HIV specific binding molecules
Specific binding molecules, including 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 are presented. TCRs of the present invention comprise non-natural mutations within the alpha and/or beta variable domains relative to a native TCR. The specific binding molecules of the invention have improved stability and/or yield and yet unexpectedly retain the advantageous properties of the specific binding molecules from which they are derived. Such specific binding molecules are particularly useful in the development of soluble immunotherapeutic reagents for the treatment of HIV infected individuals.
1. A specific binding molecule having the property of binding to SLYNTVATL (SEQ ID NO: 1) in complex with HLA-A*02 and comprising a TCR alpha chain variable domain and a TCR beta chain variable domain,
wherein the alpha chain variable domain comprises an amino acid sequence selected from:
a)
(SEQ ID NO: 2)
AKEVEQNSGP LSVPEGAIAS LNCTYSSWEG QSFFWYRQYS
GKSPELIMKL YADPDKEDGR FTAQLNKASQ YVSLLIRDSQ
PSDSATYLCA VRTNSGYALN FGKGTSLLVT P;
b)
(SEQ ID NO: 3)
AKEVEQNSGP LSVPEGAIAS LNCTYSSWEG QSFFWYRQYS
GKSPELIMFL YADPDKEDGR FTAQLNKASQ YVSLLIRDSQ
PSDSATYLCA VRTNAGYALN FGKGTSLLVT P;
and
c)
(SEQ ID NO: 4)
AKEVEQNSGP LSVPEGAIAS LNCTYSSWEG QSFFWYRQYS
GKSPELIMKL YADPDKEDGR FTAQLNKASQ YVSLLIRDSQ
PSDSATYLCA VRTNAGYALN FGKGTSLLVT P;
optionally with an N-terminal methionine (SEQ ID NOs: 41-43), and
the beta chain variable domain comprises the amino acid sequence:
(SEQ ID NO: 5)
DAGVTQSPTH LIKTRGQQVT LRCSPKSGHD TVSWYQQALG
QGPQFIFQAV RGVERQRGNF PDRFSGHQFP NYSSELNVNA
LLLGDSALYL CASSDTVSYE QYFGPGTRLT VT,
optionally with an N-terminal methionine (SEQ ID NO: 44).
2. The specific binding molecule as claimed in claim 1 , wherein the alpha chain variable domain comprises the amino acid sequence:
(SEQ ID NO: 2)
AKEVEQNSGP LSVPEGAIAS LNCTYSSWEG QSFFWYRQYS
GKSPELIMKL YADPDKEDGR FTAQLNKASQ YVSLLIRDSQ
PSDSATYLCA VRTNSGYALN FGKGTSLLVT P.
3. The specific binding molecule as claimed in claim 1 , wherein the alpha chain variable domain comprises the amino acid sequence:
(SEQ ID NO: 3)
AKEVEQNSGP LSVPEGAIAS LNCTYSSWEG QSFFWYRQYS
GKSPELIMFL YADPDKEDGR FTAQLNKASQ YVSLLIRDSQ
PSDSATYLCA VRTNAGYALN FGKGTSLLVT P.
4. The specific binding molecule as claimed in claim 1 , wherein the alpha chain variable domain comprises the amino acid sequence:
(SEQ ID NO: 4)
AKEVEQNSGP LSVPEGAIAS LNCTYSSWEG QSFFWYRQYS
GKSPELIMKL YADPDKEDGR FTAQLNKASQ YVSLLIRDSQ
PSDSATYLCA VRTNAGYALN FGKGTSLLVT P.
5. The specific binding molecule as claimed in claim 1 , which is an alpha-beta heterodimer, having an alpha chain TRAC constant domain and a beta chain TRBC1 or TRBC2 constant domain.
6. The specific binding molecule as claimed in claim 5 , wherein the alpha and beta chain constant domain amino acid 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 TRBC1 or TRBC2.
7. The specific binding molecule as claimed in claim 6 , wherein the alpha and beta chain constant domain amino acid 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.
8. The specific binding molecule as claimed in claim 1 , which is in single chain format of the type Vα-L-Vβ, Vβ-L-Vα, Vα-Cα-L-Vβ, Vα-L-Vβ-Cβ, Vα-Cα-L-Vβ-CB or Vβ-Cβ-L-Vα-Cα wherein Vα and Vβ are TCR alpha and beta variable regions respectively, Cα and Cβ are TCR α and β constant regions respectively, and L is a linker sequence.
9. The specific binding molecule as claimed in claim 1 , which is associated with a detectable label, a therapeutic agent, or a PK modifying moiety.
10. The specific binding molecule as claimed in claim 9 , which is associated with an anti-CD3 antibody covalently linked to the C- or N-terminus of the TCR alpha or beta chain.
11. The specific binding molecule as claimed in claim 10 , wherein the anti-CD3 antibody is covalently linked to the C- or N-terminus of the TCR beta chain via a linker sequence.
12. The specific binding molecule as claimed in claim 11 , wherein the linker sequence is selected from the group consisting of GGGGS (SEQ ID NO: 16), GGGSG (SEQ ID NO: 17), GGSGG (SEQ ID NO: 18), GSGGG (SEQ ID NO: 19), GSGGGP (SEQ ID NO: 20), GGEPS (SEQ ID NO: 21), GGEGGGP (SEQ ID NO: 22), and GGEGGGSEGGGS (SEQ ID NO: 23).
