U.S. patent application number 17/836052 was filed with the patent office on 2022-09-29 for genetically modified cells and uses thereof.
This patent application is currently assigned to CARTHERICS PTY. LTD.. The applicant listed for this patent is CARTHERICS PTY. LTD.. Invention is credited to Richard BOYD, Hiroshi KAWAMOTO, Alan TROUNSON.
Application Number | 20220305106 17/836052 |
Document ID | / |
Family ID | 1000006381124 |
Filed Date | 2022-09-29 |
United States Patent
Application |
20220305106 |
Kind Code |
A1 |
BOYD; Richard ; et
al. |
September 29, 2022 |
GENETICALLY MODIFIED CELLS AND USES THEREOF
Abstract
The present invention relates generally to a population of stem
cells (e.g., iPSCs or HSCs) that comprise nucleic acids encoding a
T cell receptor and a chimeric antigen receptor directed to
multiple distinct antigenic determinants, for example two distinct
tumour antigenic determinants. The present invention is also
directed to a population of T cells that co-express a T cell
receptor and a chimeric antigen receptor directed to multiple
distinct antigenic determinants, such as two distinct tumour
antigenic determinants. The cells of the present invention can be
derived from chosen donors whose HLA type is compatible with
significant sectors of the populations, and are useful in a wide
variety of applications, in particular in the context of the
therapeutic treatment of neoplastic conditions.
Inventors: |
BOYD; Richard; (Carlton,
AU) ; TROUNSON; Alan; (Carlton, AU) ;
KAWAMOTO; Hiroshi; (Carlton, AU) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
CARTHERICS PTY. LTD. |
Carlton |
|
AU |
|
|
Assignee: |
CARTHERICS PTY. LTD.
Carlton
AU
|
Family ID: |
1000006381124 |
Appl. No.: |
17/836052 |
Filed: |
June 9, 2022 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15778836 |
May 24, 2018 |
11400145 |
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PCT/AU2016/051141 |
Nov 23, 2016 |
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17836052 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 14/7051 20130101;
C07K 16/2803 20130101; A61K 2039/5158 20130101; A61P 35/00
20180101; A61K 39/001153 20180801; A61K 38/1774 20130101; A61K
35/17 20130101; C12N 2506/45 20130101; C07K 14/70517 20130101; C07K
2319/02 20130101; C12N 5/0636 20130101; C07K 2319/33 20130101; C12N
2510/00 20130101; C07K 16/3092 20130101; C07K 14/4748 20130101;
A61K 39/001102 20180801; C07K 2317/622 20130101; C07K 2319/03
20130101; A61K 35/545 20130101; C12N 5/0638 20130101 |
International
Class: |
A61K 39/00 20060101
A61K039/00; C07K 14/725 20060101 C07K014/725; C07K 16/28 20060101
C07K016/28; A61K 35/17 20060101 A61K035/17; A61K 35/545 20060101
A61K035/545; A61K 38/17 20060101 A61K038/17; C07K 14/47 20060101
C07K014/47; A61P 35/00 20060101 A61P035/00; C12N 5/0783 20060101
C12N005/0783; C07K 16/30 20060101 C07K016/30; C07K 14/705 20060101
C07K014/705 |
Foreign Application Data
Date |
Code |
Application Number |
Nov 27, 2015 |
AU |
2015904933 |
Apr 11, 2016 |
AU |
2016901328 |
Claims
1.-74. (canceled)
75. A composition, comprising: (i) a nucleic acid encoding a first
chimeric antigen receptor (CAR) which comprises an antigen
recognition moiety operably linked to a T cell activation moiety
through a hinge region and a transmembrane domain and directed to a
first antigenic determinant; and (ii) a nucleic acid encoding an
antigen-binding receptor lacking a functional signaling
intracellular domain which comprises an antigen recognition moiety,
a hinge and a transmembrane region of a CAR, directed to a second
antigenic determinant.
76. The construct of claim 75, wherein the hinge region in the
antigen-binding receptor has cysteine residues either removed or
substituted to prevent the formation of dimers.
77. The construct according to claim 75, wherein the hinge region
in the antigen-binding receptor is selected from a CD8 hinge and a
CD28 hinge.
78. The construct according to claim 75, wherein the hinge region
in the CAR comprises one or more cysteine residues to direct
dimerization of the CAR.
79. The construct according to claim 75, wherein the hinge region
in the CAR is selected from a CD8 hinge and a CD28 hinge.
80. The construct according to claim 75, wherein the first
antigenic determinant is selected from TAG-72, CD19 and MAGE.
81. The construct according to claim 75, wherein the second
antigenic determinant is CD47.
82. The construct of according to claim 75, wherein the first
antigenic determinant is TAG-72 and the second antigenic
determinant is CD47.
83. The construct according to claim 75, wherein the nucleic acid
encoding the CAR is operably linked to the nucleic acid encoding
the antigen-binding receptor through a nucleotide sequence encoding
a self-cleaving peptide.
84. A cell or a population thereof, wherein (i) the cell is (a)
stem cell or (b) a derivative cell obtained from differentiating
the cell of (a); and (ii) the cell comprises the construct as
defined in claim 75.
85. The cell according to claim 84, wherein the cell expresses at
least one homozygous HLA haplotype.
86. The cell according to claim 84 for use in the treatment of a
neoplastic condition, a microorganism infection, or an autoimmune
condition.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 15/778,836, filed May 24, 2018, which claims
the benefit of priority from International Patent Application No.
PCT/AU2016/051141, filed Nov. 23, 2016 and Australian Provisional
Patent Application No. 2016901328, filed Apr. 11, 2016 and No.
2015904933, filed Nov. 27, 2015, the entire contents of which are
incorporated herein by reference.
INCORPORATION BY REFERENCE OF SEQUENCE LISTING
[0002] The Sequence Listing in the ASCII text file, named as
34247Z_SequenceListing.txt of 36 KB, created on May 9, 2022 and
submitted to the United States Patent and Trademark Office via
EFS-Web, is incorporated herein by reference.
