T cell receptors
The present invention relates to T cell receptors (TCRs) that bind the HLA-A*02 restricted peptide SLLQHLIGL (SEQ ID NO: 1) derived from the germline cancer antigen PRAME. Said TCRs may comprise non-natural mutations within the alpha and/or beta variable domains relative to a native PRAME TCR. The TCRs of the invention are particularly suitable for use as novel immunotherapeutic reagents for the treatment of malignant disease.
1. A soluble T cell receptor (TCR), comprising: a TCR alpha chain variable domain and a TCR beta chain variable domain, wherein each variable domain comprises FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 where FR is a framework region and CDR is a complementarity determining region, wherein the soluble TCR has one of the following combinations of alpha chain and beta chain CDRs:
Alpha
CDR1
CDR2
CDR3
1
TISGTDY
GLTSN
CILILGHSRAGNYIATF
(SEQ ID
(SEQ ID NO: 40)
(SEQ ID NO: 45)
NO: 39)
2
TISGTDY
GLTSN
CILILGHSRAGNYIATF
(SEQ ID
(SEQ ID NO: 40)
(SEQ ID NO: 45)
NO: 39)
3
TISGTDY
GLTSN
CILILGHSRAGNYIATF
(SEQ ID
(SEQ ID NO: 40)
(SEQ ID NO: 45)
NO: 39)
4
TISGTDY
GLTSN
CILILGHSRAGNYIATF
(SEQ ID
(SEQ ID NO: 40)
(SEQ ID NO: 45)
NO: 39)
5
TISGTDY
GLTSN
CILILGHSRAGNYIATF
(SEQ ID
(SEQ ID NO: 40)
(SEQ ID NO: 45)
NO: 39)
6
TISGTDY
GLTSN
CILILGHSRAGNYIATF
(SEQ ID
(SEQ ID NO: 40)
(SEQ ID NO: 45)
NO: 39)
7
TISGTDY
GLTSN
CILILGHSRLGNYIATF
(SEQ ID
(SEQ ID NO: 40)
(SEQ ID NO: 46)
NO: 39)
8
TISGTDY
GLTSN
CILILGHSRLGNYQATF
(SEQ ID
(SEQ ID NO: 40)
(SEQ ID NO: 47)
NO: 39)
9
TISGTDY
GLTSN
CILILGHSRLGNYIATF
(SEQ ID
(SEQ ID NO: 40)
(SEQ ID NO: 46)
NO: 39)
10
TISGTDY
GLTSN
CILILGHSRLGNYIATF
(SEQ ID
(SEQ ID NO: 40)
(SEQ ID NO: 46)
NO: 39)
11
TISGTDY
GLTSN
CILILGHSRLGNYIATF
(SEQ ID
(SEQ ID NO: 40)
(SEQ ID NO: 46)
NO: 39)
12
TISGTDY
GLTSN
CILILGHSRLGNYIATF
(SEQ ID
(SEQ ID NO: 40)
(SEQ ID NO: 46)
NO: 39)
13
TISGTDY
GLTSN
CILILGHSRLGNYIATF
(SEQ ID
(SEQ ID NO: 40)
(SEQ ID NO: 46)
NO: 39)
14
TISGTDY
GLTSN
CILILGHSRLGNYIATF
(SEQ ID
(SEQ ID NO: 40)
(SEQ ID NO: 46)
NO: 39)
15
TISGTDY
GLTSN
CILILGHSRLGNYIATF
(SEQ ID
(SEQ ID NO: 40)
(SEQ ID NO: 46)
NO: 39)
16
TISGTDY
GLTSN
CILILGHSRLGNYIATF
(SEQ ID
(SEQ ID NO: 40)
(SEQ ID NO: 46)
NO: 39)
17
TISGTDY
GLTSN
CILILGHSRLGNYIATF
(SEQ ID
(SEQ ID NO: 40)
(SEQ ID NO: 46)
NO: 39)
18
TISGTDY
GLTSN
CILILGHSRLGNYIATF
(SEQ ID
(SEQ ID NO: 40)
(SEQ ID NO: 46)
NO: 39)
19
TISGTDY
GLTSN
CILILGHSRLGNYIATF
(SEQ ID
(SEQ ID NO: 40)
(SEQ ID NO: 46)
NO: 39)
20
TISGTDY
GLTSN
