:

bxpc3 cell plating: cells BxPC3 in logarithmic growth phase were digested with pancreatin, and then diluted to 5X 10 with RPMI 1640 complete medium containing 10% FBS ⁴ Per mL,100 μl/well of 96 well plate. I.e., 5000 cells/well plates, 2 plates. 37 ℃, CO ₂ The incubator was incubated overnight.

2. The next day starvation treatment: the original culture medium in the 96-well plate was aspirated by a pipette and replaced with serum-free RPMI 1640 basal medium. Starvation was performed overnight.

3. Dosing on day 3: IGF-1 was diluted to 200ng/mL in serum-free basal medium RPMI 1640, and then the antibody to be tested was diluted with this solution, and anti-IGF 1R antibody A12 was used as a positive control. Initial concentration was 3000nM, stepwise 4-fold dilution, 11 gradients, 100. Mu.L/well into 96-well plates. I.e.the working concentration of the antibody was 1500nM, and the final IGF-1 concentration was 100ng/mL.37 ℃,5% CO ₂ The incubator continues to cultivate.

4. Color development: after 2 days of administration, the 96-well plate was removed, the medium was discarded, and 1:10 dilutions of CCK8, 100. Mu.L/well were added to 96-well plates for color development. OD450 was read with a microplate reader after incubation in an incubator at 37 ℃ for a suitable period.

Data analysis was performed using GraphPad Prism, mapping and IC50 calculation.

As shown in the experimental results in FIGS. 5A-5B, the proliferation inhibition activity of D31-1 and D31-2 on BxPC3 is superior to that of D31-3, D31-4 and positive control A12; and the activity of D31-8 is slightly better than that of D31-5-D31-7, which is equivalent to that of positive control. EC (EC) ₅₀ The values are shown in tables 4A-4B.

TABLE 4 inhibition of proliferation of BxPC3 cells in vitro by D31-1 to D31-4

Sample	D31-1	D31-2	D31-3	D31-4	A12
						IC ₅₀ (nM)	1.962	2.511	47.74	16.54	6.766

TABLE 4 inhibition of proliferation of BxPC3 cells by D31-5-D31-8 in vitro

Sample	D31-5	D31-6	D31-7	D31-8	A12
						IC ₅₀ (nM)	39.56	67.05	20.52	5.616	14.01

Example 7 synergistic effects of cellular levels of anti-PDL1×IGF1R bispecific antibodies

The experimental procedure was as follows:

1. cell plating 96-well plates: pancreatin digestion of logarithmically grown cells MCF-7, followed by dilution of the cells with MEM complete medium containing 10% FBS to 1X 10 ⁵ Per mL,100 μl/well of 96 well plate. I.e. 1×10 ⁴ Individual cells/wells were plated, two plates were plated. 37 ℃, CO ₂ The incubator was incubated overnight.

2. Cell starvation treatment: after 24 hours of plating, the original complete culture medium is sucked out, the serum-free basic culture medium is replaced at 37 ℃, CO ₂ The incubator was incubated overnight.

3. The third day of administration and PBMC were added containing IGF-I at a final concentration of 100 ng/mL.

A first plate: the antibodies D31-2, A12+anti-PDL1mAb, A12 and anti-PDL1mAb to be detected are respectively diluted by MEM culture medium. 50. Mu.L/well was added to an overnight incubation 96-well plate at an antibody working concentration of 1. Mu.M and 100nM. PBMC cells 50. Mu.L/5X 10 ⁴ And/or holes. 37 ℃,5% CO ₂ The incubator continues to cultivate.

A second plate: the antibodies D31-8, A12+anti-PDL1mAb, A12 and anti-PDL1mAb to be detected are respectively diluted by MEM culture medium. 50. Mu.L/well was added to an overnight incubation 96-well plate with working concentrations of 500nM, 100nM, 25nM of antibody to be detected. PBMC cells 50. Mu.L/1X 10 ⁵ And/or holes. 37 ℃,5% CO ₂ The incubator continues to cultivate.

4. Color development reading plate: after 3 days of administration, 80. Mu.L/well of cell titro Glo was added and after 10 minutes incubation luminescense was read.

