U.S. patent application number 15/288904 was filed with the patent office on 2017-01-26 for therapeutic and diagnostic cloned mhc-unrestricted receptor specific for the muc1 tumor associated antigen.
The applicant listed for this patent is University of Pittsburgh - Of the Commonwealth System of Higher Education. Invention is credited to Nehad M. Alajez, Mark D. Alter, Olivera J. Finn, Jan Schmielau.
Application Number | 20170022264 15/288904 |
Document ID | / |
Family ID | 37943241 |
Filed Date | 2017-01-26 |
United States Patent
Application |
20170022264 |
Kind Code |
A1 |
Finn; Olivera J. ; et
al. |
January 26, 2017 |
THERAPEUTIC AND DIAGNOSTIC CLONED MHC-UNRESTRICTED RECEPTOR
SPECIFIC FOR THE MUC1 TUMOR ASSOCIATED ANTIGEN
Abstract
The invention provides an isolated nucleic acid encoding a
receptor, other than an immunoglobulin, wherein the receptor binds
to a MUC1 tumor antigen independently of an major
histocompatibility complex (MHC). The invention provides a method
of activating a signaling pathway and/or killing a cancer cell
using a receptor that is similar to or is a T cell receptor
Inventors: |
Finn; Olivera J.;
(Pittsburgh, PA) ; Alajez; Nehad M.; (Riyadh,
SA) ; Schmielau; Jan; (Lubeck, DE) ; Alter;
Mark D.; (Brooklyn, NY) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
University of Pittsburgh - Of the Commonwealth System of Higher
Education |
Pittsburgh |
PA |
US |
|
|
Family ID: |
37943241 |
Appl. No.: |
15/288904 |
Filed: |
October 7, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13939988 |
Jul 11, 2013 |
9469684 |
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15288904 |
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12258545 |
Oct 27, 2008 |
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13939988 |
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11295767 |
Dec 7, 2005 |
7655461 |
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12258545 |
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60634072 |
Dec 7, 2004 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
G01N 33/57492 20130101;
G01N 2333/4725 20130101; A61P 31/00 20180101; C07K 14/7051
20130101; C07K 2317/34 20130101; C07K 16/3092 20130101; C12N
2799/027 20130101; A61K 38/00 20130101; A61K 35/17 20130101; C07K
14/70503 20130101; C07K 16/2809 20130101 |
International
Class: |
C07K 14/725 20060101
C07K014/725; A61K 35/17 20060101 A61K035/17; G01N 33/574 20060101
G01N033/574 |
Goverment Interests
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH AND
DEVELOPMENT
[0002] This invention was made in part with Government support
under Grant Numbers CA56103 and predoctoral training grant
DAMD17-99-1-9352 awarded by the U.S. National Institutes of Health
and the U.S. Department of Defense, respectively. The United States
Government may have certain rights in this invention.
Claims
1-49. (canceled)
50. An isolated or substantially purified receptor other than an
immunoglobulin, wherein the receptor binds to a MUC1 tumor antigen
independently of a major histocompatibility complex (MHC).
51. The isolated or substantially purified receptor of claim 50,
wherein the receptor is soluble.
52. The isolated or substantially purified receptor of claim 50,
wherein the receptor is membrane bound.
53. An immunocytochemistry stain comprising a receptor, other than
an immunoglobulin, wherein the receptor binds to a MUC1 tumor
antigen independently of a major histocompatibility complex (MHC),
complexed with a labeling agent.
54. The immunocytochemistry stain of claim 53, wherein the receptor
is soluble.
55. The immunocytochemistry stain of claim 53, wherein the receptor
is membrane bound.
56. (canceled)
57. A method of activating a signaling pathway in a cell having a
signaling pathway, the method comprising: a. transducing the cell
having a signaling pathway with at least one nucleic acid encoding
a receptor, wherein the receptor i. is expressed and displayed on
the surface of the transduced cell, ii. binds to a MUC1 tumor
antigen independently of a major histocompatibility complex (MHC),
and b. contacting the transduced cell to a cell expressing the MUC1
tumor antigen thereby activating the signaling pathway.
58. The method of claim 57, wherein the receptor is a T cell
receptor.
59. (canceled)
60. (canceled)
61. A method of activating a signaling pathway in a cell comprising
a signaling pathway, the method comprising transducing the cell
with a receptor having affinity for MUC1, wherein the affinity is
determined by a first amino acid sequence and a second amino acid
sequence, wherein the first amino acid sequence consists
essentially of the portion of MA V.alpha.23 shown in FIG. 1 (SEQ ID
NO:1), and the second amino acid sequence consists essentially of
the portion of MA V.beta.8.3 shown in FIG. 1 (SEQ ID NO:2).
62. A method of killing a cancer cell, the method comprising a.
isolating a population of cells comprising a receptor, wherein the
receptor binds to a MUC1 tumor antigen independently of a major
histocompatibility complex (MHC), and b. contacting the isolated
population of cells to a cell expressing the MUC1 tumor antigen
thereby killing the cancer cell.
63. The method of claim 62, wherein the population comprises T
cells.
64. The method of claim 62, wherein the population consists
essentially of T cells.
65. The method of claim 62, wherein the population does not
comprise cells, other than T cells, that comprise a receptor that
binds to a MUC1 tumor antigen independently of a major
histocompatibility complex (MHC).
66. The isolated or substantially purified receptor of claim 50,
wherein the receptor: a. has a first amino acid sequence that is
identical with the portion of MA V.alpha.23 shown in FIG. 1 (SEQ ID
NO:1), and b. has a second amino acid sequence that is identical
with the portion of MA V.beta.8.3 shown in FIG. 1 (SEQ ID
NO:2).
67. The isolated or substantially purified receptor of claim 50,
wherein the receptor: a. has a first amino acid sequence that
consists essentially of the portion of MA V.alpha.23 shown in FIG.
1 (SEQ ID NO:1), and b. has a second amino acid sequence that
consists essentially of the portion of MA V.beta.8.3 shown in FIG.
1 (SEQ ID NO:2).
68. The immunocytochemistry stain of claim 53, wherein the
receptor: a. has a first amino acid sequence that is identical with
the portion of MA V.alpha.23 shown in FIG. 1 (SEQ ID NO:1), and b.
has a second amino acid sequence that is identical with the portion
of MA V.beta.8.3 shown in FIG. 1 (SEQ ID NO:2).
69. The immunocytochemistry stain of claim 53, wherein the
receptor: a. has a first amino acid sequence that consists
essentially of the portion of MA V.alpha.23 shown in FIG. 1 (SEQ ID
NO:1), and b. has a second amino acid sequence that consists
essentially of the portion of MA V.beta.8.3 shown in FIG. 1 (SEQ ID
NO:2).
70. The method of claim 57, wherein the receptor: a. has a first
amino acid sequence that is identical with the portion of MA
V.alpha.23 shown in FIG. 1 (SEQ ID NO:1), and b. has a second amino
acid sequence that is identical with the portion of MA V.beta.8.3
shown in FIG. 1 (SEQ ID NO:2).
71. The method of claim 57, wherein the receptor: a. has a first
amino acid sequence that consists essentially of the portion of MA
V.alpha.23 shown in FIG. 1 (SEQ ID NO:1), and b. has a second amino
acid sequence that consists essentially of the portion of MA
V.beta.8.3 shown in FIG. 1 (SEQ ID NO:2).
72. The method of claim 62, wherein the receptor: a. has a first
amino acid sequence that is identical with the portion of MA
V.alpha.23 shown in FIG. 1 (SEQ ID NO:1), and b. has a second amino
acid sequence that is identical with the portion of MA V.beta.8.3
shown in FIG. 1 (SEQ ID NO:2).
73. The method of claim 62, wherein the receptor: a. has a first
amino acid sequence that consists essentially of the portion of MA
V.alpha.23 shown in FIG. 1 (SEQ ID NO:1), and b. has a second amino
acid sequence that consists essentially of the portion of MA
V.beta.8.3 shown in FIG. 1 (SEQ ID NO:2).
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority to U.S. Provisional Patent
Application 60/634,072, the disclosure of which is incorporated
herein in its entirety.
FIELD OF THE INVENTION
[0003] This invention pertains to the treatment or prevention of
cancer in humans involving transfer of (a) an isolated population
of cells, or (b) cells transduced with a nucleic acid encoding a
receptor, into a human in need of treatment or prophylaxis for
cancer. The present invention also pertains to a method of
activating a signaling pathway.
BACKGROUND OF THE INVENTION
[0004] Immunotherapy of cancer involving adoptive transfer of T
cells for various human tumor antigens has significantly improved
in recent years. It has also recently become clear that
immunotherapy is more potent if both the innate and the adaptive
cellular immune responses are efficiently engaged. Timely
recognition of the tumor by the cells of the innate immune system,
such as NK cells, granulocytes, and macrophages, appears to be a
prerequisite for an efficient stimulation of tumor-specific
adaptive immunity.
[0005] Tumor-specific antibodies have been transduced into T cells
(T-bodies) endowing the transduced T cells with MHC-unrestricted
tumor antigen specificity. While antibodies can have exquisite
specificity, one disadvantage is their high affinity of binding to
antigen, which could impair infiltration of tumors, result in
irreversible binding of an effector cell to a tumor cell, and
possibly result in apoptosis of effector cells following their
interaction with tumor cells. T cell receptors (TCRs), however,
have a much lower binding affinity. Therefore, a cell bearing a
tumor-specific TCR could engage and disengage from its target
multiple times and effect its function against multiple tumor
cells. Tumor specific T cells, however, are MHC-restricted.
Accordingly, a tumor specific T cell is effective only for
treatment of patients having a suitable human lymphocyte antigen
(HLA). Additionally, tumor cells frequently down-regulate MHC or
antigen processing molecules, thereby avoiding T cell recognition.
Despite these limitations, adoptive immunotherapy therapies
involving transfer of tumor-specific TCRs into T cells continue to
be developed. Accordingly, the art is in need of an adoptive
immunotherapy that can treat or prevent cancer in most or all
patients. Desirably, such a therapy would not be dependent on the
HLA or antigen processing components of target cancer cells.
Moreover, it would be desirable if such a therapy caused little or
no immune reaction with non-target cells.
BRIEF SUMMARY OF THE INVENTION
[0006] The invention provides a method of activating a signaling
pathway in a cell. The method comprises transducing the cell with
at least one nucleic acid that encodes a receptor so that the
receptor is expressed and displayed on the surface of the cell. The
encoded receptor is capable of binding to a MUC1 antigen without
requiring the MUC1 antigen to be presented in the context of a
major histocompatibility complex (MHC). The receptor either
interacts with a signaling molecule, or comprises a signaling
domain, such that when the receptor is contacted with a cell having
the MUC1 tumor antigen on its surface, the signaling pathway is
activated. In a preferred embodiment the receptor is a T cell
receptor.
[0007] The encoded and expressed receptor can have any suitable
sequence. For example, the receptor can have any sequence in which
the affinity of the receptor for MUC1 is determined by a first
amino acid sequence and a second amino acid sequence in which the
first amino acid sequence consists essentially of the portion of MA
V.alpha.23 shown in FIG. 1 (SEQ ID NO:1) and the second amino acid
sequence consists essentially of the portion of MA V.beta.8.3 shown
in FIG. 1 (SEQ ID NO:2).
[0008] The encoded receptor can also have a sequence that is at
least 85% identical, at least 90% identical, at least 95%
identical, or is essentially identical to the portions of MA
V.alpha.23 and MA V.beta.8.3 shown in FIG. 1 (SEQ ID NOs:1 and 2,
respectively).
[0009] The cell signaling can generate any useful response in the
transduced cell, such as (without limitation) cytokine secretion
and target cell killing (e.g., cancer cell killing).
[0010] The invention also provides a population of cells comprises
a method of killing a cancer cell, the method comprising isolating
a population of cells, such as T cells, that have a receptor that
binds to a MUC1 tumor antigen independently of a major
histocompatibility complex, and contacting the population of cells
to a cancer cell expressing the MUC1 tumor antigen, thereby killing
the cancer cell. The isolated population of cells preferably does
not comprise a therapeutically-effective quantity of B cells, more
preferably is substantially free of B cells, and even more
preferably does not comprise B cells.
[0011] The invention also provides isolated nucleic acids encoding
receptors that bind to the MUC1 tumor antigen even when the MUC1
tumor antigen is not presented in the context of an MHC. The
encoded receptor preferably has at least one amino acid sequence
that is homologous with, consists essentially of, or is identical
with at least one of the amino acid sequences designated MA
V.alpha.23 or MA V.beta.8.3 in FIG. 1 (SEQ ID NOs:1 and 2,
respectively). The encoded receptor can be membrane bound when
expressed in a cell or can be expressed in a water-soluble
form.
[0012] The nucleic acid encoding the receptor can be transduced
into a variety of cells. The transduced cells can be used for
therapeutic purposes (such as the treatment or prevention of
cancer), diagnostic purposes (such as the identification of the
MUC1 tumor antigen), or as a tool in the study of T cell function
and/or immunogene therapy.
[0013] The nucleic acids (e.g., alone or in the context of a gene
delivery vector), isolated populations of cells, and transduced
cells of the invention can be combined with sterile carriers,
pharmaceutically acceptable excipients, or adjuvants, each of which
is preferably suitable for administration to a human.
[0014] The receptor can also be isolated or substantially purified,
and combined with a labeling agent to provide a reagent useful in
the detection of the MUC1 tumor antigen in a suitable sample.
[0015] These and other advantages of the invention, as well as
additional inventive features, will be apparent from the
description of the invention provided herein.
