U.S. patent application number 16/275070 was filed with the patent office on 2019-09-05 for methods of treating cancer in a subject by administering a composition comprising gamma 9 delta 2 t-cell receptors.
The applicant listed for this patent is Gadeta B.V.. Invention is credited to Elsa-Cordula Grunder, Jurgen Herbert Ernst Kuball.
Application Number | 20190271688 16/275070 |
Document ID | / |
Family ID | 48096135 |
Filed Date | 2019-09-05 |
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United States Patent
Application |
20190271688 |
Kind Code |
A1 |
Kuball; Jurgen Herbert Ernst ;
et al. |
September 5, 2019 |
METHODS OF TREATING CANCER IN A SUBJECT BY ADMINISTERING A
COMPOSITION COMPRISING GAMMA 9 DELTA 2 T-CELL RECEPTORS
Abstract
The current invention provides methods to identify
.gamma.9.delta.2T-cell receptors (.gamma.9.delta.2TCR) that mediate
anti-tumour responses. Surprisingly, it was now found that the CDR3
regions of the .gamma.9-T-cell receptor chain and the
.delta.2-T-Cell receptor chain (.delta.2TCR chain) are of
importance. Based on these findings,
combinatorial-.gamma..delta.TCR-chain-exchange (CTE) is proposed as
an efficient method for identifying .gamma.9.delta.2TCRs that
mediate anti-tumour responses. Using the method of the invention,
specific sequences of the respective .gamma.9TCR and .delta.2TCR
chains were identified that mediate anti-tumour responses. Hence,
the invention further provides for specific .gamma.9.delta.2TCRs,
or fragments thereof, that may be used e.g. in diagnostics or
treatment of cancer. The invention further provides for nucleic
acid sequences, genetic constructs and retroviral vectors that can
be used to express the .gamma.9.delta.2TCRs according to the
invention.
Inventors: |
Kuball; Jurgen Herbert Ernst;
(Hilversum, NL) ; Grunder; Elsa-Cordula; (Rabenau,
DE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Gadeta B.V. |
Utrech |
|
NL |
|
|
Family ID: |
48096135 |
Appl. No.: |
16/275070 |
Filed: |
February 13, 2019 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15842784 |
Dec 14, 2017 |
10324083 |
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16275070 |
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14388675 |
Sep 26, 2014 |
9891211 |
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PCT/NL2013/050235 |
Mar 28, 2013 |
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15842784 |
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61703788 |
Sep 21, 2012 |
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61616440 |
Mar 28, 2012 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61P 35/00 20180101;
G01N 33/505 20130101; C12N 15/85 20130101; A61K 38/00 20130101;
C12N 2510/00 20130101; C07K 14/7051 20130101; A61K 35/17 20130101;
C12N 5/0636 20130101; G01N 33/5011 20130101; G01N 33/6866
20130101 |
International
Class: |
G01N 33/50 20060101
G01N033/50; C12N 5/0783 20060101 C12N005/0783; C12N 15/85 20060101
C12N015/85; C07K 14/725 20060101 C07K014/725; A61K 35/17 20060101
A61K035/17; G01N 33/68 20060101 G01N033/68 |
Claims
1. A composition comprising an engineered .alpha..beta. T-cell that
expresses an exogenous .gamma.9.delta.2T-cell receptor or a
functional fragment thereof, wherein said .gamma.9.delta.2T-cell
receptor or the functional fragment thereof increases the avidity
of the engineered .alpha..beta. T-cell to a cancer cell compared to
a non-engineered .alpha..beta. T-cell.
2. The composition of claim 1, wherein the engineered .alpha..beta.
T-cell comprises a nucleic acid encoding the .gamma.9.delta.2T-cell
receptor sequence or the functional fragment thereof.
3. The composition of claim 1, wherein said engineered cell is
modified at an .alpha..beta. T-cell receptor (TCR) locus.
4. The composition of claim 3, wherein said modification results in
a downregulation of expression of an endogenous .alpha..beta. TCR
in said engineered cell as compared to an expression of said
endogenous .alpha..beta. TCR in a comparable non-engineered
cell.
5. The composition of claim 1, wherein said cancer cell is a
hematological cancer cell or a solid cancer cell.
6. The composition of claim 1, wherein said cancer cell is a cancer
cell selected from the group consisting of multiple myeloma, Acute
Myeloid Leukemia (AML), and myelogenous leukemia.
7. The composition of claim 1, wherein said cancer cell is a cancer
cell selected from the group consisting of osteosarcoma, renal cell
carcinoma, head and neck cancer, breast cancer, glioblastoma, and
colon carcinoma.
8. The composition of claim 1, further comprising an engineered
progenitor cell, wherein said engineered progenitor cell comprises
said .gamma.9.delta.2T-cell receptor.
9. (canceled)
10. The cellular composition of claim 8, wherein said engineered
progenitor cell is a thymocyte or a blood stem cell.
11. The composition of claim 1, wherein said .gamma.9.delta.2T-cell
receptor binds to an antigen expressed on the surface of a cancer
cell.
12. The composition of claim 1, wherein said engineered
.alpha..beta. T-cell is a CD8+ T-cell or a CD4+ T-cell.
13. A pharmaceutical composition comprising an engineered
.alpha..beta. T-cell that comprises an exogenous
.gamma.9.delta.2T-cell receptor, and a pharmaceutically acceptable
adjuvant, carrier or diluent.
14. The pharmaceutical composition of claim 13, comprising an
engineered .alpha..beta. T-cell, wherein the engineered
.alpha..beta. T-cell comprises a nucleic acid sequence encoding the
.gamma.9.delta.2T-cell receptor sequence or the functional fragment
thereof.
15. (canceled)
16. The pharmaceutical composition of claim 13, wherein said
engineered .alpha..beta. T-cell is a CD8+ T-cell or a CD4+
T-cell.
17. A method for treating a cancer in a subject in need thereof,
the method comprising: administering to the subject a
pharmaceutical composition comprising an engineered .alpha..beta.
T-cell that expresses an exogenous .gamma.9.delta.2T-cell receptor
of a functional fragment thereof.
18-19. (canceled)
20. The method of claim 17, wherein the pharmaceutical composition
comprising the engineered .alpha..beta. T-cell is administered
intravenously to the subject.
21. The method of claim 17, wherein administering the
pharmaceutical composition comprising said engineered cell results
in reduction in cancer growth.
22-23. (canceled)
24. A method of making the composition of claim 1, the method
comprising: engineering an .alpha..beta. T-cell to express an
exogenous .gamma.5T cell receptor or a fragment thereof, having a
.gamma.9TCR chain and a .delta.2TCR chain or functional fragments
thereof, and wherein the engineered cell has increased avidity to a
cancer cell expressing an antigen capable of being bound by an
antigen binding domain of the .gamma.9TCR chain and .delta.2TCR
chain.
25-26. (canceled)
27. The method of claim 24, wherein the engineering an
.alpha..beta. T-cell comprises modifying an .alpha..beta. T-cell
receptor (TCR) locus, such that the modification results in a
downregulation of expression of an endogenous .alpha..beta. TCR in
said engineered cell as compared to an expression of said
endogenous .alpha..beta. TCR in a comparable non-engineered
.alpha..beta. T-cell.
28. The method of claim 24, wherein said cancer cell is selected
from the group consisting of multiple myeloma, Acute Myeloid
Leukemia (AML), myelogenous leukemia, osteosarcoma, renal cell
carcinoma, head and neck cancer, breast cancer, glioblastoma, and
colon carcinoma.
29-30. (canceled)
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 15/842,784, filed Dec. 14, 2017, which is a continuation of
U.S. application Ser. No. 14/388,675, filed Sep. 26, 2014, now U.S.
Pat. No. 9,891,211, issued Feb. 13, 2018, which in turn is the U.S.
National Stage filing under 35 U.S.C. .sctn. 371 of International
Application No. PCT/NL2013/050235, filed Mar. 28, 2013, which in
turn claims priority to U.S. Provisional Patent Application No.
61/616,440, filed Mar. 28, 2012 and also to U.S. Provisional Patent
Application No. 61/703,788, filed Sep. 21, 2012, the entire
contents of which are incorporated by reference herein.
SEQUENCE LISTING
[0002] 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, created
on Oct. 18, 2018, is named 51887_701_303_SL.txt and is 27,092 bytes
in size.
FIELD OF THE INVENTION
[0003] The invention is in the field of medicine. It relates to
immunology and to cell therapy for the treatment of cancer. The
invention further relates to the methods for identification of T
cell receptors, in particular T cell receptors that may mediate
anti-tumour responses. The invention further relates to specific
T-cell receptors that mediate anti-tumour responses and uses
thereof, in the treatment of cancer.
BACKGROUND ART
[0004] So far, allogeneic stem cell transplantation (allo-SCT) is
the only well established and proven curative cellular therapy for
patients suffering from cancer. This is demonstrated by the
observation that long term remissions can be in particular achieved
with allo-SCT in patients with "poor-risk" leukaemia. However, cure
comes at a price of severe toxicity, resulting in an overall
mortality of approximately 30% of treated patients. This hampers
broad clinical implementation as in particular patients with low
risk cancers might have a good overall survival not justifying such
aggressive therapies. The ultimate goal remains therefore the
development of a non-aggressive transplantation method. This means
the generation of cellular products equipped with molecular
features that allow selective targeting of the cancer cell while
preserving healthy tissues. This would allow a curative treatment
to a large number of patients with medical need, irrespective of
age.
[0005] To engineer a transplant, which may be allogeneic or
autologous, to facilitate the rapid generation of e.g.
tumour-reactive .alpha..beta.T-cells, it has been proposed to
reprogram .alpha..beta.T-cells with genes encoding for a
tumour-specific .alpha..beta.T-cell receptor (TCR) or a chimeric
antigen receptor. Several of such receptors are already being used
to redirect .alpha..beta.T-cells in phase I clinical trials.
However, reprogramming .alpha..beta.T-cells with defined
.alpha..beta.TCRs is substantially hampered by their restriction to
HLA types, limiting thereby the number of patients that can be
treated with one .alpha..beta.-TCR. In addition, pairing of
introduced .alpha..beta.TCR chains with endogenous .alpha..beta.TCR
chains can induce life-threatening auto-reactivity.
[0006] It has been proposed to mediate a selective
anti-tumour-reactivity with a high-affinity TCR using the ability
of .gamma.9.delta.2T-cells to mediate anti-tumour-reactivity while
ignoring healthy-environment (Fisch et al., 1990, Science 250,
1269-1273). Isolated .gamma.9.delta.2T-cells may efficiently kill
tumour-cells of haematological malignancies and solid tumours
(Kabelitz et al., 2007, Cancer Res. 67, 5-8). However, the function
and proliferation capacity of .gamma.9.delta.2T-cells is frequently
heavily impaired in cancer patients making autologous
.gamma.9.delta.2T-cells less attractive for immune
interventions.
[0007] It has been proposed to transfer a defined
.gamma.9.delta.2TCR into .alpha..beta.T-cells, which mediates a
tumour-specific proliferation of .alpha..beta.T-cells and redirects
both effector CD8+ and helper CD4+ .alpha..beta.T-cell subsets
against a broad panel of tumour-cell-lines while normal cells in
vitro and in vivo may be ignored (Marcu-Malina et al., 2011, Blood
118, 50-59). However, little knowledge is available about how
.gamma.9.delta.2TCRs mediate different activity against tumour
cells and what strategy needs to be used to isolate
.gamma.9.delta.2TCRs with high activity against cancer cells.
[0008] Hence, there is need in the art to provide for
.gamma.9.delta.2TCRs with high activity against cancer cells and
there is a need in the art to provide for improved methods for
selecting such highly active .gamma.9.delta.2TCRs.
SUMMARY OF THE INVENTION
[0009] The current invention provides methods to identify
.gamma.9.delta.2T-cell receptors (.gamma.9.delta.2TCR) that mediate
anti-tumour responses. As said, little knowledge is available about
the molecular requirements of a .gamma.9.delta.2TCRs to mediate
anti-tumour reactivity. Especially the contribution of the
variability within the complementary determining regions (CDRs) of
the .gamma.9.delta.2TCR for mediating anti-tumour responses was
regarded as negligible in the prior art. Surprisingly, it was now
found that the CDR3 regions of the .gamma.9T-cell receptor chain
(.gamma.9TCR chain) and the .delta.2T-cell receptor chain
(.delta.2TCR chain) are of importance. Based on these findings,
combinatorial-.gamma..delta.TCR-chain-exchange (CTE) is proposed as
an efficient method for identifying .gamma.9.delta.2TCRs that
mediate anti-tumour responses. In the methods of the invention, the
CDR3 regions of the .gamma.9T-cell receptor chain and the
.delta.2T-cell receptor chain are randomly modified and combined to
form a novel .gamma.9.delta.2TCR. The newly designed
.gamma.9.delta.2TCR is provided and preferably integrated into
T-cells, which subsequently express a .gamma.9.delta.2TCR at the
cell surface. Accordingly the anti-tumour response of the
CTE-engineered T-cells is determined. In this way multiple
combinations can be tested, and for each combination the
anti-tumour response can be determined. After determining the
anti-tumour response, .gamma.9.delta.2T-cell receptors that mediate
highly active anti-tumour responses can be identified. Using the
method of the invention, such as described in the examples, already
several responsive .gamma.9.delta.2TCRs were identified that
mediate increased anti-tumour responses compared to the reference
.gamma.9.delta.2TCR G115wt. Hence, the invention further provides
for .gamma.9.delta.2TCRs, or fragments thereof, that may be used
e.g. in diagnostics or the treatment of cancer. The invention
further provides for nucleic acid sequences, genetic constructs and
retroviral vectors that can be used to express the
.gamma.9.delta.2TCRs in cells, preferably T cells, according to the
invention.
FIGURES
[0010] FIGS. 1A-F: Anti-tumour reactivity mediated by
.gamma.9.delta.2TCRs. Peripheral blood T-cells were virally
transduced with indicated wildtype .gamma.9.delta.2TCRs or
CTE-engineered .gamma.9.delta.2TCRs and tested against Daudi (FIG.
1A, FIG. 1C) in a .sup.51Cr-release assay (E:T 3:1). Specific lysis
is indicated as fold change .sup.51Cr-release measured in the
supernatant after 5 h. Fold change was calculated when compared to
reactivity of .gamma.9-G115.sub.wt/.delta.2-G115.sub.wt engineered
T-cells. (FIG. 1B, FIG. 1D) in an IFN.gamma. ELISA in the presence
of indicated amounts of pamidronate or (FIG. 1E) different E:T
ratios. (FIG. 1F) Percentages of cell-cell conjugates of Daudi and
T-cells engineered with indicated .gamma.9.delta.2TCR were
determined by flow cytometry. Data represent the mean.+-.SD.
*p<0.05, **p<0.01, ***p<0.001 by 1-way ANOVA.
[0011] FIGS. 2A-D: .gamma.9.delta.2TCR expression and functional
avidity of transduced T-cells expressing single alanine mutated
.delta.2 chain of clone G115. Peripheral blood T-cells were virally
transduced with indicated .gamma.9 and .delta.2 TCR chains and
(FIG. 2A) analyzed by flow cytometry using a .delta.2-chain
specific antibody. Shown is the fold change in mean fluorescent
intensity (MFI) in comparison to wildtype control expressing
.delta.2-G115.sub.wt. (FIG. 2C) Lytic activity of transductants was
tested in a .sup.51Cr-release assay against the tumour target Daudi
(E:T 10:1). Specific lysis is indicated as fold change
.sup.51Cr-release measured in the supernatant after 5 h. Fold
change was calculated as compared to reactivity of unmutated
wildtype (.delta.2-G115.sub.wt). Arrows indicate mutations in
.delta.2-G115 that impaired receptor expression (dashed arrows) or
functional avidity (solid arrows). (FIG. 2B, FIG. 2D) Crystal
structure of .gamma.9.delta.2TCR G115 indicating relevant amino
acids (arrows).
[0012] FIGS. 3A-F: .gamma.9.delta.2TCR expression and functional
avidity of transduced T-cells expressing .gamma.9.delta.2TCR G115
with .delta.2-CDR3 length mutations. (FIG. 3A) .gamma.9.delta.2TCR
expression of indicated transductants was analyzed by flow
cytometry using a .gamma..delta.TCR-pan antibody. Shown is the fold
change in mean fluorescent intensity (MFI) in comparison to
wildtype control expressing .delta.2-G115.sub.wt. (FIG. 3B)
IFN.gamma. secretion of .delta.2-G115.sub.LM transduced T-cells
against the tumour target Daudi (E:T 1:1) was measured by ELISA
after 24 h incubation in the presence of 100 .mu.M pamidronate.
Shown is the fold change in IFN.gamma. production when compared to
reactivity of transductants expressing .delta.2-G115.sub.wt. (FIG.
3C) Transductants expressing .delta.2-G115.sub.LM0,1,4,12 were
tested in a titration assay against the tumour target Daudi with
increasing amounts of pamidronate as indicated. IFN.gamma.
production was measured after 24 h by ELISA. (FIG. 3D) Generated
.delta.2-G115.sub.LMs were matched in a BLAST search with
.gamma.9.delta.2TCRs described in the IMGT database. Shown is the
number of citations compared to .delta.2-G115.sub.LM of similar
.delta.CDR3 length. (FIG. 3E) Transductants with
.delta.2-G115.sub.LM2,4,6 were compared side-by-side to
transductants expressing individual .gamma.9.delta.2TCRs of the
same .delta.CDR3 length. IFN.gamma. secretion of transduced T-cells
against the tumour target Daudi (E:T 1:1) was measured by ELISA
after 24 h in the presence of 100 .mu.M pamidronate. Shown is the
fold change in IFN.gamma. production compared to reactivity of
transductants expressing wt .delta.2-G115.sub.wt. Data represent
the mean.+-.SD. **p<0.01, **p<0.001 by 1-way ANOVA. (FIG. 3F)
Crystal structure of .gamma.9.delta.2TCR G115; the region that was
used for alanine stretches within .delta.CDR3 is shown in white,
residual .delta.CDR3 in green, .delta. chain in blue, .gamma. chain
in brown.
[0013] FIGS. 4A-B: Functional avidity of transduced T-cells
expressing .gamma.9.delta.2TCR G115 with .gamma.9-CDR3 length
mutations.
[0014] (FIG. 4A) Peripheral blood T-cells were virally transduced
with indicated .gamma.9 and .delta.2 TCR chains. Lytic activity of
transductants was compared side-by-side to T-cells expressing
individual .gamma.9.delta.2TCRs of the same .gamma.9CDR3 length.
Specific lysis is indicated as fold change .sup.51Cr-release
measured in the supernatant after 5 h. Data represent the
mean.+-.SD. **p<0.01 by 1-way ANOVA. (FIG. 4B) Crystal structure
of .gamma.9.delta.2TCR G115 indicating .gamma.9CDR3 in gray
including amino acids .gamma.9-G115.sub.A109, 9-G115.sub.Q110 and
.gamma.9-G115.sub.Q111 (red arrows), .delta.CDR3 is shown in green;
.delta. chain in blue; .gamma. chain in brown.
[0015] FIGS. 5A-C: Anti-tumour reactivity of T-cells transduced
with CTE-engineered .gamma.9.delta.2TCRs in vitro.
[0016] Peripheral blood T-cells were virally transduced with
indicated .gamma.9.delta.2TCRs and tested against indicated tumour
cell lines and healthy control tissue. (FIG. 5A) Transductants were
incubated with target cells (E:T 1:1) in the presence of 10 .mu.M
pamidronate. IFN.gamma. production was measured after 24 h by
ELISA. Data represent the mean.+-.SD. *p<0.05, **p<0.01,
p<0.001 by 1-way ANOVA. (FIG. 5B) Transductants were incubated
with indicated tumour targets loaded with .sup.51Cr (E:T 10:1).
Percentage of specific lysis was determined by .sup.51Cr-release
measured in the supernatant after 5 h. (FIG. 5C) CTE-engineered
T-cells were tested against primary AML blasts and healthy
progenitor cells in an IFN.gamma. ELISpot assay (E:T 3:1) in the
presence of 10 .mu.M pamidronate. Data represent the mean.+-.SD.
