Chimeric antigen receptor targeting BCMA and use thereof

A technology of receptors and single-chain antibodies, applied in the direction of polypeptides containing positioning/targeting motifs, cancer antigen components, antibody medical components, etc., can solve the problems of T cell attack, high amplification, and beyond the treatment requirements

Active Publication Date: 2018-05-11
HRAIN BIOTECHNOLOGY CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this treatment has not been perfected, and T cells will go off ...

Method used

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  • Chimeric antigen receptor targeting BCMA and use thereof
  • Chimeric antigen receptor targeting BCMA and use thereof
  • Chimeric antigen receptor targeting BCMA and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0089] Example 1: Determination of BCMA-scFv-CD8α-CD28-41BB-CD3ζ gene sequence

[0090] The gene sequence information of human CD8α hinge region, human CD8α transmembrane region, 41BB intracellular region and human CD3ζ intracellular region was searched from the NCBI website database. The anti-BCMA single-chain antibody clone number is C11D5.3. These sequences are available on the website http: / Codon optimization is performed on / sg.idtdna.com / site to ensure that it is more suitable for expression in human cells without changing the encoded amino acid sequence.

[0091] Using overlapping PCR, the above sequences were sequentially connected according to anti-BCMA scFv, human CD8α hinge region gene, human CD8α transmembrane region gene, 41BB intracellular region gene, and human CD3ζ intracellular region gene sequence, and different restriction enzymes were introduced at the junction of each sequence. site to form complete BCMA-CAR gene sequence information.

[0092]The nucleot...

Embodiment 2

[0098] Embodiment 2: Determination of BCMACAR-GMCSFR leader sequence-tEGFR gene sequence

[0099] The gene sequence information of the extracellular region of human EGFR was searched from the NCBI website database, and the sequence was codon-optimized on the website http: / / sg.idtdna.com / site to ensure that it is more suitable for human cell expression under the condition that the encoded amino acid sequence remains unchanged .

[0100] Overlap PCR was used to sequentially connect the above sequences according to the BCMACAR, GMCSFR leader sequence, and tEGFR in Example 1, and introduce different restriction sites at the junctions of each sequence to form a complete BCMACAR-GMCSFR leader sequence-tEGFR gene sequence information.

[0101] The nucleotide sequence of the CAR molecule was double-digested with NotI (NEB) and EcoRI (NEB), inserted into the NotI-EcoRI site of the retrovirus MSCV (Addgene) by T4 ligase (NEB), and transformed into the competent large intestine Bacillus...

Embodiment 3

[0107] Example 3: Retroviral packaging

[0108] 1. Day 1: 293T cells should be less than 20 passages, not overgrown. Take 0.6×10 6 For cell / ml plating, add 10ml of DMEM medium to a 10cm dish, mix the cells well, and culture overnight at 37°C;

[0109] 2. Day 2: 293T cell confluency reaches about 90% for transfection (usually about 14-18 hours after plating); prepare plasmid complexes, the amount of various plasmids is MSCV backbone 12.5ug, Gag-pol 10ug, VSVg 6.25 Ug, CaCl 2 250ul,H 2 O1ml, the total volume is 1.25ml; add HBS equal to the volume of the plasmid complex in another tube, and vortex for 20 seconds while adding the plasmid complex. Gently add the mixture to the 293T dish along the side, incubate at 37°C for 4 hours, remove the medium, wash with PBS, and add pre-warmed fresh medium again.

[0110] 3. Day 4: 48 hours after transfection, collect the supernatant and filter it with a 0.45um filter, store in -80°C, and continue to add preheated fresh DMEM medium.

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Abstract

The present invention relates to a chimeric antigen receptor targeting BCMA and use thereof. In particular, the present invention provides a polynucleotide sequence selected from the group consistingof (1) a polynucleotide sequence containing sequentially linked an anti-BCMA single chain antibody coding sequence, a human CD8 alpha hinge region coding sequence, a human CD8 transmembrane domain coding sequence, a human 41 BB intracellular region coding sequence, a human CD 3 zeta intracellular region coding sequence and an optional extracellular domain III or extracellular domain IV fragment-containing coding sequence of EGFR; and (2) a complementary sequence of the polynucleotide sequence. The present invention also provides related fusion proteins, vectors containing the coding sequences,and uses of the fusion proteins, the coding sequences, and the vectors.

Description

technical field [0001] The invention belongs to the field of chimeric antigen receptors, in particular to BCMA-targeted chimeric antigen receptors and applications thereof. Background technique [0002] Multiple myeloma is a malignant plasma cell disease, manifested as malignant clonal hyperplasia of bone marrow plasma cells, secreting monoclonal immunoglobulin or its fragments (M protein), resulting in bone, kidney and other related target organs or tissue damage, common clinical Manifested as bone pain, anemia, renal insufficiency, infection, etc. [Multiple myeloma. N Engl J Med, 2011. 364(11): p. 1046-60.]. Currently, multiple myeloma is the second most malignant tumor of the hematological system, accounting for 10% of hematological malignancies, and it mostly occurs in men. Siegel, R., et al., Cancer statistics, 2014. CA Cancer J Clin, 2014. 64(1):p. 9-29.]. [0003] B-cell maturation antigen (BCMA), also known as CD269, consists of 184 amino acid residues, its intrace...

Claims

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Application Information

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IPC IPC(8): C12N15/62C07K19/00C12N15/867C12N7/01C12N5/10A61K35/17A61K39/00A61P35/00
CPCA61K35/17A61K39/0011C07K14/7051C07K14/70517C07K14/70578C07K14/71C07K16/2878C07K16/30C07K2317/622C07K2319/02C07K2319/03C12N5/0636C12N7/00C12N15/86C12N2740/10021C12N2740/10043
Inventor 黄飞金涛王海鹰何凤史子啸
Owner HRAIN BIOTECHNOLOGY CO LTD
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