Compositions and methods for selective protein expression

A protein structure, protein technology, applied in the field of compositions and methods for selective protein expression

Pending Publication Date: 2019-05-03
NOVARTIS AG +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Such disadvantages may be particularly pronounced...

Method used

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  • Compositions and methods for selective protein expression
  • Compositions and methods for selective protein expression
  • Compositions and methods for selective protein expression

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[1136] Example 1: Materials and methods for Examples 1-13.

[1137] construct

[1138] The following constructs shown in Table 22 were used in Examples 1-13.

[1139] Note that the invention encompasses both orientations of heavy and light chains.

[1140] Table 22. Sequences of various constructs (signal peptide used in bold; furin cleavage site underlined)

[1141]

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[1157] general method

[1158] The following general materials and methods were used for the assays described in Examples 1-13.

[1159] CAR construct generation

[1160]The scFv used in the CAR construct evaluated in Example 3 was synthesized independently based on the anti-CD19 scFv sequence described above. The scFv was cloned into a CD8 hinge region together with a 4-1BB molecule and a CD3δ molecule in the heavy chain to li...

Embodiment 2

[1173] Example 2: Furin is the most highly expressed proprotein convertase in primary human T cells

[1174] Literature sources show that furin (FUR; PACE; PCSK3; SPC1 ) has a broad expression profile (Seidah et al., Nature Reviews Drug Discovery 11, 367-383 (May 2012)). Expression of proprotein convertase family members was analyzed by qRT-PCR. RNA was harvested from normal donor T cells on days 0, 4, and 11 after stimulation with anti-CD3 / anti-CD28 activation beads. During the culture period, an additional group was supplemented with 100 U / mL IL-2 and RNA was harvested on day 11. These data show that after activation with anti-CD3 / anti-CD28 beads, furin mRNA is more highly expressed than other members of the proprotein convertase family ( figure 1 ).

Embodiment 3

[1175] Example 3: CD19 CAR Expression Controlled by Compound Addition

[1176] Furin degron domain (FKBP) fused to anti-CD19 scFv CAR construct in Jurkat T cells FD , ERα FD or DHFR FD ) transduction followed by treatment with the corresponding compound. FKBP were treated with 1 μM Shield1 FD transduced cells. ERα was treated with 1 μM bazedoxifene FD transduced cells. DHFR was treated with 1 mM TMP FD transduced cells. In the presence of the compound, the expression of anti-CD19 scFv was inducible ( figure 2 ). Black = UTD; gray = construct, no compound; white = construct, with compound. These data show that multiple degron domains lead to strict compound-dependent regulation of CAR 19 expression when combined with the furin cleavage domain.

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Abstract

Provided herein are fusion proteins including two protein domains separated by a heterologous protease cleavage site, wherein a first of the protein domains is a conditional expression domain. Thus, the fusion proteins comprise three essential elements: a conditional expression domain, a domain containing the protein of interest, and a protease cleavage domain separating the two. Also provided herein are methods for treating autoantibody and alloantibody-mediated diseases or conditions in a subject by targeting B cells with anti-B cell modified T cells. In one embodiment, a chimeric antigen receptor (CAR) modified T cell is selectively ablated in a subject after adoptive transfer. Pharmaceutical compositions comprising the temporally regulated CAR modified T cell are also described herein.

Description

[0001] related application [0002] This application claims priority to U.S. Serial No. 62 / 322,931, filed April 15, 2016, and U.S. Serial No. 62 / 481,094, filed April 3, 2017, the entire contents of each of which are incorporated herein by reference. [0003] sequence listing [0004] This application contains a Sequence Listing that has been filed electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy was created on April 14, 2017, named N2067-7128WO_SL.txt, and was 2,326,027 bytes in size. Background of the invention [0005] A variety of constructs have been developed that enable selective expression of the desired protein of interest. More recent constructs have been tested that incorporate domains (referred to in the art as "degrons") engineered to degrade in the absence of stabilizing ligands. Other constructs incorporate domains engineered to aggregate in the absence of a deaggregating ligand (eg, an aggregation domain)...

Claims

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Application Information

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IPC IPC(8): C12N15/62C07K14/725
CPCC07K14/7051C07K2319/03C07K2319/50C07K2319/70C07K2319/95A61P17/00A61P19/02A61P21/04A61P25/00A61P29/00A61P31/12A61P35/00A61P35/02A61P37/02A61P5/14A61P7/06A61P9/00A61P9/12C07K16/2803C07K14/70575C07K14/70517A61K35/15C07K2317/622A61K38/00
Inventor A·戈隆索夫C·吉马雷斯G·莫茨M·米伦C·T·艾利布莱希特A·S·佩恩
Owner NOVARTIS AG
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