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A kind of anti-ceacam5 humanized antibody, its conjugate and use thereof

A technology of humanized antibodies and conjugates, which is applied in the field of biomedicine, can solve the problems of limited curative effect, low side effects, and high specificity of monoclonal antibody therapeutic targets, and achieve significant effects, strong affinity, and significant proliferation inhibition effects

Active Publication Date: 2022-07-08
烟台迈其生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Monoclonal antibody therapy has attracted more and more attention due to its high target specificity and low side effects, but its curative effect is relatively limited when used alone

Method used

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  • A kind of anti-ceacam5 humanized antibody, its conjugate and use thereof
  • A kind of anti-ceacam5 humanized antibody, its conjugate and use thereof
  • A kind of anti-ceacam5 humanized antibody, its conjugate and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] Example 1 Preparation of mouse anti-CEACAM5 monoclonal antibody

[0055] Monoclonal antibodies were prepared by immunizing mice with CEACAM5 protein (ACRO) as an antigen. A total of 6 mice were immunized, including 3 Balb / c mice and 3 C57 mice. 0.15 ml of CEACAM5 protein (75 μg protein dissolved in PBS buffer) and 0.15 ml of Freund's complete adjuvant (Sigma) were mixed in equal volumes, and then the mice were taken and injected subcutaneously on the back, with 0.3 ml per mouse. The second injection was performed after a 2-week interval. The amount of antigen injected into each mouse was the same as that of the first injection. The third injection was performed after an interval of 4 weeks. Blood drawn.

[0056] The serum of the mice was detected by enzyme-linked immunosorbent assay (ELISA), and the spleen of the mouse with the highest anti-CEACAM5 antibody titer in the serum was removed (among the six mice, one of the Balb / c mice had serum anti-CEACAM5 antibody). CE...

Embodiment 2

[0060] Example 2 Humanization of anti-CEACAM5 monoclonal antibody

[0061] Humanization was performed by grafting the light and heavy chain CDRs of a murine anti-CEACAM5 monoclonal antibody into human IgG1 variable regions.

[0062] The heavy chain variable region (VH) of a humanized anti-CEACAM5 monoclonal antibody was designed by converting the amino acid residues Arg and Met in the framework region 2 (FWR2) of the human germline heavy chain into the murine isomeric residues Ser. and Ile, the conversion of amino acid residues Val, Ile and Arg in the human germline heavy chain framework region 3 (FWR3) to the murine isomeric residues Ala, Met and Val, preserves the CDRs in the reconstituted CEACAM5-binding antibody. Conformation.

[0063] At the same time, the light chain variable region (VL) of a humanized anti-CEACAM5 monoclonal antibody was designed, and an amino acid residue Phe in the human germline light chain FWR3 was converted into a murine isomeric residue Tyr. Afte...

Embodiment 3

[0070] Example 3 Preparation of Antibody Conjugates

[0071] Prepare reducing buffer: Dissolve TCEP (Tris-2-carboxyethyl-phosphine, tris(2-carboxyethyl) phosphine) and DTPA (diethylenetriaminepentaacetic acid, diethylenetriaminepentaacetic acid) in PBS, both substances in reducing buffer The concentrations were 0.26 mM and 2 mM, respectively.

[0072] Antibody reduction: 20 mg / mL mAb (in PBS buffer) was mixed with reducing buffer in a volume ratio of 1:1, and the reaction was stirred at 25 °C for 2 h.

[0073] Prepare small molecule drug solution: Dissolve small molecule toxins MC-VC-PAB-MMAE, MC-VC-PAB-MMAF or MC-MMAF in DMSO (dimethyl sulfoxide) to a final concentration of 10 mM, respectively.

[0074] Coupling: Add 25% DMSO to the reduced protein, and then slowly add the small molecule drug solution according to the molar ratio of small molecule drug to antibody 4.4 for coupling, and stir at 25 °C for 1 h. Finally, the conjugated protein was dialyzed in PBS to remove unco...

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Abstract

The invention discloses an anti-CEACAM5 humanized antibody, its conjugate and its use, and belongs to the field of biomedicine. The antibody of the present invention includes a heavy chain and a light chain, the heavy chain includes three CDR regions, and the three CDR regions of the heavy chain have the amino acid sequences shown in SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3 respectively ; The light chain comprises three CDR regions, and the three CDR regions of the light chain respectively have the amino acid sequences shown in SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO.6. The antibody has strong affinity to CEACAM5; the affinity of the three ADCs to CEACAM5 after the naked antibody is coupled to the therapeutic agent through the linker is similar to that of the naked antibody. The invention can be used to target and inhibit the proliferation of tumor cells with positive CEACAM5 expression, and then be used to prepare medicines for treating or preventing colorectal cancer and pancreatic cancer.

Description

technical field [0001] The present invention relates to the technical field of biomedicine, in particular to an anti-CEACAM5 humanized antibody, its conjugate and use thereof. Background technique [0002] CEACAM5 (Carcinoembryonic Antigen, abbreviated as CEA, also known as CD66e) is an acidic glycoprotein with a molecular weight of about 180 kDa. It is a member of the immunoglobulin superfamily. a structural domain. CEACAM5 mainly exists in adult cancer tissues and fetal gastrointestinal tract tissues, and is involved in the intercellular adhesion of cancer cell invasion and metastasis. It is a broad-spectrum tumor marker. [0003] Monoclonal antibody therapy has received more and more attention due to its high target specificity and low side effects, but its efficacy is limited when used alone. Antibody-drug conjugates belong to a new class of anti-cancer bio-missile drugs, which are composed of three parts: antibody, linker, and cytotoxin. After the monoclonal antibody...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K16/30A61K47/68A61K45/00A61P35/00
CPCC07K16/3007A61K47/6853A61K45/00A61P35/00C07K2317/24C07K2317/565C07K2317/56C07K2317/51C07K2317/515C07K2317/92
Inventor 姚雪英李乐涵
Owner 烟台迈其生物科技有限公司
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