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Methods and compositions for CAR T cell therapy

A composition and cell technology, applied in the field of CAR T cell therapy and composition

Pending Publication Date: 2019-01-11
PURDUE RES FOUND INC +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

First, 'off-target' toxicity may occur due to normal cells expressing antigens targeted by CART cells (e.g., tumor-associated antigens)

Method used

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  • Methods and compositions for CAR T cell therapy
  • Methods and compositions for CAR T cell therapy
  • Methods and compositions for CAR T cell therapy

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0309] Preparation of lentiviral vector encoding CAR gene

[0310] An overlap PCR approach was used to generate CAR constructs containing scFv against fluorescein. A scFv against fluorescein was synthesized, namely 4M5.3 (Kd = 270 fM, 762 bp) derived from an anti-fluorescein (4-4-20) antibody. Encoding human CD8α signal peptide (SP, 63 bp), hinge and transmembrane region (249 bp), cytoplasmic domain of 4-1BB (CD137, 141 bp) as shown in Figure 14A by overlap PCR pairs) and the sequence of the CD3ζ chain (336 bp) were fused to anti-fluorescein scFv. The resulting CAR construct (1551 bp) was inserted into the EcoRI / NotI cut lentiviral expression vector pCDH-EF1-MCS-(PGK-GFP) ( FIG. 14B , System Biosciences). The sequence of the CAR construct in the lentiviral vector was confirmed by DNA sequencing.

[0311] An exemplary CAR nucleic acid coding sequence may comprise:

[0312]

[0313] .

[0314] In the exemplary nucleic acid sequences shown above, the first ATG is the ...

Embodiment 2

[0322] Preparation of Lentivirus Containing CAR Gene for Transduction of Human T Cells

[0323] To generate lentivirus containing an anti-fluorescein single-chain fragment variable (scFv) CAR, the 293TN packaging cell line was co-transfected with a mixture of lentiviral vector encoding anti-fluorescein scFvCAR and a second-generation packaging plasmid (Cellecta). After 24 and 48 hours of transfection, supernatants containing lentiviruses with CAR genes were harvested and viral particles were concentrated for future human T cell transductions by standard polyethylene glycol virus enrichment methods (Clontech) .

Embodiment 3

[0325] Isolation of Human T Cells from Human PBMCs

[0326] T cells were isolated from human peripheral blood mononuclear cells (PBMC) by Ficoll density gradient centrifugation (GE Healthcare Lifesciences). After washing away the residual Ficoll solution, T cells were isolated using the EasySep™ Human T Cell Isolation Kit (STEM CELL technologies). Purified T cells were cultured in TexMACS™ medium (Miltenyi Biotech Inc) containing 1% penicillin and streptomycin sulfate in the presence of human IL-2 (100 IU / mL, Miltenyi Biotech Inc). In multi-well plates at 1x10 6 T cells were cultured at a density of 1 cell / mL. T cells were split and re-fed every 2-3 days.

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Abstract

The present disclosure relates to methods of treating a patient with a cancer by administering to the patient a composition comprising CAR T cells and a small molecule linked to a targeting moiety bya linker. The disclosure also relates to compositions for use in such methods.

Description

[0001] Cross References to Related Applications [0002] This application claims under Title 35, United States Code, Section 119(e) of U.S. Provisional Application Serial No. 62 / 320,183, filed April 8, 2016, and U.S. Provisional Application Serial No. 62 / 323,971, filed April 18, 2016 priority, both of which are hereby incorporated by reference in their entirety. technical field [0003] The present invention relates to a method of treating a patient with cancer by administering to the patient a composition comprising CAR T cells and administering to the patient a small molecule linked to a targeting moiety via a linker. The invention also relates to compositions for use in such methods. Background of the invention [0004] Immunotherapy based on the adoptive transfer of lymphocytes (eg, T cells) into patients is a valuable therapy in the treatment of cancer and other diseases. Many important advances have been made in the development of immunotherapies based on adoptive tr...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K35/17A61K39/00C07K16/00
CPCA61K35/12A61K39/39558A61K9/0019A61K48/005A61K35/17C07K16/44C12N15/86A61K2039/505C07K2317/622C07K2317/73C07K2319/03C07K2319/33C12N2740/16043A61K47/551A61K47/555A61P35/00A61K2300/00A61K39/0011A61K2039/5156C07K14/7051C07K14/70517C07K2317/92C07K2319/02C12N2740/15043
Inventor P.S.罗H.楚Y.G.李
Owner PURDUE RES FOUND INC
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