Engineered immune-mobilizing T-cell receptors with enhanced affinity for HIV-1 gag
The present invention relates to T cell receptors (TCRs) which bind the HLA-A*02 restricted peptide SLYNTVATL (SEQ ID NO: 1) derived from the HIV Gag gene product, p17. Said TCRs comprise non-natural mutations within the alpha and/or beta variable domains relative to a native HIV TCR. The TCRs of the invention possess unexpectedly high affinity, specificity and sensitivity for a complex of SEQ ID NO: 1 and HLA-A*02, and drive a particularly potent T cell response. Such TCRs are particularly useful in the development of soluble immunotherapeutic reagents for the treatment of HIV infected individuals.
1 . A soluble T cell receptor (TCR)-anti-CD3 fusion molecule that specifically binds to SLYNTVATL (SEQ ID NO: 1) in complex with HLA-A*02 comprising a TCR alpha chain comprising a TCR alpha chain variable domain and a TCR beta chain comprising a TCR beta chain variable domain, wherein
(i) the TCR alpha chain variable domain comprises an amino acid sequence comprising at least 94% sequence identity to SEQ ID NO: 6; and
(ii) the TCR beta chain variable domain comprises an amino acid sequence comprising the sequence of SEQ ID NO: 8;
wherein an anti-CD3 antibody is covalently linked to the N-terminus or the C-terminus of the TCR beta chain.
2 . The soluble TCR-anti-CD3 fusion molecule of claim 1 , wherein the TCR alpha chain variable domain comprises a CDR1 sequence of SEQ ID NO: 26 and a CDR2 sequence of SEQ ID NO: 27.
3 . The soluble TCR-anti-CD3 fusion molecule of claim 2 , wherein the TCR beta chain variable domain comprises a CDR1 sequence of SEQ ID NO: 23, a CDR2 sequence of SEQ ID NO: 28, and a CDR3 sequence of SEQ ID NO: 25.
4 . The soluble TCR-anti-CD3 fusion molecule of claim 1 , which is an alpha-beta heterodimer, having a TCR alpha chain TRAC constant domain sequence and a TCR beta chain TRBC1 or TRBC2 constant domain sequence, optionally wherein the TCR alpha and beta chain constant domain sequences are modified by truncation or substitution to delete the native disulfide bond between Cys4 of exon 2 of TRAC and Cys2 of exon 2 of TRBC 1 or TRBC2, wherein the TCR alpha and beta chain constant domain sequence(s) are modified by substitution of cysteine residues for Thr 48 of TRAC and Ser 57 of TRBC1 or TRBC2, the said cysteines forming a disulfide bond between the TCR alpha and beta constant domains of the soluble TCR-anti-CD3 fusion molecule.
5 . The soluble TCR-anti-CD3 fusion molecule of claim 1 , comprising a TCR in single chain format of the type Va-L-Vb, Vb-L-Va, Va-Ca-L-Vb, Va-L-Vb-Cb, Va-Ca-L-Vb-Cb, or Vb-Cb-L-Va-Ca, wherein Va and Vb are TCR alpha and beta variable regions respectively, Ca and Cb are TCR alpha and beta constant regions respectively, and L is a linker sequence.
6 . The soluble TCR-anti-CD3 fusion molecule of claim 1 , wherein the anti-CD3 antibody is covalently linked via a linker sequence selected from the group consisting of GGGGS (SEQ ID NO: 12), GGGSG (SEQ ID NO: 13), GGSGG (SEQ ID NO: 14), GSGGG (SEQ ID NO: 15), GSGGGP (SEQ ID NO: 16), GGEPS (SEQ ID NO: 17), GGEGGGP (SEQ ID NO: 18), and GGEGGGSEGGGS (SEQ ID NO: 19).