BINDING PROTEINS AND ENGINEERED CELLS SPECIFIC FOR NEOANTIGENS AND USES THEREOF
The present disclosure provides compositions and methods for targeting a neoantigen to, for example, treat or prevent cancer. Disclosed embodiments include binding proteins, such as T cell receptors bind to a neoantigen:HLA complex. Disclosed binding proteins are highly sensitive to antigen, capable of inducing activation of host T cells at low concentrations of peptide antigen. In certain embodiments, binding proteins of the present disclosure are non-alloreactive against, are substantially non-alloreactive against, and/or have a low risk of alloreactivity against (i) amino acid sequences from the human proteome and/or (ii) against human HLA alleles. Polynucleotides encoding such binding protein can introduced into a host cell, such as a T cell, and the cell can be used in immunotherapy for treating various cancers.
1 . A polynucleotide comprising a nucleic acid sequence encoding:
(a) a binding protein, wherein the binding protein comprises:
a T cell receptor (TCR) or a functional derivative thereof; or
a chimeric antigen receptor (CAR) or a functional derivative thereof; and
(b) a fusion protein, wherein the fusion protein comprises:
(i) an extracellular component comprising a CD95 ligand (FasL) binding domain that comprises a CD95 (Fas) ectodomain or a functional fragment thereof; and
(ii) an intracellular component comprising a CD137 (4-1BB) intracellular signaling domain, wherein the nucleic acid sequence encoding the binding protein is positioned upstream of the nucleic acid sequence encoding the fusion polypeptide.
2 . The polynucleotide of claim 1 ,
further comprising a nucleic acid sequence encoding:
(c) a CD8 co-receptor α or β chain or a portion or variant thereof, wherein the sequence encoding the binding protein is positioned upstream of the sequence encoding the extracellular portion of a CD8 co-receptor α or β chain or the portion or variant thereof; and/or
further comprising a nucleic acid sequence encoding:
(c) a CD8 co-receptor α and β chain or portions or variants thereof, wherein the sequence encoding the binding protein is positioned upstream of the sequence encoding the extracellular portion of the CD8 co-receptor α and β chains or the portions or variants thereof.
3 . The polynucleotide of claim 2 , wherein the nucleic acid sequence encoding the fusion protein further encodes:
(d) a hydrophobic component between the extracellular and intracellular components of the fusion protein.
4 . The polynucleotide of claim 1 ,
wherein the binding protein comprises a binding domain that binds to a peptide:HLA complex, wherein the complex comprises a neoantigen peptide and an HLA protein;
wherein the binding protein comprises a single-chain TCR (scTCR) or a single-chain T cell receptor variable fragment (scTv);
wherein the binding protein comprises a TCR α chain variable (Vα) domain or a TCR β chain variable (Vβ) domain; OR
wherein the binding protein comprises a TCR α chain variable (Vα) domain and a TCR β chain variable (Vβ) domain.
5 . The polynucleotide of claim 1 , wherein the CD95 (Fas) ligand binding domain is a Fas ectodomain or a functional fragment thereof and/or wherein the intracellular component is a CD137 (4-1BB) transmembrane domain or a functional fragment thereof.
6 . The polynucleotide of claim 1 , wherein the neoantigen peptide is a KRAS, HRAS, NRAS, p53, or PIK3CA mutant peptide.
7 . The polynucleotide of claim 1 , wherein the KRAS mutant peptide comprises x-V-G-A-x-G-x-x-K, wherein x denotes any amino acid; and wherein the HLA protein is encoded by an HLA-A*11 or HLA-A*11:01 allele.
8 . The polynucleotide of claim 2 ,
further comprising a nucleic acid sequence encoding a self-cleaving peptide between the nucleic acid sequence encoding the TCR receptor variable α (Vα) region and the nucleic acid sequence encoding the TCR receptor variable β (Vβ) region further comprising a nucleic acid sequence encoding a self-cleaving peptide disposed between (a) and (b) or, where (c) is present, (b) and (c);
further comprising a nucleic acid sequence encoding a self-cleaving peptide between the sequence encoding the CD8 co-receptor α chain and the sequence encoding the CD8 co-receptor β chain
further comprising a nucleic acid sequence that encodes a self-cleaving peptide that is disposed between the nucleic acid sequence encoding a binding protein and the nucleic acid sequence encoding a polypeptide comprising an extracellular portion of a CD8 co-receptor α chain; and/or the nucleic acid sequence encoding a binding protein and the nucleic acid sequence encoding a polypeptide comprising an extracellular portion of a CD8 co-receptor β chain.
