U.S. patent application number 16/154161 was filed with the patent office on 2019-05-09 for t cell receptors.
The applicant listed for this patent is Adaptimmune Limited. Invention is credited to Eleanor Bagg, William Lawrance, Nicholas Tribble.
Application Number | 20190135892 16/154161 |
Document ID | / |
Family ID | 58701591 |
Filed Date | 2019-05-09 |
United States Patent
Application |
20190135892 |
Kind Code |
A1 |
Tribble; Nicholas ; et
al. |
May 9, 2019 |
T CELL RECEPTORS
Abstract
The present invention relates to T cell receptors (TCRs) which
bind the HLA-A*0201 restricted peptide GVYDGEEHSV (SEQ ID NO: 1)
derived from the MAGE-B2 protein. The TCRs of the invention
demonstrate excellent specificity profiles for this MAGE epitope.
Also provided are nucleic acids encoding the TCRs, cells engineered
to present the TCRs, cells harbouring expression vectors encoding
the TCRs and pharmaceutical compositions comprising the TCRs,
nucleic acids or cells of the invention.
Inventors: |
Tribble; Nicholas;
(Abingdon, GB) ; Lawrance; William; (Abingdon,
GB) ; Bagg; Eleanor; (Abingdon, GB) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Adaptimmune Limited |
Abingdon |
|
GB |
|
|
Family ID: |
58701591 |
Appl. No.: |
16/154161 |
Filed: |
October 8, 2018 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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PCT/EP2017/058576 |
Apr 10, 2017 |
|
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16154161 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 14/70503 20130101;
C12N 15/85 20130101; C12N 15/62 20130101; A61K 35/17 20130101; A61K
38/00 20130101; C07K 14/70539 20130101; A61P 35/00 20180101; C07K
14/7051 20130101 |
International
Class: |
C07K 14/725 20060101
C07K014/725; C12N 15/62 20060101 C12N015/62; C07K 14/74 20060101
C07K014/74; C12N 15/85 20060101 C12N015/85; A61P 35/00 20060101
A61P035/00; A61K 35/17 20060101 A61K035/17 |
Foreign Application Data
Date |
Code |
Application Number |
Apr 8, 2016 |
GB |
1606172.3 |
Claims
1. A T cell receptor (TCR) having the property of binding to
GVYDGEEHSV (SEQ ID No: 1) in complex with HLA-A*0201 with a
dissociation constant of from about 0.05 .mu.M to about 20.0 .mu.M
when measured with surface plasmon resonance at 25.degree. C. and
at a pH between 7.1 and 7.5 using a soluble form of the TCR, and
has at least a ten-fold selectivity of binding to SEQ ID No:1 in
complex with HLA-A*0201 over binding to GVYDGREHTV (SEQ ID No 2) in
complex with HLA-A*0201 wherein the TCR comprises a TCR alpha chain
variable domain and a TCR beta chain variable domain, and wherein
the TCR variable domains form contacts with at least residues V2,
Y3 and D4 of GVYDGEEHSV (SEQ ID No: 1).
2. A TCR according to claim 1, which is an alpha-beta heterodimer,
having an alpha chain TRAV10+TRAC constant domain sequence and a
beta chain TRBV24-1+TRBC-2 constant domain sequence.
3. A TCR as claimed in claim 1, which is in single chain format of
the type V.alpha.-L-V.beta., V.beta.-L-V.alpha.,
V.alpha.-C.alpha.-L-V.beta., or V.alpha.-L-V.beta.-C.beta., wherein
V.alpha. and V.beta. are TCR .alpha. and .beta. variable regions
respectively, C.alpha. and C.beta. are TCR .alpha. and .beta.
constant regions respectively, and L is a linker sequence.
4. A TCR as claimed in claim 1, which is associated with a
detectable label, a therapeutic agent or a PK modifying moiety.
5. A TCR as claimed in claim 1, wherein the alpha chain variable
domain comprises an amino acid sequence that has at least 80%
identity to the sequence of amino acid residues 1-105 of SEQ ID No:
3 and has the following mutation: TABLE-US-00010 CDR2 I3 R
with reference to the numbering shown in SEQ ID No: 3, and/or the
beta chain variable domain comprises an amino acid sequence that
has at least 80% identity to the sequence of amino acid residues
1-123 of SEQ ID No: 4 and has at least one of the following
mutations: TABLE-US-00011 CDR1 H3 R CDR3 N10 E CDR3 N10 R
with reference to the numbering shown in SEQ ID No: 4.
6. A TCR as claimed in claim 1, wherein the alpha chain variable
domain comprises the amino acid sequence of amino acid residues
1-105 of SEQ ID No: 3 or 5 or 7 or an amino acid sequence in which
amino acid residues 1-27, 34-47, and 54-90 thereof have at least
90% or 95% identity to the sequence of amino acid residues 1-27,
34-47, and 54-90 respectively of SEQ ID No: 3 or 5 or 7 and in
which amino acid residues 28-34, 48-53 and 91-105 have at least 90%
or 95% identity to the sequence of amino acid residues 28-33, 48-53
and 91-105 respectively of SEQ ID No 3 or 5 or 7.
7. A TCR as claimed in claim 1, wherein the alpha chain variable
domain comprises the amino acid sequence of amino acid residues
1-105 of SEQ ID No: 3 or 7 or an amino acid sequence in which amino
acid residues 1-27, 34-47 and 55-89 thereof have at least 90% or
95% identity to the sequence of amino acid residues 1-27, 34-47,
and 55-89 respectively of SEQ ID No: 3 or 7 and in which amino acid
residues 28-33, 48-53 and 91-105 have at least 90% or 95% identity
to the sequence of amino acid residues 28-33, 48-53 and 91-105
respectively of SEQ ID No: 3 or 7.
8. A TCR as claimed in claim 1, wherein in the alpha chain variable
domain the sequence of (i) amino acid residues 1-27 thereof has (a)
at least 90% identity to the sequence of amino acid residues 1-26
of SEQ ID No: 3 or (b) has one, two or three amino acid residues
inserted or deleted relative to the sequence of (a); (ii) amino
acid residues 28-33 is VSPFSN; (iii) amino acid residues 34-47
thereof has (a) at least 90% identity to the sequence of amino acid
residues 34-47 of SEQ ID NO: 3 or (b) has one, two or three amino
acid residues inserted or deleted relative to the sequence of (a);
(iv) amino acid residues 48-53 is LTIMTF or LTRMTF (v) amino acid
residues 54-90 thereof has at least 90% identity to the sequence of
amino acid residues 55-89 of SEQ ID No: 3 or has one, two or three
insertions, deletions or substitutions relative thereto; (vi) amino
acids 91-105 is CVVSGGTDSWGKLQF.
9. A TCR as claimed in claim 1, wherein the beta chain variable
domain comprises the amino acid sequence of SEQ ID No: 4 or 8-10 or
an amino acid sequence in which amino acid residues 1-45, 51-67,
and 74-109 thereof have at least 90% or 95% identity to the
sequence of amino acid residues 1-45, 51-67, and 74-109
respectively of SEQ ID No: 4 or 8-10 and in which amino acid
residues 46-50, 68-73 and 109-123 have at least 90% or 95% identity
to the sequence of amino acid residues 46-50, 68-73 and 109-123
respectively of SEQ ID No: 4 or 8-10.
10. A TCR according to claim 1, wherein in the beta chain variable
domain the sequence of (i) amino acid residues 1-45 thereof has (a)
at least 90% identity to the amino acid sequence of residues 1-26
of SEQ ID No: 4 or (b) has one, two or three amino acid residues
inserted or deleted relative to the sequence of (a); (ii) amino
acid residues 46-50 is KGHDR or KGRDR (iii) amino acid residues
51-67 thereof has (a) at least 90% identity to the sequence of
amino acid residues 51-67 of SEQ ID NO: 4 or (b) has one, two or
three amino acid residues inserted or deleted relative to the
sequence of (a); (iv) amino acid residues 68-73 is SFDVK; (v) amino
acid residues 54-90 thereof has (a) at least 90% identity to the
sequence of amino acid residues 54-90 of SEQ ID NO: 4 or (b) has
one, two or three amino acid residues inserted or deleted relative
to the sequence of (a); (vi) amino acids 109-123 is CATSGQGAYNEQFF
or CATSGQGAYREQFF or CATSGQGAYKEQFF.
11. A nucleic acid encoding a TCR as claimed in claim 1.
12. An isolated or non-naturally occurring cell, especially a
T-cell, presenting a TCR as claimed in claim 1.
13. A cell harboring (a) a TCR expression vector which comprises
nucleic acid as claimed in claim 11 in a single open reading frame,
or two distinct open reading frames encoding the alpha chain and
the beta chain respectively; or (b) a first expression vector which
comprises nucleic acid encoding the alpha chain of a TCR as claimed
in claim 1, and a second expression vector which comprises nucleic
acid encoding the beta chain of a TCR as claimed in claim 1.
14. A pharmaceutical composition comprising the TCR as claimed in
claim 1, together with one or more pharmaceutically acceptable
carriers or excipients.
15. A method of treating cancer comprising administering the TCR of
claim 1.
16. A pharmaceutical composition comprising the nucleic acid as
claimed in claim 11, together with one or more pharmaceutically
acceptable carriers or excipients.
17. A pharmaceutical composition comprising the cell as claimed in
claim 13, together with one or more pharmaceutically acceptable
carriers or excipients.
18. A method of treating cancer comprising administering the
nucleic acid of claim 11.
19. A method of treating cancer comprising administering the cell
of claim 13.
Description
RELATED APPLICATIONS AND INCORPORATION BY REFERENCE
[0001] This application is a continuation-in-part application of
international patent application Serial No. PCT/EP2017/058576 filed
Apr. 10, 2017, which published as PCT Publication No. WO
2017/174822 on Oct. 12, 2017, which claims benefit of European
patent application Serial No. 1606172.3 filed Apr. 8, 2016.
[0002] The foregoing applications, and all documents cited therein
or during their prosecution ("appln cited documents") and all
documents cited or referenced in the appln cited documents, and all
documents cited or referenced herein ("herein cited documents"),
and all documents cited or referenced in herein cited documents,
together with any manufacturer's instructions, descriptions,
product specifications, and product sheets for any products
mentioned herein or in any document incorporated by reference
herein, are hereby incorporated herein by reference, and may be
employed in the practice of the invention. More specifically, all
referenced documents are incorporated by reference to the same
extent as if each individual document was specifically and
individually indicated to be incorporated by reference.
SEQUENCE LISTING
[0003] The instant application contains a Sequence Listing which
has been submitted electronically in ASCII format and is hereby
incorporated by reference in its entirety. Said ASCII copy is named
Sequence_Listing.txt and is 30 kb in size.
FIELD OF THE INVENTION
[0004] The present invention relates to T cell receptors (TCRs)
which bind the HLA-A*0201 restricted decapeptide GVYDGEEHSV derived
from the melanoma-associated antigen (MAGE) B2 protein (amino acids
230-239). The TCRs of the invention demonstrate excellent
specificity profiles for this MAGE epitope.
BACKGROUND TO THE INVENTION
[0005] Cancer testis antigens (CTA) are a subclass of
tumour-associated antigen (TAA) encoded by approximately 140 genes.
Expression of these antigens is restricted in immune privileged
sites such as the testes, placenta and fetal ovary; they are
typically not expressed in other tissues. Expression of these genes
has been observed in malignant tumors. The immunogenicity of CTA
has led to the widespread development of cancer vaccines targeting
these antigens in many solid tumors. Within this large class of
TAA, melanoma-associated antigens (MAGE) have emerged as promising
candidates for cancer immunotherapy.
[0006] More than 30 cancer testis (CT) genes have been reported as
members of multi-gene families that are organized into gene
clusters on chromosome X (CT-X antigens). The CT gene clusters are
located between Xq24 and Xq28 and include gene families such as
MAGE and NY-ESO-1. Type I MAGE gene clusters are the most
extensively characterized and include the MAGE-A, MAGE-B and MAGE-C
families. The MAGE-A proteins are encoded by 12 different MAGE-A
gene family members (MAGE-A1 to MAGE-A12) and are defined by a
conserved 165-171 amino acid base, called the MAGE homology domain
(MHD). The MHD corresponds to the only region of shared amino acids
by all of the MAGE-A family members.
[0007] T cells recognize and interact with complexes of cell
surface molecules, referred to as human leukocyte antigens ("HLA"),
or major histocompatibility complexes ("MHCs"), and peptides. The
peptides are derived from larger molecules, which are processed by
the cells which also present the HLA/MHC molecule. The interaction
of T cells and HLA/peptide complexes is restricted, requiring a T
cell specific for a particular combination of an HLA molecule and a
peptide. If a specific T cell is not present, there is no T cell
response even if its partner complex is present. Similarly, there
is no response if the specific complex is absent, but the T cell is
present. The mechanism is involved in the immune system's response
to infection, in autoimmune disease, and in responses to
abnormalities such as tumours.
[0008] Some MAGE gene family proteins are only expressed in germ
cells and cancer (MAGE-A to MAGE-C families). Others are widely
expressed in normal tissues (MAGE-D through to MAGE-H). All these
MAGE protein families have a homologous region that is closely
matched to the sequence of the other MAGE proteins and comprises
peptides displayed as HLA/peptide complexes in immune recognition.
Hence, it is important to select TCR clinical candidates that are
highly specific for the desired MAGE peptide/HLA-A2 antigen.
[0009] MAGE B2 is a CTA member of the MAGE B gene family. The
function is unknown, though it is thought that it may play a role
in embryonal development. In tumour pathogenesis, it appears to be
involved in tumor transformation or aspects of tumor progression.
MAGE B2 has been implicated in a large number of tumours. The
peptide GVYDGEEHSV (SEQ ID NO: 1) corresponds to amino acid residue
numbers 231-241 of the known MAGE-B2 protein.
[0010] MAGE A4 is a CTA of the MAGE A gene family. MAGE A4 is
expressed in testis and placenta, and in a significant fraction of
tumors of various histological types. The peptide GVVDGREHTV (SEQ
ID NO 2) shows cross-reactivity with MAGE B2, such that certain
TCRs are able to bind to HLA molecules displaying both
peptides.
SUMMARY OF THE INVENTION
[0011] We have developed a TCR which binds to HLA molecules
displaying the MAGE B2 peptide GVYDGEEHSV in preference to MAGE A4.