BRIEF DESCRIPTION OF THE DRAWINGS
[0016] FIG. 1 depicts a comparison of the CDR2 and CDR3 regions of
the MUC1-specific antibody SM3 and the MA TCR and modeling their
interactions with MUC1. FIG. 1a Amino acid sequence alignment of MA
V.alpha.23 (SEQ ID NO:1) and MA V.beta.8.3 (SEQ ID NO:2) with SM3L
and SM3H (SEQ ID NOs:23 and 24, respectively). Numbering
corresponds to MA TCR sequence. Residues in bold are hypothesized
to be involved in the binding (FIG. 1b, and FIG. 1c) SM3 and (FIG.
1d, and FIG. 1e). MA TCR computer based models of the interactions
with the MUC1 epitope. Contact residues are shown as stick
diagrams.
[0017] FIG. 2 depicts cells transfected with scTCR recognize
MUC1.sup.+ tumors and synthetic MUC1 antigen. FIG. 2a shows
mammalian expression vector encoding MA scTCR gene. The scTCR was
cloned into the pEF6 vector. FIG. 2b shows cell surface expression
of the scTCR in RBL cells and FIG. 2c shows BWZ cells stably
transfected with the scTCR-pEF6 vector. Cells were stained with
anti-TCR .beta.F1 (open histogram) or with isotype control (filled
histogram) antibody. FIG. 2d shows degranulation of RBL cells (open
bar) or RBL-scTCR cells (striped bar) following stimulation with
plate-bound .beta.F1 antibody or MUC1 140mer peptide.
Specific-degranulation is presented as percent of maximum
degranulation induced by TCR cross-linking with .beta.F1 antibody.
FIG. 2e shows IL-2 ELISA for BWZ (open bars) or BWZscTCR (striped
bar) following stimulation with plate-bound anti-TCR .beta.F1
antibody, ionomycin+PMA (UP), DM6 (MUC1.sup.- tumor), HPAF or T3M4
(MUC.sup.+ tumors). IL-2 in culture supernatant was measured by
ELISA and values were plotted on the y-axis as pg/ml. Cells were
stimulated as indicated.
[0018] FIG. 3 shows transduction of the long-term reconstituting
hematopoietic stem cell population (c-Kit.sup.+Sca.sup.-1.sup.+
Lin.sup.- Thy1.1.sup.-) with scTCR-EGFP MFG retroviral vector. FIG.
3a shows a schematic diagram of the scTCR-EGFP MFG retroviral
vector. FIG. 3b shows Bone Marrow (BM) cells transduced with the
scTCR-EGFP MFG retroviral vector were stained on day 7 in culture
for hematopoietic stem cells surface markers (c-Kit and Sca-1), and
for lineage markers (Lin). Cells that expressed high levels of
Sca-1 and c-Kit (c) and that lacked expression of Lin (d), R2 &
R3 were gated on and were plotted against EGFP (FIG. 3e). FIG. 3f
shows mock transduced BM cells. All cells in culture were Thy1. V
(not shown).
[0019] FIG. 4 shows detection of scTCR-expressing cells at various
times post reconstitution with transduced BM cells. Mice were bled
at indicated time points and leukocytes were stained for the
appropriate cell surface markers plotted on the Y-axis: (FIG. 4a)
GR-1 for granulocytes, (FIG. 4b) mac-3 or F4/80 for
monocyte/macrophages, (FIG. 4c) DX5 for NK cells, (FIG. 4d) CD3 for
T cells, and (FIG. 4e) B220 for B cells. EGFP expression is plotted
on the x-axis. Percentages of EGFP positive cells in each lineage
are indicated.
[0020] FIG. 5 shows SCID mice reconstituted with transduced BM
cells can control the growth of the MUC1.sup.+ tumor xenograft.
FIG. 5a shows control (filled triangles) or scTCR-reconstituted
(open circles) mice were injected subcutaneously with
2.times.10.sup.6 HPAF (MUC1.sup.+) tumor cells. Tumor size is shown
on the y-axis while days post-tumor challenge is plotted on the
x-axis. P-Values were calculated by running t-test using Microsoft
Excel software. Data are presented as mean.+-.S.E. FIG. 5b shows H
& E staining of HPAF tumor sections from control mice (left
panel) or from scTCR-reconstituted mice (right panel). Figure Sc
shows staining of tumor sections from scTCR-reconstituted mice for
myeloperoxidase (neutrophils marker), F4/80 (monocytes/macrophage
marker), or Granzyme B (NK cells marker). Images were taken under
20.times. magnification. Images in the lower right squares were
taken under 100.times. magnification.
[0021] FIG. 6 shows expression of scTCR on immune cells has no
deleterious effects on MUC1+ normal tissues. FIG. 6A shows C57BL/6
(wild type) and MUC1 Tg. mice were reconstituted with BM cells
transduced with the scTCR-EGFP MFG retroviral vector. Untreated
mice served as controls. Spleen, lung, and pancreas were harvested
6 weeks postreconstitution and microscopically examined for
infiltration with EGFP.sup.+ cells (FIG. 6a) or stained with
H&E (FIG. 6b) and examined for tissue destruction.
[0022] FIG. 7 shows scTCR-reconstituted MUC1 Tg. mice rejected
MUC1+ tumor challenge. MUC1 Tg. mice were reconstituted with BM
cells transduced with scTCR (filled triangle) or with control
supernatant (filled square) and challenged 6 weeks later with
MUC1.sup.- tumor (RMA) or with RMA cells transfected with MUC1
(RMA-MUC1). Data are presented as Mean.+-.S.E. The number of mice
at each time point ranged from 5-10.
[0023] FIG. 8 shows RT-PCR analyses of scTCR expression in
transduced BM cells and in splenocytes from reconstituted SCID
mice. FIG. 8a shows expression of scTCR mRNA in transduced BM cells
72 hours post-transduction. FIG. 8b shows expression of scTCR mRNA
was in splenocytes 60 days post-reconstitution. M is 1 Kb DNA
molecular weight marker. .beta.-actin is the RT-PCR control.
[0024] FIG. 9 shows long-term expression of scTCR in mice
reconstituted with scTCR-transduced BM cells. Control (FIG. 9a) or
scTCR-reconstituted mice (FIG. 9b) were bled 7 months
post-reconstitution and were stained for GR-1. Cells were gated on
GR-1+ and were plotted against EGFP.
[0025] FIG. 10 shows expression of the TCR .alpha..beta. from MA
CTL clone on the surface of a TCR-deficient Jurkat line
(JRT3-T3.5). The TCR .alpha.-IRES-.beta. cassette was cloned into
the pEF4 expression vector (FIG. 10A), Untransfected JRT3-T3.5
(FIG. 10B), JRT3-T3.5 cells transfected with the TCR .beta. chain
(FIG. 10C), or JRT3-T3.5 cells transfected with MA TCR
.alpha.-IRES-.beta. pEF4 (FIG. 10D), were stained with anti-CD3E
(open histogram) or with isotype control (filled histogram)
antibody. IRES means internal ribosomal entry site, and Zeocin is
an antibiotic resistance gene.
[0026] FIG. 11 shows the construction and expression of
MUC1-specific .alpha..zeta. and .alpha..zeta. chimeric T cell
receptors. Expression vectors for TCR at (FIG. 11A), ix (FIG. 11B),
and .alpha..zeta./.beta..zeta. (FIG. 11C). Untransfected 293H cells
(FIG. 11D), 293H cells co-transfected with the TCR .alpha..zeta.
and TCR .beta..zeta. (FIG. 11E) or cells transfected with the
.alpha..zeta.-IRES-.beta..zeta. pLNCX2 (FIG. 11F) were stained for
surface expression of the TCR using anti-TCR antibody .beta.F1
(open histogram) or isotype control antibody (filled
histogram).
[0027] FIG. 12 shows vector construction and expression of
MUC1-specific single chain T cell receptors (scTCRs). 293H cells
(FIG. 12A) were transfected with the scTCR (FIG. 12B),
scTCR-CD4TM-h.zeta. (FIG. 12C), or scTCR-CD4TM-AGD-h.zeta. (FIG.
12D) mammalian expression vectors. Cells were stained with anti-TCR
.beta.F1 (open histogram) or isotype control (filled histogram)
antibody. FIG. 12E shows quantitative comparison of TCR expression
on 293H cells transfected with different scTCR constructs.
p<0.05.
[0028] FIG. 13 shows expression of functional scTCR on the surface
of T cells and non-T immune cells. (FIG. 13A) Rat Basophilic
Leukemia (RBL) or mouse T cell tumor BWZ cells were transfected
with the scTCR-pEF6 and stained for surface expression with
anti-TCR .beta.F1 antibody (filled) or with isotype control (open)
histogram. FIG. 13B shows IL-2 secretion from BWZ cells (open bars)
or BWZ-scTCR (filled bars) following stimulation with SEE
superantigen or with anti-TCR .beta.F1 antibody.
[0029] FIG. 14 shows expression and purification of soluble scTCR
(sscTCR) following surface biotin labeling and
immmunoprecipitation. FIG. 14A shows scTCR expression vector
encoding a thrombin cleavage site, T. FIG. 14B shows RBL cells
transfected with the scTCR were stained with anti-TCR .beta.F1
antibody (open histograms) before (right) or after (left) treatment
with thrombin. The filled histogram shows staining with isotype
control antibody. FIG. 14C shows immunoprecipitation of the scTCR
from RBL (lanes 1 and 2) or RBL cells transfected with the scTCR
(Lanes 2 and 4) before (lanes 1 and 2) or after (lanes 3 and 4)
treatment with thrombin. Lane 6-8 are SA-HRP blotting of fraction
eluted with 150 mM Glycine, PH 2.2, 100 mM Glycine pH 2.2, or
diethyl amine (DEA) pH 11.2, respectively. Lane 5 is IP from
control lysate.
[0030] FIG. 15 shows expression and purification of soluble scTCR
using a mammalian expression system. FIG. 15a shows the single
chain fraction variable (scFV) domain was cloned and fused to a
C-terminus HA and c-myc epitope tags. FIG. 15b shows secreted scTCR
(sscTCR) that was cloned and fused to a C-terminus Flag and 6-His
epitope tags. FIGS. 15c and 15d show the sscTCR as described in
FIG. 15b, except fused to the leader sequence from GM-CSF (FIG.
15c) or from Ig-.kappa. light chain (FIG. 15d). FIG. 15e shows a
western blot of the culture supernatants from 293H cells
transiently transfected with constructs a-d (a'-d'),
immunoprecipitated with appropriate anti-tag antibody and blotted
with anti-c-myc antibody (a') or with anti FLAG-M2 antibody
(b'-d'). "Ctr." is supernatant from untransfected cells. (f)
Comassiee blue staining of fractions from culture supernatant b'
purified using nickel column. Lane 1 is culture supernatant before
purification, 2 is flow through, 3 is wash, and 4-6 are different
eluted fractions. FIG. 15g shows a western blot of panel fusing
anti-Flag M2 antibody.
DETAILED DESCRIPTION OF THE INVENTION
[0031] The MA TCR referenced in FIG. 1 is a T cell receptor (TCR)
that binds to the MUC1 tumor antigen. The MA TCR recognizes an
epitope located in each of the 20 amino acid long tandem repeats in
the extracellular domain of MUC1. Each molecule of MUC1 can have
more than 100 repeats. The amino acid sequence critical for
recognition by the TCR is believed to be a five amino acid residue
sequence (PDTRP SEQ ID NO: 3) which has been called the
immunodominant knob of the MUC1 protein. A large number of these
tandemly repeated and structurally stable PDTRP (SEQ ID NO: 3)
bearing knobs on a single MUC1 molecule, as well as on neighboring
MUC1 molecules on the surface of a tumor cell, can engage multiple
TCRs, and signal a T cell to effect its function. While not
desiring to be bound by any particular theory, it is believed that
the MA TCR can effectively bind with the MUC1 tumor antigen without
requiring antigen processing and presentation in an MHC, which is
required by most T cell receptors. While the MA TCR referenced in
FIG. 1 is a preferred TCR that is capable of recognizing the MUC1
tumor antigen independently of presentation by an MHC, functionally
similar TCRs can be raised against the MUC1 tumor antigen or any
other disease associated antigen with structurally stable, repeated
amino acid epitope similar to MUC1. The antigen binding domains of
these TCRs readily can be incorporated into a variety of receptor
constructs such that a cell expressing the receptor will have at
least one signaling pathway activated when contacted to a cell
having the MUC1 or other structurally stable, repeated amino acid
epitope on its surface.
[0032] Accordingly, the invention provides a method of activating a
signaling pathway in a cell. The method comprises transducing the
cell with at least one nucleic acid encoding a receptor that binds
to the MUC1 tumor antigen independently of presentation of the MUC1
tumor antigen in the context of an MHC. The signaling pathway is
activated when the receptor is expressed and displayed on the
surface of the transduced cell, and the transduced cell is
contacted to a cell having the MUC1 tumor antigen on its surface.
Of course, the transduced cell can be a T cell, but as is further
described below, any suitable cell or collection of cells can be
transduced.
[0033] The invention also provides an isolated nucleic acid
encoding the receptor. Nucleic acids suitable in the context of the
invention include natural and synthetic nucleic acids. Natural
nucleic acids can be DNA or RNA irrespective of whether they are
isolated directly from a cell, e.g., an MA T cell clone,
synthesized by chemical or other means, or carried in a vector.
Artificial nucleic acids can include regions of natural
polynucleotides and can further comprise 2' methyl nucleic acids,
2' methoxy nucleic acids, phosphorothiorated nucleic acids,
nucleotides comprising synthetic or modified bases (e.g., inosine),
peptidyl nucleic acids (PNAs) and other synthetic nucleic acids
known in the art, so long as the nucleic acid can be transcribed,
if necessary, and translated into a receptor. The receptor encoded
by the isolated nucleic acid is preferably not an antibody or
immunoglobulin. Similarly, the receptor preferably does not have a
constant region of any antibody.