Data analysis was performed using GraphPad Prism, mapping and IC calculation ₅₀ 。

The experimental results are shown in FIGS. 6A-6B, D31-2 and D31-8 have significantly better activity in killing MCF-7 cells than monoclonal antibodies.

Example 8 anti-PDL1×IGF1R bispecific antibody mediated endocytosis of cell surface receptors

anti-IGF 1R antibodies are capable of mediating endocytosis of the cell surface receptor IGF1R to lysosomes. Endocytosis of BxPC3 cell surface receptor IGF1R was detected with a fluorescently labeled antibody and a lysosomal tracer. The test steps are as follows:

1. bispecific antibody D31-8 was labeled with green fluorescent Alexu Fluor 488: concentrating D31-8 protein to 4-5 mg/mL, taking 150 mu L and adding one tenth volume of 1M NaHCO ₃ Buffer (ph=9), pH adjusted to around 8.5, adding one tenth volume of Alexu Fluor 488 dye; rotating and vibrating at room temperature, and uniformly mixing for 1.5 hours; adding 1/10 volume of Tris-hydroxylamine (pH=9), and mixing for 1 hour at room temperature by rotary shaking; the reaction solution was added to a filter column, and the dye free of unbound protein was removed by centrifugation. Repeatedly adding PBS for washing and centrifuging, replacing the fluorescent marked protein into the PBS, and measuring the concentration for standby.

2. Paving cells: spreading BxPC3 cells in logarithmic growth phase on 96-well flat base plate with 1-2×10 ⁴ And/or holes. Placing at 37deg.C, 5% CO ₂ The incubator was left overnight.

3. Fluorescent endocytosis: the next day, fluorescence labeled antibody D31-8 was added at a final concentration of 150nM; lysosomal tracers were added at a final concentration of 65nM. Placing at 37deg.C, 5% CO ₂ After incubation in the incubator for 2 hours, the supernatant was discarded, washed with PBS and cells were digested with a small amount of pancreatin. Photographing by a high content imager.

The results are shown in fig. 7, where green fluorescent-labeled antibody binds to cell surface receptor resulting in endocytosis of the receptor and overlaps with red lysosomal tracer to yellow light, indicating localization to lysosomes after endocytosis of the receptor, suggesting that anti-PDL 1 xigf 1R bispecific antibody D31-8 is capable of mediating endocytosis of cell surface receptor IGF 1R.

Example 9 anti-PDL1×IGF1R bispecific antibodies lead to degradation of cell surface receptor IGF1R and PDL1

BxPC3 cell surface expresses IGF1R and PD-L1, and addition of anti-PDL1xIGF 1R bispecific antibody can cause endocytosis of IGF1R to lysosomes. The change in the content of IGF1R and PD-L1 proteins on the cell surface was detected by Western Blot.

The experimental procedure was as follows:

1. taking BxPC3 in logarithmic growth phase, centrifuging by pancreatin digestion, removing supernatant, re-suspending cells with culture medium of RPMI1640 and 10% FBS, and spreading 6-well plate, 5×10 ⁵ /well. Placing at 37deg.C, 5% CO ₂ The incubator was incubated overnight. The medium was then discarded and replaced with serum-free medium for overnight starvation.

2. Cells starved overnight were medicated under 8 different conditions (see FIG. 8), with 100nM antibody concentration and 80ng/mL IGF-1 concentration.

3. Adding the medicine, and then placing at 37 ℃ and 5 percent CO ₂ The incubator was incubated for 24 hours.

4. Cells were washed with PBS and lysates were added. 300. Mu.L/well was added to a 6-well plate, and after 10 minutes of lysis, samples were collected and centrifuged at 12000rpm to collect the supernatant. Protein concentration was measured by BCA method and protein concentration was adjusted consistently for 8 samples.

SDS-PAGE, transfer PVDF membrane.

6. The membrane was blocked, with 2% BSA for 2 hours at room temperature.

7. Incubating the primary antibody, murine anti-IGF 1R antibody, 1:1000, overnight at 4 ℃; rabbit anti-PD-L1 antibody, 1:1000, overnight at 4 ℃; mice were resistant to β -actin at 1:1000 overnight at 4 ℃.