FIELD OF THE INVENTION
[0003] The present invention relates generally to a population of
stem cells (e.g., iPSCs or HSCs) that comprise nucleic acids
encoding a T cell receptor and a chimeric antigen receptor directed
to multiple distinct antigenic determinants, for example two
distinct tumour antigenic determinants. The present invention is
also directed to a population of T cells that co-express a T cell
receptor and a chimeric antigen receptor directed to multiple
distinct antigenic determinants, such as two distinct tumour
antigenic determinants. The cells of the present invention can be
derived from chosen donors whose HLA type is compatible with
significant sectors of the populations, and are useful in a wide
variety of applications, in particular in the context of the
therapeutic treatment of neoplastic conditions.
BACKGROUND OF THE INVENTION
[0004] Bibliographic details of the publications referred to by
author in this specification are collected alphabetically at the
end of the description.
[0005] The reference in this specification to any prior publication
(or information derived from it), or to any matter which is known,
is not, and should not be taken as an acknowledgment or admission
or any form of suggestion that that prior publication (or
information derived from it) or known matter forms part of the
common general knowledge in the field of endeavour to which this
specification relates.
[0006] Malignant tumours, or cancers, grow in an uncontrolled
manner, invade normal tissues, and often metastasize and grow at
sites distant from the tissue of origin. In general, cancers are
derived from one or only a few normal cells that have undergone a
poorly understood process called malignant transformation. Cancers
can arise from almost any tissue in the body. Those derived from
epithelial cells, called carcinomas, are the most common kinds of
cancers. Sarcomas are malignant tumours of mesenchymal tissues,
arising from cells such as fibroblasts, muscle cells, and fat
cells. Solid malignant tumours of lymphoid tissues are called
lymphomas, and marrow and blood-borne malignant tumours of
lymphocytes and other hematopoietic cells are called
leukaemias.
[0007] Cancer is one of the three leading causes of death in
industrialised nations. As treatments for infectious diseases and
the prevention of cardiovascular disease continue to improve, and
the average life expectancy increases, cancer is likely to become
the most common fatal disease in these countries. Therefore,
successfully treating cancer requires that all the malignant cells
be removed or destroyed without killing the patient. An ideal way
to achieve this would be to induce an immune response against the
tumour that would discriminate between the cells of the tumour and
their normal cellular counterparts. However, immunological
approaches to the treatment of cancer have been attempted for over
a century with unsustainable results.
[0008] Solid tumours cause the greatest number of deaths from
cancer. Solid tumours are not usually curable once they have spread
or `metastasised` throughout the body. The prognosis of metastatic
solid tumours has improved only marginally in the last 50 years.
The best chance for the cure of a solid tumour relies on early
detection followed by the use of local treatments such as surgery
and/or radiotherapy when the solid tumour is localised and has not
spread either to the lymph nodes that drain the tumour or
elsewhere. Nonetheless, even at this early stage, and particularly
if the tumour has spread to the draining lymph nodes, microscopic
deposits of cancer known as micrometastases may have already spread
throughout the body and will subsequently lead to the death of the
patient. In this sense, cancer is a systemic disease that requires
systemically administered treatments.
[0009] There is a long history of "Golden Bullet" attempts with
toxin-loaded antibodies to attack cancers, taking advantage of
their capacity to potentially target any specific molecular entity
such as carbohydrate, lipid or protein, or combinations thereof.
Antibodies, once bound to a cancer cell, can engage Complement or
FcR+NK/K cells and induce cell lysis. Unfortunately antibody
treatment of cancer has met generally only moderate success,
primarily because of low affinity binding, poor lytic efficiency
and their brief longevity. Collectively, these compromise the
ability of antibodies to rapidly destroy cancer cells, increasing
the risk of mutation and immune evasion. More recently, there have
been reports of antibody-related therapies including those based on
antibodies directed with high affinity to cancer molecules and to
immune checkpoint blockade molecules. Although there are some
clinical successes particularly with the latter, such therapies are
still associated with various limitations.
[0010] Accordingly, common methods of treating cancer continue to
follow the long used protocol of surgical excision (if possible)
followed by radiotherapy and/or chemotherapy, if necessary. The
success rate of this rather crude form of treatment is extremely
variable but generally decreases significantly as the tumour
becomes more advanced and metastasises. Further, these treatments
are associated with severe side effects including disfigurement and
scarring from surgery (eg. mastectomy or limb amputation), severe
nausea and vomiting from chemotherapy, and most significantly,
damage to normal tissues such as the hair follicles, gut and bone
marrow which is induced as a result of the relatively non-specific
targeting mechanism of the toxic drugs which form part of most
cancer treatments.
[0011] Accordingly, there is an urgent and ongoing need to develop
improved systemic therapies for cancers, in particular metastatic
cancers.
[0012] Thymic generation of mainstream T cells is fundamentally
required for defence against infection. This pool of "immune
surveillance" T cells patrols the body to remove damaged or
abnormal cells including cancers. Since thymus-based T cell
production is characterised by random generation of the T cell
receptor (TCR) repertoire, thymopoiesis must also include very
strict selection processes that eliminate or functionally silence
those developing thymus T cells with the potential to attack self.
This "self tolerance" therefore restricts autoimmune disease
(Fletcher et al (2011). However, by necessity, this very process
compromises the immune surveillance against cancers--given that
non-viral induced cancers are by definition diseases of "self".
This means that many T cells arising in the thymus, which could
potentially have been reactive with tumour-associated antigens may
be eliminated before entry into the blood. At the very least they
will be numerically deficient and perhaps have a low affinity TCR.