[0034] The sequence of the receptor does not need to be identical
to that of the MA TCR. In fact, amino acid substitutions can be
made or allowed in both the affinity determining regions of the MA
TCR and in the framework regions of the TCR. Additionally, domains
of other proteins can be incorporated into the receptor.
Accordingly, the invention also provides a method of activating a
signaling pathway comprising transducing a cell with a nucleic acid
encoding a receptor having affinity for the MUC1 tumor antigen,
wherein the affinity of the receptor for the MUC1 tumor antigen is
determined by a first amino acid sequence consisting essentially of
the portions of MA V.alpha.23 and MA V.beta.8.3 shown in FIG. 1
(SEQ ID NOs:1 and 2, respectively).
[0035] Additionally, the invention provides a method of activating
a signaling pathway or of killing a cancer cell wherein the
transduced cell, or population of cells, comprises a receptor
having at least one amino acid sequence that has at least 85%,
optionally at least 90%, or optionally at least 95% identity with
the portion of MA V.alpha.23 shown in FIG. 1 (SEQ ID NO:1). The
receptor can also have a sequence that is identical with the
portion of MA V.alpha.23 shown in FIG. 1 (SEQ ID NO:1). The
receptor preferably also has at least one amino acid sequence that
has at least 85%, optionally at least 90%, or optionally at least
95% identity with the portion of MA V.beta.8.3 shown in FIG. 1 (SEQ
ID NO:2), or which is identical with the portion of MA V.beta.8.3
shown in FIG. 1 (SEQ ID NO:2). More preferably, the receptor
comprises both amino acid sequences.
[0036] As used herein, a sequence is 85% identical to another
sequence if, in a window comprising the number of amino acids
present in the named sequence, at least 85% of the amino acids,
allowing gaps or insertions within the window (but not allowing the
number of amino acids in the window to exceed the number of amino
acid residues in the test sequence) are the same as those in the
other sequence. For example, the portion of MA V.alpha.23 shown in
FIG. 1 has 110 amino acid residues. Accordingly, a sequence is 85%
identical with the portion of MA V.alpha.23 if at least 94 residues
in any 110 consecutive residues of one sequence can be exactly
aligned with 110 consecutive residues of the other sequence.
[0037] The receptor can also be any receptor having an amino acid
sequence comprising the complementarity determining regions (CDRs)
of MA V.alpha.23 or MA V.beta.8.3 or both.
[0038] The invention also provides a method of killing a cancer
cell. The cancer cell can be present in a culture of cells in vitro
or found in an animal's (e.g., a human's) body. The method of
killing the cancer cell involves isolating a population of cells
comprising a receptor that binds to a MUC1 tumor antigen
independently of an MHC and contacting a portion of the cells from
the isolated population of cells to a cancer cell. The population
of cells can be created by transduction of one or more cells of an
animal with a nucleic acid of the invention. In another
alternative, an animal can be immunized with the MUC1 tumor antigen
to raise lymphocytes comprising a receptor that mediates the death
of cells expressing the MUC1 tumor antigen. While not desiring to
be bound by any particular theory, it is believed that the highly
repetitive nature of the MUC1 epitope enables the routine
generation of T cells expressing receptors for the MUC1 antigen
that are MHC unrestricted. Of course, the population of cells can
comprise T cells or consist essentially of T cells. The population
of cells also can be isolated or manipulated in such a way that the
population does not comprise any cells other than T cells that
comprise a receptor that binds to a MUC1 tumor antigen
independently of an MHC. In one embodiment, for example, the
population of cells does not comprise B cells having a B cell
receptor (BCR) that is specific for the MUC1 tumor antigen.
[0039] The receptor can have any suitable affinity for the MUC1
tumor antigen. For example, the affinity of the receptor can be
higher than the affinity of the MA TCR for the MUC1 tumor antigen.
Receptors having lower affinity for the MUC1 tumor antigen than the
MA TCR are also useful in the context of the present invention.
However, the receptor preferably has about the same affinity of the
MUC1 tumor antigen as the MA TCR because this level of affinity is
high enough to efficiently invoke effector functions of T cells,
while simultaneously allowing efficient tumor infiltration and
avoiding apoptosis of the cell on which it is displayed. While
receptors having other affinities for the MUC1 tumor antigen are
useful, receptors having a K.sub.d of between 0.2 and 200 .mu.M,
are among the preferred embodiments. The affinity can be measured
with a variety of conventional techniques. Preferably, however, the
receptor is converted into a soluble form (if necessary) to measure
its affinity. The skilled artisan will appreciate that conventional
techniques employing a Biacore.RTM. device are particularly well
suited to the measurement of receptor affinities for the MUC1 tumor
antigen (Molloy et al., Molecular Immunology, 35: 73-81
(1998)).
[0040] The transduced cell, or a cell of the isolated population of
cells, desirably has a suitable avidity for a cancer cell
expressing the MUG1 tumor antigen. For example, the avidity between
the transduced cell, or cell of the isolated population, can be
between 1.times.10.sup.-5 M and 1.times.10.sup.-12 M.
[0041] The receptor can be encoded by 1, 2, or more nucleic acids.
Any receptor (or nucleic acid encoding a receptor) having the
antigen determining regions of the MA TCR is suitable in the
context of the invention, including (but not limited to) a receptor
encoded by a single nucleic acid, such as, but not limited to, a
single-chain receptor in which the regions of the receptor having
homology to the .alpha.-chain and then .beta.-chain of the MA TCR
are encoded as a single polypeptide. The receptor can also be an
scFv. In other embodiments, the receptor can be encoded as at least
two subunits. The receptor desirably has amino acid sequences
derived from the V.alpha., a J.alpha., V.beta., D.beta., and
J.beta. mature gene segments of the MA receptor. Additionally, the
receptor preferably does not have light and heavy chains or an
antibody, nor truncated polypeptides derived from light and heavy
chains or the nucleic acids encoding the same.
[0042] In a preferred embodiment the receptor comprises at least
one polypeptide that is fused to, or connected with, a portion of
the zeta-chain of CD3. Many suitable configurations exist. In a
preferred embodiment, the transmembrane and cytoplasmic domains of
the zeta-chain of a CD3 molecule are fused to the .alpha.-constant
domain of a TCR or, more preferably, a .beta.-constant domain of a
TCR. While not desiring to be bound by any particular theory, it is
believed that the fusion of a portion of the CD3 zeta-chain to the
remainder of the receptor facilitates surface expression of the
receptor on a transduced cell. In a more preferred embodiment, the
constant domain of the TCR and the portion of the CD3 zeta-chain
are separated by an amino acid linker (also known as an amino acid
spacer) encoded by a nucleic acid encoding the entire receptor or a
portion of the receptor. The linker can be any suitable sequence,
but is preferably selected to have a flexible structure. Any number
of amino acid residues can be included in the linker but typically
the linker will comprise at least one, more typically at least
three, yet more typically at least about eight, and commonly at
least about 12 amino acid residues. Additionally, the linker will
typically not comprise more than about 30 amino acid residues, more
commonly not more than about 22 amino acid residues, and commonly
not more than about 18 amino acid residues. A linker having about
15 amino acid residues is among the more preferred embodiments.
Additionally, the linkers primary function is to provide a flexible
linkage between two portions of the receptor. Accordingly, the
linker can optionally not have one or more functions selected from
the group consisting of an immunological function, a membrane
anchoring function, a membrane spanning function, a dimerization
function, a signaling function, and an intracellular trafficking
function. In contrast, however, the receptor can have amino acid
sequences in additional to the linker which provide at least one of
these functions. The linker preferably allows a high level of
receptor expression when the receptor is expressed in a T cell or
other suitable cell in comparison to an otherwise identical
receptor that lacks the linker.
[0043] In another embodiment, the receptor comprises the
transmembrane region from a CD4 molecule.
[0044] In embodiments comprising portions of a CD3 or a CD4
molecule the portions of these molecules can contain substantial
variations from their natural sequences. For example, the portions
of the CD3 or CD4 molecules can have at least about 65% identity,
at least about 80% identity, or be essentially identical to a
consensus sequence (also known as "wild type") human CD3 or CD4
molecule.
[0045] The nucleic acid encoding the receptor can be carried by any
suitable vector. The vector is preferably a gene transfer vector
(also known as a gene delivery vector). Suitable gene transfer
vectors include both viral and non-viral vectors. Suitable viral
vectors include, but are not limited to, retroviral vectors.
Suitable viral vectors include, but are not limited to adenoviral
vectors, adeno-associated viral vectors, and herpes viral vectors.
Lentiviral vectors, particularly those having the ability to
efficiently transduce quiescent cells are among the more preferred
viral vectors. Other preferred vectors in the context of the
present invention include liposomal vectors and MFG vectors.
[0046] The nucleic acids encoding a receptor having affinity for
the MUC1 tumor antigen of the invention can be transduced into a
stem cell, preferably a bone marrow stem cell. Advantageously, this
enables the generation of a wide variety of cell types expressing
the receptor, each of which has a high potential for propagation.
Similarly, the cells can be removed from an animal and transduced
in vitro. When the cells are transduced in vitro they need not be,
but optionally can be, expanded (i.e., propagated) in vitro prior
to their transfer into a host animal such as a human. Any suitable
technique can be used to propagate the cells in vitro. Suitable
methods include, but are not limited to culturing the cells with
cytokines, stimulatory molecules (e.g., stimulatory antibodies),
live, attenuated, or killed cells having the MUC1 tumor antigen on
their surface. Preferably, the cells are autologous, which means
that they are transferred into the host from which they were
obtained. Transduced autologous cells are believed to be less
likely to be rejected by the host animal into which they are
transduced.
[0047] Conveniently, the nucleic acid(s) encoding the receptor can
be transduced into the cells in vivo. While not desiring to be
bound by any particular theory, it is believed that the transduced
cells will find cells expressing the MUC1 tumor antigen on their
surface, if present, and be stimulated to expand to reach
therapeutic levels when a therapeutic quantity of cells is not
transduced directly.
[0048] The cell can be transduced in a mixed population of cells,
or can be partially or substantially purified prior to transduction
with the nucleic acid encoding the receptor. A preferred cell type
for transduction is the fibroblast. Fibroblasts are capable of
rapid expansion and transduced fibroblasts treat and prevent cancer
in patients having cancer cells that expresses the MUC1 tumor
antigen.
[0049] The transduced cells can also be of the hematopoietic
lineage. Hematopoietic cells that can be usefully transduced in the
context of the present invention include T cells, B cells, NK
cells, macrophages, granulocytes, and dendritic cells.
[0050] In a preferred embodiment, the cell to be transduced is
removed from an animal (e.g., a human or other animal), the animal
is treated for cancer, e.g., with chemotherapy or radiation or
both. The chemotherapy and/or radiation optionally can be potent
enough to decimate or destroy the patient's lymphocyte population.
Then, the transduced cells are returned to the patient. Before,
during, or after the transduced cells are transferred to the
patient or other animal, the patient or other animal can be treated
with cytokines that expand the population of lymphocytes (e.g.,
IL-2). Additionally, other therapeutic agents such as antiemetics,
erythropoietin or other red blood cell boosters, hormones, and
antibodies can also be administered to the patient or animal
before, during, or after the transfer of the transduced cells to
the patient or animal. Similarly, the animal can be treated with a
vaccine, which vaccine is preferably a vaccine promoting a reaction
against a tumor or cancer cell, and more preferably is a MUC1
vaccine. The MUC1 vaccine stimulates cells reactive with MUC1 to
persist, or preferably, to propagate. Suitable MUC1 vaccines can
comprise at least one PDTRP (SEQ ID NO: 3) sequence, which
pentapeptide optionally can be found in the context of from about 5
to about 20 amino acids having identity with a naturally-occurring
MUC-1 polypeptide known in the art. Such a vaccine preferably would
comprise a multiplicity of PDTRP (SEQ ID NO: 3) amino acid
sequences so that a MUC1 reactive cell would be presented with an
array of reactive epitopes. For example, the vaccine can be
configured to present reactive cells with 2, 3, 4, 5, 6-10, 11-20,
or more PDTRP (SEQ ID NO: 3) epitopes in an array or complex,
preferably on the surface of a cell. The vaccine can further
comprise an excipient, sterile carrier or adjuvant.
[0051] In embodiments in which contact of the receptor with the
cell having a MUC1 tumor antigen on its surface activates a
signaling pathway, the signaling pathway can be of any suitable
type. For example, the signaling pathway can cause the secretion of
a cytokine or induce a cytotoxic response leading to the death of
the cell displaying the MUC1 tumor antigen. Similarly, activation
of the signaling pathway can lead to the secretion of a
biomolecule, such as a endocrine, paracrine, autocrine, whether
steroidal, peptidyl, or other, an enzyme, a carbohydrate, a
proinflammatory or anti-inflammatory molecule, or a drug agent. In
a preferred embodiment, the biomolecule causes the death of a
cancer cell or attenuates the symptoms of cancer.
[0052] The receptor encoded by any nucleic acid of the invention
can also be substantially purified or isolated from the cell
expressing it, irrespective of whether the receptor is
water-soluble or has a high affinity for cellular membranes (or
artificial membranes such as micelles).
[0053] The inventive receptor can also be complexed with a labeling
agent. Such receptor-labeling agents complexes are useful for the
visualization or detection of cells expressing a MUC1 tumor
antigen. A labeling agent can be directly detectable or indirectly
detectable. For example, directly detectable labeling agents
include (without limitation) a protein such as green fluorescent
protein (i.e., GFP), a radioactive atom, or a gold microparticle
can be attached to the receptor. Each of these moieties can be
directly detected with suitable, conventional methods.
Alternatively, the label can be an indirect label such as (without
limitation) an enzyme (e.g., horse radish peroxidase), an antibody
hapten (e.g., the well-known FLAG epitope, biotin, avidin, or
streptavidin, or the like. The complex can be covalently or
non-covalently bound as long as the complex remains associated
during any detection steps. Additionally, the complex can be, but
need not be, purified or substantially isolated from a cell
expressing the receptor.