further comprising, operably linked in-frame:
(iii)(pnBP)-(pnSCP 1 )-(pnCD8α)-(pnSCP 2 )-(pnCD8β)-(pnFP); or
(iv)(pnBP)-(pnSCP 1 )-(pnCD8β)-(pnSCP 2 )-(pnCD8α)-(pnFP);
(iii)(pnBP)-(pnSCP 1 )-(pnFP)-(pnSCP 1 )-(pnCD8α)-(pnSCP 2 )-(pnCD8β); or
(iv)(pnBP)-(pnSCP 1 )-(pnFP)-(pnSCP 1 )-(pnCD8β)-(pnSCP 2 )-(pnCD8α);
wherein pnCD8α is the nucleic acid sequence encoding a polypeptide that comprises an extracellular portion of a CD8 co-receptor α chain,
wherein pnCD8β is the nucleic acid sequence encoding a polypeptide that comprises an extracellular portion of a CD8 co-receptor α chain,
wherein pnBP is the nucleic acid sequence encoding a binding protein,
wherein pnFP is the nucleic acid sequence encoding a fusion protein, and
wherein pnSCP 1 and pnSCP 2 are each independently a polynucleotide encoding a self-cleaving peptide, wherein the polynucleotides and/or the encoded self-cleaving peptides are optionally the same or different.
9 . The polynucleotide of claim 8 , wherein the self-cleaving peptide is a P2A, T2A, E2A, or a furin peptide.
10 . A vector comprising the polynucleotide of claim 1 .
11 . A host cell comprising the polynucleotide of claim 1 .
12 . A method for treating a disease or disorder associated with a KRAS G12V mutation or a NRAS G12V mutation or a HRAS G12V mutation in a subject, the method comprising administering to the subject an effective amount of the host cell of claim 11 .
13 . A method of eliciting an immune reaction against a cell expressing a neoantigen, the method comprising contacting the cell with the cell comprising the polynucleotide of claim 1 .
14 . A method of eliciting an immune reaction against a cell expressing a neoantigen, the method comprising contacting the cell with the host cell of claim 11 .
15 . A method of genetically engineering an immune cell, the method comprising contacting the cell with a polynucleotide comprising a nucleic acid sequence encoding a T cell receptor (TCR) or functional fragment or variant thereof, a CD8α and/or a CD8β co-receptor or functional fragment or variant thereof, and a fusion protein comprising a CD95 (Fas) ectodomain or a functional fragment thereof and an intracellular component comprising a CD137 (4-1BB) intracellular signaling domain, and expanding the immune cell.
16 . A host cell comprising:
(a) a fusion protein, wherein the fusion protein comprises:
(i) an extracellular component comprising a CD95 ligand (FasL) binding domain that comprises a CD95 (Fas) ectodomain or a functional fragment thereof; and
(ii) an intracellular component comprising a CD137 (4-1BB) intracellular signaling domain, wherein the nucleic acid sequence encoding the binding protein is positioned upstream of the nucleic acid sequence encoding the fusion polypeptide; and
(b) an exogenous CD8 co-receptor α or β chain or a portion or variant thereof.
17 . A method for treating a cancer in a subject, comprising administering to the subject an effective amount of the host cell of claim 11 .
18 . A composition comprising a plurality of host cell, wherein the host cells comprise T-cells directed against a mutant KRAS peptide wherein the composition:
(a) comprises at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or greater CD3 + cells that stain with dextramer specific for mutant KRAS peptide as assessed by flow cytometry;
(b) comprises at least 80%, 85%, 90%, 92%, 94%, 96%, 98%, or greater T cells that are CD3-positive as assessed by flow cytometry;
(c) comprises at least 70%, 75%, 80%, 85%, 90%, or greater viable cells as assessed by automated cell counting.
19 . A composition comprising the host cells of claim 11 and a pharmaceutically acceptable excipient.
20 . A composition comprising the polynucleotide of claim 1 and a pharmaceutically acceptable excipient.