*p<0.05, **p<0.01, p<0.001 by 1-way ANOVA.
[0017] FIGS. 6A-C: Anti-tumour reactivity of T-cells transduced
with CTE-engineered .gamma.9.delta.2TCRs in vivo.
[0018] The functional avidity of T-cells expressing
CTE-.gamma.9.delta.2TCR .gamma.9-G115.sub.wt/.delta.2-cl5.sub.wt or
control .gamma.9.delta.2TCR
(.gamma.9-G115.sub.wt/.delta.2-G115.sub.wt) was studied in
Rag2.sup.-/--yc.sup.-/- double knockout mice (4-7 mice per group).
After total body irradiation (2Gy) on day 0, mice were
intravenously injected with 0.5.times.10.sup.6 Daudi-luciferase or
5.times.10.sup.6 RPM18226/S-luciferase cells and 10.sup.7
CTE-.gamma.9.delta.2TCR transduced T-cells at day 1. Additionally,
6.times.10.sup.5IU IL2 in IFA and pamidronate (10 mg/kg body
weight) were injected at day 1 and every 3 weeks until the end of
the experiment. (FIG. 6A, FIG. 6B) Tumour outgrowth was assessed in
vivo by bioluminescence imaging (BLI) by measuring the entire area
of mice on both sides. Data represent the mean of all animals
measured (Daudi: n=4, RPM18226/S: n=7). *p<0.05, **p<0.01 by
1-way ANOVA (Daudi: day 42; RPM18226/S: day 35). (FIG. 6C) Overall
survival of treated Daudi mice was monitored for 72 days.
*p<0.05, **p<0.01 by logrank (Mantel-Cox) test.
[0019] FIGS. 7A-F. Combinatorial-.gamma..delta.TCR-chain-exchange
(CTE)
[0020] Individual .gamma.9.delta.2T-cell clones are isolated (FIG.
7A) and the sequence of the .gamma.9TCR and .delta.2TCR chain is
determined (FIG. 7B). By Combinatorial-.gamma..delta.TCR-chain
exchange novel .gamma.9.delta.2TCRs are designed whereby .gamma.9-
and .delta.2CDR3 regions are randomly modified (e.g. via amino acid
substitutions, deletions and/or insertions and/or shortening or
stretching of the CDR3 length) (FIG. 7C) and newly combined (FIG.
7D). Subsequently novel .gamma.9.delta.2TCRs are introduced into
cells, preferably T-cells such as .alpha..beta.T-cells (e.g. via
transduction using a retroviral vector encoding the .gamma.9TCR and
.delta.2TCR chains) and CTE-engineered T-cells are provided (FIG.
7E). Finally, CTE-engineered T-cells are tested against tumour
cells in functional assays to determine .gamma.9.delta.2TCRs that
mediate high anti-tumour responses. Hence, .gamma.9.delta.2TCRs
with e.g. the highest anti-tumour response and/or lowest side
effects can be selected (FIG. 7F).
[0021] FIG. 8: CDR3 sequence of .gamma.9.delta.2TCR G115, clone 3
and clone 5 (FIG. 8 discloses SEQ ID NOS 23-28, respectively, in
order of appearance)
DEFINITIONS
[0022] In the following description and examples a number of terms
are used. In order to provide a clear and consistent understanding
of the specifications and claims, including the scope to be given
to such terms, the following definitions are provided. Unless
otherwise defined herein, all technical and scientific terms used
have the same meaning as commonly understood by one of ordinary
skill in the art to which this invention belongs. The disclosures
of all publications, patent applications, patents and other
references are incorporated herein in their entirety by
reference.
[0023] Methods of carrying out the conventional techniques used in
methods of the invention will be evident to the skilled worker. The
practice of conventional techniques in molecular biology,
biochemistry, computational chemistry, cell culture, recombinant
DNA, bioinformatics, genomics, sequencing and related fields are
well-known to those of skill in the art and are discussed, for
example, in the following literature references: Sambrook et al.,
Molecular Cloning. A Laboratory Manual, 2nd Edition, Cold Spring
Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989; Ausubel et
al., Current Protocols in Molecular Biology, John Wiley & Sons,
New York, 1987 and periodic updates; and the series Methods in
Enzymology, Academic Press, San Diego.
[0024] In this document and in its claims, the verb "to comprise"
and its conjugations is used in its non-limiting sense to mean that
items following the word are included, but items not specifically
mentioned are not excluded. It encompasses the verbs "consisting
essentially of" as well as "consisting of".
[0025] As used herein, the singular forms "a," "an" and "the"
include plural referents unless the context clearly dictates
otherwise. For example, a method for isolating "a" DNA molecule, as
used above, includes isolating a plurality of molecules (e.g. 10's,
100's, 1000's, 10's of thousands, 100's of thousands, millions, or
more molecules).
[0026] Aligning and alignment: With the term "aligning" and
"alignment" is meant the comparison of two or more nucleotide
sequences based on the presence of short or long stretches of
identical or similar nucleotides. Several methods for alignment of
nucleotide sequences are known in the art, as will be further
explained below. With the term "aligning" and "alignment" is also
meant the comparison of two or more amino acid sequences based on
the presence of short or long stretches of identical or similar
amino acids. Several methods for alignment of amino acid sequences
are known in the art, as will be further explained below.
[0027] "Expression of a gene" refers to the process wherein a DNA
region, which is operably linked to appropriate regulatory regions,
particularly a promoter, is transcribed into an RNA, which is
biologically active, i.e. which is capable of being translated into
a biologically active protein or peptide (or active peptide
fragment) or which is active itself (e.g. in posttranscriptional
gene silencing or RNAi). An active protein in certain embodiments
refers to a protein being constitutively active. The coding
sequence is preferably in sense-orientation and encodes a desired,
biologically active protein or peptide, or an active peptide
fragment.
[0028] As used herein, the term "operably linked" refers to a
linkage of polynucleotide elements in a functional relationship. A
nucleic acid is "operably linked" when it is placed into a
functional relationship with another nucleic acid sequence. For
instance, a promoter, or rather a transcription regulatory
sequence, is operably linked to a coding sequence if it affects the
transcription of the coding sequence. Operably linked means that
the DNA sequences being linked are typically contiguous and, where
necessary to join two or more protein encoding regions, contiguous
and in reading frame.
[0029] The term "genetic construct" means a DNA sequence comprising
a region (transcribed region), which is transcribed into an RNA
molecule (e.g. an mRNA) in a cell, operably linked to suitable
regulatory regions (e.g. a promoter). A genetic construct may thus
comprise several operably linked sequences, such as a promoter, a
5' leader sequence comprising e.g. sequences involved in
translation initiation, a (protein) encoding region, splice donor
and acceptor sites, intronic and exonic sequences, and a 3'
non-translated sequence (also known as 3' untranslated sequence or
3'UTR) comprising e.g. transcription termination sequence
sites.
[0030] "Identity" is a measure of the identity of nucleotide
sequences or amino acid sequences. In general, the sequences are
aligned so that the highest order match is obtained. "Identity" per
se has an art-recognized meaning and can be calculated using
published techniques. See, e.g.: (COMPUTATIONAL MOLECULAR BIOLOGY,
Lesk, A. M., ed., Oxford University Press, New York, 1988;
BIOCOMPUTING: INFORMATICS AND GENOME PROJECTS, Smith, D. W., ed.,
Academic Press, New York, 1993; COMPUTER ANALYSIS OF SEQUENCE DATA,
PART I, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New
Jersey, 1994; SEQUENCE ANALYSIS IN MOLECULAR BIOLOGY, von Heinje,
G., Academic Press, 1987; and SEQUENCE ANALYSIS PRIMER; Gribskov,
M. and Devereux, J., eds., M Stockton Press, New York, 1991). While
a number of methods exist to measure identity between two
polynucleotide or polypeptide sequences, the term "identity" is
well known to skilled artisans (Carillo, H., and Lipton, D., SIAM
J. Applied Math (1988) 48:1073). Methods commonly employed to
determine identity or similarity between two sequences include, but
are not limited to, those disclosed in GUIDE TO HUGE COMPUTERS,
Martin J. Bishop, ed., Academic Press, San Diego, 1994, and
Carillo, H., and Lipton, D., SIAM J. Applied Math (1988) 48:1073.
Methods to determine identity and similarity are codified in
computer programs. Preferred computer program methods to determine
identity and similarity between two sequences include, but are not
limited to, GCS program package (Devereux, J., et al., Nucleic
Acids Research (1984) 12(1):387), BLASTP, BLASTN, FASTA (Atschul,
S. F. et al., J. Molec. Biol. (1990) 215:403).
[0031] As an illustration, by a polynucleotide having a nucleotide
sequence having at least, for example, 95% "identity" to a
reference nucleotide sequence encoding a polypeptide of a certain
sequence it is intended that the nucleotide sequence of the
polynucleotide is identical to the reference sequence except that
the polynucleotide sequence may include up to five point mutations
per each 100 nucleotides of the reference polypeptide sequence.
Hence, the percentage of identity of a nucleotide sequence to a
reference nucleotide sequence is to be calculated over the full
length of the reference nucleotide sequence. In other words, to
obtain a polynucleotide having a nucleotide sequence at least 95%
identical to a reference nucleotide sequence, up to 5% of the
nucleotides in the reference sequence may be deleted and/or
substituted with another nucleotide, and/or a number of nucleotides
up to 5% of the total nucleotides in the reference sequence may be
inserted into the reference sequence. These mutations of the
reference sequence may occur at the 5' or 3' terminal positions of
the reference nucleotide sequence, or anywhere between those
terminal positions, interspersed either individually among
nucleotides in the reference sequence or in one or more contiguous
groups within the reference sequence.
[0032] Similarly, by a polypeptide having an amino acid sequence
having at least, for example, 95% "identity" to a reference amino
acid sequence of SEQ ID NO: 1 or 2 is intended that the amino acid
sequence of the polypeptide is identical to the reference sequence
except that the polypeptide sequence may include up to five amino
acid alterations per each 100 amino acids of the reference amino
acid of SEQ ID NO: 1 or 2. Hence, the percentage of identity of an
amino acid sequence to a reference amino acid sequence is to be
calculated over the full length of the reference amino acid
sequence. In other words, to obtain a polypeptide having an amino
acid sequence at least 95% identical to a reference amino acid
sequence, up to 5% of the amino acid residues in the reference
sequence may be deleted or substituted with another amino acid, or
a number of amino acids up to 5% of the total amino acid residues
in the reference sequence may be inserted into the reference
sequence. These alterations of the reference sequence may occur at
the amino- or carboxy-terminal positions of the reference amino
acid sequence or anywhere between those terminal positions,
interspersed either individually among residues in the reference
sequence or in one or more contiguous groups within the reference
sequence.
[0033] A "nucleic acid" or "nucleic acid sequence" according to the
present invention may include any polymer or oligomer of pyrimidine
and purine bases, preferably cytosine, thymine, and uracil, and
adenine and guanine, respectively (See Albert L. Lehninger,
Principles of Biochemistry, at 793-800 (Worth Pub. 1982), which is
herein incorporated by reference in its entirety for all purposes).
The present invention contemplates any deoxyribonucleotide,
ribonucleotide or peptide nucleic acid component, and any chemical
variants thereof, such as methylated, hydroxymethylated or
glycosylated forms of these bases, and the like. The polymers or
oligomers may be heterogenous or homogenous in composition, and may
be isolated from naturally occurring sources or may be artificially
or synthetically produced. In addition, the nucleic acids may be
DNA or RNA, or a mixture thereof, and may exist permanently or
transitionally in single-stranded or double-stranded form,
including homoduplex, heteroduplex, and hybrid states.
[0034] As used herein, the term "promoter" refers to a nucleic acid
sequence that functions to control the transcription of one or more
genes, located upstream with respect to the direction of
transcription of the transcription initiation site of the gene, and
is structurally identified by the presence of a binding site for
DNA-dependent RNA polymerase, transcription initiation sites and
any other DNA sequences, including, but not limited to
transcription factor binding sites, repressor and activator protein
binding sites, and any other sequences of nucleotides known to one
of skill in the art to act directly or indirectly to regulate the
amount of transcription from the promoter. Optionally the term
"promoter" includes herein also the 5' UTR region (5' Untranslated
Region) (e.g. the promoter may herein include one or more parts
upstream (5') of the translation initiation codon of a gene, as
this region may have a role in regulating transcription and/or
translation). A "constitutive" promoter is a promoter that is
active in most tissues under most physiological and developmental
conditions. An "inducible" promoter is a promoter that is
physiologically (e.g. by external application of certain compounds)
or developmentally regulated. A "tissue specific" promoter is only
active in specific types of tissues or cells. A "promoter active in
a particular cell type, e.g. in T cells" refers to the general
capability of the promoter to drive transcription within such a
cell type. It does not make any implications about the
spatio-temporal activity of the promoter.
[0035] The terms "amino acid sequence" or "protein" or "peptide"
refer to molecules consisting of a chain of amino acids, without
reference to a specific mode of action, size, 3 dimensional
structure or origin. A "fragment" or "portion" of thereof may thus
still be referred to as an "amino acid sequence" or "protein" or
"peptide". An "isolated amino acid sequence" is used to refer to an
amino acid sequence which is no longer in its natural environment,
for example in vitro or in a recombinant bacterial or human host
cell.
[0036] "Engineered cells" refers herein to cells having been
engineered, e.g. by the introduction of an exogenous nucleic acid
sequence or specific alteration of an endogenous gene sequence.
Such a cell has been genetically modified for example by the
introduction of for example one or more mutations, insertions
and/or deletions in the endogenous gene and/or insertion of a
genetic construct in the genome. An engineered cell may refer to a
cell in isolation or in culture. Engineered cells may be
"transduced cells" wherein the cells have been infected with e.g. a
modified virus, for example, a retrovirus may be used, such as
described in the examples, but other suitable viruses may also be
contemplated such as lentiviruses. Non-viral methods may also be
used, such as transfections. Engineered cells may thus also be
"stably transfected cells" or "transiently transfected cells".
Transfection refers to non-viral methods to transfer DNA (or RNA)
to cells such that a gene is expressed. Transfection methods are
widely known in the art, such as calciumphosphate transfection, PEG
transfection, and liposomal or lipoplex transfection of nucleic
acids. Such a transfection may be transient, but may also be a
stable transfection wherein cells can be selected that have the
gene construct integrated in their genome.
[0037] "Randomly modifying" includes generating random sequences,
and also includes selecting .delta.2-CDR3 encoding regions and/or
.gamma.9-CDR-3 encoding regions that are occurring in nature and
can be derived from .gamma.9.delta.2T cell receptor sequences
derived from subjects, e.g. from humans, such as described in the
example section wherein such .gamma.9-CDR3 and .delta.2-CDR3
sequences were selected from a database. Generating random
sequences includes modifying one or several amino acids in a
starting .gamma.9-CDR-3 and .delta.2-CDR3 sequence, up to the point
wherein all the amino acids from the starting sequences may be
modified. Modifying amino acid sequences may be done by changing
nucleotide sequences of a codon such that the amino acid encoded by
that codon is altered.
[0038] The term "corresponding to" with regard to amino acid
sequences means that when one sequence is aligned with a reference
sequence which comprises a corresponding sequence, for example
amino acid residues 50-70, the amino acids aligning with the
example amino acid residues 50-70 are the amino acid residues of
the one sequence that are corresponding. An example of an alignment
in which corresponding amino acids are aligned from .gamma.9-CDR3
and .delta.2-CDR3 regions is depicted in FIG. 8.
[0039] The term "selectable marker" is a term familiar to one of
ordinary skill in the art and is used herein to describe any
genetic entity which, when expressed, can be used to select for a
cell or cells containing the selectable marker. Selectable marker
gene products confer for example antibiotic resistance, or another
selectable trait or a nutritional requirement. Selectable markers
such as well-known in the art include green fluorescent protein
(GFP), eGFP, luciferase, GUS and the like.
DETAILED DESCRIPTION OF THE INVENTION
[0040] In a first aspect, the invention relates to a method for
identifying .gamma.9.delta.2T-cell receptors that mediate
anti-tumour responses comprising the steps of: [0041] a) providing
T-cells; [0042] b) providing a nucleic acid sequence encoding a
.gamma.9-T-cell receptor chain comprising a .gamma.9-CDR3 encoding
region, wherein the .gamma.9-CDR3 encoding region is randomly
modified, and a nucleic acid sequence encoding a .delta.2-T-cell
receptor chain comprising a .delta.2-CDR3 encoding region, wherein
the .delta.2-CDR3 encoding region is randomly modified; [0043] c)
introducing the nucleic acid sequences of step b) into the T-cells
to provide for an engineered T-cell with a .gamma.9.delta.2T-cell
receptor comprising the .gamma.9-T-cell receptor chain of step b)
and the .delta.2-T-cell receptor chain of step b); [0044] d)
optionally, repeating steps b) and c); [0045] e) determining the
anti-tumour response of the engineered T-cells provided in steps c)
and d); [0046] f) identifying the .gamma.9.delta.2T-cell receptors
of the engineered T-cells that mediate anti-tumour responses.
[0047] T cells, or T lymphocytes, belong to a group of white blood
cells named lymphocytes, which play a role in cell-mediated
immunity. T cells originate from hematopoietic stem cells in the
bone marrow, mature in the thymus (that is where the T is derived
from), and gain their full function in peripheral lymphoid tissues.
During T-cell development, CD4.sup.-CD8.sup.- T-cells (negative for
both the CD4 and CD8 co-receptor) are committed either to an
.alpha..beta. or .gamma..delta. fate as a result of an initial
.beta. or .delta. TCR gene rearrangement. Cells that undergo early
.beta. chain rearrangement express a pre-TCR structure composed of
a complete .beta. chain and a pre-TCR.alpha. chain on the cell
surface. Such cells switch to a CD4.sup.+CD8.sup.+ state, rearrange
the TCR.alpha. chain locus, and express a mature .alpha..beta.TCR
on the surface. CD4.sup.-CD8.sup.- T cells that successfully
complete the .gamma. gene rearrangement before the .beta. gene
rearrangement express a functional .gamma..delta.TCR and remain
CD4.sup.-CD8.sup.-. (Claudio Tripodo et al. Gamm delta T cell
lymphomas Nature Reviews Clinical Oncology 6, 707-717 (December
2009). The T cell receptor associates with the CD3 protein complex.
Mature T cells, i.e. expressing a .alpha..beta.TCR or a
.gamma..delta.TCR, express the T cell receptor complex on the cell
surface. The .gamma..delta.T-cells, which constitute about 1-5% of
the total population of T cells, can be divided in further
subpopulations. A subpopulation of .gamma..delta.T-cells
constitutes .gamma.9.delta.2T-cells, which express a
.gamma.9.delta.2TCR. Within the extracellular domain of a T cell
receptor three complementarity determining regions (CDR1, CDR2,
CDR3) are located. These regions are in general the most variable
domains and contribute significantly to the diversity among TCRs.
CDR regions are composed during the development of a T-cell where
so-called Variable-(V), Diverse-(D), and Joining-(J)-gene segments
are randomly combined to generate diverse TCRs.
[0048] .alpha..beta.T cells may be defined with respect of function
as T lymphocytes that express an .alpha..beta.TCR, which recognises
peptides bound to MHC molecules (major histocompatibility complex),
which are expressed on the surface of various cells. MHCs present
peptides derived from the proteins of a cell. When for example a
cell is infected with a virus, the MHC will present viral peptides,
and the interaction between the .alpha..beta.TCR and the
MHC-complex activates specific types of T-cells which initiate and
immune responses to eliminate the infected cell. Hence,
.alpha..beta.T cells may be functionally defined as being cells
capable of recognizing peptides bound to MHC molecules.
.alpha..beta.T cells may be selected from peripheral blood for
example via the CD3 antigen as described below and in the examples,
as the large majority of T cells have the .alpha..beta.TCR.