[0054] In view of the foregoing, the invention also provides an
isolated cell that is optionally transduced with a nucleic acid of
the invention, and encodes a receptor of the invention.
[0055] Any of the nucleic acids, cells, or population of cells of
the invention can be combined with a sterile carrier,
pharmaceutically acceptable excipient, or adjuvant, each of which
is preferably suitable for administration to a mammal, and in
particular a human. The composition can comprise a buffer, and the
buffer or composition is preferably substantially isotonic with
human blood (e.g., is isotonic with 0.7 M to 1.1 M NaCl.sub.2).
[0056] Suitable methods of administering the nucleic acids of the
invention to a mammal for purposes of gene therapy are known (see,
e.g., Rosenfeld et al., Science, 252, 431-434 (1991); Jaffe et al.,
Clin. Res., 39, 302A (1991); Rosenfeld et al., Clin. Res., 39, 311A
(1991); Berkner, BioTechniques, 6, 616-629 (1988); Crystal et al.,
Human Gene Ther., 6, 643-666 (1995); Crystal et al., Human Gene
Ther., 6, 667-703 (1995)). Innate and adaptive cells can be found
in most locations in the mammalian body. Accordingly, any suitable
route of administration can be used. Intravenous administration of
cells is preferred when the mammal is human. A particular route can
provide a more immediate and more effective reaction than another
route. Pharmaceutically acceptable excipients also are well-known
to those who are skilled in the art, and are readily available. The
choice of excipient will be determined in part by the particular
method used to administer the recombinant vector. Accordingly,
there is a wide variety of suitable formulations for use in the
context of the invention.
[0057] Moreover, to optimize the ability of vectors, and
particularly viral vectors, to enter a cell by the method of the
invention, preferably the method is carried out in the absence of
neutralizing antibodies directed against the particular vector
being introduced intracellularly, which could impede transduction
of target cells. The ordinarily skilled artisan can routinely test
for the presence of such neutralizing antibodies. Techniques are
also known in the art to prevent the presence of neutralizing
antibodies from impeding effective protein production (see, e.g.,
International Patent Application WO 96/12406).
[0058] The following methods, formulations, and excipients for
administering the inventive nucleic acids, vectors, and cells are
merely exemplary and are in no way limiting.
[0059] Formulations suitable for oral administration of the nucleic
acids and vectors can consist of (a) liquid solutions, such as an
effective amount of the compound dissolved in diluents, such as
water, saline, or orange juice; (b) suspensions in an appropriate
liquid; and (c) suitable emulsions. Tablet forms can include one or
more of lactose, mannitol, corn starch, potato starch,
microcrystalline cellulose, acacia, gelatin, colloidal silicon
dioxide, croscarmellose sodium, talc, magnesium stearate, stearic
acid, and other excipients, colorants, diluents, buffering agents,
moistening agents, preservatives, flavoring agents, and
pharmacologically compatible excipients.
[0060] Preferred formulations include aqueous and non-aqueous,
isotonic sterile injection solutions, which can contain
anti-oxidants, buffers, bacteriostats, and solutes that render the
formulation isotonic with blood, and aqueous and non-aqueous
sterile suspensions that can include suspending agents,
solubilizers, thickening agents, stabilizers, and preservatives.
The inventive nucleic acids and vectors can be stored in a
freeze-dried (lyophilized) condition requiring only the addition of
the sterile liquid excipient, for example, water, for injections,
immediately prior to use. Extemporaneous injection solutions and
suspensions can be prepared from sterile powders, granules, and
tablets of the kind previously described.
[0061] The nucleic acids, vectors and cells of the invention can be
formulated in unit-dose or multi-dose sealed containers, such as
ampules and vials, and can be stored frozen. These nucleic acids,
vectors and cells of the invention can be stored in light-resistant
packaging, employing for example, colored glass vials or cardboard
boxes. Similarly, instructions for use of the compositions, which
preferably comply with the regulations of the U.S. Food and Drug
Administration, and more preferably also with its European and
Japanese equivalent agencies, can be included with these
compositions. These nucleic acids, vectors and cells of the
invention are preferably also free from non-recombinant microbes
(including without limitation fungi and mycobacteria) and
non-recombinant viruses. Preferably, the instructions suggest the
use a certain quantity of one of these compositions (or range of
quantities), or suggest administration of the composition to a
mammal for research or therapy via a particular route of
administration.
[0062] Additionally, a cell, and more preferably, a nucleic acid or
vector of the invention can be made into suppositories by mixing
with a variety of bases such as emulsifying bases or water-soluble
bases. Formulations suitable for vaginal administration can be
presented as pessaries, tampons, creams, gels, pastes, foams, or
spray formulas containing, in addition to the active ingredient,
such carriers as are known in the art to be appropriate.
[0063] The dose administered to an animal, particularly a human, in
the context of the invention will vary with the inventive
embodiment, the composition employed, the method of administration,
and the particular site and organism being treated. However, the
dose should be sufficient to provide a therapeutic response.
[0064] Any suitable number of transduced cells, or isolated cells,
can be administered to a mammal. While a single cell of the innate
or adaptive immune system is capable of expanding and providing a
benefit, when the transduced or isolated cell is not a stem cell,
it is preferable to administer at least 10.sup.3, more preferably
at least 10.sup.5, even more preferably at least 10.sup.8 and
optionally 10.sup.12 or more transduced T cells. One preferred
embodiment of the invention comprises administration of from about
10.sup.8 to about 10.sup.12 transduced cells to a human. There is
no theoretical upper limit on the number of transduced T cells that
can be administered to a mammal or the number of times that T cells
can be administered to a mammal. The ordinarily skilled artisan
will appreciate, however, that the excessive quantities of
administered transduced or isolated cells (e.g., in some
embodiments more than 10.sup.18 transduced cells) can exceed the
mammal's ability to support them, lead to undesirable clinical
sequelae, and unnecessarily increase costs. Similarly, excessive
administrations of therapeutic compositions to mammals can lead to
undesirable effects such as allergic responses and infection, and
so are preferably avoided.
[0065] A composition comprising transduced cells can be prepared so
that it does not contain living cells other than blood cells and
lymphocytes. That is, the composition can be sterile except for the
transduced or isolated cells. Such compositions can be readily
prepared by positive and negative selection of the desired cells
from a population of cells transduced with the inventive nucleic
acids or vectors. Suitable positive selection techniques include
bioaffinity separations, which are well known in the art. For
example, an antibody specific for a cell surface antigen of a
desired cell can be linked to a magnetic bead, incubated with the
transduced population, separated therefrom and optionally washed.
Similarly, undesired cells can be eliminated from the composition
by any suitable technique. Suitable negative selection techniques
include immunomagnetic removal of undesired cells, and the use of
antibiotics to destroy microbes. Moreover, leukophoresis, other
filtration techniques, sterile technique, differential
centrifugation, and other conventional methods can be used to
produce a composition suitable for administration to a human.
[0066] In embodiments in which the mammal is subjected to
lymphodepletion and cytokine or growth factor stimulation, any
suitable regimen can be used. Dudley et al., Science, 298: 850-854
(2002), Rosenberg et al., J. Natl. Cancer Inst., 86:
1159-1166(1994) and Dudley et al., J. Immunother., 25: 243-251
(2002), as well as other references described in these references,
discuss one suitable lymphodepletion and IL-2 stimulation regimen.
These references suggest, for example, treatment of a human with
cyclophosphamide (about 60 mg/kg) and fludarabine (about 25
mg/m.sup.2) and high-doses of IL-2 (i.e., about 720,000 IU/kg).
Administration of IL-2 is preferably repeated multiple times and
more preferably repeated 3 to 15 times, and is preferably
administered 1-5 times daily, which numbers can be selected and
adjusted within the discretion of the skilled medical artisan.
Example 1
[0067] This example shows that a TCR with unique MHC-unrestricted
antigen binding properties for the tumor antigen MUC1 binds its
epitope on the MUC1 protein without the requirement of processing
and presentation. This example also shows that a single chain
V.alpha./V.beta./C.beta. (scTCR) fused to a CD3 zeta-chain
(.zeta.-chain) facilitates expression on the surface of cells of
the innate immune system (e.g., granulocytes, macrophages, natural
killer cells (NK cells)) as well as the adaptive immune system
(e.g., T cells and B cells). This example additionally shows that
cells of the innate immune system reject a tumor when provided with
a tumor-antigen specific TCR. Also, this example shows that bone
marrow cells transduced with a gene transfer vector encoding a T
cell-related receptor specific for the MUC1 tumor antigen can
improve the health of a mammal having cancer in which the cancer
expresses a MUC1 tumor antigen. This example also shows that
expression of the MUC1 tumor antigen specific receptor of the
invention on large percentages of cells does not result in
infiltration or destruction of tissues expressing normal MUC1.
Additionally, this example shows that long-term expression of the
inventive receptor can be achieved, particularly by transducing a
suitable stem cell.
[0068] The following sections describe the Materials and Methods
used in this example.
[0069] Computer Modeling.
[0070] The amino acid sequence of the SM3 antibody shown in FIG. 1
was provided by Dr. J. Taylor-Papadimitriou, of the Imperial Cancer
Research Fund. The Protein Data Bank at the Research Collaboratory
for Structural Bioinformatics (www.rcsb.org/pdb) was searched for
best-fit sequence alignments with the binding domain of the SM3
antibody using BLAST. Coordinates of a crystallized human
.alpha./.beta. TCR heterodimer were provided by Dr. Ian Wilson, of
the Scripps Clinic. Modeling studies were conducted on a Silicon
Graphics Indigo workstation. Homolog templates were mutated and
initial models were constructed using the program O (Jones, et al.,
Acta Crystallogr A 47 (Pt 2):110-119 (1991)). The programs LEaP
(Schafmeister, et al., "LeAP", University of California, San
Francisco (1995)) and AMBER/Interface were used to import protein
database (PDB) files created by O into AMBER, which was used for
global energy minimizations and molecular dynamics of selected
loops and modeling of ligand binding (Pearlman, et al. Computer
Physics Communications 91: 1-41(1995)). The previously solved
structure of the MUC1 epitope, PDTRP (SEQ ID NO: 3), and its 4
flanking amino acids (Fontenot et al., J Biomol Struct Dyn 13:
245-260 (1995)) was initially positioned in the antigen-binding
region by rigid body docking using O. This positioning was followed
by energy minimization and molecular dynamics in AMBER to allow the
MUC1 epitope to position itself within the antigen-binding cleft.
Figures were constructed using Molscript and Raster3D (Kraulis, et
al., Journal of Applied Crystallography 24: 946-950 (1991),
Merritt, et al. Methods in Enzymology 277: 505-524 (1997)).
[0071] Construction of scTCR and Expression in RBL and BWZ Cells.
The RNA encoding the V.alpha.23.1J.alpha.14.3 of the MA TCR was
cloned from the MA CTL clone by RT-PCR and ligated in frame to the
TCR V.beta.8.3D.beta.J.beta.1.2 region using a 15 amino acid (aa)
flexible linker encoding sequence GGG GSG GGG SGG GUS (SEQ ID NO:
4). The cloned sequence encoding the
V.alpha.23.1J.alpha.14.3-linker-V.beta.8.3D.beta.J.beta.1.2
polypeptide was then cloned into a vector encoding the human TCR
fragment C.beta.2, followed by a polypeptide linker, GDLVPRGSSRLD
(SEQ ID NO: 5) and the murine CD3 .zeta.-chain (obtained from Dr.
A. J. McMichael, Weatherall Institute of Molecular Medicine, John
Radcliffe Hospital, Oxford, UK) (Callan, et al., Eur J Immunol 25:
1529-1534 (1995)). The last cysteine in the C.beta.2 region was
mutated to alanine using site directed mutagenesis to prevent
dimerization of the scTCR. The construct was then cloned into the
pEF6 mammalian expression vector (Invitrogen, Carlsbad, Calif.).
RBL cells were obtained from Dr. Richard Klausner (while at NCI),
and BWZ murine T cells were obtained from Dr. Nilabh Shastri
(University of California, Berkeley). RBL cells were grown in
cDMEM-10 medium (Mediatech Inc., Herndon, Va.; DMEM+10% FBS, 2 mM
L-glutamine, 100 U/ml Penicillin, 100 U/ml streptomycin, 10 .mu.M
2-ME, 1.times. non-essential amino acids, and 1.times. sodium
pyruvate), while BWZ cells were grown in cRPMI-10. RBL and BWZ
cells were transfected by electroporation using Bio-Rad Gene Pulser
II (Bio-Rad Laboratories, Hercules, Calif.) at 960 .mu.F and 200 V
settings.
[0072] Basophil Degranulation Assay.
[0073] RBL cells or RBL-scTCR were incubated with .sup.3H-serotonin
(New England Nuclear Corp., Boston, Mass.) for 24 hours. After
washing, cells were transferred to plates coated with .beta.F1
antibody or with MUC1 140mer synthetic peptide (seven repeats of
the sequence PDTRPAPGSTAPPAHGVTSA (SEQ ID NO: 6)). Plates were
centrifuged briefly and incubated for 30 minutes at 37.degree. C.
Ice-cold PBS (Sigma) was added and the supernatant was harvested
after additional centrifugation. Radioactivity was measured using a
Wallac 1205 betaplate liquid scintillation counter (Gaithersburg,
Md.).
[0074] IL-2 ELISA.
[0075] BWZ or BWZ-scTCR cells were plated in U-bottom 96 well
plates at 1.times.10.sup.5/200 .mu.l of cRPMI-10 medium, and
2.times.10.sup.4 tumor cells (irradiated 6000 rad) were added as
stimulators. Thirty six hours later, the amount of mIL-2 released
in the medium was measured using a mouse IL-2 OptEIA kit (BD
Pharmingen), according to the manufacturer's recommendations.
