Chimeric antigen receptor and methods of use thereof

a technology of chimeric antigen and receptor, applied in the field can solve the problem that the car is not pharmacologically controlled, and achieve the effect of reducing the number of chimeric antigen receptors

Inactive Publication Date: 2015-12-24
RGT UNIV OF CALIFORNIA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, such CARs are not capable o

Method used

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  • Chimeric antigen receptor and methods of use thereof
  • Chimeric antigen receptor and methods of use thereof
  • Chimeric antigen receptor and methods of use thereof

Examples

Experimental program
Comparison scheme
Effect test

example 1

Generation of CAR

[0309]Materials and Methods

[0310]The anti-human CD19 scFv was selected as the antigen recognition domain in CARs throughout the design optimization process. FIGS. 18A and 18B summarize the molecular structure of each CAR consisting of two numerically identified polypeptides. All membrane-anchored polypeptides are di-sulfide bonded homo-dimers. The membrane-anchored polypeptides are depicted as monomers for graphical simplicity.

[0311]Generation of CAR Constructs

[0312]Sequence encoding the anti-human CD19 scFv was cloned from a construct. The human 4-1BB co-stimulation and CD3 zeta ITAM signaling chains were cloned from cDNAs supplied by Open Biosystems. FKBP- and FRB-encoding sequences were cloned from plasmids supplied by Addgene.

[0313]Standard molecular cloning techniques (polymerase chain reaction (PCR), restriction digestion, ligation, etc.) were applied to generate lentiviral expression plasmids.

[0314]Effector and Target Cell Culturing Conditions

[0315]Human prim...

example 2

CARs Targeting Mesothelin

[0331]Materials and Methods

[0332]A number of chimeric antigen receptor constructs were made and tested. The constructs shown here encode three different anti-human mesothelin scFv as the antigen recognition domains. FIGS. 19A, 19B, and 19C summarize the molecular structure of each anti-human mesothelin CAR, with each CAR comprising two polypeptides. The intercellular portion of each anti-human mesothelin CAR comprises two 4-1BB co-stimulatory domains, an FKBP and FRB dimerizer-binding pair, and an ITAM intracellular signaling domain. The three different antigen recognition domains shown here are anti-mesothelin HN1 scFv, SS1 scFv, and m912 scFv. All membrane-anchored polypeptides are di-sulfide bonded homo-dimers.

[0333]Generation of CAR Constructs

[0334]Sequences encoding the anti-mesothelin were cloned from constructs or synthesized via gene assembly by PCR. The human 4-1BB co-stimulation and CD3 zeta ITAM signaling chains were cloned from cDNAs supplied by ...

example 3

Gibberellic Acid as a Dimerizer of On-Switch CARs

[0346]Materials and Methods

[0347]FIG. 20A summarizes the molecular structure of the subject gibberellic acid dimerizer CAR. The antigen binding portion comprises the anti-human CD19 scFv. The intracellular portion comprises two 4-1BB co-stimulatory domains, a GID1 and GAI dimerizer-binding pair, and an ITAM intracellular signaling domain. All membrane-anchored polypeptides are di-sulfide bonded homo-dimers.

[0348]Generation of CAR Constructs

[0349]Sequences encoding the gibberellic acid dimerizer CAR were cloned from constructs. The anti-CD 19 scFv was cloned from a plasmid. The human 4-1BB co-stimulation and CD3 zeta ITAM signaling chains were cloned from cDNAs supplied by Open Biosystems. GID1- and GAI-encoding sequences were cloned from Addgene plasmids. Standard molecular cloning techniques (polymerase chain reaction (PCR), restriction digestion, ligation, etc.) were applied to generate lentiviral expression plasmids.

[0350]Effector ...

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Abstract

The present disclosure provides a heterodimeric, conditionally active chimeric antigen receptor (CAR), and a nucleic acid comprising a nucleotide sequence encoding the CAR. The present disclosure provides cells genetically modified to produce the CAR. A CAR of the present disclosure can be used in various methods, which are also provided.

Description

CROSS-REFERENCE[0001]This application claims the benefit of U.S. Provisional Patent Application No. 61 / 765,585, filed February 15, 2013, which application is incorporated herein by reference in its entirety.STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH[0002]This invention was made with government support under Grant Nos. EY016546 and GM101782 awarded by the National Institutes of Health. The government has certain rights in the invention.INCORPORATION BY REFERENCE OF SEQUENCE LISTING PROVIDED AS A TEXT FILE[0003]A Sequence Listing is provided herewith as a text file, “UCSF-464WO SeqList_ST25.txt” created on Feb. 13, 2014 and having a size of 153 KB. The contents of the text file are incorporated by reference herein in their entirety.INTRODUCTION[0004]In cell-based adoptive immunotherapy, immune cells isolated from a patient can be modified to express synthetic proteins that enable the cells to perform new therapeutic functions after they are subsequently transferred back into the...

Claims

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Application Information

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IPC IPC(8): C07K16/28C12N9/90A61K47/48C07K14/705C07K14/725C12N15/11
CPCC07K16/2803C07K14/70578C07K14/7051A61K47/48723C12Y502/01008A61K2039/505C07K2319/03C07K2319/33C12N9/90C07K14/705C07K16/18C07K16/2866C07K2317/622C07K2319/00C07K2319/20A61K47/6891C12N9/14A61P35/00A61P37/04A61P43/00C07K16/2878C07K2319/70C07K14/70517C07K16/30C12N5/0636A61K35/17C12N2510/00C07K14/70521C07K14/70575C07K2319/74
Inventor WU, CHIA-YUNGONUFFER, JAMESLIM, WENDELL A.
Owner RGT UNIV OF CALIFORNIA
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