.alpha..beta.T cells may also be selected with an antibody specific
for the .alpha..beta.TCR, such as described below. From such
selected cells, the nucleic acid (or amino acid) sequence
corresponding to the .alpha.T-cell receptor chain and the
.beta.T-cell receptor chain may be determined. Hence,
.alpha..beta.T-cells may also be defined as being cells comprising
a nucleic acid (or amino acid) sequence corresponding to the
.alpha.T-cell receptor chain and/or the .beta.T-cell receptor
chain.
[0049] .gamma.9.delta.2T-cells may be functionally defined in that
they are specifically and rapidly activated by a set of
non-peptidic phosphorylated isoprenoid precursors, collectively
named phosphoantigens. Phosphoantigens are produced by virtually
all living cells. The most common phosphoantigen found in animal
and human cells (including cancer cells) is isopentenyl
pyrophosphate (IPP) and its isomer dimethylallyl pyrophosphate
(DMAPP). Activation of .gamma.9.delta.2T-cells comprises clonal
expansion, cytoxic activity and expression of cytokine.
.gamma.9.delta.2T-cells are also defined by expression of the
.gamma.9.delta.2T-cell receptor. For example, cells may be selected
using an antibody specific for the .gamma.9.delta.2T-cell receptor
such as described below. From such selected cells, the nucleic acid
(or amino acid sequence) sequence corresponding to the
.gamma.9T-cell receptor chain and/or the .delta.2T-cell receptor
chain may be determined. Hence, .gamma.9.delta.2T-cells may also be
defined as being cells comprising a nucleic acid (or amino acid)
sequence corresponding to a .gamma.9T-cell receptor chain and/or a
.delta.2T-cell receptor chain.
[0050] The person skilled in the art is well capable of selecting
and/or identifying cell populations characterized by expression of
an antigen or receptor on the surface of the cell such as described
throughout herein. It is understood that with regard to expression
on the surface of cells, such as CD3, CD4, CD8, .alpha..beta.TCR,
.gamma..delta.TCR and .gamma.9.delta.2TCR, this is typically done
in a population of cells of which a portion of cells have a much
higher level of expression of the antigen when compared to cells
having a lower level of expression. Hence, the terms positive or
negative are to be understood as being relative, i.e. positive
cells have a much higher expression level as compared to cells
being negative. Cells being negative in this sense may thus still
have an expression level which may be detected. Expression on the
surface of cells may be analysed using Fluorescence Activated Cell
Sorting (FACS), and many specific antibodies are commercially
available, e.g. such as for CD3, CD4, CD8, .alpha..beta.TCR,
.gamma..delta.TCR and .gamma.9.delta.2TCR, that are suitable for
such FACS analysis, such as described in the examples and as
available. .gamma.9.delta.2T-cells can hence also be defined and
selected as being positive for .gamma.9.delta.2TCR in FACS.
Antibodies suitable for FACS or similar separation techniques (such
as e.g. antibodies conjugated to magnetic beads) are widely
available. Conditions are selected, such as provided by the
antibody manufacturer that allows the selection of negative and/or
positive cells. Examples of antibodies that may be suitable for
selection of .gamma.9.delta.2-T cells, or engineered
.gamma.9.delta.2T cells such as available from BD Pharmingen (BD, 1
Becton Drive, Franklin Lakes, N.J. USA) are V.gamma.9-PE (clone B3,
#555733), V.delta.2-FITC (clone B6, #555738), .gamma..delta.TCR-APC
(clone B1, #555718) or such as available from Beckman Coulter is
pan-.gamma..delta.TCR-PE (clone IMMU510, # IM1418U) Similarly,
suitable antibodies for .alpha..beta.-T cell selection, such as
anti-CD3 antibodies may be such as available from BD Pharmingen is
CD3-FITC (#345763) or such as anti-.alpha..beta.TCR antibodies such
as available from Beckman Coulter is pan-.alpha..beta.TCR-PE
(#A39499) or pan-.alpha..beta.TCR-PC5 (#A39500).
[0051] Accordingly, in the method of the invention, first T-cells
are provided. The T-cells may be primary cells, for example from a
subject, such as described in the examples for a human subject. The
T-cells may be .alpha..beta. T-cells. The T-cells may also be cell
lines, such as SupT-1, Jurkat, or Raji cells or any other widely
available cell line. Any cell type, being a primary cell or any
other cell line will suffice, as long as the cell population, or a
substantial part thereof, expresses the T-cell receptor, i.e. such
as being positive for the .alpha..beta.T-cell receptor in a FACS
sorting or the like as described above, such a cell population may
be contemplated. Also, any cell or cell population may be
contemplated that, when provided with a .gamma.9.delta.2TCR
receptor according to the invention is capable of forming a
functional TCR complex and exerting e.g. a functional cytoxic
response and/or cytokine production. The cell that is provided may
also be a progenitor cell, preferably a blood progenitor cell such
as a thymocyte or a blood stem cell, which after it has been
provided with the right stimuli can develop into T cells or
engineered T cells. Hence it is understood that providing T cells
and providing these with the .gamma.9.delta.2TCR receptor according
to the invention may also comprise providing progenitor cells,
providing these with the .gamma.9.delta.2TCR receptor and
stimulating these progenitor cells such that these develop into
engineered T-cells.
[0052] Also, a nucleic acid sequence is provided encoding a
.gamma.9-T-cell receptor chain wherein the .gamma.9-CDR3 encoding
region is randomly modified and a nucleic acid sequence encoding a
.delta.2-T-cell receptor chain wherein the .delta.2-CDR3 encoding
region is randomly modified. Preferably, the .gamma.9-T-cell
receptor chain and the .delta.2-T-cell receptor chain are of human
origin. The human CDR3 regions of .gamma.9 and .delta.2 are well
defined. For example, the human CDR3 region of the .gamma.9-TCR
chain of amino acid sequence SEQ ID NO.1 corresponds to amino acid
residues 118-132, which corresponds to amino acid residues
.gamma.105-.gamma.117 according to the International Immunogenetics
Information System (IMGT). The human CDR3 region of the .delta.2
TCR chain of amino acid sequence SEQ ID NO.2 corresponds to amino
acid residues 112-127 which corresponds to amino acid residues
.delta.105-.delta.117 according to the IMGT. According to the
International Immunogenetics Information System (IMGT) (Lefranc MP.
IMGT, the international ImMunoGeneTics database. Nucleic Acids Res.
2003; 31:307-310, which is incorporated herein by reference) the
human CDR3 region may be delimited by, but does not include, the
anchor positions C104 and F118, see also FIG. 8, which are referred
to for each chain as .gamma.C104 and .gamma.F118 and .delta.C104
and .delta.F118. The anchor positions .gamma.C104 corresponds to
C117 in SEQ ID NO.1 and .gamma.F118 corresponds to F133 in SEQ ID
NO.1. The anchor position .delta.C104 corresponds to C111 of amino
acid sequence SEQ ID NO.2 and the anchor position .delta.F118
corresponds to F128 of amino acid sequence SEQ ID NO.2. The IMGT
provides a common access to sequence, genome and structure for
instance of T cell receptors of different species including human T
cell receptors and allows the identification of a .gamma.9T-cell
receptor chain and a .delta.2T-cell receptor chain including the
CDR3 regions, e.g. via the anchor positions.
[0053] According to the IMGT, the CDR3 is delimited by (but does
not include) the anchor positions 2nd-CYS 104 and J-PHE or J-TRP
118. The JUNCTION includes 2nd-CYS 104 and J-PHE or J-TRP 118 and
is therefore two amino acids longer than the CDR3. The CDR3
numbering goes from 105 to 117 and, if necessary, gaps or
additional positions are added at the top of the loop. Note that,
the J-PHE or J-TRP belongs to the characteristic J-REGION motif
`F/W-G-X-G` at positions 118-121, and that the CDR3 is delimited by
the same anchor positions (2nd-CYS 104 and J-PHE or J-TRP 118),
whatever the receptor type (IG or TR), the chain type (heavy or
light for IG; alpha, beta, gamma or delta for TR) or the species.
See also
imgt.org/IMGTScientificChart/Numbering/IMGTIGVLsuperfamily.html for
an explanation of the IMGT numbering. This IMGT numbering is also
used in the examples.
[0054] Hence, by using the anchor positions, according to the IMGT
standards, CDR3 regions can easily be identified. For example via
entering an amino acid sequence (or nucleic acid sequence) using
available online tools such as provided by IMGT (for example
IMGT/V-QUEST) a .gamma.9T-cell receptor chain sequence or a
.delta.2T-cell receptor chain sequence can easily be identified as
well as the exact CDR3 regions. Alternatively, .gamma.9T-cell
receptor chain sequences or .delta.2T-cell receptor chain sequences
already publicly available can be found in the IMGT and hence, the
CDR3 regions can be easily identified therefrom. Alternatively,
using SEQ ID NO.1 and SEQ ID NO.2 as a reference sequence and the
anchor amino acid residue positions therein, the CDR3 regions of
other .gamma.9-T-cell and .delta.2-T-cell receptor chains are
easily identified via an alignment, such as depicted for different
CDR3 region in FIG. 8. Hence, the skilled person can easily
identify any CDR3 region from any .gamma.9T-cell receptor chain
sequence that corresponds with the CDR3 region from SEQ ID NO.1,
and also the skilled person can easily identify any CDR3 region
from any .delta.2T-cell receptor chain sequence that corresponds
with the CDR3 regions from SEQ ID NO.2. Hence, the skilled person
knows which amino acid sequence from any .gamma.9-T-cell and/or
.delta.2-T-cell receptor chains he needs to randomly modify.
[0055] Hence, modifying the .delta.2-CDR3 encoding region in a
.delta.2-T-cell receptor chain nucleic acid sequence may comprise
aligning the nucleic acid sequence of SEQ ID NO.4 to the
.delta.2-T-cell receptor chain nucleic acid sequence of interest,
identifying the codons in the .delta.2-T-cell receptor chain
nucleic acid sequence corresponding to the codons encoding the
anchor positions in SEQ ID NO.4, and randomly modifying the nucleic
acid sequence in between the anchor positions codons identified in
the .delta.2-T-cell receptor chain nucleic acid sequence of
interest. Also, modifying the .gamma.9-CDR3 encoding region in a
.gamma.9-T-cell receptor chain nucleic acid sequence may comprises
aligning the nucleic acid sequence of SEQ ID NO.4 to the
.gamma.9-T-cell receptor chain nucleic acid sequence of interest,
identifying the codons in the .gamma.9-T-cell receptor chain
nucleic acid sequence corresponding to the codons encoding the
anchor positions in SEQ ID NO.3, and randomly modifying the nucleic
acid sequence in between the anchor positions codons identified in
the .gamma.9-T-cell receptor chain nucleic acid sequence of
interest. The random modification of the nucleic acid sequence of
the CDR3 encoding regions is such that the amino acid sequence
encoded by the nucleic acid sequence is modified, i.e. at least one
amino acid residue is changed in another amino acid residue, or at
least one amino acid residue is inserted or deleted.
[0056] The nucleic acid sequences encoding the .gamma.9-T-cell
receptor chain and the .delta.2-T-cell receptor chain may be
introduced into T-cells to provide for an engineered T-cell with a
.gamma.9.delta.2T-cell receptor comprising the .gamma.9-T-cell
receptor chain and the .delta.2-T-cell receptor chain. By
expressing the .gamma.9-T-cell receptor chain and the
.delta.2-T-cell receptor chain of which the CDR3 regions have been
randomly modified, a specific .gamma.9.delta.2-T-cell receptor is
expressed in the cell. Optionally, the steps of providing a CDR3
randomly modified .gamma.9-T-cell receptor chain and
.delta.2-T-cell receptor chain and subsequent introduction into
T-cells may be repeated. For example, each time this is done a
different combination of randomly modified CD3 regions is used. It
is also envisioned that the steps of repeating the steps, e.g. with
different combinations of randomly modified CD3 regions, may be
performed, at least in part, simultaneously. For example, a
plurality of nucleic acid sequences encoding a .gamma.9-T-cell
receptor chain wherein the .gamma.9-CDR3 encoding region is
randomly modified and a nucleic acid sequence encoding a
.delta.2-T-cell receptor chain wherein the .delta.2-CDR3 encoding
region is randomly modified, may be provided, and this plurality of
nucleic acid sequences may be introduced in T-cells thereby
providing a plurality of engineered T-cells.
[0057] The anti-tumour response of the provided engineered T-cell
expressing a .gamma.9.delta.2T-cell receptor or an engineered
.gamma.9.delta.2T-cell receptor that mediate anti-tumour responses
is identified. In case the steps of providing the nucleic acid
sequences and introduction into the T-cells is performed in
separate steps, the step of determining the anti-tumour responses
and identifying the engineered .gamma.9.delta.2T-cell receptor may
be combined because it is known which nucleic acid sequences were
introduced in each of the corresponding engineered
.gamma.9.delta.2T-cell. Determining the anti-tumour responses may
hence be performed for each engineered .gamma.9.delta.2T-cell
separately. Alternatively, for example when engineered
.gamma.9.delta.2T-cells are combined, e.g. when repeating the steps
such as described above is performed simultaneously, and a
plurality of engineered T-cells is provided, the engineered T-cells
may be separated and the anti-tumour response determined for each
of the separated engineered T-cells. The engineered T-cells may be
separated over different compartments and in each of the
compartments the anti-tumour response may be determined. In such a
scenario, the step of identifying the engineered T-cells that
mediate anti-tumour responses may involve determining the sequence,
e.g. via sequencing, of the nucleic acid sequence encoding each of
the CDR3 regions of the .gamma.9.delta.2TCR. Hence, optionally as a
final step, identifying engineered T-cells that mediate anti-tumour
responses involves determining the nucleic acid sequence encoding
the .gamma.9-T-cell receptor chain comprising the .gamma.9-CDR3
encoding region and the nucleic acid sequence encoding the
.delta.2-T-cell receptor chain comprising the .delta.2-CDR3
encoding region. It may also be envisioned to only determine the
nucleic acid sequence of both of the .gamma.9-CDR3 and the
.delta.2-CDR3 encoding regions, i.e. it is not required to
determine the complete nucleic acid sequences of the
.gamma.9-T-cell receptor chain and/or the .delta.2-T-cell receptor
chain. As an alternative to determining the nucleic acid sequence,
the amino acid sequences of the .gamma.9-CDR3 and the .delta.2-CDR3
region may also be determined.
[0058] Preferably, the nucleic acid sequence encoding a
.gamma.9-T-cell receptor chain encodes an amino acid sequence
having at least 60% sequence identity with the amino acid sequence
of SEQ ID NO.1, and wherein the nucleic acid sequence encoding a
.delta.2-T-cell receptor chain encodes an amino acid sequence
having at least 60% sequence identity with the amino acid sequence
of SEQ ID NO.2. The nucleic acid sequence encoding a
.gamma.9-T-cell receptor chain comprises preferably a nucleic acid
sequence encoding an amino acid sequence having at least 70, 80,
90, 95 or 99% sequence identity with the amino acid sequence of SEQ
ID NO.1. The nucleic acid sequence encoding a .delta.2-T-cell
receptor chain comprises preferably a nucleic acid sequence
encoding an amino acid sequence having at least 70, 80, 90, 95 or
99% sequence identity with the amino acid sequence of SEQ ID NO.2.
The skilled person is capable of defining a .gamma.9.delta.2-T-cell
receptor with respect to function. Preferably, the percentage of
sequence identity is calculated over the entire length of SEQ ID
NO.1 or SEQ ID NO.2.
[0059] Random modification of the CDR3 regions of the .gamma.9-CDR3
and .delta.2-CDR3 may comprise the arbitrary selection of amino
acid residues. Such arbitrary selection may be done by generating
random sequences in vitro, e.g. by random chemical synthesis, such
as provided by Sloning Biotechnology using chemical synthesis
(Sloning BioTechnology GmbH--A Division of MorphoSys,
Zeppelinstrasse 4, 82178 Puchheim, Germany). Random modification of
the CDR3 regions of the .gamma.9-CDR3 and .delta.2-CDR3 may also
comprise the arbitrary selection from sequences found in natural
CDR3 regions. Such natural CDR3 amino acid sequences are generated
randomly, i.e. arbitrarily selected, by nature.
[0060] With regard to the random modification, the randomly
modified .gamma.9-CDR3 encoding region may preferably be modified
at the amino acid sequence corresponding to amino acid residues
118-132 of SEQ ID NO 1. With regard to the random modification, the
randomly modified .gamma.9-CDR3 encoding region may preferably be
modified at the amino acid sequence corresponding to amino acid
residues 122-124 of SEQ ID NO 1. As said, a .gamma.9-CDR3 encoding
region is easily identified, e.g. via alignment and/or from
information as provided by IMGT, and hence the amino acid residues
from a .gamma.9-CDR3 encoding region corresponding to the amino
acid residues 118-132 of SEQ ID NO.1 as well. Optionally, amino
acid residues C117 and F133 of SEQ ID NO.1 may further be used for
identifying the corresponding amino acid residues. In an alignment,
such as depicted in FIG. 8, the amino acid residues of a
.gamma.9-CDR3 encoding region corresponding to amino acid residues
118-132 of amino acid sequence SEQ ID NO.1 can easily be
identified, and thus amino acid residues corresponding to amino
acid residues 122-124 of SEQ ID NO 1 as well. Hence, a
corresponding sequence may be easily be identified via aligning the
amino acid sequence of interest to the reference amino acid
sequence (SEQ ID NO.1), and identifying the anchor amino acid
residues C117 and F133, so that the CDR3 region is identified.
Next, the CDR3 regions are aligned and the amino acid residues of
the amino acid sequence that correspond to 118-132 of SEQ ID NO.1
is identified. With regard to the random modification, the randomly
modified .delta.2-CDR3 encoding region may be modified at the amino
acid sequence corresponding to amino acid residues 115-122 of SEQ
ID NO.2. With regard to the random modification, the randomly
modified .delta.2-CDR3 encoding region may be modified at the amino
acid sequence corresponding to amino acid residues 112-127 of SEQ
ID NO.2. A .delta.2-CDR3 encoding region may be easily identified,
e.g. via alignment and/or from information as provided by IMGT, and
hence the amino acid residues from a .delta.2-CDR3 encoding region
corresponding to the amino acid residues 112-127 of SEQ ID NO.2 as
well. Optionally, amino acid residues C111 and F128 of SEQ ID NO.2
may further be used for identifying the corresponding amino acid
residues. In an alignment, such as depicted in FIG. 8, the amino
acid residues of a .delta.2CDR3 encoding region corresponding to
amino acid residues 112-127 of amino acid sequence SEQ ID NO.2 can
easily be identified. Hence, a corresponding sequence may be easily
identified via aligning the amino acid sequence of interest to the
reference amino acid sequence (SEQ ID NO.2), and identifying the
anchor amino acid residues C111 and F128, such that the CDR3 region
is identified. Next, the CDR3 regions are aligned and the amino
acid residues of the amino acid sequence that correspond to 112-127
of SEQ ID NO.2 can be identified, and thus the amino acid sequence
that correspond to amino acid residues 115-122 as well.
[0061] In one embodiment, randomly modifying the amino acid
sequence corresponding to amino acid residues 115-122 of SEQ ID
NO.2 comprises a modification in length.
[0062] In one embodiment, randomly modifying the amino acid
sequence corresponding to amino acid residues 112-127 of SEQ ID
NO.2 comprises a modification in length.
[0063] In one embodiment, randomly modifying the amino acid
sequence comprises introducing amino acid substitutions, deletions,
and/or insertions.
[0064] With regard to the random modification, this may include any
modification such as amino acid substitution, deletion and/or
insertion. Hence, the random modification may include a
modification in sequence and/or length. The CDR3 regions may thus
vary in length ranging from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,
13, 14, 15, 16, 17, 18, 19, 20, 21 or 22 amino acid residues.