CILILGHSRLGNYIATF
(SEQ ID
(SEQ ID NO: 40)
(SEQ ID NO: 46)
NO: 39)
Beta
CDR1
CDR2
CDR3
1
LNHDA
SQIMGDE
CASSWWTGGASPISF
(SEQ ID
(SEQ ID NO:
(SEQ ID NO: 51)
NO: 42)
48)
2
LNHDA
SQIMGDE
CASSWWTGGASEISF
(SEQ ID
(SEQ ID NO:
(SEQ ID NO: 60)
NO: 42)
48)
3
LNHDA
SQIMGDE
CASSWWTGGASPIRF
(SEQ ID
(SEQ ID NO:
(SEQ ID NO: 58)
NO: 42)
48)
4
LNHDA
SQIMGDE
CASSWWTGGSAPISF
(SEQ ID
(SEQ ID NO:
(SEQ ID NO: 61)
NO: 42)
48)
5
LNHDA
SQIMGDE
CASSWWTSGASPISF
(SEQ ID
(SEQ ID NO:
(SEQ ID NO: 52)
NO: 42)
48)
6
LNHDA
SQIMGDE
CASSWWTGGSSPISF
(SEQ ID
(SEQ ID NO:
(SEQ ID NO: 62)
NO: 42)
48)
7
LNHDA
SQIMGDE
CASSWWTGGSAPIRF
(SEQ ID
(SEQ ID NO:
(SEQ ID NO: 53)
NO: 42)
48)
8
LNHDA
SQIMGDE
CASSWWTGGSAPIRF
(SEQ ID
(SEQ ID NO:
(SEQ ID NO: 53)
NO: 42)
48)
9
LNHDA
SQIVGDE
CASSWWTGGSAPIRF
(SEQ ID
(SEQ ID NO:
(SEQ ID NO: 53)
NO: 42)
50)
10
LNHDA
SQIMNDE
CASSWWTGGSAPIRF
(SEQ ID
(SEQ ID NO:
(SEQ ID NO: 53)
NO: 42)
49)
11
LNHDA
SQIMGDE
CASSPWTGGSAPIRF
(SEQ ID
(SEQ ID NO:
(SEQ ID NO: 63)
NO: 42)
48)
12
LNHDA
SQIMGDE
CASSWWTSGSAPIRF
(SEQ ID
(SEQ ID NO:
(SEQ ID NO: 54)
NO: 42)
48)
13
LNHDA
SQIMGDE
CASSWWTGGSAEIRF
(SEQ ID
(SEQ ID NO:
(SEQ ID NO: 55)
NO: 42)
48)
14
LNHDA
SQIMGDE
CASSWWTGGSAPIYF
(SEQ ID
(SEQ ID NO:
(SEQ ID NO: 56)
NO: 42)
48)
15
LNHDA
SQIMGDE
CASSWWTGGSSPISF
(SEQ ID
(SEQ ID NO:
(SEQ ID NO: 62)
NO: 42)
48)
16
LNHDA
SQIMGDE
CASSWWTGGAAPISF
(SEQ ID
(SEQ ID NO:
(SEQ ID NO: 57)
NO: 42)
48)
17
LNHDA
SQIMGDE
CASSWWTGGASPIRF
(SEQ ID
(SEQ ID NO:
(SEQ ID NO: 58)
NO: 42)
48)
18
LNHDA
SQIMGDE
CASSWWTGGSAPISF
(SEQ ID
(SEQ ID NO:
(SEQ ID NO: 61)
NO: 42)
48)
19
LNHDA
SQIMGDE
CASSWWTGGSSPIRF
(SEQ ID
(SEQ ID NO:
(SEQ ID NO: 64)
NO: 42)
48)
20
LNHDA
SQIMGDE
CASSWWTGGAAPIRF
(SEQ ID
(SEQ ID NO:
(SEQ ID NO:
NO: 42)
48)
59).
and wherein the soluble TCR has the property of binding to SLLQHLIGL (SEQ ID NO: 1) HLA-A*02 complex.
2. The TCR of claim 1 , wherein the alpha chain variable domain framework regions comprise the following sequences:
FR1—amino acids 1-25 of SEQ ID NO: 2
FR2—amino acids 33-49 of SEQ ID NO: 2
FR3—amino acids 55-87 of SEQ ID NO: 2
FR4—amino acids 105-114 of SEQ ID NO: 2
or respective sequences having at least 90% identity to said sequences, and/or the beta chain variable domain framework regions comprise the following sequences:
FR1—amino acids 1-26 of SEQ ID NO: 3
FR2—amino acids 32-48 of SEQ ID NO: 3
FR3—amino acids 56-90 of SEQ ID NO: 3
FR4—amino acids 106-114 of SEQ ID NO: 3
or respective sequences having at least 90% identity to said sequences.