In a first aspect, the present invention provides a T cell receptor
(TCR) having the property of binding to GVYDGEEHSV (SEQ ID NO: 1)
in complex with HLA-A*0201 with a dissociation constant of from
about 0.05 .mu.M to about 20.0 .mu.M when measured with surface
plasmon resonance at 25.degree. C. and at a pH between 7.1 and 7.5
using a soluble form of the TCR, wherein the TCR comprises a TCR
alpha chain variable domain and a TCR beta chain variable domain,
and wherein the TCR variable domains form contacts with at least
residues V2, Y3 and D4 of GVYDGEEHSV (SEQ ID NO: 1).
[0012] In embodiments, the TCR according to the invention has the
property of binding to GVYDGEEHSV (SEQ ID NO: 1) in complex with
HLA-A*0201 with a dissociation constant of from about 20 .mu.M to
about 50 .mu.M when measured with surface plasmon resonance at
25.degree. C. and at a pH between 7.1 and 7.5 using a soluble form
of the TCR, wherein the TCR comprises a TCR alpha chain variable
domain and a TCR beta chain variable domain. In some embodiments,
the dissociation constant is above 50 microM, such as 100 .mu.M,
200 .mu.M, 500 .mu.M or more.
[0013] Accordingly, a TCR in accordance with the invention is
capable of binding efficiently to HLA displaying GVYDGEEHSV but not
to HLA displaying GVYDGREHTV.
[0014] In some embodiments, the alpha chain variable domain of the
TCR comprises an amino acid sequence that has at least 80% identity
to the sequence of amino acid residues 1-111 of SEQ ID NO: 3 (alpha
chain), and/or the beta chain variable domain comprises an amino
acid sequence that has at least 80% identity to the sequence of
amino acid residues 1-111 of SEQ ID NO: 4 (beta chain).
[0015] In a further aspect, the present invention provides a T cell
receptor (TCR) having the property of binding to GVYDGEEHSV (SEQ ID
NO: 1) in complex with HLA-A*0201 and comprising a TCR alpha chain
variable domain and a TCR beta chain variable domain,
[0016] the alpha chain variable domain comprising an amino acid
sequence that has at least 80% identity to the sequence of amino
acid residues 1-111 of SEQ ID NO: 3, and/or
[0017] the beta chain variable domain comprising an amino acid
sequence that has at least 80% identity to the sequence of amino
acid residues 1-111 of SEQ ID NO: 4.
[0018] The GVYDGEEHSV HLA-A2 complex provides a cancer marker that
the TCRs of the invention can target. The present invention
provides such TCRs useful for the purpose of delivering cytotoxic
or immune effector agents to the cancer cells and/or useful for use
in adoptive therapy.
[0019] TCRs are described using the International Immunogenetics
(IMGT) TCR nomenclature, and links to the IMGT public database of
TCR sequences. Native alpha-beta heterodimeric TCRs have an alpha
chain and a beta chain. Broadly, each chain comprises variable,
joining and constant regions, and the beta chain also usually
contains a short diversity region between the variable and joining
regions, but this diversity region is often considered as part of
the joining region. Each variable region comprises three CDRs
(Complementarity Determining Regions) embedded in a framework
sequence, one being the hypervariable region named CDR3. There are
several types of alpha chain variable (V.alpha.) regions and
several types of beta chain variable (V.beta.) regions
distinguished by their framework, CDR1 and CDR2 sequences, and by a
partly defined CDR3 sequence. The V.alpha. types are referred to in
IMGT nomenclature by a unique TRAV number. Thus "TRAV21" defines a
TCR V.alpha. region having unique framework and CDR1 and CDR2
sequences, and a CDR3 sequence which is partly defined by an amino
acid sequence which is preserved from TCR to TCR but which also
includes an amino acid sequence which varies from TCR to TCR. In
the same way, "TRBV5-1" defines a TCR V.beta. region having unique
framework and CDR1 and CDR2 sequences, but with only a partly
defined CDR3 sequence.
[0020] The joining regions of the TCR are similarly defined by the
unique IMGT TRAJ and TRBJ nomenclature, and the constant regions by
the IMGT TRAC and TRBC nomenclature.
[0021] The beta chain diversity region is referred to in IMGT
nomenclature by the abbreviation TRBD, and, as mentioned, the
concatenated TRBD/TRBJ regions are often considered together as the
joining region.
[0022] The .alpha. and .beta. chains of .alpha..beta. TCR's are
generally regarded as each having two "domains", namely variable
and constant domains. The variable domain consists of a
concatenation of variable region and joining region. In the present
specification and claims, the term "TCR alpha variable domain"
therefore refers to the concatenation of TRAV and TRAJ regions, and
the term TCR alpha constant domain refers to the extracellular TRAC
region, or to a C-terminal truncated TRAC sequence. Likewise, the
term "TCR beta variable domain" refers to the concatenation of TRBV
and TRBD/TRBJ regions, and the term TCR beta constant domain refers
to the extracellular TRBC region, or to a C-terminal truncated TRBC
sequence.
[0023] The unique sequences defined by the IMGT nomenclature are
widely known and accessible to those working in the TCR field. For
example, they can be found in the IMGT public database. The "T cell
Receptor Factsbook", (2001) LeFranc and LeFranc, Academic Press,
ISBN 0-12-441352-8 also discloses sequences defined by the IMGT
nomenclature, but because of its publication date and consequent
time-lag, the information therein sometimes needs to be confirmed
by reference to the IMGT database.
[0024] One TCR in accordance with the invention comprises an alpha
chain extracellular domain as shown in SEQ ID NO: 2 (TRAV10+TRAC)
and a beta chain extracellular domain as shown in SEQ ID NO: 3
(TRBV24-1+TRBC-2). The terms "parental TCR", "parental MAGE-A4
TCR", are used synonymously herein to refer to this TCR comprising
the extracellular alpha and beta chain of SEQ ID Nos.: 2 and 3
respectively. It is desirable to provide TCRs that are mutated or
modified relative to the parental TCR that have a higher affinity
and/or a slower off-rate for the peptide-HLA complex than the
parental TCR.
[0025] For the purpose of providing a reference TCR against which
the binding profile of such mutated or modified TCRs may be
compared, it is convenient to use a soluble TCR in accordance with
the invention having the extracellular sequence of the parental
MAGE-A4 TCR alpha chain given in SEQ ID NO 3 and the extracellular
sequence of the parental MAGE-A4 TCR beta chain given in SEQ ID NO:
4. That TCR is referred to herein as the "the reference TCR" or
"the reference MAGE-A4 TCR". Note that SEQ ID NO: 5 comprises the
parental alpha chain extracellular sequence of SEQ ID NO: 3 and
that C162 has been substituted for T162 (i.e. T48 of TRAC).
Likewise, SEQ ID NO: 6 is the parental beta chain extracellular
sequence of SEQ ID NO: 4 and that C169 has been substituted for
S169 (i.e. S57 of TRBC2), A187 has been substituted for C187 and
D201 has been substituted for N201. These cysteine substitutions
relative to the parental alpha and beta chain extracellular
sequences enable the formation of an interchain disulfide bond
which stabilises the refolded soluble TCR, i.e. the TCR formed by
refolding extracellular alpha and beta chains. Use of the stable
disulfide linked soluble TCR as the reference TCR enables more
convenient assessment of binding affinity and binding half life.
TCRs of the invention may comprise the mutations described
above.
[0026] TCRs of the invention may be non-naturally occurring and/or
purified and/or engineered. TCRs of the invention may have more
than one mutation present in the alpha chain variable domain and/or
the beta chain variable domain relative to the parental TCR.
"Engineered TCR" and "mutant TCR" are used synonymously herein and
generally mean a TCR which has one or more mutations introduced
relative to the parental TCR, in particular in the alpha chain
variable domain and/or the beta chain variable domain thereof.
These mutation(s) may improve the binding affinity for GVYDGEEHSV
(SEQ ID NO: 1) in complex with HLA-A*020101. In certain
embodiments, there are 1, 2, 3, 4, 5, 6, 7 or 8 mutations in alpha
chain variable domain, for example 4 or 8 mutations, and/or 1, 2,
3, 4 or 5 mutations in the beta chain variable domain, for example
5 mutations. In some embodiments, the .alpha. chain variable domain
of the TCR of the invention may comprise an amino acid sequence
that has at least 90%, at least 91%, at least 92%, at least 93%, at
least 94%, at least 95%, at least 96%, at least 97%, at least 98%
or at least 99% identity to the sequence of amino acid residues
1-105 of SEQ ID NO: 3. In some embodiments, the .beta. chain
variable domain of the TCR of the invention may comprise an amino
acid sequence that has at least 90%, at least 91%, at least 92%, at
least 93%, at least 94%, at least 95%, at least 96%, at least 97%,
at least 98% or at least 99% identity to the sequence of amino acid
residues 1-123 of SEQ ID NO: 4.
[0027] The alpha chain variable domain of a TCR of the invention
may have the following mutation:
TABLE-US-00001 CDR2 I3 R
with reference to the numbering shown in SEQ ID NO: 3, and/or
[0028] the beta chain variable domain may have at least one of the
following mutations:
TABLE-US-00002 CDR1 H3 R CDR3 N10 E CDR3 N10 R
with reference to the numbering shown in SEQ ID NO: 4.
[0029] The alpha chain variable domain of a TCR of the invention
may comprise the amino acid sequence of amino acid residues 1-105
of SEQ ID NO: 3 or 5 or 7
[0030] or an amino acid sequence in which amino acid residues 1-27,
34-47, and 54-90 thereof have at least 90% or 95% identity to the
sequence of amino acid residues 1-27, 34-47, and 54-90 respectively
of SEQ ID No: 3 or 5 or 7 and in which amino acid residues 28-33,
48-53 and 91-105 have at least 90% or 95% identity to the sequence
of amino acid residues 28-33, 48-53 and 91-105 respectively of SEQ
ID No: 3 or 5 or 7.
[0031] In the alpha chain variable domain, the sequence of
[0032] (i) amino acid residues 1-26 thereof may have (a) at least
90% identity to the sequence of amino acid residues 1-26 of SEQ ID
No: 3 or (b) may have one, two or three amino acid residues
inserted or deleted relative to the sequence of (a);
[0033] (ii) amino acid residues 28-33 is VSPFSN
[0034] (iii) amino acid residues 33-49 thereof may have (a) at
least 90% identity to the sequence of amino acid residues 34-47 of
SEQ ID NO: 3 or (b) may have one, two or three amino acid residues
inserted or deleted relative to the sequence of (a);
[0035] (iv) amino acid residues 48-53 may be LTIMTF or LTRMTF
[0036] (v) amino acid residues 55-89 thereof may have at least 90%
identity to the sequence of amino acid residues 54-90 of SEQ ID No:
3 or may have one, two or three insertions, deletions or
substitutions relative thereto;
[0037] (vi) amino acids 90-93 may be CVVSGGTDSWGKLQF
[0038] The beta chain variable domain of a TCR of the invention may
comprise the amino acid sequence of SEQ ID No: 4, 6 or 8-10
[0039] or an amino acid sequence in which amino acid residues 1-45,
51-67, 74-109 thereof have at least 90% or 95% identity to the
sequence of amino acid residues 1-45, 51-67, 74-109 respectively of
SEQ ID No: 4, 6 or 8-10 and in which amino acid residues 46-50,
68-73 and 109-123 have at least 90% or 95% identity to the sequence
of amino acid residues 46-50, 68-73 and 109-123 respectively of SEQ
ID No: 4, 6 or 8-10.
[0040] In the beta chain variable domain, the sequence of
[0041] (i) amino acid residues 1-45 thereof may have (a) at least
90% identity to the amino acid sequence of residues 1-45 of SEQ ID
No: 4 or (b) may have one, two or three amino acid residues
inserted or deleted relative to the sequence of (a);
[0042] (ii) amino acid residues 46-50 may be KGHDR or KGRDR
[0043] (iii) amino acid residues 51-67 thereof may have (a) at
least 90% identity to the sequence of amino acid residues 51-67 of
SEQ ID NO: 4 or (b) may have one, two or three amino acid residues
inserted or deleted relative to the sequence of (a);
[0044] (iv) amino acid residues 68-73 may be SVFDK;
[0045] (v) amino acid residues 54-90 thereof may have (a) at least
90% identity to the sequence of amino acid residues 54-90 of SEQ ID
NO: 4 or (b) may have one, two or three amino acid residues
inserted or deleted relative to the sequence of (a);
[0046] (vi) amino acids 109-123 is CATSGQGAYNEQFF or CATSGQGAYREQFF
or CATSGQGAYKEQFF.
[0047] A TCR of the invention may have one of the following
combinations of alpha and beta chain variable domains:
TABLE-US-00003 Alpha Chain SEQ ID No Beta Chain SEQ ID No 3 4 3 6 3
8 3 9 3 10 5 4 5 6 5 8 5 9 5 10 7 4 7 6 7 8 7 9 7 10
[0048] Within the scope of the invention are phenotypically silent
variants of any TCR of the invention disclosed herein. As used
herein the term "phenotypically silent variants" is understood to
refer to a TCR which incorporates one or more further amino acid
changes in addition to those set out above which TCR has a similar
phenotype to the corresponding TCR without said change(s). For the
purposes of this application, TCR phenotype comprises antigen
binding specificity (K.sub.D and/or binding half life) and antigen
specificity. A phenotypically silent variant may have a K.sub.D
and/or binding half-life for the GVYDGEEHSV (SEQ ID No: 1)
HLA-A*0201 complex within 10% of the measured K.sub.D and/or
binding half-life of the corresponding TCR without said change(s),
when measured under identical conditions (for example at 25.degree.
C. and on the same SPR chip). Suitable conditions are further
defined in Example 3. Antigen specificity is further defined below.
As is known to those skilled in the art, it may be possible to
produce TCRs that incorporate changes in the constant and/or
variable domains thereof compared to those detailed above without
altering the affinity for the interaction with the GVYDGEEHSV (SEQ
ID No: 1) HLA-A*0201 complex. In particular, such silent mutations
may be incorporated within parts of the sequence that are known not
to be directly involved in antigen binding (e.g. outside the CDRs).
Such trivial variants are included in the scope of this invention.
Those TCRs in which one or more conservative substitutions have
been made also form part of this invention.
[0049] Mutations can be carried out using any appropriate method
including, but not limited to, those based on polymerase chain
reaction (PCR), restriction enzyme-based cloning, or ligation
independent cloning (LIC) procedures. These methods are detailed in
many of the standard molecular biology texts. For further details
regarding polymerase chain reaction (PCR) and restriction
enzyme-based cloning, see Sambrook & Russell, (2001) Molecular
Cloning--A Laboratory Manual (3.sup.rd Ed.) CSHL Press. Further
information on ligation independent cloning (LIC) procedures can be
found in Rashtchian, (1995) Curr Opin Biotechnol 6(1): 30-6.
