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Anti-cd73 antibody and application thereof

An antibody and antibody heavy chain technology, applied in the direction of antibodies, antibody medical components, anti-tumor drugs, etc., can solve the problem that the characteristics of anti-CD73 antibodies are yet to be determined.

Active Publication Date: 2021-09-28
HARBOUR BIOMED (SHANGHAI) CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Thus, despite considerable interest in research targeting CD73, the properties of the most potent anti-CD73 antibodies remain to be determined

Method used

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  • Anti-cd73 antibody and application thereof
  • Anti-cd73 antibody and application thereof
  • Anti-cd73 antibody and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0423] Example 1: Production of anti-CD73 antibodies

[0424] immunity

[0425] In immunization using Harbor H2L2 mice, CD73 protein was used as immunogen to generate anti-CD73 antibodies. Each mouse was given a first boost of 50 μg protein followed by a subsequent boost of 25 μg protein using adjuvant (Sigma, S6322) by subcutaneous and intraperitoneal injection. This immunization was performed every two weeks for a total of 6 times. Final immunization was performed by intraperitoneal injection of immunogen diluted in PBS. Serum titers against human CD73 were tested using ELISA and FACS. At indicated time points, mouse sera were sampled and titrated for ELISA and FACS analysis to test for binding to CD73 or CD73 stable cell lines. Good serum titers were observed in the protein-immunized mice.

[0426] hybridoma

[0427] Mouse splenocytes isolated from immunized mice were fused with the mouse myeloma cell line SP2 / 0 (ATCC, CRL-1581) by an electric field-based electroporator...

Embodiment 2

[0428] Example 2 Antibody Production and Purification

[0429] Recombinant plasmids encoding the antibodies of interest were transiently co-transfected into HEK293-6E or 293-F cell cultures using PEI (Polyscience, 24885). 30 μg of plasmid was mixed with 120 μL of PEI and incubated for 15 minutes at room temperature. Next, the mixture was added dropwise at 1 x 10 6 The concentration of cells / ml was suspended in 293 cells in Opti-MEM. After transfection, the cells were cultured on a shaker at 120 rpm at 37° C. and 5% CO 2 . Cell culture supernatants collected on days 6-7 were used for purification.

[0430] The supernatant containing the antibody of interest was harvested 6-7 days after transfection by centrifugation and filtration. Monoclonal antibodies were purified by passing rProtein A (GE, 17-1279-02) prepacked columns (Bio-Rad, 7311550). By packing 0.2 mL of rProteinA resin into a column, followed by 10 column volumes of ddH 2 Prepare the Protein A column by washing ...

Embodiment 3

[0431] Example 3 Antibody Engineering

[0432] The VH and VL sequences of the anti-CD73 antibody PR000506 were further optimized by germlineization and PTM removal operations.

[0433] In the germlineization procedure, the antibody VH or VL sequence is first aligned to the closest human germline sequence by an algorithm such as NCBI / Ig-BLAST, and then the residues in the framework regions that are different from the germline sequence are reversed to Corresponding residues in the germline sequence. Then, germlined antibodies composed of sequence variants are recombinantly produced by mature molecular biology techniques.

[0434] Post-translational modifications (PTMs) are widely observed in proteins expressed in mammalian cells. In addition to the PTM sites conserved in antibodies (such as the N-glycosylation conserved sites on the CH2 domain of IgG1 antibodies), other PTM sites that occur within the antibody antigen-binding site (ie, the CDR region) may reduce the antigen B...

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Abstract

Provided an isolated antigen binding protein having one or more of the following properties: a. capable of binding to both human CD73 and Cynomolgus monkey CD73; b. capable of inhibiting 5'ectonucleotidase activity of CD73; c. capable of mediating CD73 internalization; d. capable of promoting T cell proliferation; e. with a relatively stable concentration in serum; and f. capable of inhibiting tumor growth and / or tumor cell proliferation. In addition, provided a method for producing the isolated antigen binding protein as well as pharmaceutical uses of the isolated antigen binding protein in preventing, alleviating and / or treating tumor.

Description

Background technique [0001] Leukocyte differentiation antigen 73, CD73, also known as 5'-nucleotidase, is an enzyme encoded by the NT5E gene in humans. CD73 is a dimer composed of two identical 70-kD subunits linked to the outer surface of the plasma membrane by a glycosylphosphatidylinositol. [0002] CD73 dephosphorylates extracellular monophosphate nucleotides (AMP) to produce adenosine. The accumulation of extracellular adenosine in cancer tissues constitutes an important mechanism of tumor immune evasion. In addition, tumor-derived adenosine largely inhibits infiltrating effector T cells through adenylate cyclase-activated A2A receptors (Ohta et al., (2006) Proc Natl Acad Sci USA 103:13132-13137). Studies have shown that CD73 is highly expressed on the surface of a variety of tumor cells, including leukemia, bladder cancer, glioma, glioblastoma, ovarian cancer, melanoma, prostate cancer, thyroid cancer, esophageal cancer and breast cancer (Jin et al. , Cancer Res 2010;...

Claims

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Application Information

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IPC IPC(8): C07K16/28C07K16/40A61K39/395A61P35/00
CPCC07K16/2896C07K16/40C12N15/85C12N5/0686A61P35/00C07K2317/565C07K2317/51C07K2317/515C07K2317/56C07K2317/76A61K2039/505C07K2317/92C07K2317/77A61K39/39558C07K2317/34C07K2317/24C12N15/63
Inventor 石磊甘馨单茜茜戎一平何云
Owner HARBOUR BIOMED (SHANGHAI) CO LTD
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