# US 2022/0033458 A1 · High affinity cell receptor for recognizing afp antigen

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- Number: US 2022/0033458 A1
- Document ID: US20220033458A1
- Status: Published application
- Kind: A1
- Application number: 17/278,504
- Filed: 2019-09-19
- Published: 2022-02-03
- Earliest claimed: 2018-09-21
- Assignees: Xlifesc, Ltd.
- Applicants: Xlifesc, Ltd.
- Inventors: Yi LI; Xiaolin LI; Shaopei CHEN
- Web page: https://patentgrep.com/patent/US20220033458A1

## CPC

- C07K 14/7051: {T-cell receptor (TcR)-CD3 complex}
- A61P 35/00: Antineoplastic agents
- C07K 16/2809: {against the T-cell receptor (TcR)-CD3 complex}
- A61K 38/00: Medicinal preparations containing peptides (peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00)
- C07K 2317/622: Single chain antibody (scFv)
- C07K 2319/00: Fusion polypeptide
- C07K 2319/03: containing a transmembrane segment
- C07K 2319/33: fusions for targeting to specific cell types, e.g. tissue specific targeting, targeting of a bacterial subspecies
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D)

## Abstract

Provided in the present invention is a T cell receptor (TCR) having the property of binding to a FMNKFIYEI-HLA A0201 complex; and the binding affinity of the TCR to the FMNKFIYEI-HLA A0201 complex is at least 2 times the binding affinity of the wild-type TCR to the FMNKFIYEI-HLA A0201 complex. Also provided in the present invention is a fusion molecule of such TCRs with therapeutic agents. Such TCRs can be used alone or in combination with therapeutic agents, so as to target tumor cells presenting the FMNKFIYEI-HLA A0201 complex.

## Claims

### Claim 1

A T cell receptor (TCR) having an activity of binding to FMNKFIYEI-HLA A0201 complex, and the T cell receptor comprises a TCR α chain variable domain and a TCR β chain variable domain, the TCR α chain variable domain comprises three CDR regions, and the reference sequences of the three CDR regions of the TCR α chain variable domain are listed as follows,
CDR1α: TSINN
CDR2α: IRSNERE
CDR3α: ATDPRTGANSKLT,
the TCR β chain variable domain comprises three CDR regions, and the reference sequences of the three CDR regions of the TCR β chain variable domain are listed as follows,
CDR1β: SEHNR
CDR2β: FQNEAQ
CDR3β: ASEGLAYEQY,
wherein the TCR α chain variable domain contains at least one of the following mutations:
Residue before mutation
Residue after mutation
S at position 2 of CDR1α
D or P or A or E
I at position 3 of CDR1α
V
N at position 4 of CDR1α
S or Y or D
N at position 5 of CDR1α
S or K
R at position 5 of CDR3α
A or S or V or D or E or Q or T
T at position 6 of CDR3α
M or R or L or A or N or S
G at position 7 of CDR3α
A or Q or S or E or N
A at position 8 of CDR3α
S or G or P or W
N at position 9 of CDR3α
S or T
S at position 10 of CDR3α
A or N or T or P
and/or, the TCR β chain variable contains at least one of the following mutations:
Residue before mutation
Residue after mutation
S at position 1 of CDR1β
A or Q or G
E at position 2 of CDR1β
G or T or R or L
H at position 3 of CDR1β
W or R
N at position 4 of CDR1β
A or P
Q at position 2 of CDR2β
H
N at position 3 of CDR2β
G
E at position 4 of CDR2β
D
A at position 5 of CDR2β
M or L
Q at position 6 of CDR2β
E.
and the α chain variable domain of the TCR comprises an amino acid sequence having at least 90% sequence homology with the amino acid sequence shown in SEQ ID NO: 1; and/or the β chain variable domain of the TCR comprises an amino acid sequence having at least 90% sequence homology with the amino acid sequence shown in SEQ ID NO: 2.

### Claim 2 (depends on 1)

The TCR of claim 1, wherein there are 3-8 mutations in the 3 CDR regions of the TCR α chain variable domain and/or 4-8 mutations in the 3 CDR regions of the TCR β chain variable domain; or
the affinity of the TCR for FMNKFIYEI-HLA A0201 complex is at least 2 times of that of the wild type TCR; or;
the mutated position in the α chain variable domain of the TCR includes position 7 and/or position 8 of CDR3α.