Notwithstanding this, T cells are clearly potentially a major
weapon against cancer--the challenges are thus to increase their
ability to detect cancer, numerically expand them and retain, or
better, enhance their powerful cytolytic capacity. While antibodies
and T cells are the most logical weapons against cancer, their
potential rapid and effective cancer destruction has not been
clinically realized. Advances in immunotherapy have evolved through
genetically engineering T cells to express a novel chimeric
membrane receptor consisting of a cancer antigen binding antibody
fragment, coupled cytoplasmically to T cell signal transduction
molecules. The latter are commonly one or all of the TCR chain, CD
28, or CD40-Ligand (Corrigan-Curay et al (2014); Fedorov et al
(2014); Perna et al (2014); Curran et al (2015); Curran et al
(2012); Dotti et al (2014); Han et al (2013)). Such chimeric
antigen receptor (CAR) expressing T cells (CAR-T) not only harness
the two most powerful anticancer weapons of the immune system, but
also overcome their individual inadequacies. CAR-T retain the
potent, focal, cell lytic capacity and avoid the normal reliance on
the instrinsic TCR to detect very rare "cancer peptide(s)"
expressed in HLA clefts. The repertoire of T cells specific to such
nominal peptides is very rare. The antibody portion of the CAR
endows the T cells with cancer seeking specificity and overcomes
the notoriously poor cancer destructive efficacy of circulating
antibodies. Thus cancer binding is mediated by the antibody domain
of the CAR, leading to cytoplasmic signal transduction, triggering
the T cell lytic pathways to destroy the cancer.
[0013] Although still in its clinical infancy, numerous CAR-T
trials are underway. As promising as it is though, there are
several aspects of CAR-T technology that are problematic and are
preventing its clinical efficacy to be fully realized. The most
obvious is the cytokine storm that occurs during T cell mediated
cancer destruction and is tumour load dependent. Fever is
indicative of cancer destruction, but can lead to severe clinical
side effects unless managed carefully (Davila et al (2014); Casucci
et al (2015)). Current management is by cytokine modulation
treatments such as anti-IL6. Further, there exists a significant
problem with the numerical deficiency of generated CAR-T cells to
not only attack the initial cancer, but also to be preserved in
sufficient supply in case of relapse. Currently, attempts to deal
with this problem are based on the excessive use of proliferation
inducing cytokines in vitro. Still further, as effective as CAR-T
cells are at attacking cancer, even for CD19.sup.+ cancers the
tumour destruction is not 100% effective. While up to 90%
responsiveness has been reported for B-ALL, in other CD19.sup.+
cancers the results are much less effective. Accordingly, despite
the encouraging observations in relation to the utility of CAR-T,
there are still significant issues to be overcome before this
technology can take its place as reliable, effective and the new
gold standard in relation to cancer treatment.
[0014] In work leading up to the present invention it has been
determined, inter alia, that the seemingly disparate problems
currently existing in relation to the effective therapeutic
application of CAR-T technology are resolvable where the CAR-T
cells can be derived from transfected stem cells, such as adult
stem cells, rather than transfected thymocytes or other transfected
somatic cell types. For example, by transfecting stem cells (such
as induced pluripotent stem cells ("iPSCs") derived from adult
somatic cells) with a chimeric antigen receptor, the issue of
providing sufficient present and future supplies of CAR-T cells
directed to a particular tumour is resolved due to the ongoing
source of somatic T cells derived from these self-renewing
transfected stem cells. Still further, these iPSCs, and hence the
CAR-T cells derived from them, can be prior selected from donors
expressing a homozygous HLA haplotype, in particular homozygous for
an HLA type which is expressed widely in the population, thereby
providing a means of generating a bank of cells which exhibit broad
donor suitability. Still further, it has been determined that the
generation of an iPSC from a T cell which exhibits T cell receptor
specificity directed to an antigen of interest means that the gene
rearrangements for that TCR specific for the cancer antigen will be
embedded in the iPSC. All T cells induced from that iPSC will
retain the anti-cancer TCR specificity. This can be followed by
transfection of such iPSC with a CAR, enabling the subsequent
differentiation of said iPSC to a T cell, such as a CD4+ or CD8+ T
cell, which stably exhibits dual specificity for the antigen to
which the CAR is directed and a TCR directed to the antigen to
which the original T cell was directed to. Without limiting the
present invention to any one theory or mode of action, it is
thought that this is due to the actions of epigenetic memory. Still
further, it has also been determined that dual specific NKT cells
can be similarly generated. Accordingly, there can be provided an
ongoing source of T and NKT cells which are selectively and stably
directed to multiple distinct antigenic determinants, such as
multiple distinct tumour antigenic determinants, thereby enabling a
more therapeutically effective treatment step to be effected.
SUMMARY OF THE INVENTION
[0015] Throughout this specification and the claims which follow,
unless the context requires otherwise, the word "comprise", and
variations such as "comprises" and "comprising", will be understood
to imply the inclusion of a stated integer or step or group of
integers or steps but not the exclusion of any other integer or
step or group of integers or steps.
[0016] As used herein, the term "derived from" shall be taken to
indicate that a particular integer or group of integers has
originated from the species specified, but has not necessarily been
obtained directly from the specified source. Further, as used
herein the singular forms of "a", "and" and "the" include plural
referents unless the context clearly dictates otherwise.
[0017] The subject specification contains amino acid sequence
information prepared using the program PatentIn Version 3.5,
presented herein after the bibliography. Each amino acid sequence
is identified in the sequence listing by the numeric indicator
<210> followed by the sequence identifier (eg. <210>1,
<210>2, etc). The length, type of sequence (protein, etc) and
source organism for each amino acid sequence are indicated by
information provided in the numeric indicator fields <211>,
<212> and <213>, respectively. Amino acid sequences
referred to in the specification are identified by the indicator
SEQ ID NO: followed by the sequence identifier (e.g., SEQ ID NO: 1,
SEQ ID NO: 2, etc.). The sequence identifier referred to in the
specification correlates to the information provided in numeric
indicator field <400> in the sequence listing, which is
followed by the sequence identifier (e.g., <400>1,
<400>2, etc.). That is SEQ ID NO: 1 as detailed in the
specification correlates to the sequence indicated as <400>1
in the sequence listing.
[0018] Unless otherwise defined, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs.
[0019] One aspect of the present invention is directed to a
genetically modified mammalian stem cell, or a T cell
differentiated therefrom, which cell is capable of differentiating
to a T cell expressing a TCR directed to a first antigenic
determinant, and comprises a nucleic acid molecule encoding a
chimeric antigen receptor, wherein said receptor comprises an
antigen recognition moiety directed to a second antigenic
determinant, which antigen recognition moiety is operably linked to
a T cell activation moiety. In some embodiments, the genetically
modified mammalian stem cell expresses at least one homozygous HLA
haplotype.