[0076] Construction of scTCR-EGFP MFG Retroviral Vector and
Production of Viral Supernatant.
[0077] The MFG retroviral vector was obtained from Dr. Paul Robbins
(University of Pittsburgh, Pittsburgh, Pa.). The IRES-EGFP cassette
was cloned by PCR from the pIRES2-EGFP vector (Clontech
Laboratories, Palo Alto, Calif.) into the MFG retroviral vector
downstream of the scTCR gene. The GP+E-86 ecotropic retroviral
packaging cells (American Type Culture Collection, Manassas, Va.)
were transfected with the scTCR-EGFP MFG vector and cultured for 5
days, followed by sorting the EGFP.sup.high population. Sorted
cells were then cultured in DMEM-15 (DMEM+15% FBS, 2 mM
L-glutamine, 100 U/ml Penicillin, 100 U/ml streptomycin) and thirty
six hours later, retroviral supernatant was harvested and frozen at
-80.degree. C. until use.
[0078] Retroviral Transduction of BM Cells.
[0079] All experiments in animals were performed under an approved
protocol No. 0304530A-1 of the University of Pittsburgh IACUC. Six
to eight week old SCID or Balb/c mice (Jackson Laboratory, Bar
Harbor, Me.) were injected intraperitoneally with 150 mg/kg 5-FU
(Invivogen, San Diego, Calif.). Five days later, mice were
sacrificed and BM cells were isolated and pre-stimulated for 72
hours in the presence of 50 ng/ml rSCF, 10 ng/ml rmIL-3 and 10
ng/ml rmIL-6 (PeproTech Inc., Rocky Hill, N.J.) in DMEM-15. BM
cells were resuspended in retroviral supernatant supplemented with
50 ng/ml rSCF, 10 ng/ml rmIL-3, 10 ng/ml rmIL-6 and polybrene at 8
.mu.g/ml. In all BM transduction experiments, cells were plated in
24-well plates pre-coated with recombinant fibronectin CH-296
fragment (Takara Bio Inc, Madison, Wis.). Cells were then
centrifuged for 30 minutes at RT at 800.times.g in a Sorvall T6000B
centrifuge and put back in culture at 37.degree. C. Transduction
was repeated every 12 hours for a total of 4 cycles.
[0080] Reconstitution of Irradiated Mice with Transduced BM Cells
and Flow Cytometric Analyses.
[0081] Transduced BM cells were resuspended in PBS at
1.times.10.sup.7/ml, and 200 .mu.l of cell suspension was injected
via the tail vein into irradiated recipient mice. SCID mice
received a single dose of 350 rad while Balb/c mice were given a
split dose of 900 rad total (50/50) 3 hours apart. Reconstituted
mice were maintained in a germ-free environment and were put on
acidified water (pH 2.5) for 3 weeks post-reconstitution. At 3, 6,
and 11 weeks post-reconstitution, 200 .mu.l of blood was collected
via tail artery and cells were stained with the appropriate
anti-surface marker antibody, PE-conjugated anti-CD3, -B220, -GR-1,
-Mac-3 and -DX5 and APC-conjugated anti-F4/80 (eBiosciences, San
Diego, Calif.), and analyzed on the Becton-Dickinson FACSCalibur
(BD Biosciences, San Jose, Calif.).
[0082] Tumor Challenge and Immunohistochemistry.
[0083] Mice reconstituted with BM cells transduced with scTCR-MFG
or with a control supernatant were challenged subcutaneously with
various numbers of HPAF tumor cells 5 weeks post-reconstitution.
Tumor size was measured every 2-3 days using calipers. Tumors
harvested from control or treated mice were fixed in 10% formalin,
paraffin embedded, and sections were stained with H&E,
anti-Myeloperoxidase (Labvision, Fremont, Calif.), anti-Granzyme B
(Labvision), or anti-F4/80 (ebiosciences) in the Department of
Pathology core facility, University of Pittsburgh.
[0084] Fluorescent Microscopic Analyses of Tissue Sections from
Reconstituted Mice.
[0085] Six weeks post-reconstitution, C57BL/6 mice or MUC1 Tg. mice
reconstituted with scTCREGFP transduced BM cells were sacrificed
and spleen, lung, and pancreas were harvested and fixed in 2% PFA
in PBS. Tissues were then frozen, sectioned and visualized for
infiltration with EGFP+ immune cells at the Center for Biologic
Imaging, University of Pittsburgh.
[0086] Statistics.
[0087] Statistical analysis was done using Microsoft Excel and
Graphpad Prism (GraphPad Software, San Diego, Calif.) software.
[0088] In other experiments, we used live imaging microscopy to
show the specificity of the MA TCR for MUC1 by the ability of
transfected cells to flux calcium following stimulation with MUC1+
tumor. One data set shows that control-transfected JRT3-T3.5 didn't
flux calcium in response to stimulation with MUC1+ tumor line
(HPAF). A second data set shows that JRT3-T3.5 cells transfected
with the TCR .alpha. and .beta. chain from the MUC1-specific CTL
clone (MA) fluxed calcium in response to stimulation with HPAF
tumor line. FIG. 8 shows the detection of the scTCR mRNA in
transduced BM cells as well as in splenocytes from SCID mice
reconstituted with BM cells transduced with scTCR-MFG retroviral
vector weeks post-reconstitution. FIG. 9 shows long-term expression
of the scTCR on granulocytes in reconstituted C57BL/6 mice 7 months
post-reconstitution.
[0089] The following section describes the RESULTS obtained in this
example.
[0090] Computational Modeling of MA TCR Binding to the MUC1
"Immunodominant Knob" Shows Similarity to the Binding of SM3
Antibody Specific for the Same Epitope.
[0091] MUC1 specific MHC unrestricted CTL clone MA established from
a tumor-draining lymph node of a breast cancer patient was
previously described (Magarian-Blander et al., J. Immunol. 160:
3111-3120 (1998)). This clone mediated TCR-dependent killing of
MUC1.sup.+ tumor cells that was not restricted by their HLA type.
It was also capable of binding synthetic, tandemly repeated MUC1
epitopes immobilized on the surface of Polylactide-L-Glycolide
(PLGA) beads, resulting in the influx of Ca.sup.2+.
Semi-quantitative RT-PCR analysis and DNA sequencing revealed that
the TCR responsible for this binding was composed of
V.alpha.23.1J.alpha.14.3 and V.beta.8.3D.beta.J.beta.1.2. We cloned
this TCR and transduced the TCR-deficient Jurkat cell line
(J.RT3-T3.5) (Ohashi et al., Proc Natl Acad Sci. (USA) 89:
11332-11336 (1992)) with a plasmid vector encoding the full length
TCR .alpha. and .beta. chains. Its functionality and specificity
were confirmed by live imaging microscopy, where TCR-transfected
cells, but not control cells, fluxed Ca.sup.2+ upon binding to
MUC1.sup.+ human tumor cells.
[0092] The antibody SM3 recognizes the same epitope, and blocks
tumor cell recognition and killing by MHC-unrestricted MUC1-
specific CTL. Considering the common epitope recognized by both SM3
and MA TCR, their common Ig-like fold, and similarities in the CDR3
sequences (FIG. 1a), we used the available sequence and structural
information to model antigen binding by these two functionally
related receptors. Both SM3 and the MA TCR were modeled by homology
using appropriate antibody templates. Simulated docking of the MUC1
epitope produced minimized structures of both SM3 and MA TCR with
bound ligand and predicted similar molecular determinants in the
CDR2 and CDR3 regions important for binding. Our models predict
that similarly positioned arginines in the corresponding V.beta.
and V.sub.H CDR2s interact favorably with the aspartic acid in the
MUC1 epitope PDTRP (FIGS. 1b-1e). Equivalently positioned tyrosines
in the corresponding V.alpha. and V.sub.L CDR3s stabilize the
interaction with the aspartic acid of MUC1. A glutamine in the
.alpha.CDR2 and a glutamic acid in the .beta.CDR3 interact with the
arginine in the epitope, as do an asparagine in the VL CDR2 and a
glutamine in the VH CDR3.
[0093] MA TCR as a Single-Chain (scTCR) Construct Fused with Zeta
(.zeta.) Chain is Functionally Expressed on the Surface of T and
Non-T Cells.
[0094] To create a more practical reagent for future gene
therapy/immunotherapy applications, we converted the full length
two-chain TCR, which is dependent on CD3 molecules unique to T
cells for cell surface expression, into a CD3 independent
single-chain TCR (scTCR). The V.alpha.J.alpha. segment was fused to
the V.beta.D.beta.J.beta. segment using a 15 amino acid flexible
linker. This chimeric structure was then cloned into a vector
containing the human TCR C.beta.2 and the CD3 transmembrane and
cytoplasmic domains separated by a short linker, and further
subcloned into the pEF6 plasmid to create scTCR-PEF6 mammalian
expression vector (FIG. 2a). Transfection of the scTCR-PEF6 into a
CD3.sup.- rat basophil cell line RBL (FIG. 2b) or a CD3.sup.+ mouse
T cell lymphoma line BWZ (FIG. 2c) resulted in high surface
expression of the scTCR, in both cell types.
[0095] These same two cell lines were used to test the function and
antigen specificity of the scTCR. Transfected RBL degranulated upon
cross-linking of their scTCR with the anti-TCR antibody .beta.F1 or
upon specific recognition of the MUC1 antigen (FIG. 2d). No
degranulation was seen when cells were stimulated with control
antigen ovalbumin. There was no degranulation in control
untransfected RBL upon encounter with either MUC1 or .beta.F1
antibody. Similarly, transfected BWZ cells produced a substantial
amount of IL-2 when their scTCR was cross-linked with plate-bound
.beta.F1 antibody (FIG. 2e). No significant IL-2 production was
detected when BWZ-scTCR cells were incubated with the DM6 tumor
cell line that did not express MUC1; however, substantial level of
IL-2 was produced upon encounter with MUC1.sup.+ tumor cell lines
HPAF and T3M4. These two cell lines do not share HLA alleles
confirming that cells expressing the cloned scTCR exhibit the same
MHC-unrestricted recognition Of MUC1 as the original T cell clone
from which the TCR was derived. The difference in IL-2 secretion in
response to these two tumors can be attributed to frequently
observed differences in the level of expression and the extent of
glycosylation of MUC1 on different tumor cell lines.
[0096] Transduction of Bone Marrow (BM) Cells that Differentiate In
Vivo into scTCR.sup.+ Cells of Multiple Hematopoietic Lineages.
[0097] In order to test the anti-tumor activity of this
MUC1-specific TCR in vivo, we cloned the scTCR into the MFG
retroviral vector with an EGFP gene downstream of an IRES sequence
(FIG. 3a). We used the green fluorescence of EGFP to track scTCR
transduced cells. BM cells were isolated from 5-FU treated Balb/c
mice and transduced with the scTCR-EGFP MFG retroviral vector using
fibronectin-assisted transduction protocol. Seventy-two percent of
BM cells were successfully transduced (FIG. 3b). We were especially
interested in our ability to transduce hematopoietic stem cells
contained within the population of cells that are Thy1.1.sup.-
Lin.sup.- c-Kit.sup.+ Sca-1.sup.+. Thirty-eight percent (38%) of
cells of that phenotype were successfully transduced (FIG. 3e).
[0098] Sub-lethally irradiated mice received 2.times.10.sup.6 BM
cells via tail vein injection. At 3, 6, and 11 weeks
post-injection, mice were bled and the percentages of EGFP.sup.+
cells in different cell lineages were evaluated. At week 3
post-reconstitution, 5.8% of granulocytes (GR-1.sup.+) were
positive for EGFP (FIG. 4a). This number increased to 16.3% at week
11. Reconstitution of the monocyte/macrophage (FIG. 4b) and NK cell
(FIG. 4c) lineage followed similar kinetics. Transduced T cells
(CD3) were seen in the periphery at week 6 post-reconstitution and
accounted for 5.4% (FIG. 4d) of all T cells. This number dropped to
3.4% at week 11. Similarly, at 6 weeks, 3.4% of B cells
(B220.sup.+) expressed EGFP (FIG. 4e) and this number dropped to
1.9% at 11 weeks. This is consistent with published reports that
lymphoid cells transduced with retroviral vectors have the tendency
to silence expression of genes driven by the LTR promoter.
[0099] SCID Mice Reconstituted with the scTCR Transduced BM
Controlled Outgrowth of a MUC1.sup.+ Human Tumor.
[0100] Based on our findings that the highest percent of cells
expressing the scTCR were those of the innate immune system, and
that this expression was most stable, we tested the potential of
these cells alone to exert an anti-tumor effect. We reconstituted
lethally irradiated SCID mice with 2.times.10.sup.6 BM cells
transduced with the scTCR-MFG retroviral vector or mock-transduced.
Expression of the scTCR was detected by RT-PCR in transduced BM
cells prior to injection and in splenocytes from reconstituted mice
60 days later (Figure). Mice reconstituted with BM cells transduced
with the scTCR retroviral vector or with control supernatant were
challenged one month later with subcutaneous injection of
MUC1.sup.+ human tumor cell line HPAF. Mice that received scTCR-MFG
transduced BM cells were able to inhibit growth of HPAF tumor cells
compared to control mice (FIG. 5a). The difference in tumor size
between the two groups was statistically significant at each time
point (p<0.01).
[0101] Tumor sections from control mice were intact and homogeneous
in appearance, without any infiltration by immune cells (FIG. 5b,
left panel). In contrast, tumor sections from scTCR BM
reconstituted mice were almost completely destroyed and infiltrated
with various immune cells (FIG. 5b, right panel). The predominant
cells in the infiltrate were neutrophils (FIG. 5c, left) followed
by macrophages (FIG. 5c, mid) and to a lesser extent NK cells (FIG.