[0050] The TCRs of the invention have the property of binding the
MAGE-B2 peptide, GVYDGEEHSV (SEQ ID No: 1) HLA-A2 complex. The TCRs
of the invention have been found to be highly specific for those
MAGE epitopes relative to other, irrelevant epitopes, and are thus
particularly suitable as targeting vectors for delivery of
therapeutic agents or detectable labels to cells and tissues
displaying those epitopes. Specificity in the context of TCRs of
the invention relates to their ability to recognise HLA-A*0201
target cells that are positive for the peptide GVYDGEEHSV, whilst
having minimal ability to recognise HLA-A*0201 target cells that
are negative for the peptide, or HLA cells that display the MAGE A4
peptide GVYDGREHTV. To test specificity, the TCRs may be in soluble
form and/or may be expressed on the surface of T cells. Recognition
may be determined by measuring the level of T cell activation in
the presence of a TCR and target cells. In this case, minimal
recognition of peptide negative or MAGE A4 target cells is defined
as a level of T cell activation of less than 10%, preferably less
than 5%, and more preferably less than 1%, of the level produced in
the presence of peptide positive target cells, when measured under
the same conditions. For soluble TCRs of the invention, specificity
may be determined at a therapeutically relevant TCR concentration.
A therapeutically relevant concentration may be defined as a TCR
concentration of 10.sup.-9 M or below, and/or a concentration of up
to 100, preferably up to 1000, fold greater than the corresponding
EC50 value. Peptide positive cells may be obtained by
peptide-pulsing or, more preferably, they may naturally present
said peptide. Preferably, both peptide positive and peptide
negative cells are human cells.
[0051] Certain TCRs of the invention have been found to be highly
suitable for use in adoptive therapy. Such TCRs may have a K.sub.D
for the complex of less than the 200 .mu.M, for example from about
0.05 .mu.M to about 20 .mu.M or about 100 .mu.M and/or have a
binding half-life (T1/2) for the complex in the range of from about
0.5 seconds to about 12 minutes. In some embodiments, TCRs of the
invention may have a K.sub.D for the complex of from about 0.05
.mu.M to about 20 .mu.M, about 0.1 .mu.M to about 5 .mu.M or about
0.1 .mu.M to about 2 .mu.M. Without wishing to be bound by theory,
there seems to be an optimum window of affinity for TCRs with
therapeutic use in adoptive cell therapy. Naturally occurring TCRs
recognising epitopes from tumour antigens are generally of too low
affinity (20 microM to 50 microM) and very high affinity TCRs (in
the nanomolar range or higher) suffer from cross-reactivity issues
(Robbins et al (2008) J. Immunol. 180 6116-6131; Zhao et al (2007)
J. Immunol. 179 5845-5854; Scmid et al (2010) J. Immunol 184
4936-4946).
[0052] The TCRs of the invention may be .alpha..beta. heterodimers
or may be in single chain format. Single chain formats include
.alpha..beta. TCR polypeptides of the V.alpha.-L-V.beta.,
V.beta.-L-V.alpha., V.alpha.-C.alpha.-L-V.beta. or
V.alpha.-L-V.beta.-C.beta. types, wherein V.alpha. and V.beta. are
TCR .alpha. and .beta. variable regions respectively, C.alpha. and
C.beta. are TCR .alpha. and .beta. constant regions respectively,
and L is a linker sequence. For use as a targeting agent for
delivering therapeutic agents to the antigen presenting cell the
TCR may be in soluble form (i.e. having no transmembrane or
cytoplasmic domains). For stability, soluble .alpha..beta.
heterodimeric TCRs preferably have an introduced disulfide bond
between residues of the respective constant domains, as described,
for example, in WO 03/020763. One or both of the constant domains
present in an .alpha..beta. heterodimer of the invention may be
truncated at the C terminus or C termini, for example by up to 15,
or up to 10 or up to 8 or fewer amino acids. For use in adoptive
therapy, an .alpha..beta. heterodimeric TCR may, for example, be
transfected as full length chains having both cytoplasmic and
transmembrane domains. TCRs for use in adoptive therapy may contain
a disulphide bond corresponding to that found in nature between the
respective alpha and beta constant domains, additionally or
alternatively a non-native disulphide bond may be present.
[0053] As will be obvious to those skilled in the art, it may be
possible to truncate the sequences provided at the C-terminus
and/or N-terminus thereof, by 1, 2, 3, 4, 5 or more residues,
without substantially affecting the binding characteristics of the
TCR. All such trivial variants are encompassed by the present
invention.
[0054] Alpha-beta heterodimeric TCRs of the invention usually
comprise an alpha chain TRAC constant domain sequence and a beta
chain TRBC1 or TRBC2 constant domain sequence. The alpha and beta
chain constant domain sequences may be 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. The alpha and
beta chain constant domain sequences may also be 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 alpha and beta constant domains of the TCR.
[0055] Some TCRs of the invention have a binding affinity for,
and/or a binding half-life for, the GVYDGEEHSV-HLA-A2 complex
substantially higher than that of the reference MAGE-B2 TCR,
Increasing the binding affinity of a native TCR often reduces the
specificity of the TCR for its peptide-MHC ligand, and this is
demonstrated in Zhao Yangbing et al., The Journal of Immunology,
The American Association of Immunologists, US, vol. 179, No. 9, 1
Nov. 2007, 5845-5854. However, the TCRs of the invention which are
derived from the parental TCR remain specific for the
GVYDGEEHSV-HLA-A2 complex, despite having substantially higher
binding affinity than the parental TCR. Moreover, they are
significantly more (e.g. at least ten-fold) selective for MAGE-B2
over MAGE-A4 than the parental TCR.
[0056] Binding affinity (inversely proportional to the equilibrium
constant K.sub.D) and binding half-life (expressed as T1/2) can be
determined using the Surface Plasmon Resonance (BIAcore) method of
Example 3 herein. Measurements may be carried out at 25.degree. C.
and at a pH between 7.1 and 7.5 using a soluble version of the TCR.
It will be appreciated that doubling the affinity of a TCR results
in halving the K.sub.D. T1/2 is calculated as ln 2 divided by the
off-rate (k.sub.off). So doubling of T1/2 results in a halving in
K.sub.off. K.sub.D and k.sub.off values for TCRs are usually
measured for soluble forms of the TCR, i.e. those forms which are
truncated to remove hydrophobic transmembrane domain residues.
Therefore, it is to be understood that a given TCR meets the
requirement that it has a binding affinity for, and/or a binding
half-life for, the GVYDGREHTV-HLA-A2 complex if a soluble form of
that TCR meets that requirement. Preferably the binding affinity or
binding half-life of a given TCR is measured several times, for
example 3 or more times, using the same assay protocol, and an
average of the results is taken. The reference TCR has a K.sub.D of
approximately 17 .mu.M as measured by that method, and T1/2 is
approximately 1.6 S.
[0057] In a further aspect, the present invention provides nucleic
acid encoding a TCR of the invention. In some embodiments, the
nucleic acid is cDNA. In some embodiments, the invention provides
nucleic acid comprising a sequence encoding an .alpha. chain
variable domain of a TCR of the invention. In some embodiments, the
invention provides nucleic acid comprising a sequence encoding a
.beta. chain variable domain of a TCR of the invention. The nucleic
acid may be non-naturally occurring and/or purified and/or
engineered.
[0058] In another aspect, the invention provides a vector which
comprises nucleic acid of the invention. Preferably the vector is a
TCR expression vector.
[0059] The invention also provides a cell harbouring a vector of
the invention, preferably a TCR expression vector. The vector may
comprise nucleic acid of the invention encoding in a single open
reading frame, or two distinct open reading frames, the alpha chain
and the beta chain respectively. Another aspect provides a cell
harbouring a first expression vector which comprises nucleic acid
encoding the alpha chain of a TCR of the invention, and a second
expression vector which comprises nucleic acid encoding the beta
chain of a TCR of the invention. Such cells are particularly useful
in adoptive therapy. The cells of the invention may be isolated
and/or recombinant and/or non-naturally occurring and/or
engineered.
[0060] Since the TCRs of the invention have utility in adoptive
therapy, the invention includes a non-naturally occurring and/or
purified and/or or engineered cell, especially a T-cell, presenting
a TCR of the invention. The invention also provides an expanded
population of T cells presenting a TCR of the invention. There are
a number of methods suitable for the transfection of T cells with
nucleic acid (such as DNA, cDNA or RNA) encoding the TCRs of the
invention (see for example Robbins et al., (2008) J Immunol. 180:
6116-6131). T cells expressing the TCRs of the invention will be
suitable for use in adoptive therapy-based treatment of cancer. As
will be known to those skilled in the art, there are a number of
suitable methods by which adoptive therapy can be carried out (see
for example Rosenberg et al., (2008) Nat Rev Cancer 8(4):
299-308).
[0061] Soluble TCRs of the invention are useful for delivering
detectable labels or therapeutic agents to the antigen presenting
cells and tissues containing the antigen presenting cells. The may
therefore be associated (covalently or otherwise) with a detectable
label (for diagnostic purposes wherein the TCR is used to detect
the presence of cells presenting the GVYDGEEHSV-HLA-A2 complex); a
therapeutic agent; or a PK modifying moiety (for example by
PEGylation).
[0062] Detectable labels for diagnostic purposes include for
instance, fluorescent labels, radiolabels, enzymes, nucleic acid
probes and contrast reagents.
[0063] Therapeutic agents which may be associated with the TCRs of
the invention include immunomodulators, radioactive compounds,
enzymes (perforin for example) or chemotherapeutic agents
(cisplatin for example). To ensure that toxic effects are exercised
in the desired location the toxin could be inside a liposome linked
to TCR so that the compound is released slowly. This will prevent
damaging effects during the transport in the body and ensure that
the toxin has maximum effect after binding of the TCR to the
relevant antigen presenting cells.
[0064] Other suitable therapeutic agents include: [0065] small
molecule cytotoxic agents, i.e. compounds with the ability to kill
mammalian cells having a molecular weight of less than 700 Daltons.
Such compounds could also contain toxic metals capable of having a
cytotoxic effect. Furthermore, it is to be understood that these
small molecule cytotoxic agents also include pro-drugs, i.e.
compounds that decay or are converted under physiological
conditions to release cytotoxic agents. Examples of such agents
include cis-platin, maytansine derivatives, rachelmycin,
calicheamicin, docetaxel, etoposide, gemcitabine, ifosfamide,
irinotecan, melphalan, mitoxantrone, sorfimer sodiumphotofrin II,
temozolomide, topotecan, trimetreate glucuronate, auristatin E
vincristine and doxorubicin; [0066] peptide cytotoxins, i.e.
proteins or fragments thereof with the ability to kill mammalian
cells. For example, ricin, diphtheria toxin, pseudomonas bacterial
exotoxin A, DNase and RNase; [0067] radio-nuclides, i.e. unstable
isotopes of elements which decay with the concurrent emission of
one or more of .alpha. or .beta. particles, or .gamma. rays. For
example, iodine 131, rhenium 186, indium 111, yttrium 90, bismuth
210 and 213, actinium 225 and astatine 213; chelating agents may be
used to facilitate the association of these radio-nuclides to the
high affinity TCRs, or multimers thereof; [0068] immuno-stimulants,
i.e. immune effector molecules which stimulate immune response. For
example, cytokines such as IL-2 and IFN-.gamma., [0069]
Superantigens and mutants thereof; [0070] TCR-HLA fusions; [0071]
chemokines such as IL-8, platelet factor 4, melanoma growth
stimulatory protein, etc; [0072] antibodies or fragments thereof,
including anti-T cell or NK cell determinant antibodies (e.g.
anti-CD3, anti-CD28 or anti-CD16); [0073] alternative protein
scaffolds with antibody like binding characteristics [0074]
complement activators; [0075] xenogeneic protein domains,
allogeneic protein domains, viral/bacterial protein domains,
viral/bacterial peptides.
[0076] One preferred embodiment is provided by a TCR of the
invention associated (usually by fusion to an N- or C-terminus of
the alpha or beta chain) with an anti-CD3 antibody, or a functional
fragment or variant of said anti-CD3 antibody. Antibody fragments
and variants/analogues which are suitable for use in the
compositions and methods described herein include minibodies, Fab
fragments, F(ab').sub.2 fragments, dsFv and scFv fragments,
Nanobodies.TM. (these constructs, marketed by Ablynx (Belgium),
comprise synthetic single immunoglobulin variable heavy domain
derived from a camelid (e.g. camel or llama) antibody) and Domain
Antibodies (Domantis (Belgium), comprising an affinity matured
single immunoglobulin variable heavy domain or immunoglobulin
variable light domain) or alternative protein scaffolds that
exhibit antibody like binding characteristics such as Affibodies
(Affibody (Sweden), comprising engineered protein A scaffold) or
Anticalins (Pieris (German), comprising engineered anticalins) to
name but a few.
[0077] For some purposes, the TCRs of the invention may be
aggregated into a complex comprising several TCRs to form a
multivalent TCR complex. There are a number of human proteins that
contain a multimerisation domain that may be used in the production
of multivalent TCR complexes. For example, the tetramerisation
domain of p53 which has been utilised to produce tetramers of scFv
antibody fragments which exhibited increased serum persistence and
significantly reduced off-rate compared to the monomeric scFv
fragment. (Willuda et al. (2001) J. Biol. Chem. 276 (17)
14385-14392). Haemoglobin also has a tetramerisation domain that
could potentially be used for this kind of application. A
multivalent TCR complex of the invention may have enhanced binding
capability for the GVYDGREHTV HLA-A2 complex compared to a
non-multimeric wild-type or T cell receptor heterodimer of the
invention. Thus, multivalent complexes of TCRs of the invention are
also included within the invention. Such multivalent TCR complexes
according to the invention are particularly useful for tracking or
targeting cells presenting particular antigens in vitro or in vivo,
and are also useful as intermediates for the production of further
multivalent TCR complexes having such uses.
[0078] As is well-known in the art, TCRs may be subject to post
translational modifications. Glycosylation is one such
modification, which comprises the covalent attachment of
oligosaccharide moieties to defined amino acids in the TCR chain.