[0020] In another aspect there is provided a genetically modified
mammalian stem cell, or a T cell differentiated therefrom, which
cell is capable of differentiating to a CD4.sup.+ T cell expressing
a TCR directed to a first antigenic determinant, and comprises a
nucleic acid molecule encoding a chimeric antigen receptor, wherein
said receptor comprises an antigen recognition moiety directed to a
second antigenic determinant, which antigen recognition moiety is
operably linked to a T cell activation moiety. In some embodiments,
the genetically modified mammalian stem cell expresses at least one
homozygous HLA haplotype.
[0021] In still another aspect there is provided a genetically
modified mammalian stem cell, or a T cell differentiated therefrom,
which cell is capable of differentiating to a CD8.sup.+ T cell
expressing a TCR directed to a first antigenic determinant, and
comprises a nucleic acid molecule encoding a chimeric antigen
receptor, wherein said receptor comprises an antigen recognition
moiety directed to a second antigenic determinant, which antigen
recognition moiety is operably linked to a T cell activation
moiety. In some embodiments, the genetically modified mammalian
stem cell expresses at least one homozygous HLA haplotype.
[0022] In a further aspect there is provided a genetically modified
mammalian stem cell, or a T cell differentiated therefrom, which
cell is an iPSC (induced pluripotent stem cell) or an HSC
(haemopoietic stem cell), is capable of differentiating to a T cell
expressing a TCR directed to a first antigenic determinant, and
comprises a nucleic acid molecule encoding a chimeric antigen
receptor, wherein said receptor comprises an antigen recognition
moiety directed to a second antigenic determinant, which antigen
recognition moiety is operably linked to a T cell activation
moiety. In some embodiments, the genetically modified stem cell
such as iPSC or HSC expresses at least one homozygous HLA
haplotype.
[0023] In accordance with this aspect of the invention, in one
embodiment, the stem cell (e.g., iPSC) is derived from a cell in
which the TCR genes have undergone re-arrangement.
[0024] In another embodiment, said stem cell (e.g., iPSC) is
derived from a T cell or thymocyte expressing an .alpha..beta.
TCR.
[0025] In still another embodiment, said stem cell (e.g., iPSC) is
derived from a T cell or thymocyte expressing a .gamma..delta.
TCR.
[0026] In yet another embodiment, said stem cell (e.g., iPSC) is
derived from a T cell or thymocyte expressing a TCR directed to
said first antigenic determinant, i.e., the same antigenic
determinant to which the TCR expressed on a T cell derived from
said stem cell (e.g., iPSC) is directed.
[0027] In still another embodiment, said stem cell (e.g., iPSC) is
derived from a T cell or thymocyte that is CD8.sup.+.
[0028] In yet another embodiment, said stem cell (e.g., iPSC) is
derived from a T cell or thymocyte that is CD4.sup.+.
[0029] In one embodiment, the stem cell (e.g., iPSC or HSC) is
capable of differentiating into a CD4.sup.+ T cell expressing a TCR
directed to a first antigenic determinant. In another embodiment,
the stem cell (e.g., iPSC or HSC) is capable of differentiating
into a CD8.sup.+ T cell expressing a TCR directed to a first
antigenic determinant.
[0030] In still another further aspect there is provided a
genetically modified mammalian stem cell, or a T cell
differentiated therefrom, which cell is capable of differentiating
to a T cell expressing a TCR directed to a first antigenic
determinant, and comprises a nucleic acid molecule encoding a
chimeric antigen receptor, wherein said receptor comprises an
antigen recognition moiety directed to a second antigenic
determinant, which antigen recognition moiety is operably linked to
a T cell activation moiety, and wherein said antigenic determinants
are selected from tumour antigens, microorganism antigens or
autoreactive immune cell antigens. In some embodiments, the
genetically modified mammalian stem cell expresses at least one
homozygous HLA haplotype.
[0031] In one embodiment, said stem cell is an iPSC. In another
embodiment, the stem cell is an HSC.
[0032] In another embodiment, said stem cell is capable of
differentiating to a CD4.sup.+ T cell or a CD8.sup.+ T cell.
[0033] In still another embodiment, said TCR is an .alpha..beta.
TCR.
[0034] In yet still another embodiment, said stem cell (e.g., iPSC)
is derived from a T cell or thymocyte, preferably a CD8.sup.+ T
cell or thymocyte. In some embodiments, said stem cell (e.g., iPSC)
is derived from a CD8.sup.+ T cell or thymocyte expressing a TCR
directed to said first antigenic determinant, i.e., the same
antigenic determinant to which the TCR expressed on a T cell
derived from said stem cell (e.g., iPSC) is directed.
[0035] In yet another aspect there is provided a genetically
modified mammalian stem cell, or a T cell differentiated therefrom,
which cell is capable of differentiating to a T cell expressing a
TCR directed to a first tumour antigenic determinant, and comprises
a nucleic acid molecule encoding a chimeric antigen receptor,
wherein said receptor comprises an antigen recognition moiety
directed to a second tumour antigenic determinant, which antigen
recognition moiety is operably linked to a T cell activation
moiety, and wherein said first antigenic determinant is selected
from TCR recognized peptides such as WT-1 or EbvLMP2, and said
second antigenic determinant is selected from, for example, TAG-72,
CD19, MAGE, or CD47. In some embodiments, the genetically modified
mammalian stem cell expresses at least one homozygous HLA
haplotype.
[0036] The genetically modified mammalian stem cells (e.g., iPSCs
or HSCs) disclosed herein are capable of differentiating to a T
cell expressing a TCR directed to a first antigenic determinant
(e.g., a first tumour antigenic determinant), and comprises a
nucleic acid molecule encoding a chimeric antigen receptor which
comprises an antigen recognition moiety directed to a second
antigenic determinant (e.g., a second tumour antigenic
determinant), operably linked to a T cell activation moiety. That
is, the genetically modified stem cells (e.g., iPSCs or HSCs)
disclosed herein are capable of differentiating into T cells
directed to multiple, i.e., at least two (namely two or more)
antigenic determinants. In some embodiments, the genetically
modified mammalian stem cell expresses at least one homozygous HLA
haplotype.
