Chimeric antigen receptor and application thereof

A technology of chimeric antigen receptors and antigens, applied in medicine, in the field of chimeric antigen receptors, can solve problems such as low expression levels

Active Publication Date: 2020-08-07
GUANGZHOU BIO GENE TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Related targets of B cells also include CD20 and CD22, etc., but their expression levels in B cell leukemia and lymphoma are much lower than CD19

Method used

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  • Chimeric antigen receptor and application thereof
  • Chimeric antigen receptor and application thereof
  • Chimeric antigen receptor and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1B

[0083] Example 1 Design and function of BAFF antigen-binding domain

[0084] In order to use BAFF as the antigen-binding domain of the CAR molecule, the soluble BAFF was modified in this example, and the amino acid VHVFGDEL, which is necessary for the activation of positions 217-224 of the soluble BAFF (soluble form: 134-285), was replaced with GG, The modified BAFF shown in SEQ ID NO:1 was obtained.

[0085] (1) Binding ability of modified BAFF to BAFF-R

[0086] The results are shown in Table 1. The modified BAFF (mBAFF) lost the BAFF activation ability, but retained the BAFF-R binding ability.

[0087] Table 1 Analysis of the binding of recombinant BAFF and modified protein to K562-BAFFR cells

[0088] target protein Mean Fluorescence Intensity (MFI) binding positive rate wild soluble BAFF 856 59.35％ Transformation of soluble BAFF 969 62.56％ Wild soluble BAFF trimer 2968 97.45％ Modified soluble BAFF trimer 2565 95.67％ PBS 25...

Embodiment 2

[0092] Embodiment 2 Design of CAR molecules

[0093] In this example, the engineered BAFF shown in SEQ ID NO: 1 is used as the antigen-binding domain to construct a monomeric BAFF-based chimeric antigen receptor (BAFF CAR) as shown in Figure 3 (A) and a trimeric BAFF-based The chimeric antigen receptor (TriBAFF CAR) of the body BAFF (TriBAFF CAR), the three BAFFs in the TriBAFF CAR molecule are connected by a GGGGS linker, which recognizes and binds to BAFF-R; The CD19 CAR of the CD19 antibody (FMC63) scFv specifically recognizes the CD19 antigen.

[0094] In addition to the antigen-binding domain, the above-mentioned CAR molecule also includes a CD8α signal peptide sequence (Leader), a CD8α hinge region (Hinge) and a transmembrane region (Transmembrane) sequence, a 4-1BB co-stimulatory domain sequence and a CD3ζ signaling domain sequence.

Embodiment 3

[0095] The construction of embodiment 3 lentiviral vectors

[0096] (1) Whole gene synthesis of nucleic acid molecules of the following CAR molecules:

[0097] BAFF CAR: composed of CD8a signal peptide sequence, modified BAFF sequence, CD8a hinge region and transmembrane region sequence, 4-1BB and CD3ζ in tandem (the amino acid sequence is shown in SEQ ID NO: 8);

[0098] TriBAFF CAR: consists of CD8a signal peptide sequence, three repeats of modified BAFF sequences (connected by GGGGS linker between the three BAFFs), CD8a hinge region and transmembrane region sequence, 4-1BB and CD3ζ in series (amino acid sequence such as Shown in SEQID NO:9);

[0099] CD19 CAR: composed of CD8a signal peptide sequence, CD19 scFv, CD8a hinge region and transmembrane region sequence, 4-1BB and CD3ζ in tandem (the amino acid sequence is shown in SEQ ID NO: 10);

[0100] SEQ ID NO: 10:

[0101] Amino acid sequence of CD19 CAR:

[0102] MALPVTALLLPLALLLHAARPDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNW...

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Abstract

The invention provides a chimeric antigen receptor and application thereof. According to the chimeric antigen receptor, modified BAFF is adopted as an antigen binding structural domain, the modified BAFF retains the BAFF-R bonding capability, however, malignant B cell proliferation caused by BAFF activation cannot be caused, the constructed CAR molecule can recognize and combine BAFF-R, so that the prepared CAR-T cell has a specific targeted killing effect on BAFF-R positive and CD19 negative B tumor cells, and is expected to become a new means for treating tumor recurrence caused by CD19 antigen deletion.

Description

technical field [0001] The invention belongs to the field of biological technology and the technical field of cellular immunotherapy of diseases, and relates to a chimeric antigen receptor and its application, in particular to a chimeric antigen receptor targeting BAFF-R positive tumor cells and its preparation Application of drugs for treating tumor recurrence caused by CD19 antigen deficiency. Background technique [0002] Chimeric antigen receptor (CAR) T cells have been widely used in the treatment of B-cell malignancies, and CAR-T cells targeting CD19 are the pioneers of CAR-T therapy for B-cell malignancies, providing an effective treatment for B-cell malignancies. Program. However, tumor recurrence caused by the loss of CD19 antigen greatly weakens the therapeutic effect. CD19-negative relapse has become an important problem and occurs in approximately 30% of patients. Related targets of B cells also include CD20 and CD22, etc., but their expression levels in B cel...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/705C07K19/00C12N15/62C12N15/867C12N7/01C12N5/10A61K39/00A61P35/00
CPCA61P35/00A61K39/001112C07K14/7051C07K14/70578C07K2319/02C07K2319/03C07K2319/33C12N5/0636C12N7/00C12N15/86C12N2510/00C12N2740/15021C12N2740/15043
Inventor 李光超罗敏周兆丁雯
Owner GUANGZHOU BIO GENE TECH CO LTD
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