8. The secondary antibody is incubated, the TBS rinsing buffer is used for washing the membrane three times, goat anti-mouse is diluted 1:2000, and the incubation is carried out for 1 hour. The secondary antibody against PDL1 antibody was diluted 1:2000 with goat anti-rabbit IgG and incubated for 1 hour.

9. Color development and photographing, and washing the membrane three times by TBS rinsing buffer solution. BeyoECL Star (ultra hypersensitive ECL chemiluminescent kit) developed and photographed by a BIORAD imager.

As shown in FIG. 8, the receptor IGF1R and PDL1 protein content on the surfaces of the BxPC3 cells of the D31-2 and D31-8 groups were significantly reduced compared to the blank. The experimental results in connection with example 8 speculate that: after incubation of BxPC3 cells with bispecific antibodies, the cell surface receptor IGF1R endocytoses to lysosomes and is degraded in the lysosomes. Not only is the content of IGF1R greatly reduced, but PDL1 is also obviously reduced, and it is speculated that the bispecific antibody can inhibit immune escape caused by PDL 1.

From the above examples, it can be seen that double inhibition of BxPC3 cell proliferation can activate the PD1/PDL1 signal pathway; the bispecific antibodies D31-2 and D31-8 have better activity, can bind IGF1R and PDL1 antigen simultaneously, can inhibit proliferation of BxPC3 cells, can block PD1/PDL1 signal paths, have a unique function-cause endocytosis of IGF1R receptor to lysosome to be degraded, and obviously reduce the content of PDL1 on the cell surface, and presumably can inhibit immune escape caused by PDL 1; moreover, the synergistic effect experiment of the cell level shows that the double antibody combined with PBMC has the activity of killing tumor cells obviously superior to that of monoclonal antibody.

TABLE 5 antibody sequences

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While the invention has been described with respect to preferred embodiments thereof, it will be understood by those skilled in the art that various modifications and additions may be made without departing from the scope of the invention. Equivalent embodiments of the present invention will be apparent to those skilled in the art having the benefit of the teachings disclosed herein, when considered in the light of the foregoing disclosure, and without departing from the spirit and scope of the invention; meanwhile, any equivalent changes, modifications and evolution of the above embodiments according to the essential technology of the present invention still fall within the scope of the technical solution of the present invention.

Sequence listing

<110> Zeda biological medicine Co., ltd

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

85 90 95

Thr Phe Gly Ala Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 18

<211> 11

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 18

Arg Ala Ser Gln Ser Ile Gly Thr Thr Ile His

1 5 10

<210> 19

<211> 7

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 19

Tyr Ala Ser Gln Ser Phe Ser

1 5

<210> 20

<211> 9

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 20

Gln Gln Ser Asn Ser Trp Pro Leu Thr

1 5

<210> 21

<211> 447

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 21

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

<210> 22

<211> 214

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 22

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 23

<211> 244

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 23

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Thr Phe Gly Ala Gly Thr Lys Leu Glu Ile Lys Gly Gly Gly Gly Ser