[0037] Accordingly, in a further aspect, there is provided a
genetically modified mammalian stem cell capable of differentiating
into a T cell directed to more than two antigenic determinants.
[0038] In accordance with this aspect of the invention, in some
embodiments, the genetically modified mammalian stem cell (e.g.,
iPSC or HSCs) is capable of differentiating to a T cell expressing
a TCR directed to a first antigenic determinant, and comprises
multiple (i.e., two or more) nucleic acid molecules encoding
multiple chimeric antigen receptors, wherein each chimeric antigen
receptor comprises an antigen recognition moiety directed to an
antigenic determinant, which antigen recognition moiety is operably
linked to a T cell activation moiety. In some embodiments, the
genetically modified mammalian stem cell expresses at least one
homozygous HLA haplotype.
[0039] In one embodiment, the multiple antigenic determinants which
the multiple chimeric antigen receptors are directed to are each
distinct from said first antigenic determinant to which the TCR
expressed on a T cell derived from said stem cell is directed. In
another embodiment, the multiple antigenic determinants which the
multiple chimeric antigen receptors are directed to are distinct,
one from another, and are also distinct said first antigenic
determinant to which the TCR expressed on a T cell derived from
said stem is directed.
[0040] In one embodiment, the multiple CAR-encoding nucleic acids
are included in one contiguous nucleic acid fragment. For example,
the multiple CAR-encoding nucleic acids are placed in one construct
or vector which is transfected into a cell to generate a
genetically modified mammalian stem cell comprising the multiple
CAR-encoding nucleic acids. In a specific embodiment, the multiple
CAR encoding nucleic acids can be linked to each other within one
expression unit and reading frame (for example, by utilizing a
self-cleaving peptide such as P2A), such that one single
polypeptide comprising multiple CAR polypeptide sequences is
initially produced and subsequently processed to produce multiple
CARs. In another embodiment, the multiple CAR-encoding nucleic
acids are placed in separate vectors which are used in transfection
to generate a genetically modified mammalian stem cell comprising
the multiple CAR-encoding nucleic acids. Examples of CAR-encoding
nucleic acid constructs are depicted in FIG. 11, and exemplary
sequences for a CAR and various domains suitable for use in a CAR
are provided in SEQ ID NOS: 1-2 and 7-20.
[0041] Further in accordance with the aspect of the invention
providing a genetically modified mammalian stem cell capable of
differentiating into a T cell directed to more than two antigenic
determinants, in other embodiments, the genetically modified
mammalian stem cell (e.g., iPSC or HSC) (which optionally expresses
at least one homozygous HLA haplotype), is capable of
differentiating to a T cell expressing a TCR directed to a first
antigenic determinant, comprises a nucleic acid molecule encoding a
chimeric antigen receptor which comprises an antigen recognition
moiety directed to a second antigenic determinant, operably linked
to a T cell activation moiety, and further comprises a nucleic acid
molecule encoding an antigen-binding receptor which comprises an
antigen recognition moiety directed to a third antigenic
determinant. According to these embodiments, such genetically
modified stem cell is capable of differentiating into a T cell
directed to multiple antigenic determinants, preferably multiple
antigenic determinants that are distinct one from another.
Additional antigenic specificity can be provided by employing
multiple CAR-encoding nucleic acids as described herein, and/or
utilizing multiple nucleic acids encoding antigen binding
receptors.
[0042] In one embodiment, the antigen-binding receptor is a
non-signalling antigen-binding receptor; namely, the receptor is
anchored to the cell surface and binds to the third antigenic
determinant, but does not transduce signal into the cytoplasmic
part of the cell. In one embodiment, the antigen-binding receptor
comprises an antigen recognition moiety directed to a third
antigenic determinant, operably linked to a transmembrane domain,
but lacks a T cell activation moiety.
[0043] In a specific embodiment, the antigen-binding receptor is a
non-signalling antigen-binding receptor directed to CD47. For
example, the antigen-binding receptor is a non-signalling
CD47-binding molecule, e.g., a truncated, CD47-binding
molecule.
[0044] Accordingly, there is provided a genetically modified
mammalian stem cell (e.g., iPSC or HSC), or a T cell differentiated
therefrom, which cell is capable of differentiating to a T cell
expressing a TCR directed to a first antigenic determinant,
comprises (i) a nucleic acid molecule encoding a chimeric antigen
receptor, wherein said receptor comprises an antigen recognition
moiety directed to a second antigenic determinant, which antigen
recognition moiety is operably linked to a T cell activation moiety
and (ii) a nucleic acid molecule encoding a non-signalling
CD47-binding molecule, e.g., a truncated, CD47-binding molecule. In
some embodiments, the genetically modified mammalian stem cell
(e.g., iPSC or HSC) expresses at least one homozygous HLA
haplotype.
[0045] In another aspect there is provided a method of making a
genetically modified mammalian stem cell (such as an iPSC or HSC)
disclosed herein.
[0046] In one embodiment, the subject method comprises obtaining a
mammalian stem cell (such as an iPSC or HSC) that is capable of
differentiating to a T cell expressing a TCR directed to a first
antigenic determinant, which stem cell (e.g., iPSC or HSC), in one
embodiment, expresses at least one homozygous HLA haplotype; and
introducing into the stem cell (e.g., via transfection) one or more
nucleic acid molecules encoding one or more chimeric antigen
receptors, each chimeric antigen receptor comprising an antigen
recognition moiety directed to an antigenic determinant, which
antigen recognition moiety is operably linked to a T cell
activation moiety. In another embodiment, the method further
comprises introducing into the stem cell (e.g., via transfection)
one or more nucleic acid molecules encoding one or more
antigen-binding receptors (e.g., non-signalling antigen-binding
receptors), each antigen-binding receptor comprising an antigen
recognition moiety directed to an antigenic determinant. As further
disclosed herein, the multiple receptor-encoding nucleic acids can
be introduced by way of a single vector or separate vectors.