5c, right).
[0102] Lack of Autoimmunity in MUC1 Tg. Mice Reconstituted with
scTCR-Transduced BM Cells.
[0103] In order to test whether T cells expressing this TCR can
develop normally in the presence of MUC1, we reconstituted C57BL6
wild type mice and mice transgenic for human MUC1 (MUC1 Tg.) with
scTCR-transduced BM and six weeks later compared the percentages of
scTCR-expressing cells. Table 1 shows that similar percentages of
scTCR+ T cells as well as other immune cells were seen in C57BL/6
and MUC1 Tg. mice, showing that that there was no selection against
the scTCR-expressing cells in MUC1 Tg. mice. Successful
reconstitution of MUC1 Tg. mice allowed us also to determine if the
expression of this receptor could have deleterious effects on
normal tissues expressing MUC1, such as the lung and the
pancreas.
TABLE-US-00001 TABLE I scTCR-expressing cells in reconstituted
C57BL/6 and MUC1 Tg. mice Cell Type TCR Reconstituted (Surface
Marker) Control C57BL/6 MUC1 Tg. T cells (CD3.sup.+) 0.2 .+-.
0.sup.c 6.4 .+-. 1.3 9.0 .+-. 5.7 B cells (B220.sup.+) 0.2 .+-. 0
9.7 .+-. 0.3 9.5 .+-. 1.6 Granulocytes (GR-1.sup.+) 0.2 .+-. 0.7
30.8 .+-. 21.5 27.4 .+-. 9.2 NK cells (DX5.sup.+) 0.1 .+-. 0 28.5
.+-. 4.3 18.5 .+-. 3.7 Monocytes (F4/80.sup.+) 0.1 .+-. 0.2 20.4
.+-. 5.0 14.6 .+-. 2.7
The analysis was in Table 1 was performed six weeks post
reconstitution with scTCR transduced BM. C57BL/6 refers to
untreated mice. The numerical values in Table 1 refer to the mean
%.+-.the standard deviation. FIG. 6A shows very few EGFP+ cells
infiltrating these tissue and no difference between wt and MUC1 Tg.
mice. There was also no evidence of destruction of MUC1 expressing
tissues in MUC1 Tg. Mice (FIG. 6B).
[0104] One potential explanation for the lack of autoimmunity in
reconstituted MUC1Tg mice could be that these cells are rendered
tolerant or anergic in the presence of MUC1 as a self-antigen. This
was not the case, however, since these mice successfully controlled
the growth of a mouse tumor RMA transfected with human MUC1
(RMA-MUC) much better than the control mice. There was no
inhibition of growth of the untransfected RMA control (FIG. 7).
[0105] This example shows that a suitable receptor of the invention
(such as an MHC-unrestricted TCR specific for an epitope on the
tumor antigen MUC1) expressed on cells of the innate and the
adaptive immune system is able to direct the effector functions of
these cells specifically against cancer cells displaying the MUC1
tumor antigen. The MHC-unrestricted nature of the TCR, combined
with the stable expression of the MUC1 tumor antigen on a large
number of human tumors (over 80%), makes therapy based on this TCR
(scTCR) applicable to a large number of patients with a variety of
tumors. Our experiments in SCID mice showed that the cells of the
innate system alone can control tumor growth when provided with the
tumor antigen-specific TCR. ScTCR expressing cells were seen as
early as 3 weeks post-reconstitution and were still present at high
numbers more than 7 months later. Both, the early presence of
tumor-specific cells and their permanence would be expected to
provide a beneficial anti-tumor effect. Furthermore, the
persistence of scTCR+ cells (FIG. 9) suggests that among the many
different cells that were transduced in the BM, there were also the
long-term reconstituting hematopoietic stem cells that can continue
to provide scTCR+ progenitors and mature cells throughout the life
of the animal. Advantageously, expression of this TCR on large
percentages of immune and other hematopoietic cells is not
detrimental to the well-being of the animal. Long-term follow up
(more than 12 months) of reconstituted mice transgenic for human
MUC1 showed no signs of autoimmunity and no specific infiltration
of cells into normal tissues that express MUC1. We hoped this would
be the result because the epitope recognized by the sTCR has been
shown to be expressed on the hypoglycosylated MUC1 made primarily
by tumor cells.
[0106] The studies we describe here provide a reasonable
expectation that this reagent is useful for immunotherapy of
cancer. For example, a patient can be treated by transducing this
scTCR into bone marrow or peripheral stem cells before infusing the
cells into patients who have undergone high-dose chemotherapy
(HDCT). HDCT followed by autologous stem cell or BM transplant,
primarily performed in breast cancer patients, has had a limited,
but positive, therapeutic success. The high rate of
post-transplantation relapse in these patients could be a result of
the survival of some tumor cells following HDCT treatment or could
result from infusing contaminating tumor cells with the stem cell
preparation. Vaccination trials aimed at augmenting the immune
responses to eradicate residual tumor cells following stem cell
therapy has shown minimal success, probably as a result of the poor
and slow reconstitution of the T cell compartment in these
patients. The recovery of the innate compartment of the immune
system (in particular NK cells), however, occurs very rapidly after
transplantation, reaching normal levels within a month
post-transplantation. Accordingly, transducing BM cells with a
MUC1-specific TCR or other receptor of the invention prior to
transplantation will result in the expression of a tumor-specific
receptor on a high percentage of quickly reconstituting cells of
the innate immune system, which could promptly target and destroy
residual tumor cells. Because MUC1 is expressed as a tumor antigen
on greater than 80% of all human tumors, and the inventive receptor
can direct effector cells to all such tumors in virtually all
patients, there are few limits for its clinical application. By
selecting a suitable expression vector, one can target various
effector cells for in vitro or in vivo transduction and tailor this
type of gene therapy or a immunotherapy to specific stages of
disease and combinations with other therapies.
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A., T. Demirer, S. Rowley, C. D. Buckner, G. Goodman, R. Maziarz,
J. Klarnet, N. Zuckerman, G. Harrer, R. McCloskey, R. Gersh, R.
Goldberg, W. Nichols, A. Jacobs, P. Weiden, P. Montgomery, S.
Rivkin, F. R. Appelbaum, and W. I. Bensinger. 1998. High-dose
busulfan, melphalan and thiotepa followed by 22 autologous
peripheral blood stem cell (PBSC) rescue in patients with advanced
stage III/IV ovarian cancer. Bone Marrow Transplant 22:651-659.
[0147] 40. Bensinger, W. I., K. S. Schiffman, L. Holmberg, F. R.
Appelbaum, R. Maziarz, P. Montgomery, E. Ellis, S. Rivkin, P.
Weiden, K. Lilleby, S. Rowley, S. Petersdorf, J. P. Klarnet, W.
Nichols, A. Hertler, R. McCroskey, C. H. Weaver, and C. D. Buckner.
1997. High-dose busulfan, melphalan, thiotepa and peripheral blood
stem cell infusion for the treatment of metastatic breast cancer.
Bone Marrow Transplant 19:1183-1189. [0148] 41. Brenner, M. K., D.
R. Rill, M. S. Holladay, H. E. Heslop, R. C. Moen, M. Buschle, R.
A. Krance, V. M. Santana, W. F. Anderson, and J. N. Ihle. 1993.
Gene marking to determine whether autologous marrow infusion
restores long-term haemopoiesis in cancer patients. Lancet
342:1134-1137. [0149] 42. Holmberg, L. A., D. V. Oparin, T. Gooley,
K. Lilleby, W. Bensinger, M. A. Reddish, G. D. MacLean, B. M.
Longenecker, and B. M. Sandmaier. 2000. Clinical outcome of breast
and ovarian cancer patients treated with high-dose chemotherapy,
autologous stem cell rescue and THERATOPE STn-KLH cancer vaccine.
Bone Marrow Transplant 25:1233-1241. [0150] 43. Avigan, D., Z. Wu,
R. Joyce, A. Elias, P. Richardson, D. McDermott, J. Levine, L.
Kennedy, N. Giallombardo, D. Hurley, J. Gong, and D. Kufe. 2000.
Immune reconstitution following high-dose chemotherapy with stem
cell rescue in patients with advanced breast cancer. Bone Marrow
Transplant 26:169-176. [0151] 44. Koehne, G., W. Zeller, M.
Stockschlaeder, and A. R. Zander. 1997. Phenotype of lymphocyte
subsets after autologous peripheral blood stem cell
transplantation. Bone Marrow Transplant 19:149-156.
Example 2
[0152] This example describes the design and construction of
several gene transfer vectors for expression in mammalian cells of
membrane bound and soluble human T cell receptors (TCR). In
particular, this example describes a vector (TCR
.alpha.-IRES-.beta. pEF4) that encodes high-level expression of a
full-length TCR of the present invention on the surface of T cells.
Furthermore, this example describes a chimeric TCR that does not
require the presence of endogenous CD3 molecules for surface
expression, which allows the receptor to be expressed on cells
other than T cells. This example also describes a vector encoding a
single chain TCR (scTCR) as a fusion protein of
V.alpha.V.beta.C.beta. with CD3. Advantageously, this scTCR is well
suited for gene therapy because it is encoded and expressed as a
single molecule and does not require individual cells to be
transduced by multiple nucleic acids. Moreover, this example
describes a mammalian expression vector encoding a soluble human
TCR. The approaches used in this example for manipulation of a
human tumor specific TCR also can be used to study various aspects
of TCR-based immunotherapy.
[0153] The following section of this example sets forth the
MATERIALS AND METHODS used herein.
[0154] Primers.
[0155] Sequences of the oligonucleotide primers (i.e., P1-P15) used
for cloning are listed in Table 2, along with restriction enzyme
cleavage and GenBank accession number.
TABLE-US-00002 TABLE 2 Primer Accession No. No. Sequence P1
DQ269212 5'-CGGGATCCTCGAGATGGAGACCCTCTTGGGCCTGCTTA-3' (SEQ ID NO:
8) P2 DQ269213 5'-CGGGATCCGTCGACATGGCCACCAGGCTCCTCTGCTG-3' (SEQ ID
NO: 9) P3 DQ269212 5'-CGGGATCCGGAATTCTCAGCTGGACCACAGCCGCAGCGT-3'
(SEQ ID NO: 10) P4 DQ269213
5'-ATAGTTTAGCGGCCGCGGATCCTCAGAAATCCTTTCTCTTGACCA-3' (SEQ ID NO: 11)
P5 J04132 5'-GGGGATCCCAAACTCTGCTACCTGCTGG-3' (SEQ ID NO: 12) P6
J04132 5'TCCCCGCGGCGGCCGCGAATTCTTAGCGAGGGGGCAGGGCCTGCATG-3' (SEQ ID
NO: 13) P7 DQ269212 5'-CGGGATCCAGATCCCCACAGGAACTTTCTGGGCTGGGGAAG-3'
(SEQ ID NO: 14) P8 DQ269213
5'-CGGGATCCAGATCCCCACAGTCTGCTCTACCCCAGGCCTCG-3' (SEQ ID NO: 15) P9
DQ269213 5'-AGGCGCGCCCCCAGGCCTCGGCGCTGACGATC-3' (SEQ ID NO: 16) P10
BT019811 5'-AGGCGCGCCGACATGGCCCTGATTGTGCTGGGGGGC-3' (SEQ ID NO: 17)
P11 BT019811 5'-AGGCGCGCCGACGCTGGGGATATGGCCCTGATTGTGCTGGG-3' (SEQ
ID NO: 18) P12 DQ269213
5'CTAAGCGTAGTCTGGGACGTCGTATGGGTACAGATCCTCTTCTGAGATGA
GTTTTTGTTCTACAACGGTTAACCTGGTC-3' (SEQ ID NO: 19) P13 DQ269213
5'CCTGCAGGTCAATGGTGATGGTGATGATGCTTGTCATCGTCATCCTTGTA
GTCAGCGTCTGCTCTACCCCAGG-3' (SEQ ID NO: 20) P14 DQ269212
5'ATGTGGCTGCAGAGCCTGCTGCTCTTGGGCACTGTGGCCTGCAGCATCTC
TGCACCCCAGGAGGTGACGCAGATTC-3' (SEQ ID NO: 21) P15 DQ269212
5'CCATGGAGACAGACACACTCCTGCTATGGGTACTGCTGCTCTGGGTTCCA
GGTTCCACTGGTGACGCGGCCCAGGAGGTGACGCAGATTC-3' (SEQ ID NO: 22)
[0156] Cloning of Full-Length TCR .alpha. and .beta. Chains.
[0157] MA CTL clone, the source of the TCR, is described in
Magarian-Blander et al. (J. Immunol, 160: 3111-3120 (1998)). RT-PCR
was performed using GeneAmp RT-PCR kit (Applied Biosystems, Foster
City, Calif. USA) and using either V.alpha. (P1) or V.beta. (P2)
leader sequence specific forward primers and C.alpha. (P3) or
C.beta. (P4) reverse primers. The TCR .alpha. and .beta. chains
were cloned into the multiple cloning site A (MCSA) and multiple
cloning site B (MCSB) in the pIRES vector (Clontech Laboratories,
Palo Alto, Calif., USA). The TCR.alpha.-IRES-TCR.beta. cassette was
then subcloned into the pEF4 mammalian expression vector
(Invitrogen, FIG. 10A).
[0158] Construction of a Two Chain TCR (tcTCR) and a Single Chain
TCR (scTCR) Expression Vectors for Expression on T Cells and Non-T
Cells.
