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Humanized anti-CD19 antigen binding fragment

A technology of combining fragments and humanization, applied in DNA/RNA fragments, anti-receptor/cell surface antigen/cell surface determinant immunoglobulin, medical raw materials derived from mammals, etc.

Active Publication Date: 2017-11-24
GUANGZHOU BIO GENE TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Among the more than 50 CD19-targeting CAR-T clinical trials registered globally, there is only one clinical trial targeting CD19 humanized CAR-T

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0063] Example 1 Homology Modeling of Murine Antibody FMC63 scFv Chain

[0064] a. The principle of homology modeling is that similar sequences lead to similar structures, and the conservation of structures is much greater than that of sequences.

[0065] (1) First, search and compare homologous sequences in the NCBI database, and select a sequence with high homology to the amino acid sequence of the FMC63 scFv chain as a homologous template. The selected sequence should at least meet the following three conditions: ① sequence The similarity should be greater than 30%; ② High sequence identity and E value should be small enough (figure 1 shown.

[0066] The homologous template selected in the present invention is the crystal structure of the mouse monoclonal antibody Fab, and the PDB database number is: 4EDX.

[0067] After the sequence alignment was completed, the Antibody Modeler module of the MOE software was used to perform homology modeling on the FMC63 scFv chain, and A...

Embodiment 2

[0083] Example 2. IgBlast analysis and antigen affinity prediction

[0084] a. The following is the sequence of the light chain variable region of the mouse anti-CD19 antibody FMC63, the underlined part is the mouse anti-framework sequence (FR sequence), and the bold part is the CDR region sequence.

[0085] Seq ID No: 29

[0086] DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEIT

[0087] By analyzing the light chain variable region sequence of the mouse anti-CD19 antibody FMC63, the human germline antibody gene library (NCBI IgBLAST) was compared according to its light chain variable region FR1, FR2, FR3 and FR4 and the entire light chain sequence, Find the corresponding sequence of the variable region of the human germline (germline) antibody (less immunogenic than the mature antibody) similar to the sequence of the mouse anti-CD19 light chain variable region FR1, FR2, FR3 and FR4. Figure 4 It is the FR1 sequence c...

Embodiment 3

[0206] The synthesis of embodiment 3CAR gene and the construction of carrier

[0207] Select the five humanized sequences in Example 2, and add the linker sequence (gly4ser)3 between VL and VH in turn, so the structure of the scFv part designed and constructed into the chimeric antigen receptor is: VL-(gly4ser)3- VH. Specifically, the amino acid sequences of the five ScFv obtained are shown in sequence in Seq ID.No.46-Seq ID.No.50; the corresponding nucleic acid sequences are shown in SeqID.No.51-Seq ID.No.55 . details as follows:

[0208] The amino acid sequence of the scFv corresponding to the humanized sequence one (H1) is shown in Seq ID.No.46.

[0209] The amino acid sequence of the scFv corresponding to the humanized sequence 2 (H2) is shown in Seq ID.No.47.

[0210] The amino acid sequence of the scFv corresponding to the humanized sequence three (H3) is shown in Seq ID.No.48.

[0211] The amino acid sequence of the scFv corresponding to the humanized sequence four...

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Abstract

The invention provides a composition and a method for treating CD19 expression related diseases. The invention also relates to an application of humanized anti-CD19 single chain antibody with low immunogenicity as a CD19 antigen binding fragment, which is used for constructing a chimeric antigen receptor (CAR) specific to CD19, a carrier for coding the chimeric antigen receptor, and a recombined T-cell containing anti-CD19CAR. The invention also comprises a method for applying a genetically modified T-cell which expresses CAR containing the humanized anti-CD19 antigen binding fragment.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a preparation method and application of humanized anti-CD19 chimeric antigen receptor modified T cells. Background technique [0002] In recent years, tumor immunotherapy has entered a stage of rapid development, and CAR-T therapy, as one of the important methods, has achieved very encouraging results in the treatment of hematological malignancies. At present, more and more domestic enterprises, hospitals, and academic institutions are joining in this research field to jointly promote the development of CAR-T therapy and explore a wider range of potential application values. [0003] At present, the antibody recognition sequence in most CD19-targeting CAR-T technologies comes from the mouse source, which may cause strong immunogenicity after reinfusion into the human body, and is easy to be recognized and eliminated by autoimmune cells, affecting CAR- The long-term efficacy of T cel...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/28C07K19/00C12N15/13C12N15/62C12N5/10A61K35/17A61P35/00A61P35/02
CPCA61K35/17C07K14/7051C07K16/2803C07K2317/24C07K2317/56C07K2317/565C07K2317/92C07K2319/02C07K2319/03C07K2319/33
Inventor 李光超邱玉信丁雯莫文俊
Owner GUANGZHOU BIO GENE TECH CO LTD
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