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Bcma chimeric antigen receptors

a technology of chimeric antigen receptors and bcma, which is applied in the direction of viruses, vectors, fusion polypeptides, etc., can solve the problems of humanized anti-bcma cars unsuitable for t cell therapy, and achieve the effect of treating, preventing, or ameliorating b cell related conditions

Inactive Publication Date: 2017-08-10
2SEVENTY BIO INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The invention provides improved vectors for generating T cell therapies for treating B cell-related conditions. Specifically, the invention provides humanized anti-BCMA CAR molecules that elicit antigen-independent cytokine release, which makes them more suitable for use in T cell therapies. The humanized anti-BCMA antibodies or antigen binding fragments that bind to the BCMA polypeptide can be selected from a variety of sources, such as camel Ig, Ig NAR, Fab fragments, and single-domain antibodies. The CAR molecules can also include various co-stimulatory signaling domains, such as CD28, CD134, or CD152, to enhance their function. Overall, the invention provides better tools for generating effective T cell therapies for B cell-related conditions.

Problems solved by technology

Without wishing to be bound to any particular theory, the inventors have discovered that certain humanized anti-BCMA CARs elicit antigen independent (“tonic”) cytokine release, a characteristic that would make humanized anti-BCMA CARs unsuitable for use in T cell therapies.

Method used

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Examples

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example 1

Construction of Anti-BCMA Cars

[0401]CARs containing humanized anti-BCMA scFv antibodies were designed to contain an MND promoter operably linked to anti-BCMA scFv, a hinge and transmembrane domain from CD8α and a CD137 co-stimulatory domains followed by the intracellular signaling domain of the CD3ζ chain. FIG. 1. The anti-BCMA CARs comprise a CD8α signal peptide (SP) sequence for the surface expression on immune effector cells. The polynucleotide sequences of the anti-BCMA CARs are set forth in SEQ ID NOs: 30 to 44, 70, and 72; polypeptide sequences of the anti-BCMA CARs are set forth in SEQ ID NOs: 15 to 29, 71, and 73; and the vector maps is shown in FIG. 1. Tables 3 to 5 show the Identity, Genbank Reference, Source Name and Citation for the various nucleotide segments of various exemplary anti-BCMA CAR lentiviral vectors.

TABLE 3NucleotidesIdentityGenBank ReferenceSource NameCitation  1-185pUC19 plasmidAccession #L09137.2pUC19New Englandbackbonent 1-185Biolabs 185-222LinkerNot ap...

example 2

High Throughput Screen of Humanized Anti-BCMA Car

[0402]A microculture assay was developed to rapidly screen anti-BCMA CARs. The microculture assay can compare 15-20 CAR modified T cells (CART cells). Peripheral blood mononuclear cells (PBMC) were cultured in 24-well plates in media containing IL-2 (CellGenix) and antibodies specific for CD3 and CD28 (Miltenyi Biotec) to initiate outgrowth of T cells. The expanded T cells were >99% CD3 positive T cells after 7 days of culture.

[0403]T cells expanded from PBMC were modified by lentiviruses to express the anti-BCMA CARs. Transient lentiviral supernatants were added to the PBMC culture at 1:1 ratio (volume:volume) one day after culture initiation. Cultures were split as needed with fresh media containing IL-2 after becoming optically confluent using an inverted light microscope. After twelve days of culture, CAR T cells were harvested and evaluated for CAR expression and function.

[0404]Expression of CARs on the T cell surface was assayed...

example 3

Antigen Specific Reactivity of Car T Cells

[0405]Antigen-specific reactivity was examined after co-culture with BCMA-positive cell lines. Anti-BCMA CAR T cells were co-cultured with BCMA-engineered K562 (K562-BCMA) cells for 24 hours. Reactivity of the anti-BCMA CAR T cells to K562-BCMA was assayed by IFN-gamma (IFNγ) release into the supernatant by ELISA. All humanized anti-BCMA CART cells tested released similar amounts of IFNγ and the amounts were comparable the parental mouse anti-BCMA CAR. FIG. 2. In contrast, IFNγ was not detected in cultures containing either untransduced or CD19-specific CAR T cells confirming that BCMA-specificity of the CAR T cells was required for reactivity to K562-BCMA cells.

[0406]In another set of experiments, tumor cytolytic activity of T cells expressing one of five humanized anti-BCMA CARs was examined. anti-BCMA CAR T cells were co-cultured with K562-BCMA cells for four hours. The five humanized anti-BCMA CAR T cells exhibited similar cytolytic acti...

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Abstract

The invention provides improved compositions for adoptive T cell therapies for B cell related conditions.

Description

CROSS REFERENCE TO RELATED APPLICATIONS[0001]This application claims the benefit under 35 U.S.C. §119(e) of U.S. Provisional Application No. 62 / 028,664, filed Jul. 24, 2014, U.S. Provisional Application No. 62 / 044,103, filed Aug. 29, 2014, and U.S. Provisional Application No. 62 / 152,575, filed Apr. 24, 2015, each of which is incorporated by reference herein in its entirety.STATEMENT REGARDING SEQUENCE LISTING[0002]The Sequence Listing associated with this application is provided in text format in lieu of a paper copy, and is hereby incorporated by reference into the specification. The name of the text file containing the Sequence Listing is BLBD_037_03WO_ST25.txt. The text file is 124 KB, was created on Jul. 23, 2015, and is being submitted electronically via EFS-Web, concurrent with the filing of the specification.BACKGROUND[0003]Technical Field[0004]The present invention relates to improved compositions and methods for treating B cell related conditions. More particularly, the inv...

Claims

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Application Information

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IPC IPC(8): C07K16/28C12N15/86A61K39/395C07K14/705A61K38/17
CPCC07K16/2878C12N2830/60C07K14/70503C07K14/70521C07K14/70578A61K38/1774A61K38/177A61K39/3955C12N15/86C07K2317/24C07K2319/10C07K2319/03C07K2319/02C07K2317/622C07K2317/565C07K2317/56C07K2317/53C07K2317/524C07K2317/526A61K2039/572C12N2740/15043C12N2830/48C12N2830/50C07K14/70517C07K14/7051C07K2319/33C07K2317/73A61P13/12A61P17/00A61P19/02A61P21/04A61P25/00A61P29/00A61P35/00A61P35/02A61P37/00A61P37/02A61P37/06A61P37/08A61P7/00A61P7/06A61P9/00A61K39/4631A61K39/4611A61K2239/48A61K39/464417C07K2319/00A61K2039/5156
Inventor MORGAN, RICHARDFRIEDMAN, KEVIN
Owner 2SEVENTY BIO INC
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