3. The TCR of claim 1 , wherein the alpha chain variable region FR1 has a G residue at position −1 using the numbering of SEQ ID NO: 2.
4. The TCR of claim 1 , wherein the alpha chain variable domain and the beta chain variable domain are selected from one of the following:
(a) the alpha chain variable domain of SEQ ID NO: 6 and the beta chain variable domain of SEQ ID NO: 9;
(b) the alpha chain variable domain of SEQ ID NO: 6 and the beta chain variable domain of SEQ ID NO: 19;
(c) the alpha chain variable domain of SEQ ID NO: 6 and the beta chain variable domain of SEQ ID NO: 17;
(d) the alpha chain variable domain of SEQ ID NO: 6 and the beta chain variable domain of SEQ ID NO: 20;
(e) the alpha chain variable domain of SEQ ID NO: 6 and the beta chain variable domain of SEQ ID NO: 10;
(f) the alpha chain variable domain of SEQ ID NO: 6 and the beta chain variable domain of SEQ ID NO: 21;
(g) the alpha chain variable domain of SEQ ID NO: 7 and the beta chain variable domain of SEQ ID NO: 11;
(h) the alpha chain variable domain of SEQ ID NO: 8 and the beta chain variable domain of SEQ ID NO: 11;
(i) the alpha chain variable domain of SEQ ID NO: 7 and the beta chain variable domain of SEQ ID NO: 22;
(j) the alpha chain variable domain of SEQ ID NO: 7 and the beta chain variable domain of SEQ ID NO: 12;
(k) the alpha chain variable domain of SEQ ID NO: 7 and the beta chain variable domain of SEQ ID NO: 23;
(l) the alpha chain variable domain of SEQ ID NO: 7 and the beta chain variable domain of SEQ ID NO: 13;
(m) the alpha chain variable domain of SEQ ID NO: 7 and the beta chain variable domain of SEQ ID NO: 14;
(n) the alpha chain variable domain of SEQ ID NO: 7 and the beta chain variable domain of SEQ ID NO: 15;
(o) the alpha chain variable domain of SEQ ID NO: 7 and the beta chain variable domain of SEQ ID NO: 21;
(p) the alpha chain variable domain of SEQ ID NO: 7 and the beta chain variable domain of SEQ ID NO: 16;
(q) the alpha chain variable domain of SEQ ID NO: 7 and the beta chain variable domain of SEQ ID NO: 17;
(r) the alpha chain variable domain of SEQ ID NO: 7 and the beta chain variable domain of SEQ ID NO: 20;
(s) the alpha chain variable domain of SEQ ID NO: 7 and the beta chain variable domain of SEQ ID NO: 24; and
(t) the alpha chain variable domain of SEQ ID NO: 7 and the beta chain variable domain of SEQ ID NO: 18.
5. The TCR of claim 4 , wherein the TCR is an alpha-beta heterodimer further comprising an alpha chain TRAC constant domain sequence and a beta chain TRBC1 or TRBC2 constant domain sequence.
6. The TCR of claim 5 , wherein the alpha and beta chain constant domain sequences are modified by truncation or substitution to delete a native disulfide bond between Cys4 of exon 2 of TRAC and Cys2 of exon 2 of TRBC1 or TRBC2.
7. The TCR of claim 5 , wherein the alpha and/or 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 non-native disulfide bond between the alpha and beta constant domains of the TCR.
8. The TCR of claim 1 , wherein the TCR has a single chain format of the type Vα-L-Vβ, Vβ-L-Vα, Vα-Cα-L-Vβ, or Vα-L-Vβ-Cβ, wherein Vα and Vβ are TCR α and β variable regions respectively, Cα and Cβ are TCR α and β constant regions respectively, and L is a linker sequence.
9. A cell containing
(a) an expression vector encoding the TCR alpha chain variable domain and TCR beta chain variable domain of claim 1 , in a single open reading frame, or two distinct open reading frames; or
(b) a first expression vector which comprises nucleic acid encoding the alpha chain variable domain of a TCR of claim 1 , and a second expression vector which comprises nucleic acid encoding the beta chain variable domain of a TCR of claim 1 .
10. A non-naturally occurring cell presenting a TCR as claimed in claim 1 .
11. A pharmaceutical composition comprising the soluble TCR of claim 1 , together with one or more pharmaceutically acceptable carriers or excipients.
12. The pharmaceutical composition of claim 11 , wherein the pharmaceutical composition is formulated for administration to a human subject by injection.