[0047] In another embodiment, the subject method comprises
obtaining a T cell or thymocyte (preferably CD8+ T cell or
thymocyte) which expresses a TCR directed to a first antigenic
determinant, and which, in one embodiment, also expresses at least
one homozygous HLA haplotype; introducing into the T cell or
thymocyte one or more nucleic acid molecules encoding one or more
chimeric antigen receptors, each chimeric antigen receptor
comprising an antigen recognition moiety directed to an antigenic
determinant, which antigen recognition moiety is operably linked to
a T cell activation moiety; and deriving a stem cell (e.g., iPSC)
from the T cell or thymocyte. In another embodiment, the method
further comprises, before the step of deriving a stem cell from the
T cell or thymocyte, introducing into the T cell or thymocyte one
or more nucleic acid molecules encoding one or more antigen-binding
receptors (e.g., non-signalling antigen-binding receptors), each
antigen-binding receptor comprising an antigen recognition moiety
directed to an antigenic determinant.
[0048] In still another embodiment, the subject method comprises
obtaining an HSC (e.g., from the bone marrow or blood) which, in
some embodiments, expresses at least one homozygous HLA haplotype;
introducing to the HSC (i) one or more nucleic acids encoding a TCR
directed to a first antigenic determinant, (ii) one or more nucleic
acid molecules encoding one or more chimeric antigen receptors,
each chimeric antigen receptor comprising an antigen recognition
moiety directed to an antigenic determinant that is different from
said first antigenic determinant, which antigen recognition moiety
is operably linked to a T cell activation moiety; and optionally
(iii) one or more nucleic acid molecules encoding one or more
antigen-binding receptors (e.g., non-signalling antigen-binding
receptors), each antigen-binding receptor comprising an antigen
recognition moiety directed to an antigenic determinant that is
different from said first antigenic determinant and different from
the antigen determinant(s) to which the chimeric antigen
receptor(s) is(are) directed. As disclosed herein, the multiple
receptor-encoding nucleic acids can be introduced by way of a
single vector or separate vectors. Such genetically modified HSC
can be used to generate T cells having specificity to multiple
antigenic determinants.
[0049] In a further aspect there is provided a T cell that
expresses a TCR directed to a first antigenic determinant, and
expresses one or more chimeric antigen receptors, wherein each
receptor comprises an antigen recognition moiety directed to an
antigenic determinant, which antigen recognition moiety is operably
linked to a T cell activation moiety. In some embodiments, the T
cell further expresses an antigen-binding receptor which comprises
an antigen recognition moiety directed an antigenic determinant. In
some embodiments, the T cell provided therein expresses at least
one homozygous HLA haplotype.
[0050] In another aspect there is provide a method for making a T
cell that expresses a TCR directed to a first antigenic
determinant, and expresses one or more CARs wherein each CAR
comprises an antigen recognition moiety directed to an antigenic
determinant, which antigen recognition moiety is operably linked to
a T cell activation moiety, and optionally also expresses one or
more non-signalling antigen-binding receptors each of which
comprises an antigen recognition moiety directed to an antigenic
determinant. In some embodiments, the method provided herein is
directed to making a T cell that expresses at least one homozygous
HLA haplotype.
[0051] Another aspect of the present invention is directed to a
method of treating a condition characterised by the presence of an
unwanted population of cells in a mammal, said method comprising
administering to said mammal an effective number of stem cells or T
cells, as hereinbefore described.
[0052] In one embodiment, said condition is a neoplastic condition,
a microorganism infection (such as HIV, STD or antibiotic resistant
bacteria), or an autoimmune condition.
[0053] According to this embodiment, there is provided a method of
treating a neoplastic condition, said method comprising
administering to said mammal an effective number of stem cells, or
T cells, as hereinbefore defined wherein said TCR is directed to a
first tumour antigenic determinant and said CAR is directed to a
second tumour antigenic determinant.
[0054] In still another embodiment, said first tumour antigenic
determinant is WT-1.
[0055] In another embodiment, said second tumour antigenic
determinant is TAG-72, CD19, MAGE, or CD47.
[0056] Yet another aspect of the present invention is directed to
the use of stem cells or T cells, as hereinbefore defined in the
manufacture of a medicament for the treatment of a condition
characterised by the presence of an unwanted population of cells in
a mammal.
BRIEF DESCRIPTION OF THE DRAWINGS
[0057] The file of this patent contains at least one drawing
executed in color. Copies of this patent with color drawing(s) will
be provided by the Patent and Trademark Office upon request and
payment of the necessary fee.
[0058] FIGS. 1A-10. Stimulation and expansion of cytotoxic T cells
expressing a TCR specific for Wilm's Tumor 1 (WT-1) antigen. Cells
were isolated from whole blood peripheral blood mononuclear cells
(PBMCs). Cells were gated on single cells (A, F, K) where the
scatter plot is also depicted (B, G, L), followed by CD3 positive
cells (conjugated to APCCy7; C, H, M), followed by CD8 (conjugated
to PECy7) and CD4 (conjugated to PerCp; D, I, N), and finally on
CD8 cells alone (E, J, O). WT-1 staining was carried out using
HLA-A02 tetramer specific for the WT-1.sub.37 peptide.
Representations are from two separate patients (patient 1 A-E;
patient 2 F-J) that are HLA-A02 positive and compared to a
fluorescence minus one (FMO; this stain lacks the WT-1 tetramer
stain, showing the specificity stain of WT-1.sub.37, K-O).
Proportions shown are a percentage of CD3+ cells. The percentage of
WT-1 TCR T cells increased to 1.5% and 4.5% for the two samples; in
unstimulated PBMC these cells are very low (below the level of
detection using the tetramer technology herein). In other studies
(e.g., Schmeid et al (2015)) they are as few 1 per 10.sup.-6 of
CD8+ cells (range 3.times.10.sup.-7 to 3.times.10.sup.-6
cells).
[0059] FIGS. 2A-2G. CD8+ Cytotoxic T cells with TCR specific for
Wilm's Tumor 1 (WT-1) antigen are functional. Function is
represented by production of interferon gamma (IFN-.gamma.)