### Claim at position 3

3-5. (canceled)

### Claim 6 (depends on 1)

The TCR of claim 1, wherein the mutation in the α chain variable domain of the TCR includes the following group:
Residue before mutation
Residue after mutation
G at position 7 of CDR3α
S
A at position 8 of CDR3α
P
and/or the mutation in the β chain variable domain of the TCR includes the following group:
Residue before mutation
Residue after mutation
N at position 3 of CDR2β
G
E at position 4 of CDR2β
D
Q at position 6 of CDR2β
E.

### Claim 7 (depends on 1)

The TCR of claim 1, wherein the TCR comprises a TCR α chain variable domain and a TCR β chain variable domain, and said TCR α chain variable domain comprises CDR1α, CDR2α and CDR3α, wherein the amino acid sequence of CDR1α is selected from the group consisting of: TSINN, TPISS, TDIYK, TAVDS, TEISS and TDVSS; and/or the amino acid sequence of CDR2α is IRSNERE; and/or the CDR3α comprises a sequence selected from the group consisting of: ATDPSMSPNSKLT, ATDPSLSPNSKLT and ATDPSLQSNSKLT.

### Claim at position 6

8-14. (canceled)

### Claim 15 (depends on 1)

The TCR of claim 1, wherein the TCR comprises a TCR α chain variable domain and a TCR β chain variable domain, and said TCR β chain variable domain comprises CDR1I, CDR20 and CDR30, wherein the CDR2β comprises a sequence selected from the group consisting of: FQGDME, FQGDLE and FHGDLE; and/or the amino acid sequence of CDR3p is ASEGLAYEQY.

### Claim 16

(canceled)

### Claim 17 (depends on 1)

The TCR of claim 1, wherein the TCR has CDRs selected from the group consisting of:
CDR No.
CDR1α
CDR2α
CDR3α
CDR1β
CDR2β
CDR3β
1
TSINN
IRSNERE
ATDPSRQ
SEHNR
FQNEAQ
ASEGLA
SNSKLT
YEQY
2
TSINN
IRSNERE
ATDPRTG
SEHNR
FQNEAQ
ASEGLA
SSNKLT
YEQY
3
TSINN
IRSNERE
ATDPRTA
SEHNR
FQNEAQ
ASEGLA
SSAKLT
YEQY
4
TSINN
IRSNERE
ATDPAMQ
SEHNR
FQNEAQ
ASEGLA
ANSKLT
YEQY
5
TSINN
IRSNERE
ATDPVLS
SEHNR
FQNEAQ
ASEGLA
ANSKLT
YEQY
6
TSINN
IRSNERE
ATDPRTS
SEHNR
FQNEAQ
ASEGLA
GSTKLT
YEQY
7
TSINN
IRSNERE
ATDPDRS
SEHNR
FQNEAQ
ASEGLA
PNSKLT
YEQY
8
TSINN
IRSNERE
ATDPRTA
SEHNR
FQNEAQ
ASEGLA
STPKLT
YEQY
9
TSINN
IRSNERE
ATDPEAQ
SEHNR
FQNEAQ
ASEGLA
WNSKLT
YEQY
10
TSINN
IRSNERE
ATDPRTE
SEHNR
FQNEAQ
ASEGLA
STTKLT
YEQY
11
TSINN
IRSNERE
ATDPSLQ
SEHNR
FQNEAQ
ASEGLA
SNSKLT
YEQY
12
TSINN
IRSNERE
ATDPQNS
SEHNR
FQNEAQ
ASEGLA
WNSKLT
YEQY
13
TSINN
IRSNERE
ATDPASN
SEHNR
FQNEAQ
ASEGLA
WNSKLT
YEQY
14
TSINN
IRSNERE
ATDPRTG
SEHNR
FQGDME
ASEGLA
ANSKLT
YEQY
15
TDVSS
IRSNERE
ATDPAAQ
AGHAR
FQGDME
ASEGLA
WNSKLT
YEQY
16
TPISS
IRSNERE
ATDPAAQ
QGHAR
FQGDME
ASEGLA
WNSKLT
YEQY
17
TPISS
IRSNERE
ATDPATS
GTWPR
FQGDME
ASEGLA
PNSKLT
YEQY
18
TDIYK
IRSNERE
ATDPRTA
GTWPR
FQGDME
ASEGLA
SSPKLT
YEQY
19
TAVDS
IRSNERE
ATDPSLS
GTWPR
FQGDME
ASEGLA
PNSKLT
YEQY
20
TAVDS
IRSNERE
ATDPSLS
QGHAR
FQGDME
ASEGLA
PNSKLT
YEQY
21
TEISS
IRSNERE
ATDPSLS
AGHAR
FQGDME
ASEGLA
PNSKLT
YEQY
22
TAVDS
IRSNERE
ATDPSMS
AGHAR
FQGDLE
ASEGLA
PNSKLT
YEQY
23
TEISS
IRSNERE
ATDPSMS
AGHAR
FQGDME
ASEGLA
PNSKLT
YEQY
24
TEISS
IRSNERE
ATDPSMS
GTWPR
FQGDLE
ASEGLA
PNSKLT
YEQY
25
TDVSS
IRSNERE
ATDPSMS
GRWPR
FQGDLE
ASEGLA
PNSKLT
YEQY
26
TDVSS
IRSNERE
ATDPTM
GTWPR
FQGDLE
ASEGLA
SPNSKLT
YEQY
27
TAVDS
IRSNERE
ATDPTM
QGHAR
FQGDLE
ASEGLA
SPNSKLT
YEQY
28
TAVDS
IRSNERE
ATDPSLS
ARHAR
FQGDLE
ASEGLA
PNSKLT
YEQY
29
TEISS
IRSNERE
ATDPATS
AGHAR
FQGDLE
ASEGLA
PNSKLT
YEQY
30
TPISS
IRSNERE
ATDPATS
GRWPR
FQGDLE
ASEGLA
PNSKLT
YEQY
31
TAVDS
IRSNERE
ATDPSLS
AGHAR
FQGDME
ASEGLA
PNSKLT
YEQY
32
TDVSS
IRSNERE
ATDPSMS
GLWPR
FHGDLE
ASEGLA
PNSKLT
YEQY
33
TPISS
IRSNERE
ATDPSMS
AGHAR
FQGDME
ASEGLA
PNSKLT
YEQY
34
TDVSS
IRSNERE
ATDPSMS
GTWPR
FQGDME
ASEGLA
PNSKLT
YEQY
35
TAVDS
IRSNERE
ATDPTMS
QGRAR
FQGDME
ASEGLA
PNSKLT
YEQY
36
TAVDS
IRSNERE
ATDPTSS
GRWPR
FQGDLE
ASEGLA
WNSKLT
YEQY.