13. A method of producing the souble TCR of claim 1 , comprising:
a) maintaining a cell containing
(i) an expression vector which comprises nucleic acid that encodes a TCR having one of the combinations of alpha chain and beta chain CDRs of claim 1 , in a single open reading frame, or two distinct open reading frames; or
(ii) a first expression vector which comprises nucleic acid that encodes a TCR alpha chain, and a second expression vector which comprises nucleic acid that encodes a TCR beta chain under optimal conditions for expression of the TCR alpha chain and TCR beta chain, wherein the TCR has one of the combinations of alpha and beta chain CDRs of claim 1 ; and
b) isolating the TCR alpha chain and the TCR beta chain.
14. A nucleic acid encoding
(a) a TCR alpha chain variable domain comprising complementarity determining regions (CDRs) having the following amino acid sequences:
(SEQ ID NO: 39)
CDR1 - TISGTDY
(SEQ ID NO: 40)
CDR2 - GLTSN
(SEQ ID NO: 41)
CDR3 - CILILGHSGAGSYQLTF
(b) a TCR beta chain variable domain comprising CDRs having the following amino acid sequences:
(SEQ ID NO: 42)
CDR1 - LNHDA
(SEQ ID NO: 43)
CDR2 - SQIVNDF
(SEQ ID NO: 44)
CDR3 - CASSPWTSGSREQYF.
15. An expression vector comprising the nucleic acid of claim 14 .
16. A nucleic acid encoding a TCR which has one of the following combinations of alpha and beta chain CDRs:
Alpha
CDR1
CDR2
CDR3
1
TISGTDY
GLTSN
CILILGHSRAGNYIATF
(SEQ ID
(SEQ ID NO: 40)
(SEQ ID NO: 45)
NO: 39)
2
TISGTDY
GLTSN
CILILGHSRAGNYIATF
(SEQ ID
(SEQ ID NO: 40)
(SEQ ID NO: 45)
NO: 39)
3
TISGTDY
GLTSN
CILILGHSRAGNYIATF
(SEQ ID
(SEQ ID NO: 40)
(SEQ ID NO: 45)
NO: 39)
4
TISGTDY
GLTSN
CILILGHSRAGNYIATF
(SEQ ID
(SEQ ID NO: 40)
(SEQ ID NO: 45)
NO: 39)
5
TISGTDY
GLTSN
CILILGHSRAGNYIATF
(SEQ ID
(SEQ ID NO: 40)
(SEQ ID NO: 45)
NO: 39)
6
TISGTDY
GLTSN
CILILGHSRAGNYIATF
(SEQ ID
(SEQ ID NO: 40)
(SEQ ID NO: 45)
NO: 39)
7
TISGTDY
GLTSN
CILILGHSRLGNYIATF
(SEQ ID
(SEQ ID NO: 40)
(SEQ ID NO: 46)
NO: 39)
8
TISGTDY
GLTSN
CILILGHSRLGNYQATF
(SEQ ID
(SEQ ID NO: 40)
(SEQ ID NO: 47)
NO: 39)
9
TISGTDY
GLTSN
CILILGHSRLGNYIATF
(SEQ ID
(SEQ ID NO: 40)
(SEQ ID NO: 46)
NO: 39)
10
TISGTDY
GLTSN
CILILGHSRLGNYIATF
(SEQ ID
(SEQ ID NO: 40)
(SEQ ID NO: 46)
NO: 39)
11
TISGTDY
GLTSN
CILILGHSRLGNYIATF
(SEQ ID
(SEQ ID NO: 40)
(SEQ ID NO: 46)
NO: 39)
12
TISGTDY
GLTSN
CILILGHSRLGNYIATF
(SEQ ID
(SEQ ID NO: 40)
(SEQ ID NO: 46)