100 105 110

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

Thr Val Ser Ser

<210> 24

<211> 244

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 24

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

115 120 125

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

130 135 140

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

145 150 155 160

Arg Ala Ser Gln Ser Ile Gly Thr Thr Ile His Trp Tyr Gln Gln Lys

165 170 175

Pro Asp Gln Ser Pro Lys Leu Leu Ile Lys Tyr Ala Ser Gln Ser Phe

180 185 190

Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe

195 200 205

Thr Leu Thr Ile Asn Ser Val Glu Ala Glu Asp Ala Ala Thr Tyr Tyr

210 215 220

Cys Gln Gln Ser Asn Ser Trp Pro Leu Thr Phe Gly Ala Gly Thr Lys

225 230 235 240

Leu Glu Ile Lys

<210> 25

<211> 259

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 25

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

1 5 10 15

Thr Val Arg Ile Thr Cys Gln Gly Asp Ser Leu Arg Ser Tyr Tyr Ala

20 25 30

Thr Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Ile Leu Val Ile Tyr

35 40 45

Gly Glu Asn Lys Arg Pro Ser Gly Ile Pro Asp Arg Phe Ser Gly Ser

50 55 60

Ser Ser Gly Asn Thr Ala Ser Leu Thr Ile Thr Gly Ala Gln Ala Glu

65 70 75 80

Asp Glu Ala Asp Tyr Tyr Cys Lys Ser Arg Asp Gly Ser Gly Gln His

85 90 95

Leu Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gly Gly Gly

100 105 110

Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

115 120 125

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

130 135 140

Ser Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Ser

145 150 155 160

Tyr Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp

165 170 175

Met Gly Gly Ile Ile Pro Ile Phe Gly Thr Ala Asn Tyr Ala Gln Lys

180 185 190

Phe Gln Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala

195 200 205

Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr

210 215 220

Cys Ala Arg Ala Pro Leu Arg Phe Leu Glu Trp Ser Thr Gln Asp His

225 230 235 240

Tyr Tyr Tyr Tyr Tyr Met Asp Val Trp Gly Lys Gly Thr Thr Val Thr

245 250 255

Val Ser Ser

<210> 26

<211> 259

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 26

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

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Ser Tyr

20 25 30

Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Gly Ile Ile Pro Ile Phe Gly Thr Ala Asn Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Ala Pro Leu Arg Phe Leu Glu Trp Ser Thr Gln Asp His Tyr

100 105 110

Tyr Tyr Tyr Tyr Met Asp Val Trp Gly Lys Gly Thr Thr Val Thr Val

115 120 125

Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

130 135 140

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

145 150 155 160

Ser Val Ala Leu Gly Gln Thr Val Arg Ile Thr Cys Gln Gly Asp Ser

165 170 175

Leu Arg Ser Tyr Tyr Ala Thr Trp Tyr Gln Gln Lys Pro Gly Gln Ala

180 185 190

Pro Ile Leu Val Ile Tyr Gly Glu Asn Lys Arg Pro Ser Gly Ile Pro

195 200 205

Asp Arg Phe Ser Gly Ser Ser Ser Gly Asn Thr Ala Ser Leu Thr Ile

210 215 220

Thr Gly Ala Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Lys Ser Arg

225 230 235 240

Asp Gly Ser Gly Gln His Leu Val Phe Gly Gly Gly Thr Lys Leu Thr

245 250 255

Val Leu Gly

<210> 27

<211> 719

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 27

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

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Ser Tyr

20 25 30

Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Gly Ile Ile Pro Ile Phe Gly Thr Ala Asn Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Ala Pro Leu Arg Phe Leu Glu Trp Ser Thr Gln Asp His Tyr

100 105 110

Tyr Tyr Tyr Tyr Met Asp Val Trp Gly Lys Gly Thr Thr Val Thr Val

115 120 125

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

130 135 140

Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys

145 150 155 160

Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

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

450 455 460

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Ile Val Leu Thr

465 470 475 480

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

485 490 495

Thr Cys Arg Ala Ser Gln Ser Ile Gly Thr Thr Ile His Trp Tyr Gln

500 505 510

Gln Lys Pro Asp Gln Ser Pro Lys Leu Leu Ile Lys Tyr Ala Ser Gln

515 520 525

Ser Phe Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr

530 535 540

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

545 550 555 560

Tyr Tyr Cys Gln Gln Ser Asn Ser Trp Pro Leu Thr Phe Gly Ala Gly

565 570 575

Thr Lys Leu Glu Ile Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

580 585 590

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val Gln Leu Gln Gln

595 600 605

Ser Gly Gly Gly Leu Val Lys Pro Ser Gln Ser Leu Ser Leu Thr Cys

610 615 620

Thr Val Ser Gly Phe Ser Leu Thr Ser Tyr Gly Val His Trp Val Arg

625 630 635 640

Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile Gly Leu Ile Trp Ser Gly

645 650 655

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

660 665 670

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

675 680 685

Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala Arg Gln Leu Gly Leu Arg

690 695 700

Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser

705 710 715

<210> 28

<211> 719

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 28

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

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Ser Tyr

20 25 30

Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Gly Ile Ile Pro Ile Phe Gly Thr Ala Asn Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Ala Pro Leu Arg Phe Leu Glu Trp Ser Thr Gln Asp His Tyr