Preferably, the .delta.2-CDR3 encoding region may vary in length
ranging from 5-7 amino acids between positions .delta.L109 and
.delta.T113 according to the IGMT annotation. Hence, preferably the
.delta.2-CDR3 encoding region corresponding to amino acid sequence
112-127 of SEQ ID NO.2 is randomly modified between L116 and T123
of SEQ ID NO.2 such that it contains 5-7 amino acids in this
region. Hence, the entire .delta.2-CDR3 encoding region preferably
varies in length ranging from 15-17 amino acid residues. Amino acid
substitution may include any substitution such as conserved
substitutions wherein one amino acid from a group is changed for
another amino acid residue of the group, e.g. one aliphatic amino
acid residue is exchange for another aliphatic amino acid residue,
or a non-conserved substitution wherein one amino acid from a group
is changed for another amino acid residue of a different group,
e.g. one aliphatic amino acid residue is exchange for an basic
amino acid residue.
[0065] With regard to providing a nucleic acid, it is understood
that the nucleic acid is provided such that when it is introduced
in a cell that it can be expressed such that the amino acid
sequence it encodes is expressed on the surface of the cell.
Preferably this is done by integrating the nucleic acid or nucleic
acids into the genome of the cell. Preferably, the nucleic acid
sequence encoding a .gamma.9-T-cell receptor chain is provided in a
genetic construct and the nucleic acid sequence encoding a
.delta.2-T-cell receptor chain is provided in a genetic construct.
The genetic constructs may be provided on separate vectors. The
genetic constructs may also be provided on a single vector. The
nucleic acid sequence encoding a .gamma.9-T-cell receptor chain and
the nucleic acid sequence encoding a .delta.2-T-cell receptor chain
may also be provided in a single genetic construct. For example, a
single genetic construct may express a single mRNA comprising an
Internal Ribosomal Entry Site, such as described in the example,
such that each receptor chain may be transcribed from the same
mRNA.
[0066] The genetic construct and/or vectors may also comprise
selectable markers. A selectable marker may be defined as any
nucleic acid sequence and/or amino acid sequence that allows cells
that are provided therewith to be selected. For example, selectable
markers may be neomycin or puromycin resistance genes such as
described in the examples. Selection of cells to which the genetic
construct and/or vector has been transferred may than be performed
by incubating in the presence of neomycin or puromycin. Other
selectable markers may be for example any one of green, red and
yellow fluorescent proteins. Selecting may than be performed by
using FACS. It is not required to have a selectable marker, as the
cell when expressing the .gamma.9.delta.2T-cell receptor may be
selected based on that expression on itself, e.g. via selection
with an antibody directed thereto such as described above. It may
be provided that the host cell does not express a substantial
amount of the .gamma.9.delta.2T-cell receptor to allow selection of
engineered .gamma.9.delta.2T-cells, i.e. expression of the
endogenous .gamma.9.delta.2-T-cell receptor must be much lower as
compared with the engineered .gamma.9.delta.2-T-cell receptor.
[0067] Preferably, the nucleic acid sequence encoding a
.gamma.9-T-cell receptor chain is provided in a retroviral vector
and the nucleic acid sequence encoding a .delta.2-T-cell receptor
chain is provided in a retroviral vector and the steps of
introducing the nucleic acid sequences into the T-cells comprises
retroviral vector transduction of the T-cells. The transduction may
be performed simultaneously or in subsequent steps. Alternatively,
the nucleic acid sequence encoding a .gamma.9-T-cell receptor chain
and the nucleic acid sequence encoding a .delta.2-T-cell receptor
chain are provided in a single retroviral vector, and the steps of
introducing the nucleic acid sequences into the T-cells comprises
retroviral vector transduction of the T-cells. Retroviral vectors,
such as described in the examples, are highly efficient for
transferring the nucleic acid sequences to the T-cells such that
engineered T-cells can be provided. Many retroviral and lentiviral
vectors are known or such as described in the examples. Retroviral
vectors have an RNA genome which, when entered in a cell, is
reverse transcribed into DNA that is subsequently integrated into
the host genome. Integration is advantageous as it allows to
proliferation of transduced cells while maintaining the viral
vector genome comprising the genetic construct.
[0068] In one embodiment, the step of determining the anti-tumour
reactivity comprises contacting the cells with tumour cells. Tumour
cells may be any kind of tumour cells. For example, primary tumour
cells from a patient. The tumour cells may be tumour cells from
cell lines, such as the cell lines listed in the examples named
Daudi, RPM18226/S, OPM2, LME1, K562, Saos2, MZ1851RC, SCC9, Fadu,
MDA-MB231, MCF7, BT549, SW480, which are well known in the art.
Tumour cell lines may easily be obtained from the American Type
Culture Collection (ATCC, Manassas, Va.) and the like. The step of
determining anti-tumour activity may include any assay in which an
anti-tumour effect may be determined, such as having an effect on
tumour cell division rate, i.e. the speed with which the tumour
cells divide, cell death, binding to the tumour cells etc.
[0069] Determining the anti-tumour responses includes contacting
the engineered T-cell with a tumour cell and measuring its ability
to lyse the tumour cell and/or induce IFN-.gamma. production. The
ability to lyse the tumour cells include providing a fixed amount
of tumour cells with which the engineered .gamma.9.delta.2 T-cell,
i.e. an engineered T-cell expressing a .gamma.9.delta.2TCR, is
contacted and after an incubation period the number of viable
tumour cells is counted. When the number of viable cells counted is
compared to a control not contacted with the engineered
.gamma.9.delta.2 T-cell, and the number is lower, such an
engineered .gamma.9.delta.2 T-cell can be regarded to have an
anti-tumour response. In addition to counting the viable cells, one
may also perform a .sup.51Chromium-release assay similarly to what
is described in the examples. The amount of .sup.51Chromium release
being a measure of the number of cells that have been lysed.
[0070] Similarly, IFN-.gamma. production may also be determined,
e.g. via antibody staining, ELISA and/or quantitative PCR for the
expressed mRNA. Assays for determining IFN-.gamma. are commercially
widely available, such as described in the example. Engineered
.gamma.9.delta.2 T-cells are contacted with the tumour cells. The
contacting may be in the presence of a phosphoantigen, such as
pamidronate. Hence, when the amount of IFN-.gamma. produced is
higher as compared to when cells are not contacted with the
engineered .gamma.9.delta.2 T-cell, such an engineered
.gamma.9.delta.2 T-cell may be regarded as having an anti-tumour
response. In addition to comparing with a control, i.e. to cells
not contacted with the engineered .gamma.9.delta.2 T-cell, one may
also compare with a reference value. In any case, any assay in
which an effect on tumour cells may be determined involving
contacting the .gamma.9.delta.2 T-cell with the tumour cell may be
contemplated. As long as an anti-tumour effect, such as induction
of cell death, cell viability, binding to the tumour cell, and/or
IFN-.gamma. production may be determined.
[0071] Furthermore, with regard to the methods described above, the
T cells may be expanded before or after the transfer of the nucleic
acids encoding the engineered .gamma.9.delta.2 T-cell receptor,
i.e. with the randomly modified CDR3 regions. Preferably, the
expansion is after the transfer such that relatively little nucleic
acids need to be transferred. This expansion of the T cells may be
performed by stimulation with .alpha.-CD3/CD28 Dynabeads in the
presence of IL-2. A rapid expansion protocol such as described in
the examples may also be used. The expanded cells comprising the
engineered .gamma.9.delta.2 T-cell receptor, which may be selected
e.g. via a selectable marker such as described above, may be
further selected for the presence of the CD4 antigen and the CD8
antigen, e.g. using the MACS separating system as described in the
examples. The engineered T-cells may be subsequently further
expanded using the REP protocol as described by Riddel and
Greenberg, 1990 J Immunol Methods. 128(2):189-201, which is
incorporated herein by reference, or using similar further
expansion methods thereto. Briefly, the expansion method involves
using antibodies directed against T cell activation molecules, such
as TCR, CD3 and CD28 and/or feeder cells and/or stimulating
cytokines.
[0072] In another embodiment, a .gamma.9.delta.2T-cell receptor, or
a fragment thereof, is provided comprising a .gamma.9-T-cell
receptor chain comprising a .gamma.9-CDR3 region, wherein the amino
acid sequence of the .gamma.9-CDR3 region corresponding to amino
acid residues 122-124 of SEQ ID NO.1 is modified and a
.delta.2-T-cell receptor chain comprising a .delta.2-CDR3 region
wherein the amino acid sequence of the .delta.2-CDR3 region
corresponding to amino acid residues 115-122 of SEQ ID NO.2 is
modified, wherein the combinations of the amino acid modifications
within the .gamma.9.delta.2T-cell receptor, or a fragment thereof,
are as listed in Table 1. The substitutions may comprise the
combinations such as listed in table 1. The amino acid sequences of
the respective .gamma.9-CDR3 and .delta.2-CDR3 regions are
substituted with amino acid sequences selected from the group
consisting of combinations of amino acid sequences AQQ (.gamma.9)
(SEQ ID NO: 21) and ALKRTD (62) (SEQ ID NO: 15); AQQ (.gamma.9)
(SEQ ID NO: 21) and LLGY (62) (SEQ ID NO: 22); IQ (.gamma.9) (SEQ
ID NO: 20) and ALKRTD (62) (SEQ ID NO: 15); and; IQ (.gamma.9) (SEQ
ID NO: 20) and TLGMGGEY (62) (SEQ ID NO: 19). These
.gamma.9.delta.2T-cell receptors, or fragments thereof, comprising
these particular combinations of CDR3 regions, were identified
using the methods according to the invention such as described
above and as described in the examples.
[0073] Combinations of amino acid sequences of .gamma.9-CDR3 and
.delta.2-CDR3 regions that may be preferably combined are listed
below in table 1 as they show an anti-tumour effect. Table 1.
Combinations of amino acid sequences of .gamma.9-CDR3 and
.delta.2-CDR3 regions. The region corresponding to amino acid
residues of the .gamma.9-CDR3 region corresponding to number
122-124 of SEQ ID NO.1 which has been substituted, i.e. has been
modified, is listed, and the region corresponding to amino acid
residues of the .delta.2-CDR3 region corresponding to amino acid
residues 115-122 of SEQ ID NO.2 which has been substituted is
listed as well. The different combinations of .gamma.9-CDR3 and
.delta.2-CDR3 are numbered from 1-4. For example, number 2
corresponds to a .gamma.9.delta.2TCR with a .gamma.9-T-cell
receptor chain wherein the .gamma.9-CDR3 region corresponding to
number 122-124 of SEQ ID NO.1 has been substituted with AQQ (SEQ ID
NO: 21) and a .delta.2T-cell receptor chain wherein the
.delta.2-CDR3 region corresponding to amino acid residues 115-122
of SEQ ID NO.2 has been substituted with ALKRTD (SEQ ID NO:
15).
TABLE-US-00001 TABLE 1 Combinations of modified amino acid
sequences of .gamma.9-CDR3 and .delta.-CDR3 regions, wherein the
listed amino acid sequences substitute .gamma.9-CDR3 amino acid
residues corresponding with 122-124 of SEQ ID NO. 1 and substitute
.delta.2-CDR3 amino acid residues corresponding with 115-122 of SEQ
ID NO. 2. Nr. combination .gamma.9-CDR3 .delta.2-CDR3 1
.gamma.9-cI5/.delta.2-cI5 IQ (SEQ ID NO. 20) ALKRTD (SEQ ID NO. 15)
2 .gamma.9-G115/.delta.2-cI5 AQQ (SEQ ID NO. 21) ALKRTD (SEQ ID NO.
15) 3 .gamma.9-cI5/.delta.2-G115 IQ (SEQ ID NO. 20) TLGMGGEY (SEQ
ID NO. 19) 4 .gamma.9-G115/.delta.2-cI3 AQQ (SEQ ID NO. 21) LLGY
(SEQ ID NO. 22)
[0074] In another embodiment, a .gamma.9.delta.2T-cell receptor, or
a fragment thereof, is provided comprising a .gamma.9-T-cell
receptor chain comprising a .gamma.9-CDR3 region, wherein the amino
acid sequence of the .gamma.9-CDR3 region corresponding to amino
acid residues 118-132 of SEQ ID NO.1 is modified and a
.delta.2-T-cell receptor chain comprising a .delta.2-CDR3 region
wherein the amino acid sequence of the .delta.2-CDR3 region
corresponding to amino acid residues 112-127 of SEQ ID NO.2 is
modified, wherein the combinations of the amino acid modifications
within the .gamma.9.delta.2T-cell receptor, or a fragment thereof,
are as listed in Table 2. The substitutions may comprise the
combinations such as listed in table 2. The amino acid sequences of
the respective .gamma.9-CDR3 and .delta.2-CDR3 regions are
substituted with amino acid sequences selected from the group
consisting of combinations of amino acid ALWEIQELGKKIKV (.gamma.9)
(SEQ ID NO: 16) and ACDALKRTDTDKLI (62) (SEQ ID NO: 17);
ALWEAQQELGKKIKV (.gamma.9) (SEQ ID NO: 3) and ACDALKRTDTDKLI (62)
(SEQ ID NO: 17); ALWEIQELGKKIKV (.gamma.9) (SEQ ID NO: 16) and
ACDTLGMGGEYTDKLI (62) (SEQ ID NO: 4); ALWEAQQELGKKIKV (.gamma.9)
(SEQ ID NO: 3) and ACDLLGYTDKLI (62) (SEQ ID NO: 18). These
.gamma.9.delta.2T-cell receptors, or fragments thereof, comprising
these particular combinations of CDR3 regions, were identified
using the methods according to the invention such as described
above and as described in the examples.
[0075] Combinations of amino acid sequences of .gamma.9-CDR3 and
.delta.2-CDR3 regions that may be preferably combined are listed
below in table 2 as they show a strong anti-tumour effect. Table 2.
Combinations of amino acid sequences of .gamma.9-CDR3 and
.delta.2-CDR3 regions. The region corresponding to amino acid
residues of the .gamma.9-CDR3 region corresponding to number
118-132 of SEQ ID NO.1 which has been substituted, i.e. has been
modified, is listed, and the region corresponding to amino acid
residues of the .delta.2-CDR3 region corresponding to amino acid
residues 112-127 of SEQ ID NO.2 which has been substituted, i.e. is
modified, is listed as well. The different combinations of
.gamma.9-CDR3 and .delta.2-CDR3 are numbered from 1-4 in table 2.
For example, number 2 corresponds to a .gamma.9.delta.2TCR with a
.gamma.9-T-cell receptor chain wherein the .gamma.9-CDR3 region
corresponding to number 118-132 of SEQ ID NO.1 has been modified to
(substituted with) ALWEAQQELGKKIKV (SEQ ID NO: 3) and a
.delta.2T-cell receptor chain wherein the .delta.2-CDR3 region
corresponding to amino acid residues 112-127 of SEQ ID NO.2 has
been modified to (substituted with) ACDALKRTDTDKLI (SEQ ID NO:
17).
TABLE-US-00002 TABLE 2 Combinations of complete modified amino acid
sequences of .gamma.9-CDR3 and .delta.2-CDR3 regions Nr.
combination .gamma.9-CDR3 .delta.2-CDR3 1 .gamma.9-cI5/.delta.2-cI5
ALWEIQELGKKIKV ACDALKRTDTDKLI (SEQ. ID NO. 16) (SEQ. ID NO. 17) 2
.gamma.9-G115/.delta.2-cI5 ALWEAQQELGKKIKV ACDALKRTDTDKLI (SEQ. ID
NO. 3) (SEQ. ID NO. 17) 3 .gamma.9-cI5/.delta.2-G115 ALWEIQELGKKIKV
ACDTLGMGGEYTDKLI (SEQ. ID NO. 16) (SEQ. ID NO. 4) 4
.gamma.9-G115/.delta.2-cI3 ALWEAQQELGKKIKV ACDLLGYTDKLI (SEQ. ID
NO. 3) (SEQ. ID NO. 18)
[0076] In one embodiment, the .gamma.9.delta.2T-cell receptor, or a
fragment thereof, comprises a .gamma.9-T-cell receptor chain
comprising a .gamma.9-CDR3 region, wherein the amino acid sequence
of the .gamma.9-CDR3 region corresponding to amino acid residues
122-124 of the amino acid sequence of SEQ ID NO.1 is AQQ (SEQ ID
NO: 21), and a .delta.2-T-cell receptor chain comprising a
.delta.2-CDR3 region wherein the amino acid sequence of the
.delta.2-CDR3 region corresponding to amino acid residues 115-122
of the amino acid sequence of SEQ ID NO.2 is ALKRTD (SEQ ID NO:
15). In a further embodiment, the .gamma.9.delta.2T-cell receptor,
or a fragment thereof, comprises a .gamma.9-T-cell receptor chain
comprising a .gamma.9-CDR3 region, wherein the amino acid sequence
of the .gamma.9-CDR3 region corresponding to amino acid residues
118-132 of the amino acid sequence of SEQ ID NO.1 is
ALWEAQQELGKKIKV (SEQ ID NO: 3), and a .delta.2-T-cell receptor
chain comprising a .delta.2-CDR3 region wherein the amino acid
sequence of the .delta.2-CDR3 region corresponding to amino acid
residues 112-128 of the amino acid sequence of SEQ ID NO.2 is
ACDALKRTDTDKLI (SEQ ID NO: 17). This particular combination of CDR3
regions in the .gamma.9.delta.2T-cell receptor, or fragment thereof
has shown a particular high anti-tumour activity and may hence be
preferred.
[0077] In one embodiment, the .gamma.9.delta.2T-cell receptor or
fragment thereof according to invention, wherein the amino acid
sequence of the .gamma.9-CDR3 region corresponding to amino acid
residues 118-121 of the amino acid sequence of SEQ ID NO.1 is ALWE,
and wherein the amino acid sequence of the .gamma.9-CDR3 region
corresponding to amino acid residues 125-132 of the amino acid
sequence of SEQ ID NO.1 is ELGKKIKV, and wherein the amino acid
sequence of the .delta.2-CDR3 region corresponding to amino acid
residues 112-114 of the amino acid sequence of SEQ ID NO.2. is ACD,
and wherein the amino acid sequence of the .delta.2-CDR3 region
corresponding to amino acid residues 123-127 of the amino acid
sequence of SEQ ID NO.2 is TDKLI. It is understood that these amino
acid sequences correspond to the sequences which are flanking the
amino acid sequences that are to be substituted with the amino acid
sequences as listed above.
[0078] In a further embodiment, a .gamma.9.delta.2T-cell receptor,
or fragment thereof, is provided, wherein the .gamma.9-T-cell
receptor chain comprises an amino acid sequence having at least
60%, 70%, 80%, 90%, 95%, 99%, or 100% sequence identity with the
amino acid sequence of SEQ ID NO.1, and/or wherein the
.delta.2-T-cell receptor chain comprises an amino acid sequence
having at least 60%, 70%, 80%, 90%, 95%, 99%, or 100% sequence
identity with the amino acid sequence of SEQ ID NO.2. It is
understood that the amino acid sequence of the .gamma.9-CDR3 region
corresponding to amino acid residues number 122-124 of SEQ ID NO.1,
and the amino acid sequence of the .delta.2-CDR3 region
corresponding to amino acid residues 115-122 of SEQ ID NO.2, are in
this embodiment substituted in accordance with Table 1.
[0079] In one embodiment, a .gamma.9.delta.2T-cell receptor, or
fragment thereof is provided, wherein the .gamma.9-T-cell receptor
chain comprises an amino acid sequence comprising amino acid
residues 1-121 and 125-315 of the amino acid sequence of SEQ ID
NO.1 and wherein the modified amino acid residues 122-124 are as
defined Table 1, and wherein the .delta.2-T-cell receptor chain
comprises an amino acid sequence comprising amino acid residues
1-114 and 123-292 of the amino acid sequence of SEQ ID NO.2 and
wherein the modified amino acid residues 115-122 are as defined in
Table 1. It is understood that the amino acid sequence of the
.gamma.9-CDR3 region corresponding to amino acid residues number
122-124 of SEQ ID NO.1, and the amino acid sequence of the
.delta.2-CDR3 region corresponding to amino acid residues 115-122
of SEQ ID NO.2, are in this embodiment substituted in accordance
with Table 1.
