Group of chimeric antigen receptors (CARS)

A chimeric antigen receptor, antigen technology, applied in the direction of antibody medical components, antibody mimics/scaffolds, receptors/cell surface antigens/cell surface determinants, etc.

Pending Publication Date: 2021-07-23
圣安娜儿童癌症研究中心 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, further replication of this success to other tumors is presently prevented by several obstacles (Lim and June, Cell. 2017;168(4):724)

Method used

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  • Group of chimeric antigen receptors (CARS)
  • Group of chimeric antigen receptors (CARS)
  • Group of chimeric antigen receptors (CARS)

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0396] Example 1: Generation of rcSso7d-based low affinity single domain binding moieties for CAR panels according to the invention

[0397] In a first example, a strategy for generating low-affinity antigen-binding moieties suitable for use as antigen-binding moieties of a CAR panel according to the present invention is shown. Charge-reduced Sso7d (rcSso7d) is a charge-reduced version of a small (~7 kDa) DNA-binding protein from the archaea Sulfolobus solfataricus. The charge reduction minimizes nonspecific binding due to reduced electrostatic interactions. rcSso7d is a single domain protein antigen binding moiety with high thermostability and monomeric behavior, and is therefore an example suitable for binding to scaffolds. Starting from the well-characterized antigen-binding moiety rcSso7d E11.4.1, which binds to human EGFR with a K of 19 nM d combined (Traxlmayr et al., JBiol Chem. 2016;291(43):22496-22508), by performing an alanine scan, in which we replaced all amino a...

Embodiment 2

[0412] Example 2: Cysteines forming extracellular disulfide bonds prevent full exploitation of the avidity effects that reversibly control CAR function

[0413] Cysteines that form extracellular disulfide bonds in the extracellular hinge region, eg CD8α, can prevent utilization of the affinity effect according to the present invention. This is demonstrated in Example 2, wherein a low affinity mutant of the binding moiety "E11.4.1G32A" of Example 1 is fused to a CAR signaling backbone in which two extracellular cysteine ​​residues in the hinge region of CD8α (UniProt ID P01732, positions C164 and C181) were substituted or unsubstituted with serine residues, respectively. In response to target cells, however, cysteine-containing CAR variants ("Cys") effectively triggered T-cell activation, but serine-containing variants ("Ser") did not or only weakly. Thus, this example illustrates the importance of preventing disulfide bond formation when generating CAR molecules suitable for ...

Embodiment 3

[0428] Example 3: Single-chain variable fragments (scFvs) can trigger CAR aggregation in cell membranes, preventing exploitation of avidity effects that reversibly control CAR function

[0429] The third example shows that the integration of scFv-based binding moieties into CAR molecules can prevent avidity effects that exploit combinations of specific recognition antigens. Figure 4 A schematic diagram of the CAR construct shown in A shows the CAR variants tested (4D5-5-8cys-BB-3z, 4D5-5-8ser-BB-3z, 4D5-5(split)-8ser-BB-FKBP(36V )-3z) design. In the example shown, scFv 4D5-5 against HER2 was used as the antigen binding moiety and integrated into a monomeric ("Ser") or dimerized ("Cys") CAR signaling backbone. Figure 4 B shows CAR expression in primary T cells. The potent binding affinity of scFv 4D5-5 was reported as 1.1 μM (Liu et al., Cancer Res. 2015;75(17):3596-3607), which was comparable to that of E11.4.1-G32A. Jurkat T cells expressing a truncated form of HER2 (tHE...

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Abstract

Disclosed is agroup of chimeric antigen receptors (CARs) consisting of two, three or four CAR molecules, wherein the members of the group of CARs can be different or identical in their amino acid sequences to one another, and wherein each of the CAR molecules of the group comprise at least a transmembrane domain and an ectodomain comprising either an antigen binding moiety or a binding site to which another polypeptide is able to bind, wherein the another polypeptide comprises an antigen binding moiety; wherein each CAR molecule of the group comprises at least one dimerization domain, wherein this dimerization of a pair of dimerization domains is either induced by a regulating molecule and optionally reduced by another regulating molecule, or occurs in the absence of a regulating molecule and is reduced by a regulating molecule, and wherein the ectodomain of each CAR molecule of the group in its prevalent conformation is free of cysteine amino acid moieties which are able to form intermolecular disulphide bonds with other CAR molecules of the group, respectively, and wherein the antigen binding moieties of the CAR molecules of the group and of the other polypeptides being able to bind to the CAR molecules of the group are either specific for one target antigen or for a non-covalent or a covalent complex of different target antigens.

Description

technical field [0001] The present invention relates to chimeric antigen receptor (CAR) panels consisting of two, three or four CAR molecules. Background technique [0002] Immunotherapy using CAR T cells, that is, modifying T cells to express a chimeric antigen receptor (CAR), is one of the most promising approaches in cancer treatment. The high potential of this therapeutic strategy has been demonstrated to date through impressive clinical responses in patients with B-cell malignancies. However, further replication of this success to other tumors is currently held back by several obstacles (Lim and June, Cell. 2017;168(4):724). For example, CAR T cells are active drugs that replicate after administration, and their activity cannot be adequately controlled in a reversible manner. To date, several strategies for conditional CAR have been developed (Lim and June, Cell. 2017; 168(4):724). These strategies achieve reversible modulation by administration of small molecule dru...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K39/00
CPCA61P35/00A61K2039/5156A61K39/001104A61K39/001106C07K2317/31C07K2317/92C07K2318/20C07K2319/00C07K14/705C07K16/22C07K16/32C07K2317/622C07K2319/02C07K2319/03A61K35/17A61K38/00C07K14/7051
Inventor B·扎尔策M·莱纳M·特拉克斯迈尔
Owner 圣安娜儿童癌症研究中心
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