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Humanized monoclonal antibody targeting to human CD19 antigen

A monoclonal antibody, humanized technology, applied in anti-animal/human immunoglobulin, anti-receptor/cell surface antigen/cell surface determinant immunoglobulin, anti-tumor drugs, etc.

Active Publication Date: 2017-11-03
CHONGQING PRECISION BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, tumor cells can down-regulate the expression of their own MHC and ligand molecules in the tumor microenvironment, thereby inhibiting the killing activity of T cells against tumor cells.

Method used

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  • Humanized monoclonal antibody targeting to human CD19 antigen
  • Humanized monoclonal antibody targeting to human CD19 antigen
  • Humanized monoclonal antibody targeting to human CD19 antigen

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Embodiment 1, design the humanized monoclonal antibody of anti-human CD19 antigen

[0045] (1) Amino acid sequences of the CDR regions of the mouse anti-human CD19 antigen monoclonal antibody strain FMC63 light and heavy chain variable regions such as SEQ ID NO.15, SEQ ID NO.16, SEQ ID NO.17 and SEQ ID NO.18 , shown in SEQ ID NO.19, SEQ ID NO.20. The sequence was analyzed and compared with the IMGT / BLAST database, and the human antibody sequence with the highest homology was selected as the transformation template.

[0046] (2) After molecular docking simulation, the antibody amino acid residues most closely related to antigen-antibody docking are shown in Table 1. The framework sequences other than the key residues were humanized and modified with reference to the human antibody framework sequence, and then The humanized antibody is randomly mutated, and then the affinity is evaluated and predicted by the method of antigen-antibody affinity screening and molecular doc...

Embodiment 2

[0050] Example 2. Preparation of lentivirus expressing a chimeric antigen receptor targeting human CD19 antigen (CD19)

[0051] (1) Prepare the gene sequence of the chimeric antigen receptor targeting human CD19 antigen

[0052] Synthesize chimeric antigen receptor sequences containing leader peptide (also known as signal peptide), anti-human CD19 antigen single-chain antibody ScFv, hFc hinge region, transmembrane region and intracellular signal segment in sequence, its structure is as follows image 3 shown. The nucleotide sequence of the leader peptide is shown in SEQ ID NO.5, and the amino acid sequence is shown in SEQ ID NO.6; humanized single-chain antibodies hCD19-1, hCD19-2, and hCD19-3 against human CD19 antigen The amino acid sequence is shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, and the nucleotide sequence is shown in SEQ ID NO.21, SEQ ID NO.22, SEQ ID NO.23, as The amino acid sequence of the single-chain antibody CD19 derived from the control mouse anti-human C...

Embodiment 3

[0057] Example 3, Preparation of Chimeric Antigen Receptor Modified T Cells for CD19 Antigen

[0058] (1) Packaging of lentivirus

[0059] In this embodiment, the calcium phosphate method is used for packaging lentivirus, and the specific steps are as follows: 293T cells are cultivated to a better state with DMEM medium containing 10% FBS (w / v); 5 / cm 2 Density Pass up to 1 75cm 2 Incubate in a culture flask for 22 hours to ensure that the confluence of the cells is 70-80% during transfection; then replace the medium with preheated DMEM medium containing 2% FBS (w / v), and cultivate for 2 hours for later use; 680 μL ddH 2 O. 20 μg lentiviral vector (respectively mCD19, hCD19-1, hCD19-2, hCD19-3 lentiviral vector), 100 μL 2.5mM CaCl 2 Add it into a 15mL centrifuge tube, mix evenly, add 2×HBS dropwise to the mixture with a pipette after mixing, and at the same time, blow and mix with a 10mL pipette, let it stand at room temperature for 15min after mixing, and then transfer the...

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Abstract

The invention discloses a humanized monoclonal antibody targeting to human CD19 antigen. The humanized monoclonal antibody is applied to construction and preparation of chimeric antigen receptor T cells; and subsequent in-vitro experiments verify the tumor treatment effect of the chimeric antigen receptor T cells. A chimeric antigen receptor targeting to the human CD19 antigen is composed of a leader peptide, a single-chain antibody targeting to the human CD19 antigen, a hinge area for connection with ScFv, a transmembrane domain and an immune receptor tyrosine activation motif; a lentiviral expression vector is constructed from the chimeric antigen receptor and then used for infection of immune cells, so the chimeric antigen receptor targeting to the human CD19 antigen can be expressed in the immune cells; and tumors expressing the CD19 antigen are killed by recognizing the CD19 antigen on the surfaces of tumor cells, so the purpose of tumor treatment is achieved. The invention provides a novel tool for targeting therapy of tumors.

Description

technical field [0001] The invention belongs to the field of biology, and specifically relates to the design of humanized monoclonal antibodies against human CD19 antigen, and also relates to a lentiviral expression vector expressing a chimeric antigen receptor targeting human CD19 antigen, and a preparation method and application of the lentiviral expression vector . 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, which is called tumor-directed ...

Claims

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Application Information

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IPC IPC(8): C07K16/28C07K19/00C12N15/867C12N5/10A61K35/17A61P35/00A61P35/02
Inventor 张巍
Owner CHONGQING PRECISION BIOTECH CO LTD
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