[0080] It is understood that the amino acid sequences SEQ ID NO.1
and SEQ ID NO.2 each comprise a leader sequence. The leader
sequence of amino acid sequence SEQ ID NO.1 is from amino acid Nr.
1-20 of SEQ ID NO.1. The leader sequence of amino acid sequence SEQ
ID NO.2 is from amino acid Nr. 1-19 of SEQ ID NO.2. The leader
sequence may directs the amino acid chain to the surface of the
cell. The leader sequence can be cleaved from the nascent amino
acid chain and is not present in the final protein. Hence, in one
embodiment, a .gamma.9.delta.2T-cell receptor, or fragment thereof
is provided, wherein the .gamma.9-T-cell receptor chain comprises
an amino acid sequence comprising amino acid residues 21-121 and
125-315 of the amino acid sequence of SEQ ID NO.1 and wherein the
modified amino acid residues 122-124 are as defined in Table 1, and
wherein the .delta.2-T-cell receptor chain comprises an amino acid
sequence comprising amino acid residues 20-114 and 123-292 of the
amino acid sequence of SEQ ID NO.2 and wherein the modified amino
acid residues 115-122 are as defined in Table 1. It is understood
that the amino acid sequence of the .gamma.9-CDR3 region
corresponding to amino acid residues number 122-124 of SEQ ID NO.1,
and the amino acid sequence of the .delta.2-CDR3 region
corresponding to amino acid residues 115-122 of SEQ ID NO.2, are in
this embodiment substituted in accordance with Table 1. It is also
understood that it may be optional to use alternative leader
sequences, and it is also understood that this leader sequences
from SEQ ID NO.1 and SEQ ID NO.2 may be disregarded, e.g. when
comparing sequences and/determining corresponding sequences and/or
alignment and/or determining percentages of identity.
[0081] In another embodiment, a conjugate is provided comprising a
soluble fragment of the .gamma.9.delta.2T-cell receptor according
to any one of the .gamma.9.delta.2T-cell receptors as described
above. The extracellular domain of the .gamma.9.delta.2-T-cell
receptor comprises the amino acid sequence of the .gamma.9-T-cell
receptor chain corresponding to amino acid residues 21-263 of the
amino acid sequence of SEQ ID NO.1 and the amino acid sequence of
the .delta.2-T-cell receptor chain corresponding to amino acid
residues 20-249 of the amino acid sequence of SEQ ID NO.2. The
conjugate may be linked to an agent. Preferably, the agent is
selected from the group consisting of a diagnostic agent, a
therapeutic agent, an anti-cancer agent, a chemical, a
nanoparticle, a chemotherapeutic agent or a fluorochrome. Such
conjugates that may be linked to substrates (e.g. chemicals,
nanoparticles) may be used e.g. to deliver chemotherapy to a target
of interest. In addition, in diagnostics expression of defined
ligands may be tested by taking advantage of the soluble TCRs
linked to fluorochromes which are then used as staining tool or for
the biochemical isolation of the ligand.
[0082] Furthermore, in this embodiment, the invention provides for
isolated nucleic acid sequences encoding the .delta.2-T-cell
receptor chain, or fragment thereof, according to the invention,
wherein the .delta.2-CDR3 region corresponding to amino acid
residues 115-122 of the amino acid sequence SEQ ID NO.2 is
substituted with amino acid residues ALKRTD (SEQ ID NO: 15) or LLGY
(SEQ ID NO: 22). Hence, the .delta.2-CDR3 region corresponding to
amino acid residues 112-127 of the amino acid sequence SEQ ID NO.2
is substituted between D114 and T123 of SEQ ID NO.2 with amino acid
residues ALKRTD (SEQ ID NO: 15) or LLGY (SEQ ID NO: 22). The
invention further provides for an isolated nucleic acid sequence
encoding the .gamma.9-T-cell receptor chain, or fragment thereof,
according to the invention, wherein the amino acid sequence of the
.gamma.9-CDR3 region corresponding to amino acid residues 122-124
of the amino acid sequence SEQ ID NO.1 is substituted with amino
acid residues IQ (SEQ ID NO: 20). Hence, the .gamma.9-CDR3 region
corresponding to amino acid residues 118-132 of the amino acid
sequence SEQ ID NO.1 is substituted between E121 and E 125 of SEQ
ID NO 1 with amino acid residues IQ (SEQ ID NO: 20).
[0083] Also are provided genetic constructs comprising the isolated
nucleic acid sequences according to the invention, and retroviral
vectors comprising the genetic construct.
[0084] For example, isolated nucleic acid sequences are listed as
SEQ ID NO.3 and SEQ ID NO.4 and correspond respectively to open
reading frames of the .gamma.9-T-cell receptor chain (encoding
corresponding amino acid sequence SEQ ID NO.1) and the
.delta.2-T-cell receptor chain (encoding corresponding amino acid
sequence SEQ ID NO.2) of the G115wt clone, such as described in the
examples. These isolated nucleic acid sequences, when being part of
an expression cassette may be interspersed by for example intronic
encoding sequences, and have 5' and 3' non-coding regions. When
such sequences are expressed, the corresponding .gamma.9-T-cell
receptor chain and the .delta.2-T-cell receptor chain amino acid
sequences are made in the cell. Hence, the nucleic acid sequence of
SEQ ID NO.3 encodes the amino acid sequence of SEQ ID NO.1. The
nucleic acid sequence of SEQ ID NO.4 encodes the amino acid
sequence of SEQ ID NO.2. The amino acid sequences of SEQ ID NO. 1
and SEQ ID NO.2 as they are formed in the cell each comprise a
leader sequence. The leader sequence of amino acid sequence SEQ ID
NO.1 is from amino acid Nr. 1-20 of SEQ ID NO.1. The leader
sequence of amino acid sequence SEQ ID NO.2 is from amino acid Nr.
1-19 of SEQ ID NO.2. The leader sequence may direct the amino acid
chain to the surface of the cell. The leader sequence can be
cleaved from the nascent amino acid chain and is not present in the
finished protein. The constant domain of an engineered
.gamma.9.delta.2T-cell receptor may have short connecting sequences
in which cysteine residues form a disulfide bond, forming a link
between the .gamma.9-TCR chain and the .delta.2-TCR chain. For
example, C263 of amino acid sequence SEQ ID NO.1 and C249 of amino
acid sequence SEQ ID NO.2, corresponding to the .gamma.9-TCR chain
and the .delta.2-TCR chain of G115wt may form a disulphide bond. A
disulfide bond is a covalent bond formed by the coupling of two
thiol groups of the cysteins.
[0085] In another embodiment, a nucleic acid sequence encoding the
.gamma.9.delta.2T-cell receptor, or fragment thereof, according to
the invention is provided, as well as a nucleic acid sequence
comprising genetic constructs or a genetic construct encoding the
.gamma.9.delta.2T-cell receptor according to the invention. It is
understood that in this embodiment the nucleic acid sequences
encoding the .gamma.9.delta.2T-cell receptor are to be comprised in
single nucleic acid sequence. Preferably, the nucleic acid sequence
comprising the genetic construct or genetic constructs is comprised
in a retroviral vector.
[0086] In one embodiment, a cell is provided expressing the soluble
fragment of the .gamma.9.delta.2T-cell receptor according to the
invention.
[0087] In one embodiment, a T cell is provided comprising the
.gamma.9.delta.2T-cell receptor according the invention, i.e.
corresponding to the specific .gamma.9.delta.2T-cell receptors of
which the CDR3 regions are substituted with the specific amino acid
sequences such as listed and indicated above. In a further
embodiment, the T cell comprises the isolated nucleic acid
sequences as listed above, the genetic construct or genetic
constructs according as listed above or the retroviral vector or
retroviral vectors as listed above.
[0088] Furthermore, the .gamma.9.delta.2T-cell receptor, or
fragment thereof, according to the invention as described above, or
a conjugate, an isolated nucleic acid sequence, or a genetic
construct, or a retroviral vector, or a T cell, as listed above, is
for use as a medicament. Preferably, the .gamma.9.delta.2T-cell
receptor, or fragment thereof, according to the invention as
described above, or a conjugate, an isolated nucleic acid sequence,
or a genetic construct, or a retroviral vector, or a T cell, as
listed above, is for use as a medicament in the treatment of
cancer.
[0089] It is understood that the specific .gamma.9.delta.2T-cell
receptors, or fragments thereof, according the invention, i.e.
corresponding to the specific .gamma.9.delta.2T-cell receptors of
which the CDR3 regions are substituted with the specific amino acid
sequences such as listed and indicated above, are in particular
useful in medical treatments and/or in diagnostics. For example,
immune cells may be redirected against cancer cells by e.g. ex vivo
transfer of one of the specific .gamma.9.delta.2T-cell receptors as
listed into .alpha..beta.T-cells of a patient followed by expansion
and adoptive transfer of these engineered T-cells back into the
patient. Hence, immune cells may be redirected against cancer
cells. This may also be done in in combination with any other
receptors (e.g. NKG2D) in immune-cells in order to increase
anti-cancer reactivity.
Examples
[0090] Materials and Methods
[0091] Cells and Cell Lines
[0092] PBMCs were isolated from buffy coats obtained from Sanquin
Blood Bank (Amsterdam, The Netherlands). Primary AML blasts were
received after obtaining informed consent from the LML biobank UMC
Utrecht and a kind gift from Matthias Theobald (Mainz, Germany) and
were collected according to GCP and Helsinki regulations. Cell
lines are described in supplementary Material and Methods.
[0093] TCR Mutagenesis, Cloning and Sequencing
[0094] .gamma.9.delta.2TCR modifications are based on
codon-optimized genes of .gamma.9- or .delta.2-TCR chain G115
flanked by NcoI and BamHI restriction sites (synthesized by
GeneArt, Regensburg, Germany). To generate alanine-mutations,
site-directed mutagenesis was performed by overlap extension PCR 21
or whole plasmid mutagenesis 22; 23, using a proofreading
polymerase (Phusion, Bioke). Mutated NcoI-BamHI digested .gamma.9-
or .delta.2-TCR chains were ligated into the retroviral vector
pBullet and sequenced by BaseClear (Leiden, The Netherlands).
[0095] Flow Cytometry
[0096] .gamma.9.delta.2TCR expression was analyzed by flow
cytometry using a V.delta.2-FITC (clone B6, BD) or a
pan-.gamma..delta.TCR-PE antibody (clone IMMU510, Beckman Coulter).
Fold change was calculated based on MFI values of
.gamma.9-G115wt/.delta.2-G115wt transduced T-cells set to 1 and
mock transduced T-cells to 0.
[0097] Functional T-Cell Assays
[0098] 51Chromium-release assay for cell-mediated cytotoxicity was
previously described. Target cells were labeled overnight with 100
.mu.Cu 51Cr (150 .mu.Cu for primary cells) and incubated for 5 h
with transduced T-cells in five effector-to-target ratios (E:T)
between 30:1 and 0.3:1. Fold change was calculated when compared to
reactivity of engineered T-cells expressing unmutated
.gamma.9.delta.2TCR. IFN-.gamma. ELISpot was performed using
anti-hulFN-.gamma.mAb1-D1K (I) and mAb7-B6-1 (II) (Mabtech-Hamburg,
Germany) following the manufacturer's recommended procedure. Target
and effector cells (E:T 3:1) were incubated for 24 h in the
presence of pamidronate (Calbiochem, Germany) where indicated.
IFN.gamma. ELISA was performed using ELISA-ready-go! Kit
(eBioscience) following manufacturer's instructions. Effector and
target cells (E:T 1:1) were incubated for 24 h in the presence of
pamidronate as indicated. Where specified, fold change was
calculated when compared to reactivity of engineered T-cells
expressing unmutated .gamma.9.delta.2TCR.
[0099] Retroviral Transduction of T-Cells
[0100] .gamma.9.delta.2TCRs were transduced into
.alpha..beta.T-cells as previously described (Marcu-Malina et al.,
2011). In brief, packaging cells (phoenix-ampho) were transfected
with gag-pol (pHIT60), env (pCOLT-GALV) (Stanislawski et al., 2001)
and two retroviral constructs (pBullet) containing either
.gamma.9-chain-IRES-neomycine or .delta.2-chain-IRES-puromycine,
using Fugene6 reagent (Takara, Gennevilliers, France). Human PBMC
activated with .alpha.CD3 (30 ng/ml) (Orthoclone OKT3,
Janssen-Cilag, Tilburg, The Netherlands) and IL2 (50 IU/ml)
(PROLEUKIN.RTM., Novartis, Arnhem, The Netherlands) were twice
transduced with viral supernatant within 48 hours in the presence
of 50 IU/ml IL-2 and 4 .mu.g/ml polybrene (Sigma-Aldrich,
Zwijndrecht, The Netherlands). Transduced T-cells were expanded by
stimulation with .alpha.CD3/CD28 Dynabeads (0.5.times.10.sup.6
beads/10.sup.6 cells) (Invitrogen) and IL-2 (50 IU/ml) and selected
with 800 .mu.g/ml geneticin (Gibco, Karlsruhe, Germany) and 5
.mu.g/ml puromycin (Sigma-Aldrich, Zwijndrecht, The Netherlands)
for one week. Where indicated polyclonal CD4+ and CD8+ TCR
transduced T-cells with were sorted based on CD4 or CD8 expression
using CD4 and CD8 MACS separating system (Miltenyi Biothech,
Bergish Gladbach, Germany). Following selection TCR-transduced
T-cells were expanded in vitro based on a previously described REP
protocol (Riddell and Greenberg, 1990).
[0101] Functional T-Cell Assays
[0102] .sup.51Chromium-release assay for cell-mediated cytotoxicity
was previously described (Kuball et al., 2004). Target cells were
labelled overnight with 100 .mu.Cu .sup.51Cr (150 .mu.Cu for
primary cells) and subsequently incubated with transduced T-cells
in five effector to target ratios (E:T) between 30:1 and 0.3:1.
After 4-6 h .sup.51Cr-release was measured in the supernatant. Fold
change was calculated based on .sup.51Cr-release of
.gamma.9-G115wt/.delta.9-G115wt transduced T-cells normalized to 1;
for side by side experiments based on .gamma.9-3wt/59-3wt,
.gamma.9-5wt/59-5wt or .gamma.9-G115wt/59-G115wt normalized to
1.
[0103] IFN.gamma. ELISpot was performed using anti-hu IFN-.gamma.
mAb1-D1K (I) and mAb7-B6-1 (II) from Mabtech (Hamburg, Germany)
following manufactures' recommended procedure (Besold et al.,
2007). In all assays target and effector cells (E:T 3:1) were
incubated for 24 h in the presence of pamidronate (Calbiochem,
Germany) where indicated.
[0104] IFN-.gamma.ELISA was performed using ELISA-ready-go! Kit
(eBioscience) following manufacturers' instructions. Effector and
target cells (E:T 1:1) were incubated for 24 h in the presence of
pamidronate as indicated. Where specified, fold change was
calculated based on IFN-.gamma. secretion .gamma.9-G115wt/59-G115wt
transduced T-cells normalized to 1; for the side by side
experiments based on .gamma.9-3wt/59-3wt, .gamma.9-5wt/59-5wt or
.gamma.9-G115wt/59-G115wt normalized to 1.
[0105] Animal Models
[0106] To induce tumour xenografts, sublethal total body irradiated
(2Gy), 11-17 weeks old RAG-2-/-/yc-/-- BALB/C mice were injected
i.v. with 0.5.times.10.sup.6 Daudi-Luc cells (a kind gift from
Genmab Utrecht, The Netherlands) or 5.times.10.sup.6 RPM18226/S-Luc
cells (Anton Martens, Utrecht, The Netherlands) together with
10.sup.7 .gamma.9.delta.2TCR+ transduced T-cells. The
RAG-27.sup.-yc.sup.-/--BALB/C mice were originally obtained from
AMCAS b.v. (Amsterdam, the Netherlands). Mice were bred and housed
in the specific pathogen-free (SPF) breeding unit of the Central
Animal Facility of the University of Utrecht. All animal
experiments were conducted according to Institutional Guidelines
after acquiring permission from the local Ethical Committee for
Animal Experimentation, and in accordance with current Dutch laws
on Animal Experimentation. All mice developed tumours, mainly
growing in the bone marrow visualized in vivo once a week by
Biospace bioluminescent imaging. Mice were anesthetized by
isoflurane inhalation before they received an intraperitoneal
injection of 100 .mu.l of 25 mg/ml Beetle Luciferin (Promega, USA).
Bioluminescence images were acquired using a third generation
cooled GaAs intensified charge-coupled device camera, controlled by
the Photo Vision software and analyzed with M.sup.3Vision software
(all from Photon Imager; Biospace Laboratory). Mice received
0.6.times.10.sup.6 IU of IL2 (PROLEUKIN.RTM., Novartis) in IFA s.c.
on day 1 (together with tumour cells) and every 21 days till the
end of the experiment. Pamidronate (10 mg/kg body weight) was
applied in the indicated groups at day 1 i.v. and every 21 days
i.p. Outgrowing tumours were visualized in vivo by Biospace
bioluminescent imaging (BLI). Mice were anesthetized by isoflurane
before they received an intraperitoneal injection (100 .mu.l) of 25
mg/ml Beetle Luciferin (Promega). Bioluminescence images were
acquired and analyzed with M3Vision software (Photon Imager,
Biospace Laboratory).
[0107] Results
Anti-Tumour Reactivity of Individual .gamma.9.delta.2T-Cell
Clones
[0108] To investigate whether individual .gamma.9.delta.2T-cell
clones mediate differential activity against tumour cells compared
to the parental .gamma.9.delta.2T-cell population,
.gamma.9.delta.2T-cells from a healthy donor were cloned by
limiting dilution and tested against a broad panel of tumour cells
in an IFN.gamma. ELISpot. High variability in tumour recognition in
terms of specificity and functional avidity was observed between
individual .gamma.9.delta.2T-cell clones (cl); compared to the
original bulk population, cl5 and cl13 produced twice as many
IFN.gamma. spots in response to Daudi and selectively generated
significant amounts IFN.gamma. when challenged with K562, BT549 and
MCF-7. In contrast, cl3 and cl15 recognized solely Daudi cells.
Surface expression of .gamma.9.delta.2TCR, NKG2D, CD158a, NKAT-2
and NKB-1 was examined.
[0109] Anti-Tumour Reactivity Mediated by Individual
.gamma.9.delta.2TCRs
[0110] To elucidate differences among .gamma.9.delta.2TCRs of
tumour-reactive clones, sequences of wildtype (wt) .gamma.9- and
.delta.2-TCR chains of cl3
(.gamma.9-cl3.sub.wt/.delta.2-cl3.sub.wt) and cl5
(.gamma.9-cl5.sub.wt /.delta.2-cl5.sub.wt) were determined and
aligned with .gamma.9.delta.2TCR G115. All three
.gamma.9.delta.2TCRs differed in their CDR3 domains: 1-3 amino
acids between position .gamma.109 and .gamma.111 in .gamma.CDR3 and
4-8 amino acids between .delta.108 and .delta.112 in .delta.CDR3.
To determine whether distinct .gamma.9.delta.2TCRs mediate
differential anti-tumour reactivity, individual .gamma.9.delta.2TCR
chains were cloned into the retroviral vector pBullet and linked to
a selection marker as described. The wildtype-combinations
.gamma.9-cl3.sub.wt/.delta.2-cl3.sub.wt,
.gamma.9-cl5.sub.wt/.delta.2-cl5.sub.wt and
.gamma.9-G115.sub.wt/.delta.2-G115.sub.wt were transduced into
peripheral blood .alpha..beta.T-cells, selected by antibiotics and
further expanded. .gamma.9.delta.2TCR G115
(.gamma.9-G115.sub.wt/.delta.2-G115.sub.wt) served as control, as
did cells transduced with an empty vector cassette (mock).
