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Chimeric antigen receptor of anti-human CD19 antigen and application thereof

A technology of chimeric antigen receptors and antigens, which is applied in the field of genetic engineering, can solve problems such as inability to mediate, achieve a wide range of selection, enhance the ability to proliferate and kill tumors, and have high efficiency

Active Publication Date: 2017-10-03
CHONGQING PRECISION BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] However, although there are more antibodies for clinical application, they are still far from meeting the needs of clinical application, and there are still many technical problems: the degree of humanization, the optimal combination and specificity of CAR, stability and better Considering the ability to clear tumor cells, use non-human monoclonal antibodies (e.g., murine monoclonal antibodies). On the one hand, murine antibodies usually cannot mediate complement-dependent lysis or through antibody-dependent cytotoxicity or Fc receptor-mediated phagocytosis to lyse human target cells

Method used

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  • Chimeric antigen receptor of anti-human CD19 antigen and application thereof
  • Chimeric antigen receptor of anti-human CD19 antigen and application thereof
  • Chimeric antigen receptor of anti-human CD19 antigen and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0062] Example 1. Screening of humanized monoclonal antibodies against human CD19 antigen

[0063] The humanized sequence of the murine anti-CD19 antibody FMC635 was replaced by screening the humanized sequence (Part 1) of the declared patent 201710301492.1, and the humanized IgG antibody molecule was designed by random mutation, as shown in Table 1. Cloned antibody random mutation results.

[0064] Table 1

[0065]

Embodiment 2

[0066] Example 2, Purification of Humanized Monoclonal Antibody Targeting Human CD19 Antigen

[0067] Construct a stably transfected cell line that stably expresses the humanized antibody, pass the harvested cell supernatant through a 0.45um filter, and take the filtrate. Dilute with an equal volume of equilibration buffer and measure the pH. See the GE product manual for the specific steps of purification with AKTA Prime, concentrate by ultrafiltration, sterilize and filter with a 0.22 μm syringe filter after completion, subpackage, label and store in a -80°C refrigerator. Sampling, testing. Use SDS and Western blot detection, see GE Antibody Purification Handbook for specific steps. Test results such as figure 1 As shown, four monoclonal antibodies are preferred to obtain antibodies with higher purity, which are ScFv-humanized4 amino acid sequence SEQ ID NO.1, ScFv-humanized5 amino acid sequence SEQ ID NO.2, ScFv-humanized9 amino acid sequence SEQ ID NO.3, ScFv - humaniz...

Embodiment 3

[0068] Example 3. Specific detection of humanized monoclonal antibody targeting human CD19 antigen

[0069] The monoclonal antibody scFV was labeled with a His tag and cloned into a plasmid vector and transfected into HEK293 cells. The cell supernatant was harvested through a 0.45um filter, and the filtrate was taken. Dilute with an equal volume of equilibration buffer and measure the pH. After ultrafiltration and concentration, use a 0.22μm syringe filter to sterilize and filter, aliquot, label and store in a -80°C refrigerator. Different humanized antibodies were incubated with CD19-positive cells Raji and CD19-negative cells K562 for 30 minutes, and then the excess antibodies were washed away. After staining with FITC-His antibody for 30 minutes, the unlabeled antibodies were washed away, and flow cytometric detection was performed. The results are shown in the table As shown in 2, there are four monoclonal antibodies that can recognize the positive cell Raji and do not re...

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Abstract

The invention belongs to the field of gene engineering, and relates to chimeric antigen receptor of anti-human CD19 antigen and application thereof. The chimeric antigen receptor of anti-human CD19 antigen comprises polypeptide (scFv) for recognizing human CD19 antigen, a hinge region, a transmembrane region and an intracellular signal domain which are connected in sequence, wherein an amino acid sequence of the polypeptide (scFv) for recognizing human CD19 antigen is as shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3 or SEQ ID NO.4. The chimeric antigen receptor can be more stably expressed in T lymphocyte, has the capacity for properly removing cancer cell, can not only maintain the positive rate of CD19 targeted chimeric antigen receptor during the cell culture of a patient, but also enhance the capacity for CAR-T multiplication and tumor killing, does not have toxic and / or side effects on antigen-negative cell, and can be applied to the targeted therapy of tumor.

Description

technical field [0001] The invention belongs to the field of genetic engineering, relates to a chimeric antigen receptor against human CD19 antigen and its application, and also relates to a lentiviral vector containing the chimeric antigen receptor against human CD19 and its application. Background technique [0002] The production technology of monoclonal antibodies has gone through three stages: heterologous polyclonal antibodies produced by classical immunization methods; mouse monoclonal antibodies produced by cell engineering and human monoclonal antibodies produced by genetic engineering. Combining a monoclonal antibody against a certain tumor antigen with chemotherapeutic drugs or radiotherapy substances, using the guiding effect of the monoclonal antibody to carry the drug or radiotherapy substances to the target organ and directly kill the target cells is called tumor-directed therapy. In addition, radioactive markers are linked to monoclonal antibodies and injecte...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/867C12N15/66C12N5/10A61K35/17A61P35/00A61P35/02
CPCA61K35/17C07K14/7051C07K16/2803C07K2317/622C07K2319/02C07K2319/03C07K2319/33C12N15/66C12N15/86C12N2740/15043
Inventor 不公告发明人
Owner CHONGQING PRECISION BIOTECH CO LTD
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