SEMA4D antibody, preparation method and application thereof

A technology of antibody and antibody conjugate, applied in the field of SEMA4D antibody and its preparation, can solve problems such as single method, inability to effectively inhibit tumor growth, and poor effect of in vitro activity experiments

Active Publication Date: 2020-01-21
JIANGSU HYAMAB PHARMA CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] In order to overcome the shortcomings of the single screening method for anti-human SEMA4D antibodies, the poor effect of in vitro activity experiments, and the inability to effectively inhibit the growth of tumors after the combination of existing SEMA4D antibodies and immune checkpoint PD-1 antibodies, the present invention uses a variety of antibodies Screening Techniques Gain Diversity in Antibody Sequences

Method used

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  • SEMA4D antibody, preparation method and application thereof
  • SEMA4D antibody, preparation method and application thereof
  • SEMA4D antibody, preparation method and application thereof

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preparation example Construction

[0243] The preparation method of the nucleic acid is a conventional preparation method in the art. Preferably, it includes the following steps: obtaining the nucleic acid molecule encoding the above-mentioned protein by gene cloning technology, or obtaining the nucleic acid molecule encoding the above-mentioned protein by artificial full-sequence synthesis .

[0244] Those skilled in the art know that substitutions, deletions, alterations, insertions or additions can be appropriately introduced into the base sequence encoding the amino acid sequence of the above protein to provide a polynucleotide homologue. The homologue of the polynucleotide in the present invention can be prepared by replacing, deleting or adding one or more bases in the gene encoding the protein sequence within the scope of maintaining antibody activity.

[0245] carrier

[0246] The invention also provides a recombinant expression vector comprising the nucleic acid.

[0247] The recombinant expression v...

Embodiment 1

[0321] Embodiment 1 hybridoma technology prepares SEMA4D antibody

[0322] (1) Preparation of immunogen

[0323] A. Protein immunogen:

[0324] The nucleotide sequence (as shown in sequence table SEQ ID No.421) containing the coding human SEMA4D protein extracellular region (hSEMA4D ECD) Met22-Arg734 was cloned into the pCP vector with His tag (the cloning step was provided by Shanghai Ruizhi Chemical Co., Ltd. Research Co., Ltd.), National Research Counceil Canada) and prepared plasmids according to established standard molecular biology methods. For specific methods, see [Sambrook, J., Fritsch, E.F., and Maniatis, T. (1989). Molecular Cloning: A Laboratory Manual, Second Edition (Plainview, New York: Cold Spring Harbor Laboratory Press)]. CHO-S cells (purchased from Gibco) were transiently transfected with polyetherimide (PEI, purchased from Polyscience), and expanded at 37° C. using CDFortiCHO medium (purchased from Gibco). After 8-10 days, the cell culture fluid is coll...

Embodiment 2

[0352] Example 2 Preparation of SEMA4D antibody by phage display technology

[0353] (1) Biotinylation of SEMA4D protein

[0354] With 0.15M Na 2 HCO 3The protein immunogen prepared in Example 1 (ie hSEMA4D ECD-His) was dialyzed to a final concentration of 1 mg / mL. Biotin-NHS (purchased from Sigma Aldrich) was dissolved in DMF to a final concentration of 10 mg / mL. Mix Biotin-X-X-NHS and protein immunogen at a molar ratio of 8:1. After standing at room temperature for 30 minutes, add 1M NH 4 Cl terminates the reaction. Then, it was dialyzed against PBS phosphate buffer (pH 7.4) overnight at 4° C. to remove free biotin to obtain a biotinylated immunogen (ie, biotinylated hSEMA4D ECD-His). The concentration of biotinylated hSEMA4D ECD-His was measured with BCA protein concentration assay kit (purchased from Pierce).

[0355] The activity of biotinylated hSEMA4D ECD-His was determined by FACS method. The stable transgenic cell line 293F hPlexin B1 expressing the human SEMA4...

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Abstract

The present invention discloses an antibody targeting SEMA4D, a preparation method and an application of the antibody, specifically discloses a mouse-derived antibody or a fully human monoclonal antibody targeting the SEMA4D, and also discloses a method for preparing the monoclonal antibody. The monoclonal antibody can bind SEMA4D antigen with high specificity, has very high affinity and significant antitumor activity, etc.

Description

technical field [0001] The invention belongs to the field of antibodies, and in particular relates to a SEMA4D antibody and its preparation method and application. Background technique [0002] In recent years, tumor immunotherapy has become the focus in the field of tumor therapy, in which therapeutic monoclonal antibodies targeting immune checkpoints have shown anti-tumor activity in the treatment of some tumor types such as melanoma and non-small cell lung cancer, targeting To cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) and programmed cell death 1 and its ligand (programmed cell death 1 / programmed cell death ligand 1, PD-1 / PD- L1) immune checkpoint antibody has been approved by the US FDA. [0003] However, the low response rate of single drug is the main problem of existing tumor immunotherapy. In 2014, the US FDA first approved the use of PD-1 antibodies for the treatment of melanoma. Relevant clinical trial data showed that the objective response rate of pa...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/18C12N15/13G01N33/68A61K39/395A61K47/68A61P35/00
CPCC07K16/18G01N33/68A61K47/6851A61P35/00C07K2317/56C07K2317/565A61K2039/505C07K16/2803C07K2317/92C07K2317/622C07K2317/76C07K2317/33C07K2317/73G01N2333/4704G01N33/6854
Inventor 胡颖莹曹晓丹金姿含王荔娜王远东邵小慧胡少平潘明明刘妍邵微李妍妍蓝小绚顾莹朱思然刘礼乐段清
Owner JIANGSU HYAMAB PHARMA CO LTD
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