.gamma.9.delta.2TCR-transduced T-cells showed similar
.gamma.9.delta.2TCR expression and were tested for their lytic
activity against the tumour target Daudi in a .sup.51Cr-release
assay (FIG. 1A). T-cells expressing
.gamma.9-cl3.sub.wt/.delta.2-cl3.sub.wt had a 50 percent reduced
ability to lyse tumour cells (p<0.01), whereas T-cells with
.gamma.9-cl5.sub.wt/.delta.2-cl5.sub.wt were nearly twice as potent
(p<0.01) as the control
.gamma.9-G115.sub.wt/.delta.2-G115.sub.wt. To determine whether the
phenotypes of .gamma.9.delta.2TCR-transduced cells with decreased
or increased functional avidity are also present on cytokine level
a pamidronate-titration assay was performed. Pamidronate treatment
of Daudi cells blocks the mevalonate-pathway downstream to IPP
causing the accumulation of IPP and an enhanced cytokine secretion
of responsive T-cells. To exclude NK-like activation
CD4+.gamma.9.delta.2TCR-transduced T-cells, which lack the
expression of major NK-receptors like NKG2D, were selected by
MACS-sorting. Transductants were tested at different concentrations
of pamidronate against the tumour target Daudi. Mock-transduced
T-cells which underwent equivalent stimulation but express an
irrelevant .alpha..beta.TCR served as control. IFN.gamma. secretion
was measured by ELISA and the half maximal effective concentration
(EC50) was calculated (FIG. 1B). In line with changes observed for
lytic capacity, T-cells transduced with
.gamma.9-cl3.sub.wt/.delta.2-cl3.sub.wt secreted lower amounts of
IFN.gamma. (max. 600 .mu.g/ml), while T-cells expressing
.gamma.9-cl5.sub.wt/.delta.2-cl5.sub.wt produced higher levels of
IFN.gamma. (max. 1300 .mu.g/ml) at all pamidronate concentrations,
relative to control .gamma.9-G115.sub.wt/.delta.2-G115.sub.wt (max.
800 .mu.g/ml). Despite different plateaus in IFN.gamma. secretion,
all selected mutants and the wildtype control had a comparable
pamidronate-EC50 (-30 .mu.g/ml). These results indicate that
distinct .gamma.9.delta.2TCR clones mediate different functional
avidity and the high variability among parental
.gamma.9.delta.2T-cell clones in tumour recognition seems to be
substantially regulated by the CDR3 domains of individual
.gamma.9.delta.2T-cell receptors.
[0111] Combinatorial-.gamma..delta.TCR-chain-exchange (CTE) as
rapid method to modulate functional avidity of engineered
T-cells.
[0112] To make the above determination, we devised a strategy named
combinatorial-.gamma..delta.TCR-chain-exchange (CTE), which results
in the expression of newly combined .gamma.9- and .delta.2-TCR
chains on engineered T-cells. During this process,
.gamma.9-G115.sub.wt was combined with .delta.2-cl3.sub.wt or
.delta.2-cl5.sub.wt and .delta.2-G115.sub.wt with
.gamma.9-cl3.sub.wt or .gamma.9-cl5.sub.wt. These combinations were
retrovirally transduced into .alpha..beta.T-cells. In all
transductants equivalent .gamma..delta.TCR expression was detected
while the endogenous .alpha..beta.TCR was clearly down regulated.
This resulted not only into a nearly abolished allo-reactivity of
.alpha..beta.T-cells expressing
.gamma.9-G115.sub.wt/.delta.2-G115.sub.wt but also of selected
CTE-engineered .alpha..beta.T-cells when compared to
mock-transduced cells. Thus, reactivity of CTE-engineered T-cells
primarily depends on expressed .gamma..delta.TCRs and not on
residual endogenous .alpha..beta.TCRs. Next, transductants were
functionally tested against the tumour target Daudi in a
.sup.51Cr-release assay (FIG. 1C). The exchange of .gamma.9- or
.delta.2-chains indeed caused notable differences. Compared to the
original TCR .gamma.9-G115.sub.wt/.delta.2-G115.sub.wt, the
combination of .gamma.9-G115.sub.wt/.delta.2-cl3.sub.wt,
.gamma.9-G115.sub.wt/.delta.2-cl5.sub.wt or
.gamma.9-cl5.sub.wt/.delta.2-G115.sub.wt mediated 40 to 70 percent
increased specific lysis of tumour cells (all p<0.05). The same
magnitude of recognition was observed when IFN.gamma. production of
CD4.sup.+.gamma.5TCR-transduced T-cells was tested in a pamidronate
titration assay (FIG. 1 D). Moreover, only the combination
.gamma.9-cl3.sub.wt/.delta.2-G115.sub.wt led to decreased
IFN.gamma. production of transduced cells at all pamidronate
concentrations (max. 100 .mu.g/ml), while all other
CTE-.gamma.9.delta.2TCRs mediated an increased IFN.gamma.-secretion
(max. .gtoreq.1000 .mu.g/ml) as compared to control TCR
.gamma.9-G115.sub.wt/.delta.2-G115.sub.wt (max. 800 .mu.g/ml).
Equal pamidronate-EC50s of .about.30 .mu.g/ml were calculated for
all responsive .gamma.9.delta.2TCR-transduced cells.
[0113] To determine whether cell-cell interaction influences the
response-kinetics differently than pamidronate stimulation,
CTE-.gamma.9.delta.2TCR .gamma.9-G115.sub.wt/.delta.2-cl5.sub.wt
which mediates improved functional avidity and control TCR
.gamma.9-G115.sub.wt/.delta.2-G115.sub.wt were tested in an
effecter-to-target ratio (E:T) titration assay (FIG. 1 E), and an
E:T-50 was calculated. Interestingly, T-cells with
.gamma.9-G115.sub.wt/.delta.2-cl5.sub.wt responded differently with
an E:T-50 of 0.3:1, compared to an E:T-50 of 1:1 calculated for
control cells expressing .gamma.9-G115.sub.wt/.delta.2-G115.sub.wt.
To test whether the interaction between different TCRs and
ligands--thus the affinity--is indeed increased, cell-cell
conjugates between Daudi and T-cells expressing either potentially
high (.gamma.9-G115.sub.wt/.delta.2-cl5.sub.wt) or low
(.gamma.9-cl3.sub.wt/.delta.2-G115.sub.wt) affinity TCRs were
measured by flow cytometry. Significantly more cell-cell
interactions were observed when
.gamma.9-G115.sub.wt/.delta.2-cl5.sub.wt was expressed as compared
to .gamma.9-cl3.sub.wt/.delta.2-G115.sub.wt and mock-transduced
T-cells (FIG. 1F). This effect did not depend on the presence of
pamidronate. G115.sub.wt/.delta.2-cl5.sub.wt is therefore a high
affinity .gamma.9.delta.2TCR. Hence, CTE is an efficient method to
rapidly engineer .gamma.9.delta.2TCRs with increased affinity,
mediating improved functional avidity in transduced T-cells.
[0114] Residues in .delta.CDR3 and J.delta.1 are involved in
.gamma.9.delta.2TCR stability and in mediating functional avidity
of engineered .alpha..beta.T-cells
[0115] To elucidate the molecular requirements of .delta.CDR3 to
mediate optimal functional avidity, alanine-mutagenesis of a model
.delta.CDR3 (clone G115) was performed including the whole
J.delta.1 segment, as important residues have also been reported
within J.gamma.1. During an initial screening, five sequence areas
were found to either influence TCR expression or functional avidity
of .gamma.9.delta.2TCR transduced T-cells. To clarify the degree to
which single residues are responsible for impaired
.gamma.9.delta.2TCR expression and lower TCR-mediated functional
avidity, single alanine mutations were generated. The mutated and
wildtype .delta.2-G115 chains were expressed in combination with
.gamma.9-G115.sub.wt in .alpha..beta.T-cells and tested for
.gamma.9.delta.2TCR expression using a .delta.2-chain specific
antibody (FIG. 2A). Three single alanine mutations caused a 70
percent lower TCR expression when compared to the unmutated
.delta.2-G115.sub.wt, namely .delta.2-G115.sub.L116A,
.delta.2-G115F.sub.118A and .delta.2-G115v.sub.124A (FIG. 8).
Comparable results were observed using antibodies directed against
the .gamma.9-chain or the constant domain of the .gamma..delta.TCR,
indicating the importance of .delta.2-G115.sub.L116,
.delta.2-G115.sub.F118 and .delta.2-G115.sub.V124 for stable TCR
expression. The crystal structure of .gamma.9.delta.2TCR G115
supports our findings: .delta.2-G115.sub.L116,
.delta.2-G115.sub.F118 and .delta.2-G115.sub.V124 are located in
hydrophobic cores (FIG. 2B) and could thus be crucial for the
structural stability of the .gamma.9.delta.2TCR G115.
[0116] To address the impact of single alanine mutations on
functional avidity, a .sup.51Cr-release assay was performed (FIG.
2C). Transductants with low TCR expression (.delta.2-G115L116A,
.delta.2-G115F118A and .delta.2-G115V124A) could not lyse tumour
cells effectively, as they demonstrated an 80 percent lower lytic
capacity when compared to cells transduced with
.delta.2-G115.sub.wt. T-cells with mutation .delta.2-G115L109A and
.delta.2-G115.sub.l117A (FIG. 8) properly expressed the TCR but
showed a 70 percent reduced lytic activity when compared to
.delta.2-G115.sub.wt expressing cells. Similar results were
obtained when TCR mutants were transduced into CD4.sup.+ Jurkat
cells and IL-2 production was measured (data not shown). Reduction
of lytic activity was also seen when alanine substitutions
.delta.2-G115L109A and .delta.2-G115.sub.l117A were introduced into
the .delta.2-chain of .gamma..delta.TCR clone 3. These results
indicate that not only residue .delta.L109, but also .delta.l117 in
.delta.CDR3 may be generally important for .gamma.9.delta.2TCRs to
mediate functional avidity (FIG. 2D). Sequence alignments between
.delta.2-chains of clones 3, 5 and G115 indicated that .delta.L109
and .delta.l117 may be conserved (Table 2).
[0117] FIG. 8 shows an alignment of three clones and the numbering
listed is in accordance with IGMT. The .gamma.9-G115.sub.wt
sequence listed corresponds to amino acids C117-T142 of amino acid
sequence SEQ ID NO.1..gamma.112.1L, of either clone 3, 5 or
G115.sub.wt thus corresponds to L126 of amino acid sequence SEQ ID
NO.1. The .delta.2-G115.sub.wt sequence listed corresponds to amino
acids C111-C138 of amino acid sequence SEQ ID NO.2. Hence, as this
alignment shows, corresponding CDR3 regions of clones 3 and 5 can
easily be identified via alignment.
[0118] Influence of CDR3 length on functional avidity of
.gamma.9.delta.2TCR transduced T-cells
[0119] Alanine substitutions during alanine-scanning mutagenesis of
.gamma.9.delta.2TCR G115 could replace large parts of the
.delta.CDR3 domain without functional consequences. That raises the
possibility that the crucial factor for the differing functional
avidities of distinct .gamma.9.delta.2TCR combinations may also
involve the relative length between the functionally important
residues .delta.2-G115.sub.L109 and the structurally important
residue .delta.2-G115.sub.L116. Therefore, different .delta.2-G115
length mutants were generated. Since the triple
.delta.2-G115.sub.T113-K115 is also important for stable surface
expression (data not shown), nine length mutants
(.delta.2-G115.sub.LM) with 0 to 12 alanine between
.delta.2-G115.sub.L109 and .delta.2-G115.sub.T113 were generated
and equally expressed in .alpha..beta.T-cells, again in combination
with .gamma.9-G115.sub.wt (FIG. 3A). To test the functional avidity
of .delta.2-G115.sub.LM transduced T-cells, CD4.sup.+
TCR-transduced T-cells were selected by MACS-sorting and an
IFN.gamma. ELISA in response to Daudi was performed in the presence
of pamidronate (FIG. 3B). Engineered T-cells expressing
.delta.2-G115.sub.LM0 and .delta.2-G115.sub.LM1 were unable to
produce IFN.gamma. and T-cells expressing .delta.-G115.sub.LM4 or
.delta.-G115.sub.LM12 secreted only about half the amount of
IFN.gamma. compared to .delta.2-G115.sub.wt transduced cells. All
other mutants (.delta.2-G115.sub.LM2, 3, 5, 6, 9) induced
comparable amounts of IFN.gamma. in engineered T-cells relative to
transductants expressing .delta.2-G115.sub.wt. Mutants with
functional impairment (.delta.2-G115.sub.LM0,1,4,12, Table 3) were
further tested against increasing pamidronate concentrations and an
EC50 was calculated. Despite different plateaus in maximal
IFN.gamma. secretion, all selected .delta.2-G115.sub.LM transduced
cells and the wildtype control had a comparable pamidronate-EC50
(.about.30 .mu.g/ml) (FIG. 3C). Length mutations were also studied
in .gamma.CDR3 of .gamma.9.delta.2TCR G115 by engineering stretches
of 1-6 alanines between .gamma.9-G115.sub.E108 and
.gamma.9-G115.sub.E111.1 (.gamma.9-G115.sub.LM1-6). However, this
did not affect functional avidity.
[0120] This indicates that considerable alanine stretches within
.gamma.9 and .delta.2CDR3 domains can be tolerated. However, too
short and very long alanine stretches between
.delta.2-G115.sub.L109 and .delta.2-G115.sub.T113 in particular, as
well as stretches with four alanines, may be associated with
reduced or absent function of a .gamma.9.delta.2TCR (FIGS. 3B and
3C).
[0121] Consequences for the Physiological .gamma.9.32T-Cell
Repertoire
[0122] The ImMunoGeneTics (IMGT) database was searched for reported
stretches between .gamma.9-G115.sub.E109 and
.gamma.9-G115.sub.E1111 as well as .delta.2-G115.sub.L109 and
.delta.2-G115.sub.T113. A preferential length for reported
.gamma.9-chains was found for CDR3 regions corresponding to
.gamma.9-G115.sub.LM2 and .gamma.9-G115.sub.LM3, but shorter
stretches were also reported. In contrast, .delta.2-chains with
short .delta.CDR3 domains, such as .delta.2-G115.sub.LM1 or
.delta.2-G115.sub.LM0, were not reported (FIG. 3D), in line with
our observation that such chains may not be functional. The
majority of listed .gamma.9.32TCRs contain .delta.CDR3 lengths
which correspond to .delta.2-G115.sub.LM5,6,7. These findings
support a preference to select .gamma.9.delta.2TCRs with a
.delta.CDR3 length of 5-7 residues between .delta.2-G115-L109 and
.delta.2-G115.sub.T113. Nevertheless, individual sequence
differences can still play a role in .gamma.9.delta.2TCR mediated
functional avidity.
[0123] Influence of the CDR3 sequence on .gamma.9.delta.2TCR
mediated functional avidity.
[0124] To test both the length and sequence of .delta.CDR3 for
mediating optimal functional avidity, .gamma.9.delta.2TCR length
mutants .delta.2-G115.sub.LM2, .delta.2-G115.sub.LM4, and
.delta.2-G115.sub.LM6 were transduced into .alpha..beta.T-cells in
combination with .gamma.9-G115.sub.wt. IFN.gamma.-secretion of
transductants in response to Daudi was compared to cells transduced
with wildtype sequences from .delta.2-cl3.sub.wt (corresponds in
length to .delta.2-G115.sub.LM2), .delta.2-cl5.sub.wt (corresponds
in length to .delta.2-G115.sub.LM4), and .delta.2-G115.sub.wt
(corresponds in length to .delta.2-G115.sub.LM6) (FIG. 8). T-cells
transduced with .delta.2-G115.sub.LM6 and .delta.2-G115.sub.wt did
not differ in the amount of cytokine secretion, all other
combinations of wildtype chains showed a more than two-fold
increase in IFN.gamma. when compared to the length mutant that
selectively contained alanines (FIG. 3E). These results were
confirmed when the lytic capacity of transduced cells was tested.
The sequence in .delta.CDR3 may therefore also be a significant
factor for the functioning of a .gamma.9.delta.2TCR.
[0125] Accordingly, the sequential importance of .gamma.CDR3 was
studied. Thereby, .gamma.9-G115.sub.LM1-3 were transduced into
T-cells in combination with 82-G115.sub.wt. IFN-secretion of
transductants in response to Daudi was compared to cells transduced
with .gamma.9-cl3.sub.wt (corresponds in length to
.gamma.9-G115.sub.LM1), .gamma.9-cl5.sub.wt (corresponds in length
to .gamma.9-G115.sub.LM2) and .gamma.9-G115.sub.wt (corresponding
to .gamma.9-G115.sub.LM3) (FIG. 8). T-cells expressing
.gamma.9-cl3.sub.wt/.delta.2-G115.sub.wt selectively produced lower
amounts of IFN.gamma. when compared to their equivalent
.gamma.9-G115.sub.LM1 (FIG. 4A). Previously, the same
.gamma.9.delta.2TCR combination was also found to mediate reduced
functional avidity (FIGS. 1C and 1D). Loss of activity could be
restored to normal levels (referred to .gamma.9.delta.2TCR
G115.sub.wt) by mutating .gamma.CDR3.sub.E109 in
.gamma.9-cl3.sub.wt to .gamma.CDR3.sub.A109, demonstrating that a
single change in the variable sequence of .gamma.9CDR3 may be
sufficient to regulate functional avidity of .gamma.9.delta.2TCR
transduced T-cells tested here.
[0126] In summary, the length and sequence of the .delta.2CDR3
domain between L109 and T113 (FIG. 8) can play a role in
.gamma.9.delta.2TCR-mediated functional avidity. In addition, the
individual sequence between E108 and E111.1 in .gamma.9CDR3 may
hamper activity of a .gamma.9.delta.2TCR, and in G115
.gamma.CDR3.sub.A109 may be of importance for ligand interaction
(FIG. 8 and FIG. 4B). Combined, this provides a rationale for
CTE-engineered .gamma.9.delta.2TCRs but also for random mutagenesis
within both .gamma.9 and .delta.2CDR3 regions.
[0127] CTE-engineered T-cells as a tool for cancer
immunotherapy.
[0128] CTE-engineered .gamma.9.delta.2TCRs with increased activity
against tumour cells are interesting candidates for TCR-gene
therapeutic strategies. Changes in functional avidity mediated by
CTE-.gamma.9.delta.2TCRs may constitute a unique phenomenon of a
defined .gamma.9.delta.2TCR pair in response to the B-lymphoblastic
cell line Daudi, or this may be a general response to most tumour
targets. Therefore, CTE-.gamma.9.delta.2TCRs that mediated
increased (.gamma.9-G115, .delta.2-cl5.sub.wt) or reduced
(.gamma.9-cl3.sub.wt/.delta.2-G115.sub.wt) activity were tested
against various tumours in an IFN.gamma. ELISA in the presence of
pharmacological concentrations of pamidronate (10 .mu.M) (FIG. 5A).
Tumour reactivity was significantly increased against a whole range
of different tumour entities including other hematological cancers
such as RPM18226/S, OPM2, LME1 (all multiple myeloma), K562
(myelogenous leukemia) as well as solid cancer cell lines such as
Saos2 (osteosarcoma), MZ1851RC (renal cell carcinoma), SCC9, Fadu
(head and neck cancer), MDA-MB231, MCF7, BT549 (all breast cancer),
and SW480 (colon carcinoma) when taking advantage of
.gamma.9-G115.sub.wt/.delta.2-cl5.sub.wt, as compared to
.gamma.9-G115.sub.wt/.delta.2-G115.sub.wt, and was significantly
reduced or even absent for all other targets using
.gamma.9-cl3.sub.wt/.delta.2-G115.sub.wt. Moreover, CTE-engineered
T-cells with increased activity against tumour cells still did not
show any reactivity towards healthy tissue such as PBMCs and
fibroblasts. Superior lytic activity of T-cells engineered with
.gamma.9-G115.sub.wt/.delta.2-cl5.sub.wt was also observed for
hematological cancer cells like RPM18226/S, OPM2, L363 as well as
solid cancer cell lines Saos2, MZ1851RC, SCC9, MDA-MB231, and SW480
when compared to control T-cells expressing
.gamma.9-G115.sub.wt/.delta.2-G115.sub.wt (FIG. 5B). Therefore,
CTE-engineered .gamma.9.delta.2TCRs can provide higher anti-tumour
response against a broad panel of tumour cells while not affecting
normal tissue, and thus have the potential to increase efficacy of
TCR-engineered T-cells.