### Claim 18 (depends on 1)

The TCR of claim 1, wherein the TCR is soluble.

### Claim 19 (depends on 1)

The TCR of claim 1, wherein the TCR is an αβ heterodimeric TCR or a single chain TCR; and preferably, the TCR comprises α chain constant region sequence TRAC*01 and β chain constant region sequence TRBC1*01 or TRBC2*01.

### Claim 20 (depends on 19)

The TCR of claim 19, wherein the TCR comprises (i) all or part of the TCR α chain except for its transmembrane domain, and (ii) all or part of the TCR β chain except for its transmembrane domain, wherein both of (i) and (ii) comprise the variable domain and at least a portion of the constant domain of the TCR chain.

### Claim 21 (depends on 20)

The TCR of claim 20, wherein an artificial interchain disulfide bond is contained between the α chain variable region and the P chain constant region of the TCR; preferably cysteine residues forming an artificial interchain disulfide bond between the α chain variable region and the β chain constant region of the TCR are substituted for one or more groups of amino acids selected from the following:
Thr48 of TRAC*01 exon 1 and Ser57 of TRBC1*01 or TRBC2*01 exon 1;
Thr45 of TRAC*01 exon 1 and Ser77 of TRBC1*01 or TRBC2*01 exon 1;
Tyr10 of TRAC*01 exon 1 and Ser17 of TRBC1*01 or TRBC2*01 exon 1;
Thr45 of TRAC*01 exon 1 and Asp59 of TRBC1*01 or TRBC2*01 exon 1;
Ser15 of TRAC*01 exon 1 and Glu15 of TRBC1*01 or TRBC2*01 exon 1;
Arg53 of TRAC*01 exon 1 and Ser54 of TRBC1*01 or TRBC2*01 exon 1;
Pro89 of TRAC*01 exon 1 and Ala19 of TRBC1*01 or TRBC2*01 exon 1; and
Tyr10 of TRAC*01 exon 1 and Glu20 of TRBC1*01 or TRBC2*01 exon 1.