[0159] Human CD3 .zeta. chain was cloned using forward primer (P5)
and reverse primer (P6). This cloning strategy maintained an
endogenous BamH I site at nucleotide number 80 in the extracellular
domain of human CD3 .zeta.. The PCR product was cloned into the
pCDNA3.1 TA vector (Invitrogen). The extracellular domains of the
TCR .alpha. and .beta. chains were cloned using (P1) or (P2)
forward primers and (P7) or (P8) reverse primers. The CD3
.zeta./pCDNA3.1 vector was digested with BamHI restriction enzyme
(New England BioLabs, Beverly, Mass., USA) and the TCR .alpha. or
.beta. chains were cloned in-frame with the CD3 .zeta. chain at the
BamH I site (FIG. 11A and FIG. 11B). The TCR .alpha..zeta. and
.beta..zeta. were subcloned into the pIRES vector and finally the
.alpha..zeta.-IRES-Pt cassette was subcloned into the pLNCX2
(Clontech) expression vector (FIG. 11C). The scTCR was constructed
by cloning the TCR V.alpha.J.alpha. and joining into the TCR
V.beta.D.beta.J.beta.C.beta. region using flexible linker
(GGGGS).sub.3. The TCR
V.alpha.J.alpha.-V.beta.D.beta.J.beta.C.beta. was then ligated
in-frame to the murine CD3 .zeta. chain. A linker encoding a
thrombin cleavage site, GDLVPRGSSRLD (SEQ ID NO: 7), was introduced
between the TCR .beta. constant and the CD3 .zeta. transmembrane
domain. The scTCR was cloned into the pEF6 vector.
[0160] Construction of scTCR-CD4TM-h.zeta. Mammalian Expression
Vectors.
[0161] The scTCR V.alpha.J.alpha.-V.beta.D.beta.J.beta.C.beta.
extracellular domain was amplified using V.alpha. forward (P1) and
C.beta. reverse (P9) and cloned into the pEF6 vector. An Asc I site
was introduced at the C-terminus in the TCR C.beta. region. The
human CD4 transmembrane (TM) domain fused to the human CD3 .zeta.
cytoplasmic domain was amplified from the hCD4.zeta. vector
(obtained from Dr. Margo R. Roberts, University of Virginia, VA,
USA) using forward (P10) and reverse (P6) primers. Asc I and Sac II
sites were introduced into the forward and reverse primers,
respectively that allowed in-frame ligation to the scTCR
extracellular domain. A modified version of this vector was created
by inserting a three amino acid (AGD) linker between the TCR
C.beta. region and the CD4 TM domain. In the latter, PCR was done
using (P6) and (P11) primers.
[0162] Surface Biotin Labeling and Thrombin Cleavage of scTCR.
[0163] Surface biotin labeling and thrombin cleavage was performed
using convention methods as described in Engel et al. (Science 256:
1318-1321 (1992)).
[0164] Construction of Secreted scTCRs.
[0165] A soluble, single chain fraction variable domain (sscFV)
encoding the TCR V.alpha.J.alpha.V.beta.D.beta.J.beta. or soluble
scTCR (sscTCR) domain consisting of the TCR
V.alpha.J.alpha.V.beta.D.beta.J.beta.C.beta. was cloned into
pCDNA3.1 vector by RT-PCR using (P1) and (P12) or (P2) and (P13)
primers, respectively. Modified versions of the sscTCR vector were
created by fusing the sscTCR to the GM-CSF, PCR was done using P14
and P13, or Ig-.kappa. light chain leader sequences (PCR was done
using P15 and P13).
[0166] Cell Transfection. Human embryonic kidney cells, HEK 29311,
were transfected using lipofectamine 2000 (Invitrogen) according to
manufacturer's instruction. Cells were analyzed for protein
expression 48-72 hours post-transfection. Jurkat cells were
electroporated using a BioRad Gene Pulser II (Bio-Rad Laboratories,
Hercules, Calif., USA) at 960 .mu.F and 200 V settings.
[0167] Stimulation with Superantigen. BWZ and BWZ-scTCR cells were
stimulated with SEE (Toxin Technology, Sarasota, Fla.). Thirty-six
hours later, IL-2 in culture supernatant was measured using murine
IL-2 ELISA kit (BD Pharmingen, San Diego, Calif.) according to
manufacturer's recommendations.
[0168] Purification of Soluble scTCR and Western Blotting.
[0169] Transfected 293H cells were grown in DMEM-10. Seventy-two
hours after replacing with fresh medium, culture medium was
harvested and used for protein purification. Anti-HA, c-Myc, and
6-His antibodies were purchased from Santa Cruz Biotechnology,
Santa Cruz, Calif., USA. Anti-Flag M2 antibody was purchased from
Sigma. For constructs encoding c-Myc or HA tagged proteins, sscTCR
was purified using Protein G Sepharose beads (Amersham Biosciences)
coated with the appropriate anti-tag antibody. For vectors encoding
6-His tagged proteins, sscTCR was purified using Nickel-Agarose
column (Qiagen) according to manufacturer's recommendations.
[0170] The following section sets forth the RESULTS obtained in
this example.
[0171] Reconstitution of the TCR/CD3 Complex on the Surface of
JRT3-T3.5 Jurkat Line Transfected with the TCR .alpha. and .beta.
Chain Construct.
[0172] J.RT3-T3.5 cells lack the TCR transcript and have low levels
of the TCR .alpha. chain message, and are therefore useful host
cells for testing the expression of transfected TCR. The pEF4
mammalian expression vector was chosen because expression of cloned
genes is driven by the human elongation factor-1 alpha
(EF-1.alpha.) promoter, which is expected to be more
transcriptionally active and stable in T cells than viral
promoters. In addition, the presence of the IRES sequence permits
expression of the TCR .alpha. and .beta. chain genes from the same
message. This is expected to result in similar levels of expression
of both genes, in contrast to genes driven by different promoters.
As shown in FIG. 10, untransfected JRT3-T3.5 did not express the
TCR/CD3 complex on their surface (FIG. 10A). Transfection of the
TCR .beta. chain alone didn't reconstitute the TCR/CD3 complex on
cell surface (FIG. 10B); however, stable transfection of the
TCR.alpha.-IRES-.beta. pEF4 vector into J.RT3-T3.5 cells resulted
in high levels of TCR/CD3 complex surface expression (FIG. 10C).
Cells transfected with the TR-ALPHA-IRES-TR-BETA pEF4 vector
recognized MUC1+ tumors in vitro.
[0173] Engineered MUC1-Specific Two Chain TCRs (tcTCR) Allows
Expression on the Surface of Non-T Cells.
[0174] The requirement for CD3 molecules for expression of the TCR
on the cell surface limits its expression to T cells. Engineered
vectors that would bypass this requirement were constructed and
allow expression of TCRs and TCR like receptors on other cell types
(Engel et al., Science 256: 1318-1321 (1992)). In T cells, the
engineered vector eliminates the possibility that the transferred
TCRs could pair with endogenous TCR .alpha. or .beta. chains that
could reduce the level of the specific receptor on the surface as
well as form a potentially autoreactive TCR. Construction of the
TCR .alpha..zeta. and .beta..zeta. was done as detailed in the
Materials and Methods. As shown in FIG. 11, transfection of the TCR
at (A) and TCR .beta..zeta.(B) constructs into human epithelial
kidney HEK 293H (non-T cell) cells resulted in surface expression
of the TCR .alpha./.beta. heterodimer (D and E). Similar levels of
surface expression were also obtained when 293H cells were
transfected with the TCR .alpha..zeta.-IRES-.beta..zeta. pLNCX2
vector (FIGS. 11C and 11F).
[0175] Different Configurations of Single Chain TCRs (scTCRs) were
Expressed at Various Levels on the Surface of 293H Cells.
[0176] For clinical application, transfection with multiple
plasmids can be disadvantageous. Single chain (sc)TCR and single
chain antibodies obviate this hurdle and are known in the art. We
made three different MUC1 tumor antigen specific scTCR constructs
and compared the levels of their surface expression (FIG. 12). FIG.
12 depicts the level of surface expression of scTCR (FIG. 12B),
scTCR-CD4TM-11 (FIG. 12C) and the scTCR-AGD-CD4TM-h.zeta. (FIG.
12D). These scTCRs consist of the TCR antigen binding domain and a
signaling component from the CD3 .zeta. chain. As shown in FIG. 3B,
transfection of the scTCR into 293H cells resulted in a substantial
increase in surface expression compared to control (A), and
transfection with the seTCR-CD4TM-h.zeta. construct gave a much
lower level of expression (FIG. 3C). However, inserting a 3 amino
acids (AGD) linker between the TCR C.beta. chain and the CD4 TM
domain restored high level of surface expression of the scTCR (FIG.
3D). While not desiring to be bound by any particular theory, it is
possible that these 3 amino acids provided enough flexibility to
the scTCR to allow proper folding of the molecule and normal level
of surface expression. FIG. 12E shows the same results in a more
quantitative way. High surface expression of the scTCR was detected
on both T cells (BWZ murine thymoma) and non-T cells (RBL rat
basophilic leukemia) transfected with TCR-pEF6 vector (FIG. 13A).
The TCR is functional as shown by the ability of the transfected
BWZ cells to be stimulated with plate-bound anti-TCR .beta.F1
antibody or with SEE superantigen, which binds specifically to the
human TCR V.beta.8 region (FIG. 13B). These cells also recognized
MUC1+ tumor cells in vitro (see Example 1).
[0177] Multiple Expression Vectors for Soluble scTCR
Production.
[0178] Soluble single chain T cell receptors (sscTCRs) can be used
as vehicles to deliver and target therapeutic drugs to the site of
their specific antigen (e.g., a tumor expressing MUC1 tumor
antigen). Additionally, sscTCR can be used to study the affinity of
interaction between the TCR and its ligand using, e.g., by Biacore
analysis. We successfully generated sscTCR by inserting a thrombin
cleavage site between the scTCR C.beta. region and the CD3
transmembrane domain (FIG. 14A). FIG. 14B shows that the scTCR
containing the thrombin cleavage site can be cleaved from the
surface of transfected cells. Following thrombin cleavage, the
scTCR could be purified from the soluble fraction (FIG. 14C, lane
4) using an affinity column. As expected, the sscTCR has a lower
molecular weight than the membrane bound scTCR (FIG. 14C, lanes 2
and 4). The sscTCR can be eluted from the affinity column under
high pH elution conditions (FIG. 14C, lane 8). Even though this
approach was successful in generating sscTCR, the amount that was
obtained was extremely low. Other groups have reported expression
of a soluble, single chain fraction variable (sscFV) domain of both
antibody and T cell receptor. FIG. 15a shows the design of the
sscFV construct that encodes the TCR
V.alpha.J.alpha.-V.beta.D.beta.J.beta.. FIG. 15b shows the same
sscTCR construct that was terminated just before the last cysteine
in the TCR C.beta. region. Two other constructs were designed as
described in FIG. 15b, with the exception of replacing the V.alpha.
leader sequence in the sscTCR with either a GM-CSF (FIG. 15c) or
Ig-.kappa. light chain (FIG. 15d) signal peptide. Various epitope
tags were inserted at the C terminus to facilitate protein
expression and purification. When the sscFV construct was
transfected into 293H cells, no recombinant sscFV protein could be
detected in the culture supernatant (FIG. 15e, lane a'). However,
transfection of the sscTCR construct into 293H cells resulted in
significant amounts of recombinant protein secreted in culture
supernatants (FIG. 15e, lane b'). Transfection of the sscTCR that
was fused to the Ig-.kappa. light chain leader sequence gave a
lower level of protein expression than was seen in b' (FIG. 15e,
lane d'), and transfection of the sscTCR construct fused to the
GM-CSF leader sequence yielded no protein secretion (FIG. 15e, lane
c'). These results showed that the presence of the TCR .beta. chain
constant region is absolutely required for expression of the
sscTCR. While not desiring to be bound by any particular theory, we
hypothesized that the TCR .beta. chain constant region must be
important for the proper folding of the protein or that it
interacts with and masks other hydrophobic amino acid residues in
the TCR .beta. chain VDJ region, otherwise the scFV is rendered
insoluble. As shown in FIG. 15f, the recombinant sscTCR could be
purified from culture supernatant using a nickel column. FIG. 15g
shows the western blot analysis of purified fractions obtained in
FIG. 15f, using anti-FLAG M2 antibody. The expression of soluble
form scTCR in these mammalian cells appears to be sufficiently
robust to produce this reagent for therapeutic purposes or for
biophysical analyses.
[0179] Thus this example describes several mammalian expression
vectors useful for functional high-level expression of human TCR
.alpha. and .beta. chains that are useful for biological and
biochemical analyses, as well as immunotherapy. Our TCR
.alpha.-IRES-.beta. pEF4 vector encoding the tumor antigen-specific
TCR generated high levels and stable expression of the TCR
.alpha..beta./CD3 complex on the surface of transfected T cells. We
also constructed chimeric TCR .alpha..zeta. and TCR .beta..zeta.
that were successfully expressed on the surface of 293H cells (a
non T cell line that doesn't express the CD3 complex).
Additionally, we showed that surface expression of the TCR was
dependent on the co-expression of the TCR .alpha..zeta. and
TCR.beta..zeta.. We hypothesized that pairing of the TCR at to the
TCR .beta..zeta. was crucial for proper folding and transport of
the heterodimer through the endoplasmic reticulum (ER) and Golgi
and eventually to the cell surface.
[0180] In contrast to the expression of two chain TCRs, functional
scTCRs can be expressed on the cell surface from a single mRNA
transcript. In the example shown here we constructed a single chain
TCR specific for the tumor antigen MUC1 and expressed it on the
surface of different cell types. The expression of the scTCR on the
surface of transfected cells was lower than the level of expression
of the native TCR, which we attributed to the presence of charged
amino acids in the transmembrane (TM) domain of the CD3 zeta chain
that might cause dimerization and retention of the scTCR in the
endoplasmic reticulum. In an attempt to increase the level of
surface expression of the scTCR, we replaced the TM domain of
CD3.zeta. in the scTCR with the TM domain of human CD4. However,
this new construct was expressed at very low level until we
inserted a 3 amino acid (AGD) linker between the scTCR constant
region and the CD4 TM domain. High level of surface expression of
the scTCR was restored in this new construct. The short linker
provided either flexibility or sufficient spacing between the TCR
constant region and CD4 TM to allow normal surface expression.