For example, asparagine residues, or serine/threonine residues are
well-known locations for oligosaccharide attachment. The
glycosylation status of a particular protein depends on a number of
factors, including protein sequence, protein conformation and the
availability of certain enzymes. Furthermore, glycosylation status
(i.e. oligosaccharide type, covalent linkage and total number of
attachments) can influence protein function. Therefore, when
producing recombinant proteins, controlling glycosylation is often
desirable. Controlled glycosylation has been used to improve
antibody-based therapeutics. (Jefferis R., Nat Rev Drug Discov.
2009 March; 8(3):226-34.). For soluble TCRs of the invention
glycosylation may be controlled in vivo, by using particular cell
lines for example, or in vitro, by chemical modification. Such
modifications are desirable, since glycosylation can improve
phamacokinetics, reduce immunogenicity and more closely mimic a
native human protein (Sinclair AM and Elliott S., Pharm Sci. 2005
August; 94(8):1626-35).
[0079] For administration to patients, the TCRs, nucleic acids
and/or cells of the invention (usually associated with a detectable
label or therapeutic agent), may be provided in a pharmaceutical
composition together with a pharmaceutically acceptable carrier or
excipient. Therapeutic or imaging TCRs in accordance with the
invention will usually be supplied as part of a sterile,
pharmaceutical composition which will normally include a
pharmaceutically acceptable carrier. This pharmaceutical
composition may be in any suitable form, (depending upon the
desired method of administering it to a patient). It may be
provided in unit dosage form, will generally be provided in a
sealed container and may be provided as part of a kit. Such a kit
would normally (although not necessarily) include instructions for
use. It may include a plurality of said unit dosage forms.
[0080] The pharmaceutical composition may be adapted for
administration by any appropriate route, preferably a parenteral
(including subcutaneous, intramuscular, or preferably intravenous)
route. Such compositions may be prepared by any method known in the
art of pharmacy, for example by mixing the active ingredient with
the carrier(s) or excipient(s) under sterile conditions.
[0081] Dosages of the substances of the present invention can vary
between wide limits, depending upon the disease or disorder to be
treated, the age and condition of the individual to be treated,
etc. and a physician will ultimately determine appropriate dosages
to be used.
[0082] TCRs, pharmaceutical compositions, vectors, nucleic acids
and cells of the invention may be provided in substantially pure
form, for example at least 80%, at least 85%, at least 90%, at
least 91%, at least 92%, at least 93%, at least 94%, at least 95%,
at least 96%, at least 97%, at least 98%, at least 99% or 100%
pure.
[0083] Also provided by the invention are: [0084] A TCR, nucleic
acid or cell of the invention for use in medicine, preferably for
use in a method of treating cancer, such as solid tumours (e.g.,
lung, liver and gastric metastases) and/or squamous cell
carcinomas. [0085] the use of a TCR, nucleic acid or cell of the
invention in the manufacture of a medicament for treating cancer.
[0086] a method of treating cancer in a patient, comprising
administering to the patient a TCR, nucleic acid or cell of the
invention.
[0087] Preferred features of each aspect of the invention are as
for each of the other aspects mutatis mutandis. The prior art
documents mentioned herein are incorporated to the fullest extent
permitted by law.
[0088] The invention is further described in the following
non-limiting examples.
[0089] Reference is made to the enclosed sequences, in which:
[0090] SEQ ID NO 1 is the MAGE B2 peptide
[0091] SEQ ID NO 2 is the MAGE A4 peptide
[0092] SEQ ID No: 3 is the amino acid sequence of the extracellular
part of the alpha chain of a parental MAGE-A4-specific TCR, and SEQ
ID No: 4 shows_the amino acid sequence of the extracellular part of
the beta chain of a parental MAGE-A4-specific TCR beta chain amino
acid sequence.
[0093] SEQ ID No: 5 shows the amino acid sequence of the alpha
chain of a native Lenti TCR (referred to herein as the "reference
TCR"). The sequence is the same as that of The parental TCR except
that a cysteine is substituted for T162 (i.e. T48 of the TRAC
constant region). SEQ ID No: 6 is the beta chain of a native Lenti
TCR (referred to herein as the "reference TCR). The sequence is the
same as that of the parental TCR except that a_cysteine is
substituted for S169 (i.e. S57 of the TRBC2 constant region) and
A187 is substituted for C187 and D201 is substituted for N201.
[0094] SEQ ID No: 7 show the sequence of alpha chains which may be
present in TCRs of the invention. The subsequences forming the CDR
regions, or substantial parts of the CDR regions, are
underlined.
[0095] SEQ ID Nos: 8, 9 and 10 show the sequence of the beta chain
which may be present in TCRs of the invention. The subsequences
forming the CDR regions, or substantial parts of the CDR regions
are underlined.
[0096] SEQ ID Nos: 11-16 show the sequences of TCRs which could not
be improved by selection and mutation in accordance with the
present invention.
EXAMPLES
Example 1--Cloning of the Reference MAGE-A4 TCR Alpha and Beta
Chain Variable Region Sequences into pGMT7-Based Expression
Plasmids
[0097] The parental MAGE-A4 TCR variable alpha and TCR variable
beta domains of SEQ ID NOS: 3 and 4 respectively were cloned into
pGMT7-based expression plasmids containing either C.alpha. or
C.beta. by standard methods described in (Molecular Cloning a
Laboratory Manual Third edition by Sambrook and Russell). Plasmids
were sequenced using an Applied Biosystems 3730x1 DNA Analyzer. The
reference MAGE-A4 TCR variable alpha and TCR variable beta domains
of SEQ ID NOS: 4 and 5 respectively were cloned in the same
way.
[0098] The DNA sequence encoding the TCR alpha chain variable
region was ligated into pEX956, which was cut with restriction
enzymes. The DNA sequence encoding the TCR beta chain variable
region was ligated into pEXb21, which was also cut with restriction
enzymes.
[0099] Ligated plasmids were transformed into competent E. coli
strain XL1-blue cells and plated out on LB/agar plates containing
100 .mu.g/mL ampicillin. Following incubation overnight at
37.degree. C., single colonies were picked and grown in 5 mL LB
containing 100 .mu.g/mL ampicillin overnight at 37.degree. C. with
shaking. Cloned plasmids were purified using a Miniprep kit
(Qiagen) and the plasmids were sequenced using an Applied
Biosystems 3730x1 DNA Analyzer.
Example 2--Expression, Refolding and Purification of Soluble
Reference MAGE-A4 TCR
[0100] The expression plasmids containing the reference TCR
.alpha.-chain and .beta.-chain respectively, as prepared in Example
1, were transformed separately into E. coli strain BL21pLysS, and
single ampicillin-resistant colonies were grown at 37.degree. C. in
TYP (ampicillin 100 .mu.g/ml) medium to OD.sub.600 of -0.6-0.8
before inducing protein expression with 0.5 mM IPTG. Cells were
harvested three hours post-induction by centrifugation for 30
minutes at 4000 rpm in a Beckman J-6B. Cell pellets were lysed with
25 ml Bug Buster (NovaGen) in the presence of MgCl.sub.2 and
DNaseI. Inclusion body pellets were recovered by centrifugation for
30 minutes at 13000 rpm in a Beckman J2-21 centrifuge. Three
detergent washes were then carried out to remove cell debris and
membrane components. Each time the inclusion body pellet was
homogenised in a Triton buffer (50 mM Tris-HCl pH 8.0, 0.5%
Triton-X100, 200 mM NaCl, 10 mM NaEDTA,) before being pelleted by
centrifugation for 15 minutes at 13000 rpm in a Beckman J2-21.
Detergent and salt was then removed by a similar wash in the
following buffer: 50 mM Tris-HCl pH 8.0, 1 mM NaEDTA. Finally, the
inclusion bodies were divided into 30 mg aliquots and frozen at
-70.degree. C. Inclusion body protein yield was quantified by
solubilising with 6 M guanidine-HCl and an OD measurement was taken
on a Hitachi U-2001 Spectrophotometer. The protein concentration
was then calculated using the extinction coefficient.
[0101] Approximately 15 mg of TCR .alpha. chain and 15 mg of TCR
.beta. chain solubilised inclusion bodies were thawed from frozen
stocks and diluted into 10 ml of a guanidine solution (6 M
Guanidine-hydrochloride, 50 mM Tris HCl pH 8.1, 100 mM NaCl, 10 mM
EDTA, 10 mM DTT), to ensure complete chain denaturation. The
guanidine solution containing fully reduced and denatured TCR
chains was then injected into 0.5 litre of the following refolding
buffer: 100 mM Tris pH 8.1, 400 mM L-Arginine, 2 mM EDTA, 5 M Urea.
The redox couple (cysteamine hydrochloride and cystamine
dihydrochloride) to final concentrations of 6.6 mM and 3.7 mM
respectively, were added approximately 5 minutes before addition of
the denatured TCR chains. The solution was left for .about.30
minutes. The refolded TCR was dialysed in Spectrapor 1 membrane
(Spectrum; Product No. 132670) against 10 L H.sub.2O for 18-20
hours. After this time, the dialysis buffer was changed twice to
fresh 10 mM Tris pH 8.1 (10 L) and dialysis was continued at
5.degree. C. 3.degree. C. for another .about.8 hours.
[0102] Soluble TCR was separated from degradation products and
impurities by loading the dialysed refold onto a POROS 50HQ anion
exchange column and eluting bound protein with a gradient of 0-500
mM NaCl in 10 mM Tris pH 8.1 over 50 column volumes using an Akta
purifier (GE Healthcare). Peak fractions were pooled and a cocktail
of protease inhibitors (Calbiochem) were added. The pooled
fractions were then stored at 4.degree. C. and analysed by
Coomassie-stained SDS-PAGE before being pooled and concentrated.
Finally, the soluble TCR was purified and characterised using a GE
Healthcare Superdex 75HR gel filtration column pre-equilibrated in
PBS buffer (Sigma). The peak eluting at a relative molecular weight
of approximately 50 kDa was pooled and concentrated prior to
characterisation by BIAcore surface plasmon resonance analysis.
Example 3--Binding Characterisation
BIAcore Analysis
[0103] A surface plasmon resonance biosensor (BIAcore 3000.TM.) can
be used to analyse the binding of a soluble TCR to its peptide-MHC
ligand. This is facilitated by producing soluble biotinylated
peptide-HLA ("pHLA") complexes which can be immobilised to a
streptavidin-coated binding surface (sensor chip). The sensor chips
comprise four individual flow cells which enable simultaneous
measurement of T-cell receptor binding to four different pHLA
complexes. Manual injection of pHLA complex allows the precise
level of immobilised class I molecules to be manipulated
easily.
[0104] Biotinylated class I HLA-A*0201 molecules were refolded in
vitro from bacterially-expressed inclusion bodies containing the
constituent subunit proteins and synthetic peptide, followed by
purification and in vitro enzymatic biotinylation (O'Callaghan et
al. (1999) Anal. Biochem. 266: 9-15). HLA-A*0201-heavy chain was
expressed with a C-terminal biotinylation tag which replaces the
transmembrane and cytoplasmic domains of the protein in an
appropriate construct. Inclusion body expression levels of
.about.75 mg/litre bacterial culture were obtained. The MEW
light-chain or .beta.2-microglobulin was also expressed as
inclusion bodies in E. coli from an appropriate construct, at a
level of .about.500 mg/litre bacterial culture.
[0105] E. coli cells were lysed and inclusion bodies are purified
to approximately 80% purity. Protein from inclusion bodies was
denatured in 6 M guanidine-HCl, 50 mM Tris pH 8.1, 100 mM NaCl, 10
mM DTT, 10 mM EDTA, and was refolded at a concentration of 30
mg/litre heavy chain, 30 mg/litre .beta.2m into 0.4 M L-Arginine,
100 mM Tris pH 8.1, 3.7 mM cystamine dihydrochloride, 6.6 mM
cysteamine hydrochloride, 4 mg/L of the MAGE-A4 GVYDGREHTV or
MAGE-B2 GVYDGEEHSV peptide required to be loaded by the HLA-A*02
molecule, by addition of a single pulse of denatured protein into
refold buffer at <5.degree. C. Refolding was allowed to reach
completion at 4.degree. C. for at least 1 hour.
[0106] Buffer was exchanged by dialysis in 10 volumes of 10 mM Tris
pH 8.1. Two changes of buffer were necessary to reduce the ionic
strength of the solution sufficiently. The protein solution was
then filtered through a 1.5 .mu.m cellulose acetate filter and
loaded onto a POROS 50HQ anion exchange column (8 ml bed volume).
Protein was eluted with a linear 0-500 mM NaCl gradient in 10 mM
Tris pH 8.1 using an Akta purifier (GE Healthcare).
HLA-A*0201-peptide complex eluted at approximately 250 mM NaCl, and
peak fractions were collected, a cocktail of protease inhibitors
(Calbiochem) was added and the fractions were chilled on ice.
[0107] Biotin-tagged pHLA molecules were buffer exchanged into 10
mM Tris pH 8.1, 5 mM NaCl using a GE Healthcare fast desalting
column equilibrated in the same buffer. Immediately upon elution,
the protein-containing fractions were chilled on ice and protease
inhibitor cocktail (Calbiochem) was added. Biotinylation reagents
were then added: 1 mM biotin, 5 mM ATP (buffered to pH 8), 7.5 mM
MgCl.sub.2, and 5 .mu.g/ml BirA enzyme (purified according to
O'Callaghan et al. (1999) Anal. Biochem. 266: 9-15). The mixture
was then allowed to incubate at room temperature overnight.
[0108] The biotinylated pHLA-A*0201 molecules were purified using
gel filtration chromatography. A GE Healthcare Superdex 75 HR 10/30
column was pre-equilibrated with filtered PBS and 1 ml of the
biotinylation reaction mixture was loaded and the column was
developed with PBS at 0.5 ml/min using an Akta purifier (GE
Healthcare). Biotinylated pHLA-A*0201 molecules eluted as a single
peak at approximately 15 ml. Fractions containing protein were
pooled, chilled on ice, and protease inhibitor cocktail was added.
Protein concentration was determined using a Coomassie-binding
assay (PerBio) and aliquots of biotinylated pHLA-A*01 molecules
were stored frozen at -20.degree. C.
[0109] Such immobilised complexes are capable of binding both
T-cell receptors and the coreceptor CD8.alpha..alpha., both of
which may be injected in the soluble phase. The pHLA binding
properties of soluble TCRs are observed to be qualitatively and
quantitatively similar if the TCR is used either in the soluble or
immobilised phase. This is an important control for partial
activity of soluble species and also suggests that biotinylated
pHLA complexes are biologically as active as non-biotinylated
complexes.