### Claim 22

(canceled)

### Claim 23 (depends on 1)

The TCR of claim 1, wherein the amino acid sequence of the α chain variable domain of the TCR is selected from the group consisting of: SEQ ID NO: 51-77 and 91-92; and/or the amino acid sequence of the β chain variable domain of the TCR is selected from the group consisting of: SEQ TD NO: 78-88 and 93-101.

### Claim 24 (depends on 1)

The TCR of claim 1, wherein the TCR is selected from the group consisting of:
Sequence of α chain
Sequence of β chain
TCR
variable domain
variable domain
No.
SEQ ID NO:
SEQ ID NO:
1
54
2
2
52
2
3
51
2
4
53
2
5
55
2
6
56
2
7
57
2
8
58
2
9
59
2
10
60
2
11
61
2
12
62
2
13
63
2
14
1
78
15
64
79
16
65
80
17
66
81
18
67
81
19
68
81
20
68
80
21
69
79
22
70
82
23
71
79
24
71
83
25
72
84
26
73
83
27
74
85
28
68
86
29
75
82
30
66
84
31
68
79
32
72
87
33
76
79
34
72
81
35
74
88
36
77
84
37
1
93
38
1
94
39
1
95
40
1
96
41
1
97
42
1
98
43
1
99
44
1
100
45
91
101
46
92
101.

### Claim 25 (depends on 1)

The TCR of claim 1, wherein the TCR is a single-chain TCR; preferably the TCR is a single-chain TCR consisting of an α variable domain and a β variable domain, and the ac variable domain and the β variable domain are connected by a flexible short peptide sequence (linker).

### Claim at position 18

26-27. (canceled)

### Claim 28

A TCR, wherein the TCR is selected from the group consisting of:
Sequence of α chain
Sequence of β chain
TCR
variable domain
variable domain
No.
SEQ ID NO:
SEQ ID NO:
s-1
12
4
s-2
10
4
s-3
9
4
s-4
11
4
s-5
13
4
s-6
14
4
s-7
15
4
s-8
16
4
s-9
17
4
s-10
18
4
s-11
19
4
s-12
20
4
s-13
21
4
s-14
3
36
s-15
22
37
s-16
23
38
s-17
24
39
s-18
25
39
s-19
26
39
s-20
26
38
s-21
27
37
s-22
28
40
s-23
29
37
s-24
29
41
s-25
30
42
s-26
31
41
s-27
32
43
s-28
26
44
s-29
33
40
s-30
24
42
s-31
26
37
s-32
30
45
s-33
34
37
s-34
30
39
s-35
32
46
s-36
35
42.

### Claim 29 (depends on 1)

The TCR of claim 1, wherein a conjugate binds to the α chain and/or β chain of the TCR at C- or N-terminal; the conjugate is a detectable label or a therapeutic agent is an anti-CD3 antibody.

### Claim at position 21

30-31. (canceled)

### Claim 32 (depends on 1)

A multivalent TCR complex comprising at least two TCR molecules, and at least one TCR molecule is the TCR of claim 1.

### Claim at position 23

33-35. (canceled)

### Claim 36 (depends on 1)

An isolated cell, expressing the TCR claim 1.

### Claim 37 (depends on 1)

A pharmaceutical composition, comprising a pharmaceutically acceptable carrier, and the TCR of claim 1, or a TCR complex comprising at least two TCR molecules, wherein at least one TCR molecule is the TCR of claim 1, or a cell expressing the TCR of claim 1.

### Claim 38 (depends on 1)

A method for treating a disease, comprising administering the TCR of claim 1, or a TCR complex comprising at least two TCR molecules, wherein at least one TCR molecule is the TCR of claim 1, or a cell of claim 1, or a pharmaceutical composition comprising a pharmaceutically acceptable carrier and the TCR, TCR complex or cell to a subject in need thereof, preferably the disease is a tumor, and more preferably, the tumor is liver cancer.

### Claim at position 27

39-40. (canceled)

### Claim 41 (depends on 1)

A method for preparing the T cell receptor of claim 1, comprising the steps of:
(i) culturing a host cell to express the T cell receptor of claim 1 wherein the host cell comprises a vector, and the vector comprises a nucleic acid molecule comprising a nucleic acid sequence encoding the TCR of claim 1 or a complement sequence thereof, or the host cell has the exogenous nucleic acid molecule integrated into its genome;
(ii) isolating or purifying the T cell receptor.