[0181] Most previous attempts to generate soluble TCR were made
using prokaryotic expression systems. However, proteins expressed
in prokaryotic cells lack post-translational modifications and may
be improperly folded. In order to avoid these potential problems,
we chose to express soluble MUC1-specific scTCR using mammalian
expression systems. We terminated the scTCR construct just before
the last cysteine in the TCR.quadrature. constant region. Following
transfection into 293H cells, large amount of soluble scTCR was
detected in culture supernatants.
[0182] In conclusion the various constructs we adapted and tested
for the expression of the MUC1-specific TCR can be of interest and
help to other investigators interested in TCR immunotherapy or in
studying TCR-antigen interactions.
[0183] The following references are useful in understanding the
invention, and in particular this example: [0184] 1. Dudley M E,
Wunderlich J R, Robbins P F, Yang J C, Hwu P, Schwartzentruber D J,
Topalian S L, Sherry R, Restifo N P, Hubicki A M et al: Cancer
regression and autoimmunity in patients after clonal repopulation
with antitumor lymphocytes. Science 2002, 298(5594):850-854. [0185]
2. Rubinstein M P, Kadima A N, Salem M L, Nguyen C L, Gillanders W
E, Nishimura M I, Cole D J: Transfer of TCR genes into mature T
cells is accompanied by the maintenance of parental T cell avidity.
J Immunol 2003, 170(3):1209-1217. [0186] 3. Morgan R A, Dudley M E,
Yu Y Y, Zheng Z, Robbins P F, Theoret M R, Wunderlich J R, Hughes M
S, Restifo N P, Rosenberg S A: High efficiency TCR gene transfer
into primary human lymphocytes affords avid recognition of melanoma
tumor antigen glycoprotein 100 and does not alter the recognition
of autologous melanoma antigens. J Immunol 2003, 171(6):3287-3295.
[0187] 4. Aarnoudse C A, Kruse M, Konopitzlcy R, Brouwenstijn N,
Schrier P I: TCR reconstitution in Jurkat reporter cells
facilitates the identification of novel tumor antigens by cDNA
expression cloning. Int J Cancer 2002, 99(1):7-13. [0188] 5. Derby
M A, Wang J, Margulies D H, Berzofslcy J A: Two
intermediate-avidity cytotoxic T lymphocyte clones with a disparity
between functional avidity and MHC tetramer staining. Int Immunol
2001, 13(6):817-824. [0189] 6. Snyder J T, Alexander-Miller M A,
Berzofskyl J A, Belyakov I M: Molecular mechanisms and biological
significance of CTL avidity. Curr HIV Res 2003, 1(3):287-294.
[0190] 7. Weijtens M E, Hart E H, Bolhuis R L: Functional balance
between T cell chimeric receptor density and tumor associated
antigen density: CTL mediated cytolysis and lymphokine production.
Gene Ther 2000, 7(1):35-42. [0191] 8. Yang S, Linette G P,
Longerich S, Haluska F G: Antimelanoma activity of CTL generated
from peripheral blood mononuclear cells after stimulation with
autologous dendritic cells pulsed with melanoma gp100 peptide
G209-2M is correlated to TCR avidity. J Immunol 2002,
169(1):531-539. [0192] 9. Magarian-Blander J, Ciborowski P. Hsia S,
Watkins S C, Finn O J: Intercellular and intracellular events
following the MHC-unrestricted TCR recognition of a tumor-specific
peptide epitope on the epithelial antigen MUC1. J Immunol 1998,
160(7):3111-3120. [0193] 10. Engel I, Ottenhoff T H, Klausner R D:
High-efficiency expression and solubilization of functional T cell
antigen receptor heterodimers. Science 1992, 256(5061):1318-1321.
[0194] 11. Mittelbrunn M, Yanez-Mo M, Sancho D, Ursa A,
Sanchez-Madrid F: Cutting edge: dynamic redistribution of
tetraspanin CD81 at the central zone of the immune synapse in both
T lymphocytes and APC. J Immunol 2002, 169(12):6691-6695. [0195]
12. Willemsen R A, Weijtens M E, Ronteltap C, Eshhar Z, Gratama J
W, Chames P, Bolhuis R L: Grafting primary human T lymphocytes with
cancer-specific chimeric single chain and two chain TCR. Gene Ther
2000, 7(16):1369-1377. [0196] 13. Novotny J, Ganju R K, Smiley S T,
Hussey R E, Luther M A, Recny M A, Siliciano R F, Reinherz E L: A
soluble, single-chain T-cell receptor fragment endowed with
antigen-combining properties. Proc Natl Acad Sci USA 1991,
88(19):8646-8650. [0197] 14. Eshhar Z, Bach N, Fitzer-Attas C J,
Gross G, Lustgarten J, Waks T, Schindler D G: The T-body approach:
potential for cancer immunotherapy. Springer Semin Immunopathol
1996, 18(2):199-209. [0198] 15. Gregoire C, Lin S Y, Mazza G, Rebai
N, Luescher I F, Malissen B: Covalent assembly of a soluble T cell
receptor-peptide-major histocompatibility class I complex. Proc
Natl Acad Sci USA 1996, 93(14):7184-7189. [0199] 16. Pavlinkova G,
Colcher D, Booth B J, Goel A, Batra S K: Pharmacokinetics and
biodistribution of a light-chain-shuffled CC49 single-chain Fv
antibody construct. Cancer Immunol Immunother 2000,
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[0200] All references, including publications, patent applications,
and patents, cited herein are hereby incorporated by reference to
the same extent as if each reference were individually and
specifically indicated to be incorporated by reference and were set
forth in its entirety herein.
[0201] The use of the terms "a" and "an" and "the" and similar
referents in the context of describing the invention (especially in
the context of the following claims) are to be construed to cover
both the singular and the plural, unless otherwise indicated herein
or clearly contradicted by context. The terms "comprising,"
"having," "including," and "containing" are to be construed as
open-ended terms (i.e., meaning "including, but not limited to,")
unless otherwise noted. Recitation of ranges of values herein are
merely intended to serve as a shorthand method of referring
individually to each separate value falling within the range,
unless otherwise indicated herein, and each separate value is
incorporated into the specification as if it were individually
recited herein. All methods described herein can be performed in
any suitable order unless otherwise indicated herein or otherwise
clearly contradicted by context. The use of any and all examples,
or exemplary language (e.g., "such as") provided herein, is
intended merely to better illuminate the invention and does not
pose a limitation on the scope of the invention unless otherwise
claimed. No language in the specification should be construed as
indicating any non-claimed element as essential to the practice of
the invention.
[0202] Preferred embodiments of this invention are described
herein, including the best mode known to the inventors for carrying
out the invention. Variations of those preferred embodiments may
become apparent to those of ordinary skill in the art upon reading
the foregoing description. The inventors expect skilled artisans to
employ such variations as appropriate, and the inventors intend for
the invention to be practiced otherwise than as specifically
described herein. Accordingly, this invention includes all
modifications and equivalents of the subject matter recited in the
claims appended hereto as permitted by applicable law. Moreover,
any combination of the above-described elements in all possible
variations thereof is encompassed by the invention unless otherwise
indicated herein or otherwise clearly contradicted by context.
Sequence CWU 1
1
241110PRTArtificialMA V-alpha 23 1Gln Glu Val Thr Gln Ile Pro Ala
Ala Leu Ser Val Pro Glu Gly Glu 1 5 10 15 Asn Leu Val Leu Asn Cys
Ser Phe Thr Asp Ser Ala Ile Tyr Asn Leu 20 25 30 Gln Trp Phe Arg
Gln Asp Pro Gly Lys Gly Leu Thr Ser Leu Leu Leu 35 40 45 Ile Gln
Ser Ser Gln Arg Glu Gln Thr Ser Gly Arg Leu Asn Ala Ser 50 55 60
Leu Asp Lys Ser Ser Gly Arg Thr Thr Leu Tyr Ile Ala Ala Ser Gln 65
70 75 80 Pro Gly Asp Ser Ala Thr Tyr Leu Cys Ala Val Thr Ser Ser
Tyr Gly 85 90 95 Lys Leu Thr Phe Gly Gln Gly Thr Ile Leu Thr Val
His Pro 100 105 110 2112PRTArtificialMA V-beta 8.3 2Asp Ala Arg Val
Thr Gly Thr Pro Arg His Lys Val Thr Glu Met Gly 1 5 10 15 Gln Glu
Val Thr Met Arg Cys Gln Pro Ile Leu Gly His Asn Thr Val 20 25 30
Phe Trp Tyr Arg Gln Thr Met Met Gln Gly Leu Glu Leu Leu Gln Tyr 35
40 45 Phe Arg Asn Arg Ala Pro Leu Asp Asp Ser Gly Met Pro Lys Asp
Arg 50 55 60 Phe Ser Ala Glu Met Pro Asp Ala Thr Leu Ala Thr Leu
Lys Ile Gln 65 70 75 80 Pro Ser Glu Pro Arg Asp Ser Ala Val Tyr Phe
Cys Ala Ser Gly Leu 85 90 95 Gly Glu Ala Ser Gly Tyr Thr Phe Gly
Ser Gly Thr Arg Leu Thr Val 100 105 110 35PRTArtificialMuc-1
epitope 3Pro Asp Thr Arg Pro 1 5 415PRTArtificiallinker 4Gly Gly
Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 1 5 10 15
512PRTArtificiallinker 5Gly Asp Leu Val Pro Arg Gly Ser Ser Arg Leu
Asp 1 5 10 620PRTArtificialrepeat unit of 140mer synthetic MUC1
peptide 6Pro Asp Thr Arg Pro Ala Pro Gly Ser Thr Ala Pro Pro Ala
His Gly 1 5 10 15 Val Thr Ser Ala 20 712PRTArtificialthrombin
cleavage site 7Gly Asp Leu Val Pro Arg Gly Ser Ser Arg Leu Asp 1 5
10 838DNAArtificialprimer 8cgggatcctc gagatggaga ccctcttggg
cctgctta 38937DNAArtificialprimer 9cgggatccgt cgacatggcc accaggctcc
tctgctg 371039DNAArtificialprimer 10cgggatccgg aattctcagc
tggaccacag ccgcagcgt 391145DNAArtificialprimer 11atagtttagc
ggccgcggat cctcagaaat cctttctctt gacca 451228DNAArtificialprimer
12ggggatccca aactctgcta cctgctgg 281347DNAArtificialprimer
13tccccgcggc ggccgcgaat tcttagcgag ggggcagggc ctgcatg
471441DNAArtificialprimer 14cgggatccag atccccacag gaactttctg
ggctggggaa g 411541DNAArtificialprimer 15cgggatccag atccccacag
tctgctctac cccaggcctc g 411632DNAArtificialprimer 16aggcgcgccc
ccaggcctcg gcgctgacga tc 321736DNAArtificialprimer 17aggcgcgccg
acatggccct gattgtgctg gggggc 361841DNAArtificialprimer 18aggcgcgccg
acgctgggga tatggccctg attgtgctgg g 411979DNAArtificialprimer
19ctaagcgtag tctgggacgt cgtatgggta cagatcctct tctgagatga gtttttgttc
60tacaacggtt aacctggtc 792073DNAArtificialprimer 20cctgcaggtc
aatggtgatg gtgatgatgc ttgtcatcgt catccttgta gtcagcgtct 60gctctacccc
agg 732176DNAArtificialprimer 21atgtggctgc agagcctgct gctcttgggc
actgtggcct gcagcatctc tgcaccccag 60gaggtgacgc agattc
762290DNAArtificialprimer 22ccatggagac agacacactc ctgctatggg
tactgctgct ctgggttcca ggttccactg 60gtgacgcggc ccaggaggtg acgcagattc
9023110PRTArtificialSM3L 23Asp Ile Val Val Thr Gly Glu Ser Ala Leu
Thr Thr Ser Pro Gly Glu 1 5 10 15 Thr Val Thr Leu Thr Cys Arg Ser
Ser Thr Gly Ala Val Thr Thr Ser 20 25 30 Asn Tyr Ala Asn Trp Val
Gln Glu Lys Pro Asp His Leu Phe Thr Gly 35 40 45 Leu Ile Gly Gly
Thr Asn Asn Arg Ala Pro Gly Val Pro Ala Arg Phe 50 55 60 Ser Gly
Ser Leu Ile Gly Asp Lys Ala Ala Leu Thr Ile Thr Gly Ala 65 70 75 80
Gln Thr Glu Asp Glu Ala Ile Tyr Phe Cys Ala Leu Trp Tyr Ser Asn 85
90 95 His Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly 100
105 110 24110PRTArtificialSM3H 24Gln Glu Ser Gly Gly Gly Leu Val
Gln Pro Gly Ser Met Lys Leu Ser 1 5 10 15 Cys Val Ala Ser Gly Phe
Thr Phe Ser Asn Tyr Trp Met Asn Trp Val 20 25 30 Arg Gln Ser Pro
Glu Lys Gly Leu Glu Trp Val Ala Glu Ile Arg Leu 35 40 45 Lys Ser
Asn Asn Tyr Ala Thr His Tyr Ala Glu Ser Val Lys Gly Arg 50 55 60
Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Ser Val Tyr Leu Gln Met 65
70 75 80 Asn Asn Leu Arg Ala Glu Asp Thr Gly Ile Tyr Tyr Cys Thr
Gly Val 85 90 95 Gly Gln Phe Ala Tyr Trp Gly Gln Gly Thr Thr Val
Thr Val 100 105 110
* * * * *