(Ghanekar et al 2001). IFN-.gamma. expression was found after WT-1
specific stimulation. Activated cells were gated on the CD8+ and
HLA-A02 tetramer to the WT-1.sub.37 peptide--PE conjugated
fluorochrome. These cytotoxic T cells with TCR specific for WT-1,
when stimulated with WT-1, demonstrated intracellular cytokine
stain of IFN-.gamma. (conjugated to the pacific blue fluorochrome).
Representations are from two separate patients (Wt-1 #1 and WT-1
#2) (patient 1: A-B; patient 2: C-D) that are HLA-A02 positive and
compared to a fluorescence minus one (FMO; E-F; this stain lacked
the WT-1 tetramer stain, showing the specificity stain of
WT-1.sub.37 (G). Proportions shown are a percentage of WT1+CD8+
cells. Over 80% of the WT-1 TCR T cells produced IFN.gamma..
[0060] FIG. 2H. Addition of the LAG 3 inhibitor (IMP 321) increases
the frequency of WT-1 specific T cells after 4 days stimulation. In
this experiment, purified but unseparated cord blood mononuclear
cells were plated either alone, with either anti CD28 alone, with
WT-1 peptide (Miltenyi BioTech) and CD28 .mu.g/ml) or with WT-1
peptide plus IMP 321, for 24 hours and 4 days. No effects were
observed by 24 hours (data not shown) but, consistent with the
kinetics of IMP 321 effect on activation dendritic cells (Brigone
et al (2007)), there was a doubling of WT-1 specific CD8+ T cells
after 4 days.
[0061] FIG. 3. Production of iPSC from cancer specific (eg WT-1)
TCR T cells. Cancer antigen specific T cells are extremely rare in
normal blood; they are revealed by stimulation in vitro with WT-1
peptide bound to autologous B cells acting as antigen presenting
cells (formed into lymphoblast cells lines (LCL) using EBV), in the
presence of cytokines. Cancer antigen specific T cells are shown as
double labelled with CD8 (for cytotoxic T cells) and tetramer for
HLA-WT-1 binding to the TCR of these CD8+ cells. These cells were
then converted into iPSC using the Yamanaka reprogramming factors.
The rearranged TCR genes specific for WT-1 were embedded in the TCR
locus of the iPSCs.
[0062] FIG. 4. Morphological progression of iPSC colonies to
haemopoietic lineage and lymphoid progenitors after culture for 1,
5, 9 and 13 days on OP9 support cells. Note large numbers of single
haemopoietic-like cells by day 13.
[0063] FIG. 5. Flow cytometric analysis of iPSC-derived cells after
culture for 13 days on OP9 cells, clearly shows evidence of
haemopietic specialisation with the presence of haemopoietic stem
cells (HSC) (CD34+CD43+).
[0064] FIG. 6. Flow cytometry for HSC in iPSC-derived cells after
13 days culture on OP9 cells followed by 9 days culture on OP9
DL-L1 cells. Cells were gated on viability, CD45 expressions,
single cells then examined for HSC content by staining for CD34 and
CD43. Note the reduction in HSC from >90% pre OPDL-L1 culture
(FIG. 5), to .about.60% after 9 days culture on OP9DL-L1 cells.
[0065] FIG. 7. Flow cytometry for T cell development of
iPSC-derived cells after 13 days culture on OP9 cells followed by 9
days culture on OP9 DL-L1 cells. There is clear evidence of
commitment to the T cell lineage with expression of CD5 and CD7 and
the first stages of thymocyte development with immature (i.e.,
lacking CD3; data not shown) CD4+, CD8+"single positive" cells and
CD4+CD8+"double positive" cells.
[0066] FIG. 8. Flow cytometry for HSC and T cell differentiation in
iPSC-derived cells after 13 days culture on OP9 cells followed by
16 days culture on OP9 DL-L1 cells. Immature T cells expressing CD4
and/or CD8 were still clearly present and there was further
reduction of HSC from .about.60% to .about.25%. Most importantly
mature CD8+ cells were present and expressed CD3, .alpha..beta. TCR
and the CD8.beta. chain (in addition to CD8a--not shown)
[0067] FIG. 9. Schematic representation of the induction of WT-1
specific TCR, CD8.alpha..beta. T cells from iPSC derived from in
vitro expanded WT-1 specific TCR T cells. The treatment of the
CD4+CD8+ cells with (low levels) anti CD3 antibody mimic the
signalling that occurs within the thymus during positive selection;
this increased CD8+ T cells expressing both the CD8.alpha. and
CD8.beta. chains.
[0068] FIG. 10. WT-1 specific TCR, CD8.alpha..beta. T cells induced
from iPSC derived from in vitro expanded WT-1 specific TCR T cells,
retained full function (e.g., cytotoxicity to WT-1 expressing
targets) equivalent to the original T cells. The effector:target
ratio was 3:1; graded concentrations of WT-1 peptides were
tested.
[0069] FIG. 11. Schematic diagram of chimeric antigen receptor and
antigen-binding receptor constructs. A panel of Chimeric Antigen
Receptor (CAR) constructs have been developed--with scFv for either
TAG 72 or CD19 (as a positive control). The constructs used either
human CD8 or CD28 as hinge and transmembrane regions and CD28,
CD3.zeta. chain or 4-1BB cytoplasmic activation signalling domains.
P2A is a signal sequence directing proteolytic cleavage, which in
the top five constructs shown in FIG. 11 releases EGFP as a
fluorescent reporter of expression, and in the lower (sixth)
construct shown in FIG. 11, releases a second CAR receptor
construct shown as Leader (CD8)-scFv (anti-CD47)-hinge/TM
(CD28)-endodomain tail (CD8) in which the Leader will be processed
to release the anti-CD47 scFv on the surface anchored by the
hinge/TM and the endodomain tail contains no signalling sequences.
Any CD47-binding ectodomain could be used for the purpose of
binding to CD47 on target cells, including for example SIRP-alpha.
