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METHODS FOR ENHANCING TCR[alpha][beta]+ CELL DEPLETION EFFICIENCY

A cell, cell population technology for depleting cells expressing endogenous TCRs to address safety or efficacy barriers

Pending Publication Date: 2021-10-08
ALLOGENE THERAPEUTICS INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the possibility of developing graft-versus-host disease (GvHD) or host-versus-graft disease (HvGD) is a major safety or efficacy hurdle for the widespread use of engineered allogeneic CAR-T cells in cancer therapy

Method used

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  • METHODS FOR ENHANCING TCR[alpha][beta]+ CELL DEPLETION EFFICIENCY
  • METHODS FOR ENHANCING TCR[alpha][beta]+ CELL DEPLETION EFFICIENCY
  • METHODS FOR ENHANCING TCR[alpha][beta]+ CELL DEPLETION EFFICIENCY

Examples

Experimental program
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example

[0216] As shown in the examples below, the disclosed combination antibody approach resulted in a significant increase in the efficiency of TCR+ cell depletion, which unexpectedly reduced residual TCR+% levels from 0.1%-1% to 0.1%-0.01% compared to TCR antibodies alone % (measured on day 1 after depletion). These examples demonstrate that the disclosed depletion methods offer significant advantages over current methods for depleting TCR+ cells. These advantages may provide benefits to patients receiving allogeneic therapy in the form of a reduced likelihood of the patient exhibiting GvHD.

example 1

[0217] Example 1. The combination of anti-TCR antibody and anti-CD3 antibody increases the efficiency of TCR+ cell depletion

[0218] Using CAR-engineered immune cells, by targeting TRAC and CD52 genes Electroporation knockdown of endogenous TCR and CD52 gene expression. TCR and CD52 knockout cells were then exposed to TCR depleting reagents. Cells were contacted with primary anti-TCR antibody conjugated to biotin, alone or in combination with anti-CD3 antibody or anti-CD52 antibody, as indicated in Table 1 .

[0219] Next, a secondary anti-biotin antibody conjugated to magnetic microbeads (nanoparticles with a diameter of approximately 50 nm) was further added to the primary antibody-labeled cells, so that the magnetic microbeads and Any residual TCR+ cell binding. Then use CliniMACS The instrument applies the labeled cells to the magnetic column. TCR+ cells are retained inside the magnetic column, while unlabeled TCR- cells pass through to the product collection bag. ...

example 2

[0229] Example 2. Post-depletion culture

[0230] This example demonstrates that depleted immune cell populations maintain the same low residual TCR+ levels during post-depletion culture. On day 1 post-depletion, depleted cells were frozen and later thawed for post-depletion culture. TCR and CD3 expression levels were measured before freezing and immediately after thawing. Such as Figures 5A-5E As shown, neither TCR frequency nor CD3 frequency was significantly different due to freeze-thaw cycles; Figure 5A The frequency of TCR+, CD3+, CD3+ / TCR- and CD3+ / TCR+ cells before freezing and after thawing was plotted by FACS plot using anti-TCR, anti-CD3 single stain, or anti-TCR / CD3 double stain, Figure 5B Digitally plotted the frequency of TCR+ cells before freezing and after thawing using an anti-TCR antibody, Figure 5C Digitally plotted the frequency of CD3+ cells before freezing and after thawing using anti-CD3 antibody, Figure 5D Digitally plotted the frequency of CD3...

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Abstract

Provided herein are improved methods for robust TCR+ cell depletion and production of populations of TCR- cells, which can be beneficial to minimize the GvHD risk in patients receiving allogeneic CAR T cell therapy. Provided herein are methods that increase the efficiency of depleting TCR+ cells from a population of cells in order to significantly reduce any residual levels of TCR+ cells present in cell populations in which expression of endogenous TCR has been reduced or eliminated. Associated kits and cell populations are also provided.

Description

[0001] Cross References to Related Applications [0002] This application claims the benefit of priority to U.S. Provisional Application No. 62 / 821,768, filed March 21, 2019, the contents of which are hereby incorporated by reference in their entirety. technical field [0003] The present disclosure relates to methods for depleting endogenous TCR (eg, TCRαβ) expressing cells from engineered immune cell populations, including those comprising chimeric antigen receptors (CARs). Background technique [0004] Adoptive transfer of immune cells genetically modified to recognize malignancy-associated antigens shows promise as a new approach for the treatment of cancer (see e.g. Brenner et al., Current Opinion in Immunology, 22(2) : 251-257 (2010); Rosenberg et al., Nature Reviews Cancer, 8(4): 299-308 (2008)). Immune cells can be genetically modified to express a chimeric antigen receptor (CAR) (see, e.g., Eshhar et al., Proc. Natl. Acad. Sci. USA, 90(2): 720- 724 (1993) and Sad...

Claims

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Application Information

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IPC IPC(8): A61K35/17A61K48/00C07K16/28C12N5/0783C12N15/90
CPCA61K48/00C12N15/90C12N5/0636C07K16/2893C07K16/2809C12N2510/00C12N5/0081C07K14/7051A61P35/00C07K2319/03C07K2319/33A61K39/4631A61K39/4611C12N5/0634A61K39/4621A61K39/464434A61K2239/26C12N15/113A61K2039/5158C12N2501/515
Inventor 倪亚瑾宁红秀J·M·李M·W·伦纳德
Owner ALLOGENE THERAPEUTICS INC
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