PARALLEL CHIMERIC ANTIGEN RECEPTORS (pCARs) AND ADAPTOR CHIMERIC ANTIGEN RECEPTORS COMPRISING ALTERNATIVE SIGNALLING DOMAINS AND METHODS OF USE THEREOF
AssigneeKing's College London
InventorsPhoebe Dunbar, Marc Davies, Maya Glover, John Maher
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Abstract
Provided herein are second generation chimeric antigen receptors (CARs), parallel CARs (pCARs), and adaptor CARs comprising an intracellular signalling domain modified for optimal signalling. Also provided herein are compositions and methods for improving anti-tumour efficacy and restimulation capacity of CAR T-cells, pCAR T-cells, and adaptor CAR T-cells.
Claims35
A parallel chimeric antigen receptor (pCAR) comprising:
- (1) a second-generation chimeric antigen receptor (second-generation CAR) comprising:
- a) an intracellular signalling domain comprising a modified CD3ζ polypeptide;
- b) a co-stimulatory signalling region;
- c) a transmembrane domain;
- and
- d) a first binding element that specifically interacts with a first epitope on a first target antigen,
- wherein the modified CD3ζ polypeptide comprises an unmodified immunoreceptor tyrosine-based activation motif 1 (ITAM1), and one or more mutations in ITAM2 and/or ITAM3;
- and
- (2) a chimeric co-stimulatory receptor (CCR) comprising at least one CCR polypeptide comprising:
- e) a co-stimulatory signalling region which is different from that of (b);
- f) a transmembrane domain;
- and
- g) a second binding element that specifically interacts with a second epitope on a second target antigen.
The pCAR of any one of the preceding claims, wherein the modified CD3ζ polypeptide comprises a sequence selected from SEQ ID NOs:
- 48-53.
The pCAR of any one of the preceding claims, wherein the first target antigen is selected from an NKG2D ligand (e.g., MICA, MICB, ULBP1-ULBP6), MUC1, αvβ6 integrin, ErbB, HER2, B7-H3, Claudin 18.2, Claudin 18.6, Glypican 3, ALK, CD70, and prostate-specific membrane antigen (PSMA).
The pCAR of any one of the preceding claims, wherein the first binding element comprises the CDRs of the HMFG2 antibody, the VH and VL domains of HMFG2 antibody or HMFG2 single-chain variable fragment (scFv).
The pCAR of any one of the preceding claims, wherein the second-generation CAR has at least 90% sequence identity to the amino acid sequence of SEQ ID NO:
- 59.
The pCAR of any one of the preceding claims, wherein the co-stimulatory signalling region is selected from (i) co-stimulatory signalling regions of members of the B7/CD28 family, the ILT/CD85 family, the tumour necrosis factor (TNF) superfamily, the SLAM family, and the TIM family, (ii) a CD28 co-stimulatory domain, a 4-1BB co-stimulatory domain, an OX40 co-stimulatory domain, and an ICOS co-stimulatory domain, or (iii) a co-stimulatory molecule selected from CD7, CD96, CD160, CD200, CD300a, CRTAM, DAP10, DAP12, Dectin-1, DPPIV, EphB6, LAG-3, TSLP R, or a variant thereof.
The pCAR of any one of the preceding claims, wherein the co-stimulatory signalling region of (e) is a 4-1BB co-stimulatory domain.
The pCAR of any one of the preceding claims, wherein the transmembrane domain is selected from the transmembrane domains of CD8α, CD28, CD4, CD3ζ, FcεR1γ.
The pCAR of any one of the preceding claims, wherein the transmembrane domain of (c) is a CD28 transmembrane domain and/or the transmembrane domain of (f) is a CD8α transmembrane domain.
The pCAR of any one of the preceding claims, wherein the second target antigen is selected from an NKG2D ligand (e.g., MICA, MICB, ULBP1-ULBP6), MUC1, αvβ6 integrin, ErbB, HER2, B7-H3, GD2, Claudin 18.2, Claudin 18.6, Glypican 3, ALK, CD47, CD70, prostate-specific membrane antigen (PSMA), a PD-1 ligand (e.g., PD-L1), NKp44 ligands (e.g., NKp44L (MLL5), PCNA, viral haemagglutinins, nidogen-1, galectin-3, other proteoglycans) and NKp30 ligands (e.g., BAG6, B7-H6).
The pCAR of any one of the preceding claims, wherein the second binding element specifically interacts with an ErbB antigen, an NKG2D ligand, a PD-1 ligand, an NKp44 ligand, or an NKp30 ligand.
The pCAR of any one of the preceding claims, wherein the second binding element is any one of a T1E peptide, an NKG2D polypeptide, a PD-1 polypeptide, an NKp44 polypeptide, an NKp30 polypeptide, or a fragment, portion, or variant thereof.
The pCAR of any one of the preceding claims, wherein the CCR of (2) comprises two or more CCR polypeptides.
The pCAR of any one of the preceding claims, wherein the CCR of (2) comprises three CCR polypeptides.
The pCAR of any one of the preceding claims, wherein the second-generation CAR has at least 90% sequence identity to the amino acid sequence of SEQ ID NO:
- 59 and the CCR has at least 90% sequence identity to the amino acid sequence of any one of SEQ ID NO:
- 24, SEQ ID NO:
- 77, SEQ ID NO:
- 79, SEQ ID NO:
- 81, and SEQ ID NO:
- 83.
One or more polynucleotide constructs encoding the pCAR of any one of the preceding claims.
A fusion polypeptide comprising (i) a DNAX-activating protein 10 (Dap10) polypeptide, or a functional variant thereof and (ii) a modified CD3ζ polypeptide that comprises an unmodified immunoreceptor tyrosine-based activation motif 1 (ITAM1), and one or more mutations in ITAM2 and/or ITAM3.
Dependencies are inferred from the claim text.