[0110] The BIAcore 3000.TM. surface plasmon resonance (SPR)
biosensor measures changes in refractive index expressed in
response units (RU) near a sensor surface within a small flow cell,
a principle that can be used to detect receptor ligand interactions
and to analyse their affinity and kinetic parameters. The BIAcore
experiments were performed at a temperature of 25.degree. C., using
PBS buffer (Sigma, pH 7.1-7.5) as the running buffer and in
preparing dilutions of protein samples. Streptavidin was
immobilised to the flow cells by standard amine coupling methods.
The pHLA complexes were immobilised via the biotin tag. The assay
was then performed by passing soluble TCR over the surfaces of the
different flow cells at a constant flow rate, measuring the SPR
response in doing so.
Equilibrium Binding Constant
[0111] The above BIAcore analysis methods were used to determine
equilibrium binding constants. Serial dilutions of the disulfide
linked soluble heterodimeric form of the reference MAGE-A4 TCR were
prepared and injected at constant flow rate of 5 .mu.l min.sup.-1
over two different flow cells; one coated with .about.1000 RU of
specific GVYDGREHTV HLA-A*0201 complex, the second coated with
.about.1000 RU of non-specific complex. Response was normalised for
each concentration using the measurement from the control cell.
Normalised data response was plotted versus concentration of TCR
sample and fitted to a non-linear curve fitting model in order to
calculate the equilibrium binding constant, K.sub.D. (Price &
Dwek, Principles and Problems in Physical Chemistry for Biochemists
(2.sup.nd Edition) 1979, Clarendon Press, Oxford). The disulfide
linked soluble form of the reference MAGE-A4 TCR (Example 2)
demonstrated a K.sub.D of approximately 2.00 .mu.M. From the same
BIAcore data the T1/2 was approximately 0.95 s.
Kinetic Parameters
[0112] The above BIAcore analysis methods were also used to
determine equilibrium binding constants and off-rates.
[0113] For high affinity TCRs (see Example 4 below) K.sub.D was
determined by experimentally measuring the dissociation rate
constant, k.sub.off, and the association rate constant, k.sub.on.
The equilibrium constant K.sub.D was calculated as
k.sub.off/k.sub.on.
[0114] TCR was injected over two different cells one coated with
.about.1000 RU of specific GVYDGREHTV HLA-A*0201 complex, the
second coated with .about.1000 RU of non-specific complex. Flow
rate was set at 50 .mu.l/min. Typically 250 .mu.l of TCR at
.about.1 .mu.M concentration was injected. Buffer was then flowed
over until the response had returned to baseline or >2 hours had
elapsed. Kinetic parameters were calculated using BIAevaluation
software. The dissociation phase was fitted to a single exponential
decay equation enabling calculation of half-life.
Example 4--Preparation of High Affinity TCRs of the Invention
[0115] Expression plasmids containing the TCR .alpha.-chain and
.beta.-chain respectively were prepared as in Example 1:
TABLE-US-00004 TCR ID Alpha Chain SEQ ID No Beta Chain SEQ ID No
TCR1 (parental) 3 4 TCR2 958 7i3r 4 TCR3 962 3 8h3r TCR4 1028 3
9N10R TCR5 1030 7i3r 9n10r TCR6 1034 3 10N10K
[0116] The plasmids were transformed separately into E. coli strain
BL21pLysS, and single ampicillin-resistant colonies grown at
37.degree. C. in TYP (ampicillin 100 .mu.g/ml) medium to OD.sub.600
of .about.0.6-0.8 before inducing protein expression with 0.5 mM
IPTG. Cells were harvested three hours post-induction by
centrifugation for 30 minutes at 4000 rpm in a Beckman J-6B. Cell
pellets were lysed with 25 ml Bug Buster (Novagen) in the presence
of MgCl.sub.2 and DNaseI. Inclusion body pellets were recovered by
centrifugation for 30 minutes at 13000 rpm in a Beckman J2-21
centrifuge. Three detergent washes were then carried out to remove
cell debris and membrane components. Each time the inclusion body
pellet was homogenised in a Triton buffer (50 mM Tris-HCl pH 8.0,
0.5% Triton-X100, 200 mM NaCl, 10 mM NaEDTA,) before being pelleted
by centrifugation for 15 minutes at 13000 rpm in a Beckman J2-21.
Detergent and salt was then removed by a similar wash in the
following buffer: 50 mM Tris-HCl pH 8.0, 1 mM NaEDTA. Finally, the
inclusion bodies were divided into 30 mg aliquots and frozen at
-70.degree. C. Inclusion body protein yield was quantified by
solubilising with 6 M guanidine-HCl and an OD measurement was taken
on a Hitachi U-2001 Spectrophotometer. The protein concentration
was then calculated using the extinction coefficient.
[0117] Approximately 10 mg of TCR .alpha. chain and 10 mg of TCR
.beta. chain solubilised inclusion bodies for each TCR of the
invention were diluted into 10 ml of a guanidine solution (6 M
Guanidine-hydrochloride, 50 mM Tris HCl pH 8.1, 100 mM NaCl, 10 mM
EDTA, 10 mM DTT), to ensure complete chain denaturation. The
guanidine solution containing fully reduced and denatured TCR
chains was then injected into 0.5 litre of the following refolding
buffer: 100 mM Tris pH 8.1, 400 mM L-Arginine, 2 mM EDTA, 5 M Urea.
The redox couple (cysteamine hydrochloride and cystamine
dihydrochloride) to final concentrations of 6.6 mM and 3.7 mM
respectively, were added approximately 5 minutes before addition of
the denatured TCR chains. The solution was left for .about.30
minutes. The refolded TCR was dialysed in Spectrapor 1 membrane
(Spectrum; Product No. 132670) against 10 L H.sub.2O for 18-20
hours. After this time, the dialysis buffer was changed twice to
fresh 10 mM Tris pH 8.1 (10 L) and dialysis was continued at
5.degree. C. 3.degree. C. for another .about.8 hours.
[0118] Soluble TCR was separated from degradation products and
impurities by loading the dialysed refold onto a POROS 50HQ anion
exchange column and eluting bound protein with a gradient of 0-500
mM NaCl in 10 mM Tris pH 8.1 over 15 column volumes using an Akta
purifier (GE Healthcare). The pooled fractions were then stored at
4.degree. C. and analysed by Coomassie-stained SDS-PAGE before
being pooled and concentrated. Finally, the soluble TCRs were
purified and characterised using a GE Healthcare Superdex 75HR gel
filtration column pre-equilibrated in PBS buffer (Sigma). The peak
eluting at a relative molecular weight of approximately 50 kDa was
pooled and concentrated prior to characterisation by BIAcore
surface plasmon resonance analysis.
[0119] The affinity profiles of the thus-prepared TCRs for the
MAGE-A4 epitope or MAGE-B2 epitope were assessed using the method
of Example 3, and compared with the reference TCR. The results are
set forth in the following table:
TABLE-US-00005 MAGE A4 MAGE-B2 K.sub.D (.mu.M) K.sub.D (.mu.M)
Reference (TCR1) 65.1 17 TCR2 208.4 17.8 TCR3 195.5 16.61 TCR4
183.3 11.27 TCR5 484.0 18.77 TCR6 305.3 6.03
[0120] Attempts were also made to prepare high affinity TCRs based
on combinations of SEQ ID Nos 11/12, 13/14, 15/16.
[0121] In the case of TCR A, which combines alpha chain of SEQ ID
NO 11 and the Beta chain of SEQ ID NO 12, cross-reactivity was
noted between MAGE-A1, MAGE-A10 and PRAME. It was not possible to
remove this cross-reactivity by mutation and selection.
[0122] TCR B combines the alpha chain of SEQ ID NO 12 and the Beta
chain of SEQ ID NO 14. TCR B could not be folded to form a soluble
TCR, so no binding characterisation was possible.
[0123] TCR C combines the alpha chain of SEQ ID NO 15 and the Beta
chain of SEQ ID No 16. This TCR was soluble when expressed and
could bid to antigen. However, when expressed in T-cells, TCR C
showed no activity.
Example 5--Transfection of T-Cells with Parental and Variant
MAGE-A4 TCRs
(a) Lentiviral Vector Preparation by Express-in Mediated Transient
Transfection of 293T Cells
[0124] A 3rd generation lentiviral packaging system was used to
package lentiviral vectors containing the gene encoding the desired
TCR. 293T cells were transfected with 4 plasmids (one lentiviral
vector containing the TCR alpha chain-P2A-TCR beta chain single ORF
gene described in Example 5c (below), and 3 plasmids containing the
other components necessary to construct infective but
non-replicative lentiviral particles) using Express-In mediated
transfection (Open Biosystems).
[0125] For transfection one T150 flask of 293T cells in exponential
growth phase was taken, with cells evenly distributed on the plate,
and slightly more than 50% confluent. Express-In aliquots were
brought to room temperature. 3 ml Serum-Free Medium (RPMI 1640+10
mM HEPES) were placed in a sterile 15 ml conical tube. 174 .mu.l of
Express-In Reagent were added directly into the Serum-Free Medium
(this provides for a 3.6:1 weight ratio of Reagent to DNA). This
was mixed thoroughly by inverting tubes 3-4 times and incubated at
room temperature for 5-20 minutes.
[0126] In a separate 1.5 ml microtube was added 15 .mu.g plasmid
DNA to premixed packaging mix aliquots (containing 18 .mu.g
pRSV.REV (Rev expression plasmid), 18 .mu.g pMDLg/p.RRE (Gag/Pol
expression plasmid), 7 .mu.g pVSV-G (VSV glycoprotein expression
plasmid), usually .about.22 and pipetted up and down to ensure
homogeneity of the DNA mix. Approx 1 mL of Express-In/Serum-Free
Medium was added to the DNA mix dropwise then pipetted up and down
gently before transferring back to the remainder of the
Express-In/Serum-Free Medium. The tube was inverted ube 3-4 times
and incubated at room temperature for 15-30 minutes. Old culture
medium was removed from the flask of cells. Express-In/medium/DNA
(3 mL) complex was added directly into the bottom of an upright
flask of 293T cells. Slowly, the flask was placed flat to cover the
cells and very gently rocked to ensure even distribution. After 1
minute 22 ml fresh culture medium (R10+HEPES: RPMI 1640, 10%
heat-inactivated FBS, 1% Pen/Strep/L-glutamine, 10 mM HEPES) was
added and the flask carefully returned to the incubator. This was
incubated overnight at 37.degree. C./5% CO2. After 24 hours, the
medium containing packaged lentiviral vectors was harvested.
[0127] To harvest the packaged lentiviral vectors, the cell culture
supernatant was filtered through a 0.45 micron nylon syringe
filter, the culture medium centrifuged at 10,000 g for 18 hours (or
112,000 g for 2 hours), most of the supernatant removed (taking
care not to disturb the pellet) and the pellet resuspended in the
remaining few mL of supernatant (usually about 2 ml from a 31 ml
starting volume per tube). This was snap frozen on dry ice in 1 ml
aliquots and stored at -80.degree. C.
(b) Transduction of T Cells with Packaged Lentiviral Vectors
Containing Gene of Interest
[0128] Prior to transduction with the packaged lentiviral vectors,
human T cells (CD8 or CD4 or both depending on requirements) were
isolated from the blood of healthy volunteers. These cells were
counted and incubated overnight in R10 containing 50 U/mL IL-2 at
1.times.10.sup.6 cells per ml (0.5 mL/well) in 48 well plates with
pre-washed anti-CD3/CD28 antibody-coated microbeads (Dynabeads.RTM.
T cell expander, Invitrogen) at a ratio of 3 beads per cell.
[0129] After overnight stimulation, 0.5 ml of neat packaged
lentiviral vector was added to the desired cells. This was
incubated at 37.degree. C./5% CO2 for 3 days. 3 days
post-transduction the cells were counted and diluted to
0.5.times.10.sup.6 cells/ml. Fresh medium containing IL-2 was added
as required. Beads were removed 5-7 days post-transduction. Cells
were counted and fresh medium containing IL-2 replaced or added at
2 day intervals. Cells were kept between 0.5.times.10.sup.6 and
1.times.10.sup.6 cells/mL. Cells were analysed by flow cytometry
from day 3 and used for functional assays (e.g. ELISpot for
IFN.gamma. release, see Example 6) from day 5. From day 10, or when
cells are slowing division and reduced in size, cells are frozen in
aliquots of at least 4.times.10.sup.6 cells/vial (at
1.times.10.sup.7 cells/ml in 90% FBS/10% DMSO) for storage.
Example 6--Activation of MAGE B2 TCR Engineered T Cells
[0130] The following assay was carried out to demonstrate the
activation of TCR-transduced cytotoxic T lymphocytes (CTLs) in
response to tumour cell lines. IFN-.gamma. production, as measured
using the ELISPOT assay, was used as a read-out for cytotoxic T
lymphocyte (CTL) activation.
ELISPOTs
Reagents
[0131] Assay media: 10% FCS (Gibco, Cat#2011-09), 88% RPMI 1640
(Gibco, Cat#42401), 1% glutamine (Gibco Cat#25030) and 1%
penicillin/streptomycin (Gibco Cat#15070-063).
[0132] Wash buffer: 0.01M PBS/0.05% Tween 20
[0133] PBS (Gibco Cat#10010)
[0134] The Human IFN.gamma. ELISPOT kit (BD Bioscience; Cat#551849)
containing capture and detection antibodies and Human IFN-.gamma.
PVDF ELISPOT 96 well plates, with associated AEC substrate set (BD
Bioscience, Cat#551951)
Methods
Target Cell Preparation
[0135] The target cells used in this method were natural
epitope-presenting cells: A375 human melanoma cells which are both
HLA-A2.sup.+MAGE A10.sup.+. HCT116 human colon cancer, which are
HLA-A2.sup.+MAGE A10.sup.+, were used as a negative control.
Sufficient target cells (50,000 cells/well) were washed by
centrifugation three times at 1200 rpm, 10 min in a Megafuge.RTM.
1.0 (Heraeus). Cells were then re-suspended in assay media at
10.sup.6 cells/ml.
