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Bicistronic chimeric antigen receptors and their uses

A chimeric antigen receptor, antigen technology, applied in the direction of antibodies, antibody mimics/scaffolds, receptors/cell surface antigens/cell surface determinants, etc., can solve the problems of unmet cancer treatment and poor prognosis

Pending Publication Date: 2020-01-03
UNITED STATES OF AMERICA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Despite advances in treatments such as chemotherapy, the prognosis for many cancers, including hematological malignancies, can be poor
Therefore, there is an unmet need for additional treatments of cancer, especially hematological malignancies

Method used

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  • Bicistronic chimeric antigen receptors and their uses
  • Bicistronic chimeric antigen receptors and their uses
  • Bicistronic chimeric antigen receptors and their uses

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0300] This example demonstrates the production of CAR constructs, lentiviral vectors encoding the CAR constructs, and CAR-expressing T cells according to embodiments of the invention, as well as the production of other CARs for comparison.

[0301] The CAR construct was synthesized by GENEWIZ (South Plainfield, NJ, USA) and then subcloned into the lentiviral plasmid backbone between the NhE1 site and the HincII site.

[0302] Lentiviral vectors encoding CAR constructs were generated by transient transfection of the 293T cell line. Briefly, 293T cells were seeded into poly-D-lysine-coated 15 cm plates (BD Biosciences, San Jose, CA, USA). The next day, plasmids encoding the CAR constructs together with packaging and envelope vectors (pMDLg / pRRE, pMD-2G, and pRSV-Rev) were transfected into 293T using lipofectamine 3000 (Life Technologies, Carlsbad, CA, USA). cell. Lentiviral supernatants were collected 48-72 hours after transfection, centrifuged at 3000 RPM for 10 minutes to r...

Embodiment 2

[0311] This example demonstrates surface expression on human T cells of a CAR cleaved from a CAR construct according to an embodiment of the invention compared to other CARs.

[0312] The surface expression of anti-CD19 CAR and anti-CD22 CAR on V1-transduced T cells was about 15%, while the expression of anti-CD19 CAR from vectors encoding only a single anti-CD19 CAR was 61%, and that from vectors encoding only a single anti-CD22 The expression of the anti-CD22 CAR of the CAR vector was 56% ( Figures 2A-2C ).

[0313] Human PBMC from healthy donors were activated for 24 hours with CD3 / CD28 microbeads. Activated T cells were then transduced with the vector alone or co-transduced with both a single anti-CD19 CAR and a single anti-CD22 CAR vector. Surface expression of anti-CD19 CAR and anti-CD22 CAR was analyzed on day 8. Co-transduced T cells had much lower expression of both anti-CD19 and anti-CD22 CARs compared to the bispecific cyclic CAR6. Anti-CD19 and anti-CD22 CARs ...

Embodiment 3

[0316] This example demonstrates the in vitro activity of CAR constructs according to embodiments of the present invention compared to other CARs based on cytokine production.

[0317] CAR-transduced T cells (1E5) were washed 3 times with 1×PBS, and then co-incubated with an equal amount of target cells in 200ml RPMI medium in a 96-well plate in a 37°C incubator for 15 to 20 hours. Target cells were K562 expressing CD19 or CD22 or both CD19 and CD22. K562 cells were used as a negative control. Cytokines levels of IL2 and IFNγ in culture supernatants were measured with ELISA kits (R&D Systems, Minneapolis, MN, USA). All tests were set up in triplicate. V1 CAR T cells produced large amounts of IL2 and IFNg when co-cultured with CD22-expressing target cells, but produced only low levels of IL2 and IFNg when co-cultured with CD19-expressing target cells ( Figure 5A and 5B ).

[0318] The CML cell line K562 was artificially transduced with CD19 or CD22 or both to express the ...

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Abstract

An embodiment of the invention provides bicistronic chimeric antigen receptor (CAR) amino acid constructs. Nucleic acids, recombinant expression vectors, host cells, populations of cells, and pharmaceutical compositions relating to the CAR constructs are disclosed. Methods of detecting the presence of cancer in a mammal and methods of treating or preventing cancer in a mammal are also disclosed. Methods of making the CAR constructs are disclosed.

Description

[0001] Cross References to Related Applications [0002] This patent application claims the benefit of U.S. Provisional Patent Application No. 62 / 506,268, filed May 15, 2017, which is hereby incorporated by reference in its entirety. [0003] Statement Regarding Federally Funded Research and Development [0004] This invention was made with Government support under Grant No. Z01 BC011565 from the National Institutes of Health, National Cancer Institute. The government has certain rights in this invention. [0005] Incorporation by Reference of Electronically Submitted Materials [0006] Incorporated herein by reference in its entirety is the computer readable nucleotide / amino acid sequence listing filed contemporaneously with this document and identified as follows: dated May 15, 2018 by An ASCII (text) file of "739267_ST25.TXT" of 180,939 bytes. [0007] Background of the invention [0008] Cancer is a public health problem. Despite advances in treatments such as chemothe...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/28A61K39/395A61P35/00
CPCC07K16/2803A61K2039/505C07K2317/31C07K2317/622C07K2317/73C07K2319/00C07K2319/03C07K2319/33A61P35/00A61K39/464412A61K2239/38A61K39/464413A61K39/4611A61K2239/48A61K39/4631A61K2239/31G01N33/574G01N2333/70503A61K35/17C07K14/395C07K14/7051C12N15/85
Inventor 秦海瑛克里斯特尔·L·麦考尔特里·J·弗莱
Owner UNITED STATES OF AMERICA
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