阅读说明：本技术 抗tip-1抗体及其用途 (anti-TIP-1 antibodies and uses thereof ) 是由 王明晗 邹晖 J·奥克斯 于 2019-03-06 设计创作，主要内容包括：本发明描述了抗TIP-1抗体及其抗原结合片段。本发明还描述了编码所述抗体的核酸、包含所述抗体的组合物、制备所述抗体的方法,以及使用所述抗体治疗或预防疾病,例如癌症的方法。(anti-TIP-1 antibodies and antigen-binding fragments thereof are described. The invention also describes nucleic acids encoding the antibodies, compositions comprising the antibodies, methods of making the antibodies, and methods of using the antibodies to treat or prevent diseases, such as cancer.)

1. An isolated monoclonal antibody or antigen-binding fragment thereof comprising heavy chain complementarity determining region 1(HCDR1), HCDR2, HCDR3, light chain complementarity determining region 1(LCDR1), LCDR2, and LCDR3 having the following polypeptide sequences, respectively:

seq ID NO: 3. 4, 5, 6, 7 and 8;

wherein the antibody or antigen-binding fragment thereof specifically binds to TIP-1, preferably human TIP-1.

2. The isolated monoclonal antibody or antigen-binding fragment thereof of claim 1, comprising a heavy chain variable region sequence substantially identical to SEQ ID NO: 1, or a heavy chain variable region comprising a polypeptide sequence having at least 95% identity to SEQ ID NO: 2 a light chain variable region of a polypeptide sequence having at least 95% identity.

3. The isolated monoclonal antibody or antigen-binding fragment thereof of claim 1 or 2, comprising:

a. has the sequence shown in SEQ ID NO: 1 and a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 2, or a light chain variable region of the polypeptide sequence of seq id No. 2.

4. The isolated monoclonal antibody or antigen binding fragment thereof of any one of claims 1 to 3, which is capable of inducing effector-mediated tumor cell lysis.

5. The isolated monoclonal antibody or antigen-binding fragment thereof of any one of claims 1 to 4, which is chimeric.

6. The isolated monoclonal antibody or antigen-binding fragment thereof of any one of claims 1 to 5, which is human or humanized.

7. An isolated nucleic acid encoding the monoclonal antibody or antigen binding fragment thereof of any one of claims 1-6.

8. A vector comprising the isolated nucleic acid of claim 7.

9. A host cell comprising the vector of claim 8.

10. A pharmaceutical composition comprising the isolated monoclonal antibody, or antigen-binding fragment thereof, of any one of claims 1-6, and a pharmaceutically acceptable carrier.

11. A method of treating cancer in a subject in need thereof, comprising administering to the subject the pharmaceutical composition of claim 10.

12. A method of targeting TIP-1 on the surface of a cancer cell in a subject in need thereof, the method comprising administering to the subject the pharmaceutical composition of claim 10.

13. A method of producing the monoclonal antibody or antigen-binding fragment thereof of any one of claims 1-6, comprising culturing a cell comprising a nucleic acid encoding the monoclonal antibody or antigen-binding fragment thereof under conditions in which the monoclonal antibody or antigen-binding fragment thereof is produced, and recovering the antibody or antigen-binding fragment from the cell or culture.

14. A method of making a pharmaceutical composition comprising the monoclonal antibody, or antigen-binding fragment thereof, of any one of claims 1-6, comprising combining the monoclonal antibody, or antigen-binding fragment thereof, with a pharmaceutically acceptable carrier to obtain the pharmaceutical composition.

15. A method of determining a level of TIP-1 in a subject, the method comprising:

a. obtaining a sample from a subject;

b. contacting a sample with the isolated monoclonal antibody or antigen binding fragment thereof of any one of claims 1-6; and

c. determining the level of TIP-1 in the subject.

16. The method of claim 15, wherein the sample is a tissue sample.

17. The method of claim 16, wherein the tissue sample is a cancer tissue sample.

18. The method of claim 15, wherein the sample is a blood sample.

Technical Field

The present invention relates to monoclonal anti-TIP-1 antibodies, nucleic acids and expression vectors encoding the antibodies, recombinant cells comprising the vectors, and compositions comprising the antibodies. The invention also provides methods of making the antibodies, and methods of using the antibodies to treat diseases, including cancer.

Reference to electronically submitted sequence Listing

This application contains a sequence listing submitted electronically via EFS-Web as a sequence listing in ASCII format with the file name "689204-8 WO sequence listing", creation date 3 months 1 days 2019, and size 4 kb. The sequence listing submitted by EFS-Web is part of this specification, which is incorporated herein by reference in its entirety.

Background

Tax interacting protein 1(TIP-1, also known as Tax1bp3 or glutaminase interacting protein, GIP) is an intracellular protein (124 amino acids in humans and mice) comprising a PDZ (PSD-95/Discs large/ZO-1 homologous) domain. The single PDZ domain containing residues 13-112 of the 124 amino acid protein (Olala et al, FEBS Lett 2001; 488: 116-122) is the only functional and structural unit that TIP-1 can recognize. This suggests that TIP-1 has a unique role in PDZ-containing proteins and can exert its function only through protein-protein interactions.

TIP-1 interacts with a number of intracellular proteins through the PDZ domain, including glutaminase L, β -Catenin (β -Catenin), FAS, HTLV Tax, HPV E6, Rhotekin and Kir2.3 (Zoetewey et al, biochemistry 2011; 50: 3528-3539). However, the exact biological function of TIP-1 is still unclear. Increased expression of TIP-1 was detected in human invasive breast cancer cells and was shown to be associated with tumor cell adhesion, migration and lung metastasis (Han et al, Biochemical and biophysical research letters 2012; 422: 139-145).