Effector Cell Preparation
[0136] The effector cells (T cells) used in this method were
peripheral blood lymphocytes (PBL), obtained by negative selection
using CD14 and CD25 microbead kits (Miltenyi Biotech
Cat#130-050-201 and 130-092-983 respectively) from freshly isolated
peripheral blood mononuclear cells (PBMC) from the venous blood of
healthy volunteers. Cells were stimulated with antiCD3/CD28 coated
beads (Dynabeads.RTM. T cell expander, Invitrogen), transduced with
lentivirus carrying the gene encoding the full .alpha..beta. TCR of
interest (based on the construct described in Example 5) and
expanded in assay media containing 50 U/mL IL-2 until between 10
and 13 days post transduction. These cells were then placed in
assay media prior to washing by centrifugation at 1200 rpm, 10 min
in a Megafuge.RTM. 1.0 (Heraeus). Cells were then re-suspended in
assay media at a 4.times. the final required concentration.
[0137] Plates were prepared as follows: 100 .mu.L anti-IFN-.gamma.
capture antibody was diluted in 10 ml sterile PBS per plate. 100
.mu.L of the diluted capture antibody was then dispensed into each
well. The plates were then incubated overnight at 4.degree. C.
Following incubation the plates were washed (programme 1, plate
type 2, Ultrawash Plus 96-well plate washer; Dynex) to remove the
capture antibody. Plates were then blocked by adding 200 .mu.L of
assay media to each well and incubated at room temperature for two
hours. The assay media was then washed from the plates (programme
1, plate type 2, Ultrawash Plus 96-well plate washer, Dynex) and
any remaining media was removed by flicking and tapplng the ELISPOT
plates on a paper towel.
[0138] The constituents of the assay were then added to the ELISPOT
plate in the following order:
[0139] 50 .mu.L of target cells 10.sup.6 cells/ml (giving a total
of 50,000 target cells/well)
[0140] 50 .mu.L media (assay media)
[0141] 50 .mu.L effector cells (20,000 TCR-transduced PBL
cells/well)
[0142] The plates were then incubated overnight (37.degree. C./5%
CO2). The next day the plates were washed three times (programme 1,
plate type 2, Ultrawash Plus 96-well plate washer, Dynex) with wash
buffer and tapped dry on paper towel to remove excess wash buffer.
100 .mu.l of primary detection antibody was then added to each
well. The primary detection antibody was diluted into 10 mL of
dilution buffer (the volume required for a single plate) using the
dilution specified in the manufacturer's instructions. Plates were
then incubated at room temperature for at least 2 hours prior to
being washed three times (programme 1, plate type 2, Ultrawash Plus
96-well plate washer, Dynex) with wash buffer; excess wash buffer
was removed by tapplng the plate on a paper towel.
[0143] Secondary detection was performed by adding 100 .mu.L of
diluted streptavidin-HRP to each well and incubating the plate at
room temperature for 1 hour. The streptavidin-HRP was diluted into
10 mL dilution buffer (the volume required for a single plate),
using the dilution specified in the manufacturer's instructions.
The plates were then washed three times (programme 1, plate type 2,
Ultrawash Plus 96-well plate washer, Dynex) with wash buffer and
tapped on paper towel to remove excess wash buffer. Plates were
then washed twice with PBS by adding 200 .mu.L to each well,
flicking the buffer off and tapplng on a paper towel to remove
excess buffer. No more than 15 min prior to use, one drop (20 uL)
of AEC chromogen was added to each 1 ml of AEC substrate and mixed.
10 ml of this solution was prepared for each plate; 100 .mu.L was
added per well. The plate was then protected from light using foil,
and spot development monitored regularly, usually occurring within
5-20 min. The plates were washed in tap water to terminate the
development reaction, and shaken dry prior to their disassembly
into three constituent parts. The plates were then allowed to dry
at room temperature for at least 2 hours prior to counting the
spots using an Immunospot.RTM. Plate reader (CTL; Cellular
Technology Limited).
Example 7--Identification of the Binding Motif by Substitution with
all Alternative Amino Acids
[0144] Variants of the native MAGE-B2 peptide were obtained in
which the amino acid residue at each position was sequentially
replaced with all 19 alternative naturally-occurring amino acid,
such that 171 peptides were prepared in total. The native and
amino-acid substituted peptides were pulsed on to antigen
presenting cells, and interferon .gamma. (IFN.gamma.) production,
as measured using the ELISpot assay, used as a read-out for the
activation of T cells transduced with TCR1. Essential positions
were defined by a greater than 50% reduction in T cell activity
relative to the native peptide.
[0145] ELISpot assays were carried as described in Example 6.
[0146] The tolerated residues at each position of the peptide are
shown below. Underlined amino acids represent the native residue at
the corresponding position in the peptide.
TABLE-US-00006 Position Tolerated residues 1 G 2 V 3 FY 4 D 5 GN 6
DAES 7 YSWTFQMHPLANGDICE 8 FWVLMAYRKCTIQSHGPN 9 TVIASPGKEQNH 10
FMVAI
[0147] It is therefore apparent that the MAGE B2 TCR4 makes contact
with at least V2 Y3 and D4 of the peptide (SEQ ID No: 1) when in
complex with HLA-A*0201 on the surface of antigen presenting
cells.
[0148] The invention is further described by the following numbered
paragraphs:
[0149] 1. A T cell receptor (TCR) having the property of binding to
GVYDGEEHSV (SEQ ID No: 1) in complex with HLA-A*0201 with a
dissociation constant of from about 0.05 .mu.M to about 20.0 .mu.M
when measured with surface plasmon resonance at 25.degree. C. and
at a pH between 7.1 and 7.5 using a soluble form of the TCR, and
has at least a ten-fold selectivity of binding to SEQ ID No:1 in
complex with HLA-A*0201 over binding to GVYDGREHTV (SEQ ID No 2) in
complex with HLA-A*0201 wherein the TCR comprises a TCR alpha chain
variable domain and a TCR beta chain variable domain, and wherein
the TCR variable domains form contacts with at least residues V2,
Y3 and D4 of GVYDGEEHSV (SEQ ID No: 1).
[0150] 2. A TCR according to numbered paragraph 1, which is an
alpha-beta heterodimer, having an alpha chain TRAV10+TRAC constant
domain sequence and a beta chain TRBV24-1+TRBC-2 constant domain
sequence.
[0151] 3. A TCR as claimed in numbered paragraph 1, which is in
single chain format of the type V.alpha.-L-V.beta.,
V.beta.-L-V.alpha., V.alpha.-C.alpha.-L-V.beta., or
V.alpha.-L-V.beta.-C.beta., wherein V.alpha. and V.beta. are TCR
.alpha. and .beta. variable regions respectively, C.alpha. and
C.beta. are TCR .alpha. and .beta. constant regions respectively,
and L is a linker sequence.
[0152] 4. A TCR as claimed in any preceding numbered paragraph,
which is associated with a detectable label, a therapeutic agent or
a PK modifying moiety.
[0153] 5. A TCR as claimed in any preceding numbered paragraph,
wherein the alpha chain variable domain comprises an amino acid
sequence that has at least 80% identity to the sequence of amino
acid residues 1-105 of SEQ ID No: 3 and has the following
mutation:
TABLE-US-00007 CDR2 I3 R
with reference to the numbering shown in SEQ ID No: 3, and/or the
beta chain variable domain comprises an amino acid sequence that
has at least 80% identity to the sequence of amino acid residues
1-105 of SEQ ID No: 5 and has at least one of the following
mutations:
TABLE-US-00008 CDR1 H3 R CDR3 N10 E CDR3 N10 R
with reference to the numbering shown in SEQ ID No: 4.
[0154] 6. A TCR as claimed in any preceding numbered paragraph,
wherein the alpha chain variable domain comprises the amino acid
sequence of amino acid residues 1-105 of SEQ ID No: 3 or 7 or 8 or
[0155] an amino acid sequence in which amino acid residues 1-27,
34-47, and 54-90 thereof have at least 90% or 95% identity to the
sequence of amino acid residues 1-27, 34-47, and 54-90 respectively
of SEQ ID No: 3 or 7 or 8 and in which amino acid residues 28-34,
48-53 and 91-105 have at least 90% or 95% identity to the sequence
of amino acid residues 28-33, 48-53 and 91-105 respectively of SEQ
ID No 3 or 7 or 8.
[0156] 7. A TCR as claimed in any one of numbered paragraphs 1-7,
wherein the alpha chain variable domain comprises the amino acid
sequence of amino acid residues 1-105 of SEQ ID No: 3 or 7 or an
amino acid sequence in which amino acid residues 1-27, 34-47 and
55-89 thereof have at least 90% or 95% identity to the sequence of
amino acid residues 1-27, 34-47, and 55-89 respectively of SEQ ID
No: 3 or 7 and in which amino acid residues 28-33, 48-53 and 91-105
have at least 90% or 95% identity to the sequence of amino acid
residues 28-33, 48-53 and 91-105 respectively of SEQ ID No: 3 or
7.
[0157] 8. A TCR as claimed in any preceding numbered paragraph,
wherein in the alpha chain variable domain the sequence of [0158]
(i) amino acid residues 1-27 thereof has (a) at least 90% identity
to the sequence of amino acid residues 1-26 of SEQ ID No: 3 or (b)
has one, two or three amino acid residues inserted or deleted
relative to the sequence of (a); [0159] (ii) amino acid residues
28-33 is VSPFSN; [0160] (iii) amino acid residues 34-47 thereof has
(a) at least 90% identity to the sequence of amino acid residues
34-47 of SEQ ID NO: 3 or (b) has one, two or three amino acid
residues inserted or deleted relative to the sequence of (a);
[0161] (iv) amino acid residues 48-53 is LTIMTF or LTRMTF [0162]
(v) amino acid residues 54-90 thereof has at least 90% identity to
the sequence of amino acid residues 55-89 of SEQ ID No: 3 or has
one, two or three insertions, deletions or substitutions relative
thereto; [0163] (vi) amino acids 91-105 is CVVSGGTDSWGKLQF
[0164] 9. A TCR as claimed in any preceding numbered paragraph,
wherein the beta chain variable domain comprises the amino acid
sequence of SEQ ID No: 4 or 8-10 or an amino acid sequence in which
amino acid residues 1-45, 51-67, and 74-109 thereof have at least
90% or 95% identity to the sequence of amino acid residues 1-45,
51-67, and 74-109 respectively of SEQ ID No: 4 or 8-10 and in which
amino acid residues 46-50, 68-73 and 109-123 have at least 90% or
95% identity to the sequence of amino acid residues 46-50, 68-73
and 109-123 respectively of SEQ ID No: 4 or 8-10.
[0165] 10. A TCR according to any preceding numbered paragraph,
wherein in the beta chain variable domain the sequence of [0166]
(i) amino acid residues 1-45 thereof has (a) at least 90% identity
to the amino acid sequence of residues 1-26 of SEQ ID No: 4 or (b)
has one, two or three amino acid residues inserted or deleted
relative to the sequence of (a); [0167] (ii) amino acid residues
46-50 is KGRDR or KGRDR [0168] (iii) amino acid residues 51-67
thereof has (a) at least 90% identity to the sequence of amino acid
residues 51-67 of SEQ ID NO: 4 or (b) has one, two or three amino
acid residues inserted or deleted relative to the sequence of (a);
[0169] (iv) amino acid residues 68-73 is SFDVK; [0170] (v) amino
acid residues 54-90 thereof has (a) at least 90% identity to the
sequence of amino acid residues 54-90 of SEQ ID NO: 4 or (b) has
one, two or three amino acid residues inserted or deleted relative
to the sequence of (a); [0171] (vi) amino acids 109-123 is
CATSGQGAYNEQFF or CATSGQGAYREQFF or CATSGQGAYKEQFF
[0172] 11. Nucleic acid encoding a TCR as claimed in any one of the
preceding numbered paragraphs.
[0173] 12. An isolated or non-naturally occurring cell, especially
a T-cell, presenting a TCR as claimed in any one of numbered
paragraphs 1 to 12.
[0174] 13. A cell harbouring [0175] (a) a TCR expression vector
which comprises nucleic acid as claimed in numbered paragraph 13 in
a single open reading frame, or two distinct open reading frames
encoding the alpha chain and the beta chain respectively; or [0176]
(b) a first expression vector which comprises nucleic acid encoding
the alpha chain of a TCR as claimed in any of numbered paragraphs 1
to 12, and a second expression vector which comprises nucleic acid
encoding the beta chain of a TCR as claimed in any of numbered
paragraphs 1 to 12.
[0177] 14. A pharmaceutical composition comprising a TCR as claimed
in any one of numbered paragraphs 1 to 10, nucleic acid of numbered
paragraph 11 or a cell as claimed in numbered paragraph 12 or
numbered paragraph 13, together with one or more pharmaceutically
acceptable carriers or excipients.
[0178] 15. The TCR of any one of numbered paragraphs 1 to 12,
nucleic acid of numbered paragraph 13 or cell of numbered paragraph
14 or numbered paragraph 15 for use in medicine.
[0179] 16. The TCR, nucleic acid or cell for use as claimed in
numbered paragraph 15, for use in a method of treating cancer.