100 105 110

Tyr Tyr Tyr Tyr Met Asp Val Trp Gly Lys Gly Thr Thr Val Thr Val

115 120 125

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

130 135 140

Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys

145 150 155 160

Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

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

450 455 460

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val Gln Leu Gln

465 470 475 480

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

485 490 495

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

500 505 510

Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile Gly Leu Ile Trp Ser

515 520 525

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

530 535 540

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

545 550 555 560

Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala Arg Gln Leu Gly Leu

565 570 575

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

580 585 590

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

595 600 605

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

610 615 620

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

625 630 635 640

Ile Gly Thr Thr Ile His Trp Tyr Gln Gln Lys Pro Asp Gln Ser Pro

645 650 655

Lys Leu Leu Ile Lys Tyr Ala Ser Gln Ser Phe Ser Gly Val Pro Ser

660 665 670

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Asn

675 680 685

Ser Val Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Ser Asn

690 695 700

Ser Trp Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Ile Lys

705 710 715

<210> 29

<211> 719

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 29

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

115 120 125

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

130 135 140

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

145 150 155 160

Arg Ala Ser Gln Ser Ile Gly Thr Thr Ile His Trp Tyr Gln Gln Lys

165 170 175

Pro Asp Gln Ser Pro Lys Leu Leu Ile Lys Tyr Ala Ser Gln Ser Phe

180 185 190

Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe

195 200 205

Thr Leu Thr Ile Asn Ser Val Glu Ala Glu Asp Ala Ala Thr Tyr Tyr

210 215 220

Cys Gln Gln Ser Asn Ser Trp Pro Leu Thr Phe Gly Ala Gly Thr Lys

225 230 235 240

Leu Glu Ile Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly

245 250 255

Gly Gly Ser Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys

260 265 270

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

275 280 285

Ser Ser Tyr Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu

290 295 300

Glu Trp Met Gly Gly Ile Ile Pro Ile Phe Gly Thr Ala Asn Tyr Ala

305 310 315 320

Gln Lys Phe Gln Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser

325 330 335

Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val

340 345 350

Tyr Tyr Cys Ala Arg Ala Pro Leu Arg Phe Leu Glu Trp Ser Thr Gln

355 360 365

Asp His Tyr Tyr Tyr Tyr Tyr Met Asp Val Trp Gly Lys Gly Thr Thr

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

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

450 455 460

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

465 470 475 480

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

485 490 495

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

500 505 510

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

515 520 525

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

530 535 540

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

545 550 555 560

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

565 570 575

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

580 585 590

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

595 600 605

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

610 615 620

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

625 630 635 640

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

645 650 655

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

660 665 670

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

675 680 685

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

690 695 700

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

705 710 715

<210> 30

<211> 719

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 30

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Thr Phe Gly Ala Gly Thr Lys Leu Glu Ile Lys Gly Gly Gly Gly Ser