[0129] To assess the potential clinical impact of CTE-engineered
.gamma.9.delta.2TCRs, it was tested whether an increased efficacy
of CTE-.gamma.9.delta.2TCRs may also be present when primary blasts
of AML patients are chosen as targets. Therefore,
CTE-.gamma.9.delta.2TCR transduced T-cells were tested against 11
primary AML blasts and healthy CD34+ progenitor cells in an
IFN.gamma.-ELISpot (FIG. 5C). Transductants expressing
.gamma.9-G115.sub.wt/.delta.2-cl5.sub.wt recognized 8 out of 11
primary AML samples equally or superiorly compared to control
.gamma.9-G115.sub.wt/.delta.2-G115.sub.wt. Furthermore, CD34.sup.+
progenitor cells were not recognized by T-cells expressing either
.gamma.9-G115.sub.wt/.delta.2-cl5.sub.wt or
.gamma.9-G115.sub.wt/.delta.2-G115.sub.wt. In light of these
findings, CTE-engineered TCR
.gamma.9-G115.sub.wt/.delta.2-cl5.sub.wt appears to be a promising
candidate for clinical application.
[0130] Finally, to demonstrate that CTE-.gamma.9.delta.2TCRs are
safe and function with increased efficacy when compared to the
original constructs in vivo, adoptive transfer of T-cells
engineered with CTE-TCRs was studied in a humanized mouse model:
protection against outgrowth of Daudi or RPM18226/S in
Rag2.sup.-/-yc.sup.-/- double knockout mice. Therefore peripheral
blood .alpha..beta.T-cells were transduced with CTE-TCR
.gamma.9-G115.sub.wt/.delta.2-cl5.sub.wt or control TCR
.gamma.9-G115.sub.wt/.delta.2-G115.sub.wt. CTE-TCR transduced
T-cells showed similar expression of homing markers including
L-selectin and CCR7. Irradiated Rag2.sup.-/-yc.sup.-/- mice
received luciferase-transduced Daudi (0.5.times.10.sup.6) or
RPM18226/S cells (5.times.10.sup.6) and 10.sup.7 CTE-engineered
T-cells by intravenous injection. The frequency of T-cell infusion
was reduced to one intravenous injection relative to our previously
reported model were two infusions were given in order to test
superiority of CTE-TCR transduced T-cells under suboptimal
conditions. Consequently, this resulted in loss of protection with
TCR G115.sub.wt-engineered T-cells when tumour growth was measured
by bioluminescence imaging (BLI) (FIGS. 6A and 6B). However,
CTE-engineered T-cells expressing
.gamma.9-G115.sub.wt/.delta.2-cl5.sub.wt clearly reduced tumour
outgrowth for Daudi (20.000 counts/min, day 42, n=4) and RPM18226/S
(80.000 counts/min, day 35, n=7) as compared to TCR
G115.sub.wt-engineered T-cells (Daudi: 180.000 counts/min, day 42;
RPM18226/S: 210.000 counts/min, day 35). T-cells could be found in
the periphery until 1-2 weeks after infusion in mice, but frequency
of T-cells did not correlate with tumour regression. Finally, in
the rapidly lethal Daudi-model only mice treated with
CTE-engineered T-cells had a significant increased overall survival
of -2 months relative to mice treated with T-cells expressing
.gamma.9-G115.sub.wt/.delta.2-G115.sub.wt (FIG. 6C). These results
indicate that CTE-engineered .gamma.9.delta.2TCRs efficiently
mediate anti-tumour reactivity in vivo, which points to CTE as a
potential tool to optimize .gamma.9.delta.2TCRs for clinical
application.
[0131] In table 3 below, results with regard to methods performed
according to the invention are listed. The delta-CDR3 domain was
tested for TCR function by alanine-mutagenesis (FIG. 8, mutation)
of the CDR3-domain (including the whole J-segment) of the well
studied clone G115 and amino acids that may be relevant for TCR
stability and TCR function are found. In addition, the amino acid
sequence length (FIG. 8, length mutations) between .gamma.E108 and
.gamma.E111.1 of the .gamma.9-chain and between .delta.L109 and
.delta.T113 of the .delta.2-chain was tested. The individual
sequences of .gamma.9-CDR3 and .delta.2-CDR3 domains are of
importance for function (FIG. 8, sequence). By CTE-engineering the
.gamma.9- and .delta.2-TCR chains of clone G115 was combined with
.gamma.9- and .delta.2-TCR chains of clone 3 and clone 5,
respectively. CTE-engineering resulted in 4 newly designed
.gamma..delta.TCRs. The original TCRs and the new CTE-engineered
TCRs (FIG. 8, combination) were transduced into
.alpha..beta.T-cells and tested for their function
TABLE-US-00003 TABLE 3 Effect of mutations and chain-combinations
on .gamma.9.delta.2-TCR function IFN-.gamma. cytotoxic
.gamma.9.delta.2TCR expression response activity mutation
.gamma.9-G115/.delta.2-G115 original +++ +++ +++
.gamma.9-G115/.delta.2-G115.sub.T108A .delta.T108
.fwdarw..delta.A108 ++++ +++ +++
.gamma.9-G115/.delta.2-G115.sub.L109A .delta.L109
.fwdarw..delta.A109 ++++ - - .gamma.9-G115/.delta.2-G115.sub.G110A
.delta.G110 .fwdarw..delta.A110 +++ nd +++
.gamma.9-G115/.delta.2-G115.sub.M111A, G111.1A, G112.2A .delta.M111
.fwdarw..delta.A111 +++ +++ +++ .delta.G111.1 .fwdarw..delta.A111.1
.delta.G112.2 .fwdarw..delta.A112.2
.gamma.9-G115/.delta.2-G115.sub.E112.1A, Y112A, T113A .delta.E112.1
.fwdarw..delta.A112.1 +++ +++ +++ .delta.Y112 .fwdarw..delta.A112
.delta.T113 .fwdarw..delta.A113
.gamma.9-G115/.delta.2-G115.sub.D114A .delta.D114
.fwdarw..delta.A110 +++ nd +++
.gamma.9-G115/.delta.2-G115.sub.K115A .delta.K115
.fwdarw..delta.A110 +++ nd +++
.gamma.9-G115/.delta.2-G115.sub.L116A .delta.L116
.fwdarw..delta.A116 - nd - .gamma.9-G115/.delta.2-G115.sub.I117Aa
.delta.I117 .fwdarw..delta.A117 +++ - -
.gamma.9-G115/.delta.2-G115.sub.F118A .delta.F118
.fwdarw..delta.A118 - nd - .gamma.9-G115/.delta.2-G115.sub.G119A
.delta.G119 .fwdarw..delta.A119 +++ nd +++
.gamma.9-G115/.delta.2-G115.sub.K120A, G121A, T122A .delta.K120
.fwdarw..delta.A120 +++ +++ +++ .delta.G121 .fwdarw..delta.A121
.delta.T122 .fwdarw..delta.A122
.gamma.9-G115/.delta.2-G115.sub.R123A .delta.R123
.fwdarw..delta.A123 +++ nd +++
.gamma.9-G115/.delta.2-G115.sub.V124A .delta.V124
.fwdarw..delta.A124 - nd - .gamma.9-G115/.delta.2-G115.sub.T125A
.delta.T125 .fwdarw..delta.A125 +++ nd +++
.gamma.9-G115/.delta.2-G115.sub.V126A .delta.V126
.fwdarw..delta.A126 +++ nd +++
.gamma.9-G115/.delta.2-G115.sub.E127A .delta.E127
.fwdarw..delta.A127 +++ nd +++
.gamma.9-G115/.delta.2-G115.sub.P128A .delta.P128
.fwdarw..delta.A128 +++ nd +++
.gamma.9-G115/.delta.2-G115.sup.deletion.sub.T113-K115 deletion
.delta.T113, + nd nd .delta.D114 & .delta.K115 length mutation
.gamma.9-G115.sub.LM1/.delta.2-G115 .gamma.E108-1A-.gamma.E111.1
+++ +++ +++ .gamma.9-G115.sub.LM2/.delta.2-G115
.gamma.E108-2A-.gamma.E111.1 +++ +++ +++
.gamma.9-G115.sub.LM3/.delta.2-G115 .gamma.E108-3A-.gamma.E111.1
+++ +++ +++ .gamma.9-G115.sub.LM4/.delta.2-G115
.gamma.E108-4A-.gamma.E111.1 +++ +++ +++
.gamma.9-G115.sub.LM5/.delta.2-G115 .gamma.E108-5A-.gamma.E111.1
+++ +++ +++ .gamma.9-G115.sub.LM6/.delta.2-G115
.gamma.E108-6A-.gamma.E111.1 +++ +++ +++
.gamma.9-G115/.delta.2-G115.sub.LM0 .delta.L109-0A-.delta.T113 +++
- - .gamma.9-G115/.delta.2-G115.sub.LM1 .delta.L109-1A-.delta.T113
+++ - - .gamma.9-G115/.delta.2-G115.sub.LM2
.delta.L109-2A-.delta.T113 +++ +++ +++
.gamma.9-G115/.delta.2-G115.sub.LM3 .delta.L109-3A-.delta.T113 +++
+++ +++ .gamma.9-G115/.delta.2-G115.sub.LM4
.delta.L109-4A-.delta.T113 +++ + +++
.gamma.9-G115/.delta.2-G115.sub.LM5 .delta.L109-5A-.delta.T113 +++
+++ +++ .gamma.9-G115/.delta.2-G115.sub.LM6
.delta.L109-6A-.delta.T113 +++ +++ +++
.gamma.9-G115/.delta.2-G115.sub.LM9 .delta.L109-9A-.delta.T113 +++
++ +++ .gamma.9-G115/.delta.2-G115.sub.LM12
.delta.L109-12A-.delta.T113 +++ + +++ sequence
.gamma.9-G115/.delta.2-cl3 original +++ +++ +++
.gamma.9-G115/.delta.2-G115.sub.LM2 .delta.L109-2A-.delta.T113 +++
+ + .gamma.9-G115/.delta.2-cl5 original +++ +++ +++
.gamma.9-G115/.delta.2-G115.sub.LM4 .delta.L109-4A-.delta.T113 +++
+ + .gamma.9-cl3/.delta.2-G115 original +++ nd +
.gamma.9-cl3.sub.E109A/.delta.2-G115 .gamma.E109
.fwdarw..gamma.A109 +++ nd +++ combination
.gamma.9-G115/.delta.2-G115 original +++ +++ +++
.gamma.9-cl3/.delta.2-cl3 original +++ + +
.gamma.9-cl5/.delta.2-cl5 original +++ +++++ +++++
.gamma.9-cl3/.delta.2-G115 CTE +++ + + .gamma.9-G115/.delta.2-cl3
CTE +++ ++++ ++++ .gamma.9-cl5/.delta.2-G115 CTE +++ ++++ ++++
.gamma.9-G115/.delta.2-cl5 CTE +++ +++++ +++++
TABLE-US-00004 SEQ LISTING SEQ ID description type 1
.gamma.9-T-cell receptor chain (G115) AA 2 .delta.2-T-cell receptor
chain (G115) AA 3 .gamma.9-T-cell receptor chain CDR3 region AA
(G115) 4 .delta.2-T-cell receptor chain CDR3 region AA (G115) 5
.gamma.9-T-cell receptor chain (G115) NA 6 .delta.2-T-cell receptor
chain (G115) NA 7 .gamma.9-T-cell receptor chain (clone 3) NA 8
.gamma.9-T-cell receptor chain (clone 3) AA 9 .delta.2-T-cell
receptor chain (clone 3) NA 10 .delta.2-T-cell receptor chain
(clone 3) AA 11 .gamma.9-T-cell receptor chain (clone 5) NA 12
.gamma.9-T-cell receptor chain (clone 5) AA 13 .delta.2-T-cell
receptor chain (clone 5) NA 14 .delta.2-T-cell receptor chain
(clone 5) AA 15 ALKRTD AA 16 ALWEIQELGKKIKV AA 17 ACDALKRTDTDKLI AA
18 ACDLLGYTDKLI AA 19 TLGMGGEY AA 20 IQ AA 21 AQQ AA 22 LLGY AA AA
= amino acid sequence NA = nucleic acid sequence
Sequence CWU 1
1
281315PRTHomo sapiens 1Met Val Ser Leu Leu His Ala Ser Thr Leu Ala
Val Leu Gly Ala Leu1 5 10 15Cys Val Tyr Gly Ala Gly His Leu Glu Gln
Pro Gln Ile Ser Ser Thr 20 25 30Lys Thr Leu Ser Lys Thr Ala Arg Leu
Glu Cys Val Val Ser Gly Ile 35 40 45Thr Ile Ser Ala Thr Ser Val Tyr
Trp Tyr Arg Glu Arg Pro Gly Glu 50 55 60Val Ile Gln Phe Leu Val Ser
Ile Ser Tyr Asp Gly Thr Val Arg Lys65 70 75 80Glu Ser Gly Ile Pro
Ser Gly Lys Phe Glu Val Asp Arg Ile Pro Glu 85 90 95Thr Ser Thr Ser
Thr Leu Thr Ile His Asn Val Glu Lys Gln Asp Ile 100 105 110Ala Thr
Tyr Tyr Cys Ala Leu Trp Glu Ala Gln Gln Glu Leu Gly Lys 115 120
125Lys Ile Lys Val Phe Gly Pro Gly Thr Lys Leu Ile Ile Thr Asp Lys
130 135 140Gln Leu Asp Ala Asp Val Ser Pro Lys Pro Thr Ile Phe Leu
Pro Ser145 150 155 160Ile Ala Glu Thr Lys Leu Gln Lys Ala Gly Thr
Tyr Leu Cys Leu Leu 165 170 175Glu Lys Phe Phe Pro Asp Val Ile Lys
Ile His Trp Glu Glu Lys Lys 180 185 190Ser Asn Thr Ile Leu Gly Ser
Gln Glu Gly Asn Thr Met Lys Thr Asn 195 200 205Asp Thr Tyr Met Lys
Phe Ser Trp Leu Thr Val Pro Glu Lys Ser Leu 210 215 220Asp Lys Glu
His Arg Cys Ile Val Arg His Glu Asn Asn Lys Asn Gly225 230 235
240Val Asp Gln Glu Ile Ile Phe Pro Pro Ile Lys Thr Asp Val Ile Thr
245 250 255Met Asp Pro Lys Asp Asn Cys Ser Lys Asp Ala Asn Asp Thr
Leu Leu 260 265 270Leu Gln Leu Thr Asn Thr Ser Ala Tyr Tyr Met Tyr
Leu Leu Leu Leu 275 280 285Leu Lys Ser Val Val Tyr Phe Ala Ile Ile
Thr Cys Cys Leu Leu Arg 290 295 300Arg Thr Ala Phe Cys Cys Asn Gly
Glu Lys Ser305 310 3152292PRTHomo sapiens 2Met Glu Arg Ile Ser Ser
Leu Ile His Leu Ser Leu Phe Trp Ala Gly1 5 10 15Val Met Ser Ala Ile
Glu Leu Val Pro Glu His Gln Thr Val Pro Val 20 25 30Ser Ile Gly Val
Pro Ala Thr Leu Arg Cys Ser Met Lys Gly Glu Ala 35 40 45Ile Gly Asn
Tyr Tyr Ile Asn Trp Tyr Arg Lys Thr Gln Gly Asn Thr 50 55 60Met Thr
Phe Ile Tyr Arg Glu Lys Asp Ile Tyr Gly Pro Gly Phe Lys65 70 75
80Asp Asn Phe Gln Gly Asp Ile Asp Ile Ala Lys Asn Leu Ala Val Leu
85 90 95Lys Ile Leu Ala Pro Ser Glu Arg Asp Glu Gly Ser Tyr Tyr Cys
Ala 100 105 110Cys Asp Thr Leu Gly Met Gly Gly Glu Tyr Thr Asp Lys
Leu Ile Phe 115 120 125Gly Lys Gly Thr Arg Val Thr Val Glu Pro Arg
Ser Gln Pro His Thr 130 135 140Lys Pro Ser Val Phe Val Met Lys Asn
Gly Thr Asn Val Ala Cys Leu145 150 155 160Val Lys Glu Phe Tyr Pro
Lys Asp Ile Arg Ile Asn Leu Val Ser Ser 165 170 175Lys Lys Ile Thr
Glu Phe Asp Pro Ala Ile Val Ile Ser Pro Ser Gly 180 185 190Lys Tyr
Asn Ala Val Lys Leu Gly Lys Tyr Glu Asp Ser Asn Ser Val 195 200
205Thr Cys Ser Val Gln His Asp Asn Lys Thr Val His Ser Thr Asp Phe
210 215 220Glu Val Lys Thr Asp Ser Thr Asp His Val Lys Pro Lys Glu
Thr Glu225 230 235 240Asn Thr Lys Gln Pro Ser Lys Ser Cys His Lys
Pro Lys Ala Ile Val 245 250 255His Thr Glu Lys Val Asn Met Met Ser
Leu Thr Val Leu Gly Leu Arg 260 265 270Met Leu Phe Ala Lys Thr Val
Ala Val Asn Phe Leu Leu Thr Ala Lys 275 280 285Leu Phe Phe Leu
290315PRTHomo sapiens 3Ala Leu Trp Glu Ala Gln Gln Glu Leu Gly Lys
Lys Ile Lys Val1 5 10 15416PRTHomo sapiens 4Ala Cys Asp Thr Leu Gly
Met Gly Gly Glu Tyr Thr Asp Lys Leu Ile1 5 10 155948DNAHomo sapiens
5atggtgtccc tgctgcacgc cagcaccctg gccgtgctgg gcgccctgtg cgtgtatggc
60gccggacacc tggaacagcc ccagatcagc agcaccaaga ccctgagcaa gaccgccagg
120ctggaatgcg tggtgtccgg catcaccatc agcgccacct ccgtgtactg
gtacagagag 180agacccggcg aggtcatcca gttcctggtg tccatcagct
acgacggcac cgtgcggaaa 240gagagcggca tccccagcgg caagttcgag
gtggacagaa tccccgagac cagcacctcc 300accctgacca tccacaacgt
ggagaagcag gacatcgcca cctactactg cgccctgtgg 360gaggcccagc
aggaactggg caagaaaatc aaggtgttcg gccctggcac caagctgatc
420atcaccgaca agcagctgga cgccgacgtg agccccaagc ctaccatctt
cctgcccagc 480atcgccgaga ccaagctgca gaaggccggc acctacctgt
gcctgctgga aaagttcttc 540cccgacgtga tcaagatcca ctgggaggaa
aagaagagca acaccatcct gggcagccag 600gaaggcaata ccatgaaaac