TABLE-US-00009 MAGE B2 Epitope SEQ ID NO 1 GVYDGEEHSV MAGE A4
Epitope SEQ ID NO 2 GVVDGREHTV alpha variable chain SEQ ID NO 3
MKNQVEQSPQSLIILEGKNCTLQCNYTVSPFSNLRWYKQDTGRGPVSLTI
MTFSENTKSNGRYTATLDADTKQSSLHITASQLSDSASYICVVSGGTDSW GKLQF beta
variable chain SEQ ID NO 4
MASLLFFCGAFYLLGTGSMDADVTQTPRNRITKTGKRIMLECSQTKGHDR
MYWYRQDPGLGLRLIYYSFDVKDINKGEISDGYSVSRQAQAKFSLSLESA
IPNQTALYFCATSGQGAYNEQFF alpha chain soluble form SEQ ID NO 5
MKKHLTTFLVILWLYFYRGNGKNQVEQSPQSLIILEGKNCTLQCNYTVSP
FSNLRWYKQDTGRGPVSLTIMTFSENTKSNGRYTATLDADTKQSSLHITA
SQLSDSASYICVVSGGTDSWGKLQFGAGTQVVVTPDIQNPDPAVYQLRDS
KSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAW
SNKSDFACANAFNNSIIPEDTFFPSPESS beta chain soluble form SEQ ID No 6
MASLLFFCGAFYLLGTGSMDADVTQTPRNRITKTGKRIMLECSQTKGHDR
MYWYRQDPGLGLRLIYYSFDVKDINKGEISDGYSVSRQAQAKFSLSLESA
IPNQTALYFCATSGQGAYNEQFFGPGTRLTVLEDLKNVFPPEVAVFEPSE
AEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPA
LNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVT QIVSAEAWGRAD
mutant alpha variable chain SEQ ID NO 7
MKNQVEQSPQSLIILEGKNCTLQCNYTVSPFSNLRWYKQDTGRGPVSLTR
MTFSENTKSNGRYTATLDADTKQSSLHITASQLSDSASYICVVSGGTDSW GKLQF mutant
beta variable chain SEQ ID NO 8
MASLLFFCGAFYLLGTGSMDADVTQTPRNRITKTGKRIMLECSQTKGRDR
MYWYRQDPGLGLRLIYYSFDVKDINKGEISDGYSVSRQAQAKFSLSLESA
IPNQTALYFCATSGQGAYNEQFF mutant beta variable chain SEQ ID NO 9
MASLLFFCGAFYLLGTGSMDADVTQTPRNRITKTGKRIMLECSQTKGHDR
MYWYRQDPGLGLRLIYYSFDVKDINKGEISDGYSVSRQAQAKFSLSLESA
IPNQTALYFCATSGQGAYEEQFF mutant beta variable chain SEQ ID NO 10
MASLLFFCGAFYLLGTGSMDADVTQTPRNRITKTGKRIMLECSQTKGHDR
MYWYRQDPGLGLRLIYYSFDVKDINKGEISDGYSVSRQAQAKFSLSLESA
IPNQTALYFCATSGQGAYREQFF alpha chain soluble form SEQ ID NO 11
METLLGLLILWLQLQWVSSKQEVTQIPAALSVPEGENLVLNCSFTDSAIY
NLQWFRQDPGKGLTSLLLIQSSQREQTSGRLNASLDKSSGRSTLYIAASQ
PGDSATYLCAVGGYSTLTFGKGTVLLVSPDNIQNPDPAVYQLRDSKSSDK
SVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSD
FACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGF
RILLLKVAGFNLLMTLRLWSS beta chain soluble form SEQ ID No 12
MSISLLCCAAFPLLWAGPVNAGVTQTPKFRILKIGQSMTLQCAQDMNHNY
MYWYRQDPGMGLKLIYYSVGAGITDKGEVPNGYNVSRSTTEDFPLRLELA
APSQTSVYFCASSYSRWSPLHFGNGTRLTVTEDLNKVFPPEVAVFEPSEA
EISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVSTDPQPLKEQPAL
NDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQ
IVSAEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSALVLM AMVKRKDF alpha
chain soluble form SEQ ID NO 13
MQKEVEQNSGPLSVPEGAIASLNCTYSDRGSQSFFWYRQYSGKSPELIMF
IYSNGDKEDGRFTAQLNKASQYVSLLIRDSQPSDSATYLCAVKMANQAGT
ALIFGKGTTLSVSSNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQ
SKDSDVYITDKCVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDT FFPSPESS beta
chain soluble form SEQ ID No 14
MQDGGITQSPKFQVLRTGQSMTLLCAQDMNHEYMYWYRQDPGMGLRLIHY
SVGAGITDQGEVPNGYNVSRLNKREFSLRLESAAPSQTSVYFCASLGGLA
DEQFFGPGTRLTVLEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGF
YPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALNDSRYALSSRLRVSATF
WQDPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRAD alpha chain soluble
form SEQ ID NO 15
MKTFAGFSFLFLWLQLDCMSRGEDVEQSLFLSVREGDSSVINCTYTDSSS
TYLYWYKQEPGAGLQLLTYIFSNMDMKQDQRLTVLLNKKDKHLSLRIADT
QTGDSAIYFCAERNSGAGSYQLTFGKGTKLSVIPNIQNPDPAVYQLRDSK
SSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWS
NKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLS
VIGFRILLLKVAGFNLLMTLRLWSS beta chain soluble form SEQ ID No 16
MGTSLLCWMALCLLGADHADTGVSQNPRHKITKRGQNVTFRCDPISEHNR
LYWYRQTLGQGPEFLTYFQNEAQLEKSRLLSDRFSAERPKGSFSTLEIQR
TEQGDSAMYLCASSLFSGVNTEAFFGQGTRLTVVEDLNKVFPPEVAVFEP
SEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVSTDPQPLKEQ
PALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKP
VTQIVSAEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSAL VLMAMVKRKDF
Sequence CWU 1
1
28110PRTHomo sapiens 1Gly Val Tyr Asp Gly Glu Glu His Ser Val1 5
10210PRTHomo sapiens 2Gly Val Tyr Asp Gly Arg Glu His Thr Val1 5
103105PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polypeptide" 3Met Lys Asn Gln Val Glu Gln Ser
Pro Gln Ser Leu Ile Ile Leu Glu1 5 10 15Gly Lys Asn Cys Thr Leu Gln
Cys Asn Tyr Thr Val Ser Pro Phe Ser 20 25 30Asn Leu Arg Trp Tyr Lys
Gln Asp Thr Gly Arg Gly Pro Val Ser Leu 35 40 45Thr Ile Met Thr Phe
Ser Glu Asn Thr Lys Ser Asn Gly Arg Tyr Thr 50 55 60Ala Thr Leu Asp
Ala Asp Thr Lys Gln Ser Ser Leu His Ile Thr Ala65 70 75 80Ser Gln
Leu Ser Asp Ser Ala Ser Tyr Ile Cys Val Val Ser Gly Gly 85 90 95Thr
Asp Ser Trp Gly Lys Leu Gln Phe 100 1054123PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 4Met Ala Ser Leu Leu Phe Phe Cys Gly Ala Phe Tyr Leu
Leu Gly Thr1 5 10 15Gly Ser Met Asp Ala Asp Val Thr Gln Thr Pro Arg
Asn Arg Ile Thr 20 25 30Lys Thr Gly Lys Arg Ile Met Leu Glu Cys Ser
Gln Thr Lys Gly His 35 40 45Asp Arg Met Tyr Trp Tyr Arg Gln Asp Pro
Gly Leu Gly Leu Arg Leu 50 55 60Ile Tyr Tyr Ser Phe Asp Val Lys Asp
Ile Asn Lys Gly Glu Ile Ser65 70 75 80Asp Gly Tyr Ser Val Ser Arg
Gln Ala Gln Ala Lys Phe Ser Leu Ser 85 90 95Leu Glu Ser Ala Ile Pro
Asn Gln Thr Ala Leu Tyr Phe Cys Ala Thr 100 105 110Ser Gly Gln Gly
Ala Tyr Asn Glu Gln Phe Phe 115 1205229PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 5Met Lys Lys His Leu Thr Thr Phe Leu Val Ile Leu Trp
Leu Tyr Phe1 5 10 15Tyr Arg Gly Asn Gly Lys Asn Gln Val Glu Gln Ser
Pro Gln Ser Leu 20 25 30Ile Ile Leu Glu Gly Lys Asn Cys Thr Leu Gln
Cys Asn Tyr Thr Val 35 40 45Ser Pro Phe Ser Asn Leu Arg Trp Tyr Lys
Gln Asp Thr Gly Arg Gly 50 55 60Pro Val Ser Leu Thr Ile Met Thr Phe
Ser Glu Asn Thr Lys Ser Asn65 70 75 80Gly Arg Tyr Thr Ala Thr Leu
Asp Ala Asp Thr Lys Gln Ser Ser Leu 85 90 95His Ile Thr Ala Ser Gln
Leu Ser Asp Ser Ala Ser Tyr Ile Cys Val 100 105 110Val Ser Gly Gly
Thr Asp Ser Trp Gly Lys Leu Gln Phe Gly Ala Gly 115 120 125Thr Gln
Val Val Val Thr Pro Asp Ile Gln Asn Pro Asp Pro Ala Val 130 135
140Tyr Gln Leu Arg Asp Ser Lys Ser Ser Asp Lys Ser Val Cys Leu
Phe145 150 155 160Thr Asp Phe Asp Ser Gln Thr Asn Val Ser Gln Ser
Lys Asp Ser Asp 165 170 175Val Tyr Ile Thr Asp Lys Thr Val Leu Asp
Met Arg Ser Met Asp Phe 180 185 190Lys Ser Asn Ser Ala Val Ala Trp
Ser Asn Lys Ser Asp Phe Ala Cys 195 200 205Ala Asn Ala Phe Asn Asn
Ser Ile Ile Pro Glu Asp Thr Phe Phe Pro 210 215 220Ser Pro Glu Ser
Ser2256262PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polypeptide" 6Met Ala Ser Leu Leu Phe
Phe Cys Gly Ala Phe Tyr Leu Leu Gly Thr1 5 10 15Gly Ser Met Asp Ala
Asp Val Thr Gln Thr Pro Arg Asn Arg Ile Thr 20 25 30Lys Thr Gly Lys
Arg Ile Met Leu Glu Cys Ser Gln Thr Lys Gly His 35 40 45Asp Arg Met
Tyr Trp Tyr Arg Gln Asp Pro Gly Leu Gly Leu Arg Leu 50 55 60Ile Tyr
Tyr Ser Phe Asp Val Lys Asp Ile Asn Lys Gly Glu Ile Ser65 70 75
80Asp Gly Tyr Ser Val Ser Arg Gln Ala Gln Ala Lys Phe Ser Leu Ser
85 90 95Leu Glu Ser Ala Ile Pro Asn Gln Thr Ala Leu Tyr Phe Cys Ala
Thr 100 105 110Ser Gly Gln Gly Ala Tyr Asn Glu Gln Phe Phe Gly Pro
Gly Thr Arg 115 120 125Leu Thr Val Leu Glu Asp Leu Lys Asn Val Phe
Pro Pro Glu Val Ala 130 135 140Val Phe Glu Pro Ser Glu Ala Glu Ile
Ser His Thr Gln Lys Ala Thr145 150 155 160Leu Val Cys Leu Ala Thr
Gly Phe Tyr Pro Asp His Val Glu Leu Ser 165 170 175Trp Trp Val Asn
Gly Lys Glu Val His Ser Gly Val Ser Thr Asp Pro 180 185 190Gln Pro
Leu Lys Glu Gln Pro Ala Leu Asn Asp Ser Arg Tyr Cys Leu 195 200
205Ser Ser Arg Leu Arg Val Ser Ala Thr Phe Trp Gln Asn Pro Arg Asn
210 215 220His Phe Arg Cys Gln Val Gln Phe Tyr Gly Leu Ser Glu Asn
Asp Glu225 230 235 240Trp Thr Gln Asp Arg Ala Lys Pro Val Thr Gln
Ile Val Ser Ala Glu 245 250 255Ala Trp Gly Arg Ala Asp
2607105PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polypeptide" 7Met Lys Asn Gln Val Glu Gln Ser
Pro Gln Ser Leu Ile Ile Leu Glu1 5 10 15Gly Lys Asn Cys Thr Leu Gln
Cys Asn Tyr Thr Val Ser Pro Phe Ser 20 25 30Asn Leu Arg Trp Tyr Lys
Gln Asp Thr Gly Arg Gly Pro Val Ser Leu 35 40 45Thr Arg Met Thr Phe
Ser Glu Asn Thr Lys Ser Asn Gly Arg Tyr Thr 50 55 60Ala Thr Leu Asp
Ala Asp Thr Lys Gln Ser Ser Leu His Ile Thr Ala65 70 75 80Ser Gln
Leu Ser Asp Ser Ala Ser Tyr Ile Cys Val Val Ser Gly Gly 85 90 95Thr
Asp Ser Trp Gly Lys Leu Gln Phe 100 1058123PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 8Met Ala Ser Leu Leu Phe Phe Cys Gly Ala Phe Tyr Leu
Leu Gly Thr1 5 10 15Gly Ser Met Asp Ala Asp Val Thr Gln Thr Pro Arg
Asn Arg Ile Thr 20 25 30Lys Thr Gly Lys Arg Ile Met Leu Glu Cys Ser
Gln Thr Lys Gly Arg 35 40 45Asp Arg Met Tyr Trp Tyr Arg Gln Asp Pro
Gly Leu Gly Leu Arg Leu 50 55 60Ile Tyr Tyr Ser Phe Asp Val Lys Asp
Ile Asn Lys Gly Glu Ile Ser65 70 75 80Asp Gly Tyr Ser Val Ser Arg
Gln Ala Gln Ala Lys Phe Ser Leu Ser 85 90 95Leu Glu Ser Ala Ile Pro
Asn Gln Thr Ala Leu Tyr Phe Cys Ala Thr 100 105 110Ser Gly Gln Gly
Ala Tyr Asn Glu Gln Phe Phe 115 1209123PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 9Met Ala Ser Leu Leu Phe Phe Cys Gly Ala Phe Tyr Leu
Leu Gly Thr1 5 10 15Gly Ser Met Asp Ala Asp Val Thr Gln Thr Pro Arg
Asn Arg Ile Thr 20 25 30Lys Thr Gly Lys Arg Ile Met Leu Glu Cys Ser
Gln Thr Lys Gly His 35 40 45Asp Arg Met Tyr Trp Tyr Arg Gln Asp Pro
Gly Leu Gly Leu Arg Leu 50 55 60Ile Tyr Tyr Ser Phe Asp Val Lys Asp
Ile Asn Lys Gly Glu Ile Ser65 70 75 80Asp Gly Tyr Ser Val Ser Arg
Gln Ala Gln Ala Lys Phe Ser Leu Ser 85 90 95Leu Glu Ser Ala Ile Pro
Asn Gln Thr Ala Leu Tyr Phe Cys Ala Thr 100 105 110Ser Gly Gln Gly
Ala Tyr Glu Glu Gln Phe Phe 115 12010123PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 10Met Ala Ser Leu Leu Phe Phe Cys Gly Ala Phe Tyr Leu
Leu Gly Thr1 5 10 15Gly Ser Met Asp Ala Asp Val Thr Gln Thr Pro Arg
Asn Arg Ile Thr 20 25 30Lys Thr Gly Lys Arg Ile Met Leu Glu Cys Ser