100 105 110

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly

245 250 255

Gly Gly Ser Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys

260 265 270

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

275 280 285

Ser Ser Tyr Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu

290 295 300

Glu Trp Met Gly Gly Ile Ile Pro Ile Phe Gly Thr Ala Asn Tyr Ala

305 310 315 320

Gln Lys Phe Gln Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser

325 330 335

Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val

340 345 350

Tyr Tyr Cys Ala Arg Ala Pro Leu Arg Phe Leu Glu Trp Ser Thr Gln

355 360 365

Asp His Tyr Tyr Tyr Tyr Tyr Met Asp Val Trp Gly Lys Gly Thr Thr

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

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

450 455 460

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

465 470 475 480

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

485 490 495

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

500 505 510

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

515 520 525

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

530 535 540

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

545 550 555 560

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

565 570 575

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

580 585 590

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

595 600 605

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

610 615 620

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

625 630 635 640

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

645 650 655

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

660 665 670

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

675 680 685

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

690 695 700

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

705 710 715

<210> 31

<211> 721

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 31

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ser Ser

450 455 460

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

465 470 475 480

Arg Ile Thr Cys Gln Gly Asp Ser Leu Arg Ser Tyr Tyr Ala Thr Trp

485 490 495

Tyr Gln Gln Lys Pro Gly Gln Ala Pro Ile Leu Val Ile Tyr Gly Glu

500 505 510

Asn Lys Arg Pro Ser Gly Ile Pro Asp Arg Phe Ser Gly Ser Ser Ser

515 520 525

Gly Asn Thr Ala Ser Leu Thr Ile Thr Gly Ala Gln Ala Glu Asp Glu

530 535 540

Ala Asp Tyr Tyr Cys Lys Ser Arg Asp Gly Ser Gly Gln His Leu Val

545 550 555 560

Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gly Gly Gly Gly Ser

565 570 575

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu

580 585 590

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

595 600 605

Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Ser Tyr Ala

610 615 620

Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met Gly

625 630 635 640

Gly Ile Ile Pro Ile Phe Gly Thr Ala Asn Tyr Ala Gln Lys Phe Gln

645 650 655

Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr Met

660 665 670

Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala

675 680 685

Arg Ala Pro Leu Arg Phe Leu Glu Trp Ser Thr Gln Asp His Tyr Tyr

690 695 700

Tyr Tyr Tyr Met Asp Val Trp Gly Lys Gly Thr Thr Val Thr Val Ser

705 710 715 720

Ser

<210> 32

<211> 721

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 32

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

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

450 455 460

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

465 470 475 480

Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Ser Tyr Ala Ile

485 490 495

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

500 505 510

Ile Ile Pro Ile Phe Gly Thr Ala Asn Tyr Ala Gln Lys Phe Gln Gly

515 520 525

Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr Met Glu

530 535 540

Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg

545 550 555 560

Ala Pro Leu Arg Phe Leu Glu Trp Ser Thr Gln Asp His Tyr Tyr Tyr

565 570 575

Tyr Tyr Met Asp Val Trp Gly Lys Gly Thr Thr Val Thr Val Ser Ser

580 585 590

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

595 600 605

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

610 615 620

Ala Leu Gly Gln Thr Val Arg Ile Thr Cys Gln Gly Asp Ser Leu Arg

625 630 635 640

Ser Tyr Tyr Ala Thr Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Ile

645 650 655

Leu Val Ile Tyr Gly Glu Asn Lys Arg Pro Ser Gly Ile Pro Asp Arg

660 665 670

Phe Ser Gly Ser Ser Ser Gly Asn Thr Ala Ser Leu Thr Ile Thr Gly

675 680 685

Ala Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Lys Ser Arg Asp Gly

690 695 700

Ser Gly Gln His Leu Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu

705 710 715 720

Gly

<210> 33

<211> 721

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 33

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

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Ser Tyr

20 25 30

Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Gly Ile Ile Pro Ile Phe Gly Thr Ala Asn Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Ala Pro Leu Arg Phe Leu Glu Trp Ser Thr Gln Asp His Tyr