Recently, TIP-1 was found to translocate to the surface of cancer cells following radiation induction (Yan et al, cancer target 2016; 7: 43352-43362), suggesting that TIP-1 may be based on an induced cancer neoantigen. Thus, anti-TIP-1 monoclonal antibodies (mabs) can be used to selectively target cancer cells and as potential therapeutic agents.

Summary of The Invention

In one general aspect, the present invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof that binds TIP-1.

The invention provides an isolated monoclonal antibody or antigen-binding fragment thereof comprising heavy chain complementarity determining region 1(HCDR1), HCDR2, HCDR3, light chain complementarity determining region 1(LCDR1), LCDR2, and LCDR3, having the following polypeptide sequences, respectively:

a. are respectively SEQ ID NO: 3. 4, 5, 6, 7 and 8;

wherein the antibody or antigen-binding fragment thereof specifically binds TIP-1, preferably human TIP-1.

In certain embodiments, the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region whose polypeptide sequence is identical to SEQ ID NO: 1 or a light chain variable region comprising a polypeptide sequence at least 95% identical to SEQ ID NO: 2 is at least 95% identical.

In certain embodiments, an isolated monoclonal antibody or antigen-binding fragment thereof comprises:

a. has the sequence shown in SEQ ID NO: 1 and a heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 2, or a light chain variable region of the polypeptide sequence shown in the figure.

In certain embodiments, an isolated monoclonal antibody, or antigen-binding fragment thereof, is capable of mediating antibody-dependent cellular cytotoxicity (ADCC) and/or complement-dependent cytotoxicity (CDC) by binding to TIP-1 on the surface of a cell, thereby inducing effector-mediated tumor cell lysis, and/or mediating recruitment of conjugated drugs, and/or forming a bispecific antibody with another monoclonal antibody or antigen-binding fragment thereof having an anti-cancer effect.

In certain embodiments, the isolated monoclonal antibody or antigen-binding fragment thereof is chimeric.

In certain embodiments, the isolated monoclonal antibody or antigen-binding fragment thereof is human or humanized.

The invention also provides an isolated nucleic acid encoding the monoclonal antibody of the invention or an antigen binding fragment thereof.

The invention also provides a vector comprising an isolated nucleic acid encoding the monoclonal antibody of the invention or an antigen binding fragment thereof.

The invention also provides a host cell comprising a vector comprising an isolated nucleic acid encoding a monoclonal antibody of the invention, or an antigen binding fragment thereof.

In certain embodiments, pharmaceutical compositions are provided comprising an isolated monoclonal antibody or antigen-binding fragment thereof of the invention and a pharmaceutically acceptable carrier.

The present invention also provides a method of treating cancer in a subject in need thereof comprising administering to the subject a pharmaceutical composition of the present invention. The cancer may be any liquid or solid cancer, for example, may be selected from, but not limited to, lung cancer, stomach cancer, colon cancer, hepatocellular cancer, renal cell carcinoma, urothelial carcinoma of the bladder, metastatic melanoma, breast cancer, ovarian cancer, cervical cancer, head and neck cancer, pancreatic cancer, glioma, glioblastoma and other solid tumors, as well as non-hodgkin's lymphoma (NHL), Acute Lymphocytic Leukemia (ALL), Chronic Lymphocytic Leukemia (CLL), Chronic Myelogenous Leukemia (CML), Multiple Myeloma (MM), Acute Myelogenous Leukemia (AML) and other liquid tumors.

The invention also provides methods of producing the monoclonal antibodies of the invention or antigen binding fragments thereof. The method comprises culturing a cell comprising a nucleic acid encoding the monoclonal antibody or antigen-binding fragment thereof under conditions in which the monoclonal antibody or antigen-binding fragment thereof is produced, and recovering the antibody or antigen-binding fragment from the cell or culture.

The invention also provides a method of preparing a pharmaceutical composition comprising a monoclonal antibody or antigen-binding fragment thereof of the invention, the method comprising combining the monoclonal antibody or antigen-binding fragment thereof with a pharmaceutically acceptable carrier to obtain a pharmaceutical composition.

The invention also provides methods of determining the level of TIP-1 in a subject. The method comprises (a) obtaining a sample from a subject; (b) contacting the sample with an isolated monoclonal antibody or antigen binding fragment thereof of the invention; (c) determining the level of TIP-1 in the subject. The sample may be, for example, a tissue sample or a blood sample. The tissue sample may be, for example, a cancer tissue sample.

Brief description of the drawings

The foregoing summary, as well as the following detailed description of preferred embodiments of the present application, will be better understood when read in conjunction with the appended drawings. It should be understood, however, that the present application is not limited to the precise embodiments shown in the drawings.

FIG. 1 shows the binding of purified mouse anti-TIP-1 mAb T2 to TIP1-Myc-DDK in an ELISA assay. The T2 mAb concentrations used were (left to right in the figure): 500ng/ml,20ng/ml,5ng/ml and 1ng/ml respectively; secondary antibody only, this group received no primary antibody treatment but was treated with HRP conjugated anti-mouse IgG secondary antibody; no antibody, neither primary nor secondary antibody was added.

FIG. 2 shows the binding capacity of anti-TIP-1 chimeric mAbs (mouse VH and VL fused to human IgG1 and kappa chain constant regions, respectively) T2 in an ELISA assay; the T2 chimeric mAb concentrations used were (left to right in the figure): respectively at 18ng/ml,4ng/ml and 1 ng/ml; secondary antibody only, untreated with primary antibody but with HRP-conjugated anti-mouse IgG secondary antibody; no antibody, neither primary nor secondary antibody was added.

Detailed Description

Examples

The present invention provides the following non-limiting embodiments.