13. The specific binding molecule as claimed in claim 12 , wherein the beta chain comprises the amino acid sequence:
(SEQ ID NO: 30)
AIQMTQSPS SLSASVGDRV TITCRASQDI RNYLNWYQQK
PGKAPKLLIY YTSRLESGVP SRFSGSGSGT DYTLTISSLQ
PEDFATYYCQ QGNTLPWTFG QGTKVEIKGG GGSGGGGSGG
GGSGGGGSGG GSEVQLVESG GGLVQPGGSL RLSCAASGYS
FTGYAMNWVR QAPGKGLEWV ALINPYKGVS TYNQKFKDRF
TFSVDKSKNT AYLQMNSLRA EDTAVYYCAR SGYYGDSDWY
FDVWGQGTLV TVSSGGGGSD AGVTQSPTHL IKTRGQQVTL
RCSPKSGHDT VSWYQQALGQ GPQFIFQAVR GVERQRGNFP
DRFSGHQFPN YSSELNVNAL LLGDSALYLC ASSDTVSYEQ
YFGPGTRLTV TEDLKNVFPP EVAVFEPSEA EISHTQKATL
VCLATGFYPD HVELSWWVNG KEVHSGVCTD PQPLKEQPAL
NDSRYALSSR LRVSATFWQD PRNHFRCQVQ FYGLSENDEW
TQDRAKPVTQ IVSAEAWGRA D.
14. The specific binding molecule as claimed in claim 12 , wherein the beta chain comprises the amino acid sequence:
(SEQ ID NO: 31)
AIQMTQSPS SLSASVGDRV TITCRASQDI RNYLNWYQQK
PGKAPKLLIY YTSRLESGVP SRFSGSGSGT DYTLTISSLQ
PEDFATYYCQ QGNTLPWTFG QGTKVEIKGG GGSGGGGSGG
GGSGGGGSGG GSEVQLVESG GGLVQPGGSL RLSCAASGYS
FTGYTMNWVR QAPGKGLEWV ALINPYKGVS TYNQKFKDRF
TISVDKSKNT AYLQMNSLRA EDTAVYYCAR SGYYGDSDWY
FDVWGQGTLV TVSSGGGGSD AGVTQSPTHL IKTRGQQVTL
RCSPKSGHDT VSWYQQALGQ GPQFIFQAVR GVERQRGNFP
DRFSGHQFPN YSSELNVNAL LLGDSALYLC ASSDTVSYEQ
YFGPGTRLTV TEDLKNVFPP EVAVFEPSEA EISHTQKATL
VCLATGFYPD HVELSWWVNG KEVHSGVCTD PQPLKEQPAL
NDSRYALSSR LRVSATFWQD PRNHFRCQVQ FYGLSENDEW
TQDRAKPVTQ IVSAEAWGRA D.
15. The specific binding molecule as claimed in claim 13 , wherein the alpha chain comprises the amino acid sequence:
(SEQ ID NO: 32)
AKEVEQNSGP LSVPEGAIAS LNCTYSSWEG QSFFWYRQYS
GKSPELIMKL YADPDKEDGR FTAQLNKASQ YVSLLIRDSQ
PSDSATYLCA VRTNAGYALN FGKGTSLLVT PHIQKPDPAV
YQLRDSKSSD KSVCLFTDFD SQTNVSQSKD SDVYITDKCV
LDMRSMDFKS NSAVAWSNKS DFACANAFNN SIIPEDT.
16. A pharmaceutical composition comprising a specific binding molecule having the property of binding to SLYNTVATL (SEQ ID NO: 1) in complex with HLA-A*02 and comprising a TCR alpha chain variable domain and a TCR beta chain variable domain,
wherein the alpha chain variable domain amino acid sequence comprises an amino acid sequence selected from:
a)
(SEQ ID NO: 2)
AKEVEQNSGP LSVPEGAIAS LNCTYSSWEG QSFFWYRQYS
GKSPELIMKL YADPDKEDGR FTAQLNKASQ YVSLLIRDSQ
PSDSATYLCA VRTNSGYALN FGKGTSLLVT P;
b)
(SEQ ID NO: 3)
AKEVEQNSGP LSVPEGAIAS LNCTYSSWEG QSFFWYRQYS
GKSPELIMFL YADPDKEDGR FTAQLNKASQ YVSLLIRDSQ
PSDSATYLCA VRTNAGYALN FGKGTSLLVT P;
and
c)
(SEQ ID NO: 4)
AKEVEQNSGP LSVPEGAIAS LNCTYSSWEG QSFFWYRQYS
GKSPELIMKL YADPDKEDGR FTAQLNKASQ YVSLLIRDSQ
PSDSATYLCA VRTNAGYALN FGKGTSLLVT P;
optionally with an N-terminal methionine (SEQ ID NOs: 41-43), and
the beta chain variable domain comprises the amino acid sequence:
(SEQ ID NO: 5)
DAGVTQSPTH LIKTRGQQVT LRCSPKSGHD TVSWYQQALG
QGPQFIFQAV RGVERQRGNF PDRFSGHQFP NYSSELNVNA
LLLGDSALYL CASSDTVSYE QYFGPGTRLT VT,
optionally with an N-terminal methionine (SEQ ID NO: 44), together with one or more pharmaceutically acceptable carriers or excipients.