100 105 110

Tyr Tyr Tyr Tyr Met Asp Val Trp Gly Lys Gly Thr Thr Val Thr Val

115 120 125

Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

130 135 140

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

145 150 155 160

Ser Val Ala Leu Gly Gln Thr Val Arg Ile Thr Cys Gln Gly Asp Ser

165 170 175

Leu Arg Ser Tyr Tyr Ala Thr Trp Tyr Gln Gln Lys Pro Gly Gln Ala

180 185 190

Pro Ile Leu Val Ile Tyr Gly Glu Asn Lys Arg Pro Ser Gly Ile Pro

195 200 205

Asp Arg Phe Ser Gly Ser Ser Ser Gly Asn Thr Ala Ser Leu Thr Ile

210 215 220

Thr Gly Ala Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Lys Ser Arg

225 230 235 240

Asp Gly Ser Gly Gln His Leu Val Phe Gly Gly Gly Thr Lys Leu Thr

245 250 255

Val Leu Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly

260 265 270

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

275 280 285

Ser Gln Ser Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Thr

290 295 300

Ser Tyr Gly Val His Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

Cys Ala Arg Gln Leu Gly Leu Arg Ala Met Asp Tyr Trp Gly Gln Gly

370 375 380

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

385 390 395 400

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

405 410 415

Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp

420 425 430

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

435 440 445

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

450 455 460

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

465 470 475 480

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

485 490 495

Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro

500 505 510

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

515 520 525

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

530 535 540

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

545 550 555 560

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

565 570 575

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

580 585 590

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

595 600 605

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

610 615 620

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

625 630 635 640

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

645 650 655

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

660 665 670

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

675 680 685

Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu

690 695 700

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

705 710 715 720

Lys

<210> 34

<211> 721

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 34

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

1 5 10 15

Thr Val Arg Ile Thr Cys Gln Gly Asp Ser Leu Arg Ser Tyr Tyr Ala

20 25 30

Thr Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Ile Leu Val Ile Tyr

35 40 45

Gly Glu Asn Lys Arg Pro Ser Gly Ile Pro Asp Arg Phe Ser Gly Ser

50 55 60

Ser Ser Gly Asn Thr Ala Ser Leu Thr Ile Thr Gly Ala Gln Ala Glu

65 70 75 80

Asp Glu Ala Asp Tyr Tyr Cys Lys Ser Arg Asp Gly Ser Gly Gln His

85 90 95

Leu Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gly Gly Gly

100 105 110

Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

115 120 125

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

130 135 140

Ser Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Ser

145 150 155 160

Tyr Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp

165 170 175

Met Gly Gly Ile Ile Pro Ile Phe Gly Thr Ala Asn Tyr Ala Gln Lys

180 185 190

Phe Gln Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala

195 200 205

Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr

210 215 220

Cys Ala Arg Ala Pro Leu Arg Phe Leu Glu Trp Ser Thr Gln Asp His

225 230 235 240

Tyr Tyr Tyr Tyr Tyr Met Asp Val Trp Gly Lys Gly Thr Thr Val Thr

245 250 255

Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly

260 265 270

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

275 280 285

Ser Gln Ser Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Thr

290 295 300

Ser Tyr Gly Val His Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

Cys Ala Arg Gln Leu Gly Leu Arg Ala Met Asp Tyr Trp Gly Gln Gly

370 375 380

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

385 390 395 400

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

405 410 415

Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp

420 425 430

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

435 440 445

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

450 455 460

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

465 470 475 480

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

485 490 495

Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro

500 505 510

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

515 520 525

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

530 535 540

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

545 550 555 560

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

565 570 575

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

580 585 590

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

595 600 605

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

610 615 620

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

625 630 635 640

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

645 650 655

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

660 665 670

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

675 680 685

Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu

690 695 700

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

705 710 715 720

Lys

Claims

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

a first antigen binding domain, and

a second antigen binding domain;

The second antigen binding domain is an antibody or antigen binding fragment thereof that specifically binds IGF 1R.

2. The bispecific antibody of claim 1, wherein the antigen-binding fragment has a structure selected from the group consisting of: (i) Fab fragments; (ii) a F (ab') 2 fragment; (iii) Fv fragments; or (iv) a single chain Fv (scFv); and/or the number of the groups of groups,

the bispecific antibody is a homodimer having a structure from the N-terminus to the C-terminus of formula Ia, ib, IIa, IIb, iiia, iiib, iva or ivb:

Wherein,,

VL _A representing the light chain variable region of an anti-PDL 1 antibody;

VH _A represents the heavy chain variable region of an anti-PDL 1 antibody;

VL _B representing the light chain variable region of an anti-IGF 1R antibody;

VH _B represents the heavy chain variable region of an anti-IGF 1R antibody;

CH represents a heavy chain constant region;

CL represents the light chain constant region;

l1, L2 are each independently a bond or a peptide linker;

"-" represents disulfide or covalent bonds;

"-" represents a peptide bond;

3. The bispecific antibody of claim 2, wherein the anti-PDL 1 antibody comprises the following three complementarity determining regions HCDR:

HCDR1 shown in SEQ ID NO. 14;

HCDR2 shown in SEQ ID NO. 15; and

HCDR3 shown in SEQ ID NO. 16; and

LCDR1 shown in SEQ ID NO. 18;

LCDR2 as shown in SEQ ID NO. 19; and

LCDR3 as shown in SEQ ID NO. 20.