caacgacacc tacatgaagt tcagctggct gaccgtgccc 660gagaagagcc
tggacaaaga gcacagatgc atcgtccggc acgagaacaa caagaacggc
720gtggaccagg aaatcatctt cccccccatc aagaccgatg tgatcacaat
ggaccccaag 780gacaactgca gcaaggacgc caacgatacc ctgctgctgc
agctgaccaa caccagcgcc 840tactacatgt atctcctgct gctgctgaag
agcgtggtgt acttcgccat catcacctgc 900tgtctgctgc ggcggaccgc
cttctgctgc aacggcgaga agagctga 9486879DNAHomo sapiens 6atggagcgga
tcagcagcct gatccacctg agcctgttct gggccggagt gatgagcgcc 60atcgagctgg
tgcccgagca ccagaccgtg cccgtgagca tcggcgtgcc cgccaccctg
120cggtgcagca tgaagggcga ggccatcggc aactactaca tcaactggta
cagaaagacc 180cagggcaaca ccatgacctt catctaccgg gagaaggaca
tctacggccc tggcttcaag 240gacaacttcc agggcgacat cgacatcgcc
aagaacctgg ccgtgctgaa gatcctggcc 300cccagcgaga gggacgaggg
cagctactac tgcgcctgcg acaccctggg catgggcggc 360gagtacaccg
acaagctgat cttcggcaag ggcacccggg tgaccgtgga gcccagaagc
420cagccccaca ccaagcccag cgtgttcgtg atgaagaacg gcaccaacgt
ggcctgcctg 480gtgaaagagt tctaccccaa ggacatccgg atcaacctgg
tgtccagcaa gaagatcacc 540gagttcgacc ccgccatcgt gatcagcccc
agcggcaagt acaacgccgt gaagctgggc 600aagtacgagg acagcaacag
cgtgacctgc agcgtgcagc acgacaacaa gaccgtgcac 660agcaccgact
tcgaggtgaa aaccgactcc accgaccacg tgaagcccaa agagaccgag
720aacaccaagc agcccagcaa gagctgccac aagcccaagg ccatcgtgca
caccgagaag 780gtgaacatga tgagcctgac cgtgctgggc ctgcggatgc
tgttcgccaa gacagtggcc 840gtgaacttcc tgctgaccgc caagctgttc ttcctgtga
8797943DNAHomo sapiens 7atggtgtccc tgctgcacgc cagcaccctg gccgtgctgg
gcgccctgtg cgtgtatggc 60gccggacacc tggaacagcc ccagatcagc agcaccaaga
ccctgagcaa gaccgccagg 120ctggaatgcg tggtgtccgg catcaccatc
agcgccacct ccgtgtactg gtacagagag 180agacccggcg aggtcatcca
gttcctggtg tccatcagct acgacggcac cgtgcggaaa 240gagagcggca
tccccagcgg caagttcgag gtggacagaa tccccgagac cagcacctcc
300accctgacca tccacaacgt ggagaagcag gacatcgcca cctactactg
cgccctgtgg 360gaggaggaac tgggcaagaa aatcaaggtg ttcggccctg
gcaccaagct gatcatcacc 420gacaagcagc tggacgccga cgtgagcccc
aagcctacca tcttcctgcc cagcatcgcc 480gagaccaagc tgcagaaggc
cggcacctac ctgtgcctgc tggaaaagtt cttccccgac 540gtgatcaaga
tccactggga ggaaaagaag agcaacacca tcctgggcag ccaggaaggc
600aataccatga aaaccaacga cacctacatg aagttcagct ggctgaccgt
gcccgagaag 660agcctggaca aagagcacag atgcatcgtc cggcacgaga
acaacaagaa cggcgtggac 720caggaaatca tcttcccccc catcaagacc
gatgtgatca caatggaccc caaggacaac 780tgcagcaagg acgccaacga
taccctgctg ctgcagctga ccaacaccag cgcctactac 840atgtatctcc
tgctgctgct gaagagcgtg gtgtacttcg ccatcatcac ctgctgtctg
900ctgcggcgga ccgccttctg ctgcaacggc gagaagagct gag 9438313PRTHomo
sapiens 8Met Val Ser Leu Leu His Ala Ser Thr Leu Ala Val Leu Gly
Ala Leu1 5 10 15Cys Val Tyr Gly Ala Gly His Leu Glu Gln Pro Gln Ile
Ser Ser Thr 20 25 30Lys Thr Leu Ser Lys Thr Ala Arg Leu Glu Cys Val
Val Ser Gly Ile 35 40 45Thr Ile Ser Ala Thr Ser Val Tyr Trp Tyr Arg
Glu Arg Pro Gly Glu 50 55 60Val Ile Gln Phe Leu Val Ser Ile Ser Tyr
Asp Gly Thr Val Arg Lys65 70 75 80Glu Ser Gly Ile Pro Ser Gly Lys
Phe Glu Val Asp Arg Ile Pro Glu 85 90 95Thr Ser Thr Ser Thr Leu Thr
Ile His Asn Val Glu Lys Gln Asp Ile 100 105 110Ala Thr Tyr Tyr Cys
Ala Leu Trp Glu Glu Glu Leu Gly Lys Lys Ile 115 120 125Lys Val Phe
Gly Pro Gly Thr Lys Leu Ile Ile Thr Asp Lys Gln Leu 130 135 140Asp
Ala Asp Val Ser Pro Lys Pro Thr Ile Phe Leu Pro Ser Ile Ala145 150
155 160Glu Thr Lys Leu Gln Lys Ala Gly Thr Tyr Leu Cys Leu Leu Glu
Lys 165 170 175Phe Phe Pro Asp Val Ile Lys Ile His Trp Glu Glu Lys
Lys Ser Asn 180 185 190Thr Ile Leu Gly Ser Gln Glu Gly Asn Thr Met
Lys Thr Asn Asp Thr 195 200 205Tyr Met Lys Phe Ser Trp Leu Thr Val
Pro Glu Lys Ser Leu Asp Lys 210 215 220Glu His Arg Cys Ile Val Arg
His Glu Asn Asn Lys Asn Gly Val Asp225 230 235 240Gln Glu Ile Ile
Phe Pro Pro Ile Lys Thr Asp Val Ile Thr Met Asp 245 250 255Pro Lys
Asp Asn Cys Ser Lys Asp Ala Asn Asp Thr Leu Leu Leu Gln 260 265
270Leu Thr Asn Thr Ser Ala Tyr Tyr Met Tyr Leu Leu Leu Leu Leu Lys
275 280 285Ser Val Val Tyr Phe Ala Ile Ile Thr Cys Cys Leu Leu Arg
Arg Thr 290 295 300Ala Phe Cys Cys Asn Gly Glu Lys Ser305
3109867DNAHomo sapiens 9atggagcgga tcagcagcct gatccacctg agcctgttct
gggccggagt gatgagcgcc 60atcgagctgg tgcccgagca ccagaccgtg cccgtgagca
tcggcgtgcc cgccaccctg 120cggtgcagca tgaagggcga ggccatcggc
aactactaca tcaactggta cagaaagacc 180cagggcaaca ccatgacctt
catctaccgg gagaaggaca tctacggccc tggcttcaag 240gacaacttcc
agggcgacat cgacatcgcc aagaacctgg ccgtgctgaa gatcctggcc
300cccagcgaga gggacgaggg cagctactac tgcgcctgcg acctgctggg
ctacaccgac 360aagctgatct tcggcaaggg cacccgggtg accgtggagc
ccagaagcca gccccacacc 420aagcccagcg tgttcgtgat gaagaacggc
accaacgtgg cctgcctggt gaaagagttc 480taccccaagg acatccggat
caacctggtg tccagcaaga agatcaccga gttcgacccc 540gccatcgtga
tcagccccag cggcaagtac aacgccgtga agctgggcaa gtacgaggac
600agcaacagcg tgacctgcag cgtgcagcac gacaacaaga ccgtgcacag
caccgacttc 660gaggtgaaaa ccgactccac cgaccacgtg aagcccaaag
agaccgagaa caccaagcag 720cccagcaaga gctgccacaa gcccaaggcc
atcgtgcaca ccgagaaggt gaacatgatg 780agcctgaccg tgctgggcct
gcggatgctg ttcgccaaga cagtggccgt gaacttcctg 840ctgaccgcca
agctgttctt cctgtga 86710288PRTHomo sapiens 10Met Glu Arg Ile Ser
Ser Leu Ile His Leu Ser Leu Phe Trp Ala Gly1 5 10 15Val Met Ser Ala
Ile Glu Leu Val Pro Glu His Gln Thr Val Pro Val 20 25 30Ser Ile Gly
Val Pro Ala Thr Leu Arg Cys Ser Met Lys Gly Glu Ala 35 40 45Ile Gly
Asn Tyr Tyr Ile Asn Trp Tyr Arg Lys Thr Gln Gly Asn Thr 50 55 60Met
Thr Phe Ile Tyr Arg Glu Lys Asp Ile Tyr Gly Pro Gly Phe Lys65 70 75
80Asp Asn Phe Gln Gly Asp Ile Asp Ile Ala Lys Asn Leu Ala Val Leu
85 90 95Lys Ile Leu Ala Pro Ser Glu Arg Asp Glu Gly Ser Tyr Tyr Cys
Ala 100 105 110Cys Asp Leu Leu Gly Tyr Thr Asp Lys Leu Ile Phe Gly
Lys Gly Thr 115 120 125Arg Val Thr Val Glu Pro Arg Ser Gln Pro His
Thr Lys Pro Ser Val 130 135 140Phe Val Met Lys Asn Gly Thr Asn Val
Ala Cys Leu Val Lys Glu Phe145 150 155 160Tyr Pro Lys Asp Ile Arg
Ile Asn Leu Val Ser Ser Lys Lys Ile Thr 165 170 175Glu Phe Asp Pro
Ala Ile Val Ile Ser Pro Ser Gly Lys Tyr Asn Ala 180 185 190Val Lys
Leu Gly Lys Tyr Glu Asp Ser Asn Ser Val Thr Cys Ser Val 195 200
205Gln His Asp Asn Lys Thr Val His Ser Thr Asp Phe Glu Val Lys Thr
210 215 220Asp Ser Thr Asp His Val Lys Pro Lys Glu Thr Glu Asn Thr
Lys Gln225 230 235 240Pro Ser Lys Ser Cys His Lys Pro Lys Ala Ile
Val His Thr Glu Lys 245 250 255Val Asn Met Met Ser Leu Thr Val Leu
Gly Leu Arg Met Leu Phe Ala 260 265 270Lys Thr Val Ala Val Asn Phe
Leu Leu Thr Ala Lys Leu Phe Phe Leu 275 280 28511946DNAHomo sapiens
11atggtgtccc tgctgcacgc cagcaccctg gccgtgctgg gcgccctgtg cgtgtatggc
60gccggacacc tggaacagcc ccagatcagc agcaccaaga ccctgagcaa gaccgccagg
120ctggaatgcg tggtgtccgg catcaccatc agcgccacct ccgtgtactg
gtacagagag 180agacccggcg aggtcatcca gttcctggtg tccatcagct
acgacggcac cgtgcggaaa 240gagagcggca tccccagcgg caagttcgag
gtggacagaa tccccgagac cagcacctcc 300accctgacca tccacaacgt
ggagaagcag gacatcgcca cctactactg cgccctgtgg 360gagatccagg
aactgggcaa gaaaatcaag gtgttcggcc ctggcaccaa gctgatcatc
420accgacaagc agctggacgc cgacgtgagc cccaagccta ccatcttcct
gcccagcatc 480gccgagacca agctgcagaa ggccggcacc tacctgtgcc
tgctggaaaa gttcttcccc 540gacgtgatca agatccactg ggaggaaaag
aagagcaaca ccatcctggg cagccaggaa 600ggcaatacca tgaaaaccaa
cgacacctac atgaagttca gctggctgac cgtgcccgag 660aagagcctgg
acaaagagca cagatgcatc gtccggcacg agaacaacaa gaacggcgtg
720gaccaggaaa tcatcttccc ccccatcaag accgatgtga tcacaatgga
ccccaaggac 780aactgcagca aggacgccaa cgataccctg ctgctgcagc
tgaccaacac cagcgcctac 840tacatgtatc tcctgctgct gctgaagagc
gtggtgtact tcgccatcat cacctgctgt 900ctgctgcggc ggaccgcctt
ctgctgcaac ggcgagaaga gctgag 94612314PRTHomo sapiens 12Met Val Ser
Leu Leu His Ala Ser Thr Leu Ala Val Leu Gly Ala Leu1 5 10 15Cys Val
Tyr Gly Ala Gly His Leu Glu Gln Pro Gln Ile Ser Ser Thr 20 25 30Lys
Thr Leu Ser Lys Thr Ala Arg Leu Glu Cys Val Val Ser Gly Ile 35 40
45Thr Ile Ser Ala Thr Ser Val Tyr Trp Tyr Arg Glu Arg Pro Gly Glu
50 55 60Val Ile Gln Phe Leu Val Ser Ile Ser Tyr Asp Gly Thr Val Arg
Lys65 70 75 80Glu Ser Gly Ile Pro Ser Gly Lys Phe Glu Val Asp Arg
Ile Pro Glu 85 90 95Thr Ser Thr Ser Thr Leu Thr Ile His Asn Val Glu
Lys Gln Asp Ile 100 105 110Ala Thr Tyr Tyr Cys Ala Leu Trp Glu Ile
Gln Glu Leu Gly Lys Lys 115 120 125Ile Lys Val Phe Gly Pro Gly Thr
Lys Leu Ile Ile Thr Asp Lys Gln 130 135 140Leu Asp Ala Asp Val Ser
Pro Lys Pro Thr Ile Phe Leu Pro Ser Ile145 150 155 160Ala Glu Thr
Lys Leu Gln Lys Ala Gly Thr Tyr Leu Cys Leu Leu Glu 165 170 175Lys
Phe Phe Pro Asp Val Ile Lys Ile His Trp Glu Glu Lys Lys Ser 180 185
190Asn Thr Ile Leu Gly Ser Gln Glu Gly Asn Thr Met Lys Thr Asn Asp
195 200 205Thr Tyr Met Lys Phe Ser Trp Leu Thr Val Pro Glu Lys Ser
Leu Asp 210 215 220Lys Glu His Arg Cys Ile Val Arg His Glu Asn Asn
Lys Asn Gly Val225 230 235 240Asp Gln Glu Ile Ile Phe Pro Pro Ile
Lys Thr Asp Val Ile Thr Met 245 250 255Asp Pro Lys Asp Asn Cys Ser
Lys Asp Ala Asn Asp Thr Leu Leu Leu 260 265 270Gln Leu Thr Asn Thr
Ser Ala Tyr Tyr Met Tyr Leu Leu Leu Leu Leu 275 280 285Lys Ser Val
Val Tyr Phe Ala Ile Ile Thr Cys Cys Leu Leu Arg Arg 290 295 300Thr
Ala Phe Cys Cys Asn Gly Glu Lys Ser305 31013873DNAHomo sapiens
13atggagcgga tcagcagcct gatccacctg agcctgttct gggccggagt gatgagcgcc
60atcgagctgg tgcccgagca ccagaccgtg cccgtgagca tcggcgtgcc cgccaccctg
120cggtgcagca tgaagggcga ggccatcggc aactactaca tcaactggta
cagaaagacc 180cagggcaaca ccatgacctt catctaccgg gagaaggaca
tctacggccc tggcttcaag 240gacaacttcc agggcgacat cgacatcgcc
aagaacctgg ccgtgctgaa gatcctggcc 300cccagcgaga gggacgaggg
cagctactac tgcgcctgcg acgccctgaa gagaaccgac 360accgacaagc
tgatcttcgg caagggcacc cgggtgaccg tggagcccag aagccagccc
420cacaccaagc ccagcgtgtt cgtgatgaag aacggcacca acgtggcctg
cctggtgaaa 480gagttctacc ccaaggacat ccggatcaac ctggtgtcca
gcaagaagat caccgagttc 540gaccccgcca tcgtgatcag ccccagcggc
aagtacaacg ccgtgaagct gggcaagtac 600gaggacagca acagcgtgac
ctgcagcgtg cagcacgaca acaagaccgt gcacagcacc 660gacttcgagg
tgaaaaccga ctccaccgac cacgtgaagc ccaaagagac cgagaacacc
720aagcagccca gcaagagctg ccacaagccc
aaggccatcg tgcacaccga gaaggtgaac 780atgatgagcc tgaccgtgct
gggcctgcgg atgctgttcg ccaagacagt ggccgtgaac 840ttcctgctga
ccgccaagct gttcttcctg tga 87314290PRTHomo sapiens 14Met Glu Arg Ile
Ser Ser Leu Ile His Leu Ser Leu Phe Trp Ala Gly1 5 10 15Val Met Ser
Ala Ile Glu Leu Val Pro Glu His Gln Thr Val Pro Val 20 25 30Ser Ile
Gly Val Pro Ala Thr Leu Arg Cys Ser Met Lys Gly Glu Ala 35 40 45Ile
Gly Asn Tyr Tyr Ile Asn Trp Tyr Arg Lys Thr Gln Gly Asn Thr 50 55
60Met Thr Phe Ile Tyr Arg Glu Lys Asp Ile Tyr Gly Pro Gly Phe Lys65
70 75 80Asp Asn Phe Gln Gly Asp Ile Asp Ile Ala Lys Asn Leu Ala Val
Leu 85 90 95Lys Ile Leu Ala Pro Ser Glu Arg Asp Glu Gly Ser Tyr Tyr
Cys Ala 100 105 110Cys Asp Ala Leu Lys Arg Thr Asp Thr Asp Lys Leu
Ile Phe Gly Lys 115 120 125Gly Thr Arg Val Thr Val Glu Pro Arg Ser
Gln Pro His Thr Lys Pro 130 135 140Ser Val Phe Val Met Lys Asn Gly
Thr Asn Val Ala Cys Leu Val Lys145 150 155 160Glu Phe Tyr Pro Lys
Asp Ile Arg Ile Asn Leu Val Ser Ser Lys Lys 165 170 175Ile Thr Glu
Phe Asp Pro Ala Ile Val Ile Ser Pro Ser Gly Lys Tyr 180 185 190Asn
Ala Val Lys Leu Gly Lys Tyr Glu Asp Ser Asn Ser Val Thr Cys 195 200
205Ser Val Gln His Asp Asn Lys Thr Val His Ser Thr Asp Phe Glu Val
210 215 220Lys Thr Asp Ser Thr Asp His Val Lys Pro Lys Glu Thr Glu
Asn Thr225 230 235 240Lys Gln Pro Ser Lys Ser Cys His Lys Pro Lys
Ala Ile Val His Thr 245 250 255Glu Lys Val Asn Met Met Ser Leu Thr
Val Leu Gly Leu Arg Met Leu 260 265 270Phe Ala Lys Thr Val Ala Val
Asn Phe Leu Leu Thr Ala Lys Leu Phe 275 280 285Phe Leu
290156PRTHomo sapiens 15Ala Leu Lys Arg Thr Asp1 51614PRTHomo
sapiens 16Ala Leu Trp Glu Ile Gln Glu Leu Gly Lys Lys Ile Lys Val1
5 101714PRTHomo sapiens 17Ala Cys Asp Ala Leu Lys Arg Thr Asp Thr
Asp Lys Leu Ile1 5 101812PRTHomo sapiens 18Ala Cys Asp Leu Leu Gly
Tyr Thr Asp Lys Leu Ile1 5 10198PRTHomo sapiens 19Thr Leu Gly Met
Gly Gly Glu Tyr1 5202PRTHomo sapiens 20Ile Gln1213PRTHomo sapiens
21Ala Gln Gln1224PRTHomo sapiens 22Leu Leu Gly Tyr12324PRTHomo
sapiens 23Cys Ala Leu Trp Glu Glu Glu Leu Gly Lys Lys Ile Lys Val
Phe Gly1 5 10 15Pro Gly Thr Lys Leu Ile Ile Thr 202425PRTHomo
sapiens 24Cys Ala Leu Trp Glu Ile Gln Glu Leu Gly Lys Lys Ile Lys
Val Phe1 5 10 15Gly Pro Gly Thr Lys Leu Ile Ile Thr 20
252526PRTHomo sapiens 25Cys Ala Leu Trp Glu Ala Gln Gln Glu Leu Gly
Lys Lys Ile Lys Val1 5 10 15Phe Gly Pro Gly Thr Lys Leu Ile Ile Thr
20 252624PRTHomo sapiens 26Cys Ala Cys Asp Leu Leu Gly Tyr Thr Asp
Lys Leu Ile Phe Gly Lys1 5 10 15Gly Thr Arg Val Thr Val Glu Pro
202726PRTHomo sapiens 27Cys Ala Cys Asp Ala Leu Lys Arg Thr Asp Thr
Asp Lys Leu Ile Phe1 5 10 15Gly Lys Gly Thr Arg Val Thr Val Glu Pro
20 252828PRTHomo sapiens 28Cys Ala Cys Asp Thr Leu Gly Met Gly Gly
Glu Tyr Thr Asp Lys Leu1 5 10 15Ile Phe Gly Lys Gly Thr Arg Val Thr
Val Glu Pro 20 25
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