Gln Thr Lys Gly His 35 40 45Asp Arg Met Tyr Trp Tyr Arg Gln Asp Pro
Gly Leu Gly Leu Arg Leu 50 55 60Ile Tyr Tyr Ser Phe Asp Val Lys Asp
Ile Asn Lys Gly Glu Ile Ser65 70 75 80Asp Gly Tyr Ser Val Ser Arg
Gln Ala Gln Ala Lys Phe Ser Leu Ser 85 90 95Leu Glu Ser Ala Ile Pro
Asn Gln Thr Ala Leu Tyr Phe Cys Ala Thr 100 105 110Ser Gly Gln Gly
Ala Tyr Arg Glu Gln Phe Phe 115 12011271PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 11Met Glu Thr Leu Leu Gly Leu Leu Ile Leu Trp Leu Gln
Leu Gln Trp1 5 10 15Val Ser Ser Lys Gln Glu Val Thr Gln Ile Pro Ala
Ala Leu Ser Val 20 25 30Pro Glu Gly Glu Asn Leu Val Leu Asn Cys Ser
Phe Thr Asp Ser Ala 35 40 45Ile Tyr Asn Leu Gln Trp Phe Arg Gln Asp
Pro Gly Lys Gly Leu Thr 50 55 60Ser Leu Leu Leu Ile Gln Ser Ser Gln
Arg Glu Gln Thr Ser Gly Arg65 70 75 80Leu Asn Ala Ser Leu Asp Lys
Ser Ser Gly Arg Ser Thr Leu Tyr Ile 85 90 95Ala Ala Ser Gln Pro Gly
Asp Ser Ala Thr Tyr Leu Cys Ala Val Gly 100 105 110Gly Tyr Ser Thr
Leu Thr Phe Gly Lys Gly Thr Val Leu Leu Val Ser 115 120 125Pro Asp
Asn Ile Gln Asn Pro Asp Pro Ala Val Tyr Gln Leu Arg Asp 130 135
140Ser Lys Ser Ser Asp Lys Ser Val Cys Leu Phe Thr Asp Phe Asp
Ser145 150 155 160Gln Thr Asn Val Ser Gln Ser Lys Asp Ser Asp Val
Tyr Ile Thr Asp 165 170 175Lys Thr Val Leu Asp Met Arg Ser Met Asp
Phe Lys Ser Asn Ser Ala 180 185 190Val Ala Trp Ser Asn Lys Ser Asp
Phe Ala Cys Ala Asn Ala Phe Asn 195 200 205Asn Ser Ile Ile Pro Glu
Asp Thr Phe Phe Pro Ser Pro Glu Ser Ser 210 215 220Cys Asp Val Lys
Leu Val Glu Lys Ser Phe Glu Thr Asp Thr Asn Leu225 230 235 240Asn
Phe Gln Asn Leu Ser Val Ile Gly Phe Arg Ile Leu Leu Leu Lys 245 250
255Val Ala Gly Phe Asn Leu Leu Met Thr Leu Arg Leu Trp Ser Ser 260
265 27012308PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polypeptide" 12Met Ser Ile Ser Leu
Leu Cys Cys Ala Ala Phe Pro Leu Leu Trp Ala1 5 10 15Gly Pro Val Asn
Ala Gly Val Thr Gln Thr Pro Lys Phe Arg Ile Leu 20 25 30Lys Ile Gly
Gln Ser Met Thr Leu Gln Cys Ala Gln Asp Met Asn His 35 40 45Asn Tyr
Met Tyr Trp Tyr Arg Gln Asp Pro Gly Met Gly Leu Lys Leu 50 55 60Ile
Tyr Tyr Ser Val Gly Ala Gly Ile Thr Asp Lys Gly Glu Val Pro65 70 75
80Asn Gly Tyr Asn Val Ser Arg Ser Thr Thr Glu Asp Phe Pro Leu Arg
85 90 95Leu Glu Leu Ala Ala Pro Ser Gln Thr Ser Val Tyr Phe Cys Ala
Ser 100 105 110Ser Tyr Ser Arg Trp Ser Pro Leu His Phe Gly Asn Gly
Thr Arg Leu 115 120 125Thr Val Thr Glu Asp Leu Asn Lys Val Phe Pro
Pro Glu Val Ala Val 130 135 140Phe Glu Pro Ser Glu Ala Glu Ile Ser
His Thr Gln Lys Ala Thr Leu145 150 155 160Val Cys Leu Ala Thr Gly
Phe Phe Pro Asp His Val Glu Leu Ser Trp 165 170 175Trp Val Asn Gly
Lys Glu Val His Ser Gly Val Ser Thr Asp Pro Gln 180 185 190Pro Leu
Lys Glu Gln Pro Ala Leu Asn Asp Ser Arg Tyr Cys Leu Ser 195 200
205Ser Arg Leu Arg Val Ser Ala Thr Phe Trp Gln Asn Pro Arg Asn His
210 215 220Phe Arg Cys Gln Val Gln Phe Tyr Gly Leu Ser Glu Asn Asp
Glu Trp225 230 235 240Thr Gln Asp Arg Ala Lys Pro Val Thr Gln Ile
Val Ser Ala Glu Ala 245 250 255Trp Gly Arg Ala Asp Cys Gly Phe Thr
Ser Val Ser Tyr Gln Gln Gly 260 265 270Val Leu Ser Ala Thr Ile Leu
Tyr Glu Ile Leu Leu Gly Lys Ala Thr 275 280 285Leu Tyr Ala Val Leu
Val Ser Ala Leu Val Leu Met Ala Met Val Lys 290 295 300Arg Lys Asp
Phe30513208PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polypeptide" 13Met Gln Lys Glu Val
Glu Gln Asn Ser Gly Pro Leu Ser Val Pro Glu1 5 10 15Gly Ala Ile Ala
Ser Leu Asn Cys Thr Tyr Ser Asp Arg Gly Ser Gln 20 25 30Ser Phe Phe
Trp Tyr Arg Gln Tyr Ser Gly Lys Ser Pro Glu Leu Ile 35 40 45Met Phe
Ile Tyr Ser Asn Gly Asp Lys Glu Asp Gly Arg Phe Thr Ala 50 55 60Gln
Leu Asn Lys Ala Ser Gln Tyr Val Ser Leu Leu Ile Arg Asp Ser65 70 75
80Gln Pro Ser Asp Ser Ala Thr Tyr Leu Cys Ala Val Lys Met Ala Asn
85 90 95Gln Ala Gly Thr Ala Leu Ile Phe Gly Lys Gly Thr Thr Leu Ser
Val 100 105 110Ser Ser Asn Ile Gln Asn Pro Asp Pro Ala Val Tyr Gln
Leu Arg Asp 115 120 125Ser Lys Ser Ser Asp Lys Ser Val Cys Leu Phe
Thr Asp Phe Asp Ser 130 135 140Gln Thr Asn Val Ser Gln Ser Lys Asp
Ser Asp Val Tyr Ile Thr Asp145 150 155 160Lys Cys Val Leu Asp Met
Arg Ser Met Asp Phe Lys Ser Asn Ser Ala 165 170 175Val Ala Trp Ser
Asn Lys Ser Asp Phe Ala Cys Ala Asn Ala Phe Asn 180 185 190Asn Ser
Ile Ile Pro Glu Asp Thr Phe Phe Pro Ser Pro Glu Ser Ser 195 200
20514244PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polypeptide" 14Met Gln Asp Gly Gly
Ile Thr Gln Ser Pro Lys Phe Gln Val Leu Arg1 5 10 15Thr Gly Gln Ser
Met Thr Leu Leu Cys Ala Gln Asp Met Asn His Glu 20 25 30Tyr Met Tyr
Trp Tyr Arg Gln Asp Pro Gly Met Gly Leu Arg Leu Ile 35 40 45His Tyr
Ser Val Gly Ala Gly Ile Thr Asp Gln Gly Glu Val Pro Asn 50 55 60Gly
Tyr Asn Val Ser Arg Leu Asn Lys Arg Glu Phe Ser Leu Arg Leu65 70 75
80Glu Ser Ala Ala Pro Ser Gln Thr Ser Val Tyr Phe Cys Ala Ser Leu
85 90 95Gly Gly Leu Ala Asp Glu Gln Phe Phe Gly Pro Gly Thr Arg Leu
Thr 100 105 110Val Leu Glu Asp Leu Lys Asn Val Phe Pro Pro Glu Val
Ala Val Phe 115 120 125Glu Pro Ser Glu Ala Glu Ile Ser His Thr Gln
Lys Ala Thr Leu Val 130 135 140Cys Leu Ala Thr Gly Phe Tyr Pro Asp
His Val Glu Leu Ser Trp Trp145 150 155 160Val Asn Gly Lys Glu Val
His Ser Gly Val Cys Thr Asp Pro Gln Pro 165 170 175Leu Lys Glu Gln
Pro Ala Leu Asn Asp Ser Arg Tyr Ala Leu Ser Ser 180 185 190Arg Leu
Arg Val Ser Ala Thr Phe Trp Gln Asp Pro Arg Asn His Phe 195 200
205Arg Cys Gln Val Gln Phe Tyr Gly Leu Ser Glu Asn Asp Glu Trp Thr
210 215 220Gln Asp Arg Ala Lys Pro Val Thr Gln Ile Val Ser Ala Glu
Ala Trp225 230 235 240Gly Arg Ala Asp15275PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 15Met Lys Thr Phe Ala Gly Phe Ser Phe Leu Phe Leu Trp
Leu Gln Leu1 5 10 15Asp Cys Met Ser Arg Gly Glu Asp Val Glu Gln Ser
Leu Phe Leu Ser 20 25 30Val
Arg Glu Gly Asp Ser Ser Val Ile Asn Cys Thr Tyr Thr Asp Ser 35 40
45Ser Ser Thr Tyr Leu Tyr Trp Tyr Lys Gln Glu Pro Gly Ala Gly Leu
50 55 60Gln Leu Leu Thr Tyr Ile Phe Ser Asn Met Asp Met Lys Gln Asp
Gln65 70 75 80Arg Leu Thr Val Leu Leu Asn Lys Lys Asp Lys His Leu
Ser Leu Arg 85 90 95Ile Ala Asp Thr Gln Thr Gly Asp Ser Ala Ile Tyr
Phe Cys Ala Glu 100 105 110Arg Asn Ser Gly Ala Gly Ser Tyr Gln Leu
Thr Phe Gly Lys Gly Thr 115 120 125Lys Leu Ser Val Ile Pro Asn Ile
Gln Asn Pro Asp Pro Ala Val Tyr 130 135 140Gln Leu Arg Asp Ser Lys
Ser Ser Asp Lys Ser Val Cys Leu Phe Thr145 150 155 160Asp Phe Asp
Ser Gln Thr Asn Val Ser Gln Ser Lys Asp Ser Asp Val 165 170 175Tyr
Ile Thr Asp Lys Thr Val Leu Asp Met Arg Ser Met Asp Phe Lys 180 185
190Ser Asn Ser Ala Val Ala Trp Ser Asn Lys Ser Asp Phe Ala Cys Ala
195 200 205Asn Ala Phe Asn Asn Ser Ile Ile Pro Glu Asp Thr Phe Phe
Pro Ser 210 215 220Pro Glu Ser Ser Cys Asp Val Lys Leu Val Glu Lys
Ser Phe Glu Thr225 230 235 240Asp Thr Asn Leu Asn Phe Gln Asn Leu
Ser Val Ile Gly Phe Arg Ile 245 250 255Leu Leu Leu Lys Val Ala Gly
Phe Asn Leu Leu Met Thr Leu Arg Leu 260 265 270Trp Ser Ser
27516311PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polypeptide" 16Met Gly Thr Ser Leu
Leu Cys Trp Met Ala Leu Cys Leu Leu Gly Ala1 5 10 15Asp His Ala Asp
Thr Gly Val Ser Gln Asn Pro Arg His Lys Ile Thr 20 25 30Lys Arg Gly
Gln Asn Val Thr Phe Arg Cys Asp Pro Ile Ser Glu His 35 40 45Asn Arg
Leu Tyr Trp Tyr Arg Gln Thr Leu Gly Gln Gly Pro Glu Phe 50 55 60Leu
Thr Tyr Phe Gln Asn Glu Ala Gln Leu Glu Lys Ser Arg Leu Leu65 70 75
80Ser Asp Arg Phe Ser Ala Glu Arg Pro Lys Gly Ser Phe Ser Thr Leu
85 90 95Glu Ile Gln Arg Thr Glu Gln Gly Asp Ser Ala Met Tyr Leu Cys
Ala 100 105 110Ser Ser Leu Phe Ser Gly Val Asn Thr Glu Ala Phe Phe
Gly Gln Gly 115 120 125Thr Arg Leu Thr Val Val Glu Asp Leu Asn Lys
Val Phe Pro Pro Glu 130 135 140Val Ala Val Phe Glu Pro Ser Glu Ala
Glu Ile Ser His Thr Gln Lys145 150 155 160Ala Thr Leu Val Cys Leu
Ala Thr Gly Phe Phe Pro Asp His Val Glu 165 170 175Leu Ser Trp Trp
Val Asn Gly Lys Glu Val His Ser Gly Val Ser Thr 180 185 190Asp Pro
Gln Pro Leu Lys Glu Gln Pro Ala Leu Asn Asp Ser Arg Tyr 195 200
205Cys Leu Ser Ser Arg Leu Arg Val Ser Ala Thr Phe Trp Gln Asn Pro
210 215 220Arg Asn His Phe Arg Cys Gln Val Gln Phe Tyr Gly Leu Ser
Glu Asn225 230 235 240Asp Glu Trp Thr Gln Asp Arg Ala Lys Pro Val
Thr Gln Ile Val Ser 245 250 255Ala Glu Ala Trp Gly Arg Ala Asp Cys
Gly Phe Thr Ser Val Ser Tyr 260 265 270Gln Gln Gly Val Leu Ser Ala
Thr Ile Leu Tyr Glu Ile Leu Leu Gly 275 280 285Lys Ala Thr Leu Tyr
Ala Val Leu Val Ser Ala Leu Val Leu Met Ala 290 295 300Met Val Lys
Arg Lys Asp Phe305 3101710PRTHomo sapiens 17Gly Val Val Asp Gly Arg
Glu His Thr Val1 5 10186PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 18Val Ser Pro Phe Ser Asn1 5196PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 19Leu Thr Ile Met Thr Phe1 5206PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 20Leu Thr Arg Met Thr Phe1 52115PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 21Cys Val Val Ser Gly Gly Thr Asp Ser Trp Gly Lys Leu Gln
Phe1 5 10 15225PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 22Lys Gly His Asp Arg1
5235PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 23Lys Gly Arg Asp Arg1
5245PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 24Ser Val Phe Asp Lys1
52514PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 25Cys Ala Thr Ser Gly Gln Gly Ala Tyr
Asn Glu Gln Phe Phe1 5 102614PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 26Cys Ala Thr Ser Gly Gln Gly Ala Tyr Arg Glu Gln Phe Phe1
5 102714PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 27Cys Ala Thr Ser Gly Gln
Gly Ala Tyr Lys Glu Gln Phe Phe1 5 10285PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 28Ser Phe Asp Val Lys1 5
* * * * *