4. The bispecific antibody of claim 2, wherein the anti-IGF 1R antibody comprises the following three complementarity determining regions HCDR:

HCDR1 shown in SEQ ID NO. 4;

HCDR2 shown in SEQ ID NO. 5; and

HCDR3 shown in SEQ ID NO. 6; and

LCDR1 shown in SEQ ID NO. 8;

LCDR2 as shown in SEQ ID NO. 9; and

LCDR3 as shown in SEQ ID NO. 10.

5. The bispecific antibody of claim 2, wherein the heavy chain variable region of said anti-PDL 1 antibody is set forth in SEQ ID No. 13; the light chain variable region of the anti-PDL 1 antibody is shown as SEQ ID NO. 17; and/or the heavy chain variable region of the anti-IGF 1R antibody is shown as SEQ ID NO. 3; the light chain variable region of the IGF1R antibody is shown as SEQ ID NO. 7.

6. The bispecific antibody of claim 2, wherein said bispecific antibody is selected from the group consisting of:

7. An isolated polynucleotide molecule encoding the bispecific antibody of any one of claims 1-6.

8. A vector comprising the isolated polynucleotide molecule of claim 7.

9. A host cell comprising or incorporating the isolated polynucleotide molecule of claim 7 or comprising the vector of claim 8.

10. A method of preparing a bispecific antibody according to any one of claims 1 to 6, characterized in that the method of preparation comprises the steps of:

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

(b) Isolating and purifying the bispecific antibody of step (a).

11. A conjugate comprising an antibody or antigen-binding fragment thereof and a conjugate moiety, wherein the antibody is a bispecific antibody according to any one of claims 1 to 6 and the conjugate moiety is a detectable label or a drug molecule.

12. Kit, characterized in that it comprises a bispecific antibody according to any one of claims 1 to 6, or a conjugate according to claim 11.

13. Use of the bispecific antibody of any one of claims 1-6 or the conjugate of claim 11 in the preparation of a kit for detecting the presence or level of PDL1 and/or IGF1R in a sample.

14. A pharmaceutical composition, characterized in that it comprises a bispecific antibody according to any one of claims 1-6 or a conjugate according to claim 11; optionally, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier, medium.

15. The pharmaceutical composition of claim 14, further comprising one or more agents for treating tumors.

16. Use of a bispecific antibody according to any one of claims 1-6 or a conjugate according to claim 11 or a pharmaceutical composition according to claim 14 or 15 in one or more of the following:

preparing a medicament for preventing and/or treating tumors;

preparing a medicament for diagnosing tumors;

preparing an antibody drug conjugate;

preparing a drug blocking a PDL1/PD-1 pathway;

preparing a medicament that down-regulates PDL1 activity or level;

preparing a medicament for relieving immunosuppression of PDL1 on an organism;

preparing a drug that blocks IGF1R binding to IGF 1;

preparing a medicament that down regulates IGF1R activity or IGF1R levels;

17. The use of claim 16, wherein the tumor is selected from one or more of bowel cancer, esophageal cancer, head and neck cancer, skin cancer, kidney cancer, hepatocellular carcinoma, ovarian serous cyst adenocarcinoma, thyroid cancer, lung adenocarcinoma, head and neck squamous cell carcinoma, glioblastoma multiforme, thymus cancer, brain low-grade glioma, rectal adenocarcinoma, pheochromocytoma and paraganglioma, esophageal cancer, renal clear cell carcinoma, cervical squamous carcinoma and adenocarcinoma, melanoma, renal tumor, prostate cancer, bladder cancer, colon cancer, rectal cancer, stomach cancer, liver cancer, lung cancer, ovarian cancer, leukemia, breast cancer, mesothelioma, cervical cancer, endometrial cancer, lymphoma, nasopharyngeal carcinoma, bladder urothelial cancer, renal papillary cell carcinoma, pancreatic cancer, renal chromophobe carcinoma, breast invasive carcinoma, lung squamous carcinoma, sarcoma, acute myeloid leukemia, and the like.