NO: 39)
13
TISGTDY
GLTSN
CILILGHSRLGNYIATF
(SEQ ID
(SEQ ID NO: 40)
(SEQ ID NO: 46)
NO: 39)
14
TISGTDY
GLTSN
CILILGHSRLGNYIATF
(SEQ ID
(SEQ ID NO: 40)
(SEQ ID NO: 46)
NO: 39)
15
TISGTDY
GLTSN
CILILGHSRLGNYIATF
(SEQ ID
(SEQ ID NO: 40)
(SEQ ID NO: 46)
NO: 39)
16
TISGTDY
GLTSN
CILILGHSRLGNYIATF
(SEQ ID
(SEQ ID NO: 40)
(SEQ ID NO: 46)
NO: 39)
17
TISGTDY
GLTSN
CILILGHSRLGNYIATF
(SEQ ID
(SEQ ID NO: 40)
(SEQ ID NO: 46)
NO: 39)
18
TISGTDY
GLTSN
CILILGHSRLGNYIATF
(SEQ ID
(SEQ ID NO: 40)
(SEQ ID NO: 46)
NO: 39)
19
TISGTDY
GLTSN
CILILGHSRLGNYIATF
(SEQ ID
(SEQ ID NO: 40)
(SEQ ID NO: 46)
NO: 39)
20
TISGTDY
GLTSN
CILILGHSRLGNYIATF
(SEQ ID
(SEQ ID NO: 40)
(SEQ ID NO: 46)
NO: 39)
Beta
CDR1
CDR2
CDR3
1
LNHDA
SQIMGDE
CASSWWTGGASPISF
(SEQ ID
(SEQ ID NO:
(SEQ ID NO: 51)
NO: 42)
48)
2
LNHDA
SQIMGDE
CASSWWTGGASEISF
(SEQ ID
(SEQ ID NO:
(SEQ ID NO: 60)
NO: 42)
48)
3
LNHDA
SQIMGDE
CASSWWTGGASPIRF
(SEQ ID
(SEQ ID NO:
(SEQ ID NO: 58)
NO: 42)
48)
4
LNHDA
SQIMGDE
CASSWWTGGSAPISF
(SEQ ID
(SEQ ID NO:
(SEQ ID NO: 61)
NO: 42)
48)
5
LNHDA
SQIMGDE
CASSWWTSGASPISF
(SEQ ID
(SEQ ID NO:
(SEQ ID NO: 52)
NO: 42)
48)
6
LNHDA
SQIMGDE
CASSWWTGGSSPISF
(SEQ ID
(SEQ ID NO:
(SEQ ID NO: 62)
NO: 42)
48)
7
LNHDA
SQIMGDE
CASSWWTGGSAPIRF
(SEQ ID
(SEQ ID NO:
(SEQ ID NO: 53)
NO: 42)
48)
8
LNHDA
SQIMGDE
CASSWWTGGSAPIRF
(SEQ ID
(SEQ ID NO:
(SEQ ID NO: 53)
NO: 42)
48)
9
LNHDA
SQIVGDE
CASSWWTGGSAPIRF
(SEQ ID
(SEQ ID NO:
(SEQ ID NO: 53)
NO: 42)
50)
10
LNHDA
SQIMNDE
CASSWWTGGSAPIRF
(SEQ ID
(SEQ ID NO:
(SEQ ID NO: 53)
NO: 42)
49)
11
LNHDA
SQIMGDE
CASSPWTGGSAPIRF
(SEQ ID
(SEQ ID NO:
(SEQ ID NO: 63)
NO: 42)
48)
12
LNHDA
SQIMGDE
CASSWWTSGSAPIRF
(SEQ ID
(SEQ ID NO:
(SEQ ID NO: 54)
NO: 42)
48)
13
LNHDA
SQIMGDE
CASSWWTGGSAEIRF
(SEQ ID
(SEQ ID NO:
(SEQ ID NO: 55)
NO: 42)
48)
14
LNHDA
SQIMGDE
CASSWWTGGSAPIYF
(SEQ ID
(SEQ ID NO:
(SEQ ID NO: 56)
NO: 42)
48)
15
LNHDA
SQIMGDE
CASSWWTGGSSPISF
(SEQ ID
(SEQ ID NO:
(SEQ ID NO: 62)
NO: 42)
48)
16
LNHDA
SQIMGDE
CASSWWTGGAAPISF
(SEQ ID
(SEQ ID NO:
(SEQ ID NO: 57)
NO: 42)
48)
17
LNHDA
SQIMGDE
CASSWWTGGASPIRF
(SEQ ID
(SEQ ID NO:
(SEQ ID NO: 58)
NO: 42)
48)
18
LNHDA
SQIMGDE
CASSWWTGGSAPISF
(SEQ ID
(SEQ ID NO:
(SEQ ID NO: 61)
NO: 42)
48)
19
LNHDA
SQIMGDE
CASSWWTGGSSPIRF
(SEQ ID
(SEQ ID NO:
(SEQ ID NO: 64)
NO: 42)
48)
20
LNHDA
SQIMGDE
CASSWWTGGAAPIRF
(SEQ ID
(SEQ ID NO:
(SEQ ID NO:
NO: 42)
48)
59).
17. An expression vector comprising the nucleic acid of claim 16 .