The hinge region may contain cysteine residues to direct
dimerization by disulphide bond formation between adjacent hinge
domains, which is characteristic of the natural CD8 hinge, or may
have the cysteine residues substituted by other residues, such as
serine, which do not form disulphide bonds and do not form
covalently stabilised dimers. Exemplary sequences of CAR and
CD47-binding receptor, as well as various domain sequences suitable
for use in constructing a CAR or an antigen-binding receptor, are
set forth in SEQ ID NOS: 1-20.
[0070] FIG. 12. Retrovirus Transformation scheme. Schematic of the
processes undertaken for generating CAR containing retroviral
constructs. The CAR construct is cloned into the pSAMEN plasmid
vector and is linked to the fluorescent reporter EGFP by a P2A
self-cleaving polypeptide to separate the CAR and reporter. When
transduction of the cell is successful, the P2A is expressed and
cleaved, and the EGFP is identified by flow cytometry and
immunofluorescence microscopy.
[0071] FIG. 13. Lentivirus Transformation scheme. Schematic of the
processes undertaken for generating CAR containing lentiviral
constructs. The CAR construct is cloned into the pWP1 plasmid
vector and is linked to the fluorescent reporter EGFP by a P2A
self-cleaving polypeptide to separate the CAR and reporter. When
transduction of the cell is successful the P2A is expressed and
cleaved, and the EGFP identified by flow cytometry and
immunofluorescence microscopy.
[0072] FIG. 14A. Schematic of normal second generation CAR
structure. scFv binding domains to target antigens; hinge region
(stalk) allowing integration of the CAR into the plasma membrane
(length of hinge can differentially influence scFv binding to
target cells); cytoplasmic signalling domains which induce T cell
activation upon engagement of the scFv. The CAR structure is shown
as a dimer, stabilised by disulphide bonds between adjacent
cysteine residues in the hinges region.
[0073] FIG. 14B. Schematic of a non-signalling antigen-binding
receptor, a truncated CD47 "attachment stalk". Structure shows scFv
domains or single V-domains for CD47 antigen binding, attached to a
hinge and transmembrane region but no signalling domains are
present in the endodomain. This construct would allow increased
binding affinity of the CAR-T cell to the cancer cells expressing
high levels of CD47. While this receptor could also bind to normal
cells which express lower levels of CD47, there would be no signal
transduction and hence no damage to the normal cells. The Hinge
region may contain cysteine residues to direct dimerization by
disulphide bond formation between adjacent hinge domains, or may
have the cysteine residues substituted by other residues, such as
serine, which do not form disulphide bonds and do not form
covalently stabilised dimers.
[0074] FIG. 15. Flow cytometry analysis of CAR transduced human
PBMC derived CD3+ T cells demonstrating successful transduction
with the TAG72 Lentivirus CAR construct (20.8% positive compared to
<0.1% in the controls) and CD19 lentivirus CAR construct (33.9%
positive).
[0075] FIG. 16. Western blot analysis confirming protein expression
in TAG 27 and CD19 CAR-transfected T cells.
[0076] FIG. 17. TAG-72 CAR-T mediated killing of ovarian cancer
(TAG72+) target cells. Effector:target ratio (E:T)=1:1. TAG-72
CAR-T effector cells (GFP positive cells) developed from CD3
activated normal blood T cells, were isolated as >95% pure via
FACS and subsequently stimulated for enhance cytolytic activity for
72 h in the presence of immobilised .alpha.CD.sup.3/.alpha.CD28 and
IL-2 before use. Change in cell impedance (represented here as the
arbitrary unit Cell Index) was monitored over 40 h and compared to
stimulated non-transduced CD3.sup.+ve cells isolated from PBMCs and
stimulated vector only CAR-T cells. TAG72 CAR-T cells showed the
highest killing however CD3/CD28 activated non-CAR-T cells also
showed killing, albeit to a much lesser degree.
[0077] FIG. 18. Determining the specificity of TAG-72 CAR-T
killing. TAG-72 and CD19 CAR-T respectively were isolated via FACS
and immediately added to TAG-72.sup.hi/CD19.sup.low target cells
without in vitro stimulation (E:T=5:1). Change in cell impedance
(represented here as the arbitrary unit Cell Index) was monitored
over 15 h. TAG-72 CAR-T cells showed strong killing of the cell
line. CD19 CAR-T cells were the same as non-CAR T cell
controls.
[0078] FIGS. 19A-19B. Flow cytometry analysis of CAR transduction
of WT-1 specific TCR CD8+ T cells derived from iPSC produced from
WT-1 specific T cells. FIG. 19A. WT-1 specific TCR T cells were
successfully transduced with the TAG72 Lentivirus CAR construct
(31.3% positive compared to <0.1% in the controls). FIG. 19B.
WT-1 specific TCR T cells derived from iPSC formed from WT-1
specific TCR T cells successfully transduced with dual specificity
CAR construct for TAG 72 plus non-signalling truncated CD47 (55%
transduced); transduced with TAG 72 alone 32%. These transduced T
cells contained 3 anti-cancer specificities: WT-1 (TCR); TAG72
(CAR); truncated non-signalling CD47.
[0079] FIG. 20A-20I. Cytotoxic function of WT-1 specific TCR T
cells, and dual specific TAG 72 CAR/WT-1 TCR T cells. WT-1 specific
TCR T cells and dual specific TAG72 CAR/WT-1 TCR T cells were
incubated in monolayer cultures with the ovarian cancer cell line
CAOV4 to for 24 hours to assess cytotoxicity. Despite the low
effector:target ratio of 2:1 (necessary because of the low numbers
of effectors obtained), there was specific killing with WT-1 TCR T
cells and this was increased further with transduction with the
TAG72 CAR. The technique is based on AquaAmine which stains amines
within the cell. When a cell dies or is dying the compromised cell
membrane allows the dye to infiltrate the cell and stain the amines
more intensely. Cell cytotoxicity is therefore depicted by an
increased staining intensity of cellular amines. Note: live cells
will still give some (albeit low) positive staining because some
amines reside on the cell surface. A, D, G: CAOV4 cancer cells
alone. B, E, H: CAOV4 cancer cells incubate with WT-1 TCR T cells.
C, F, I: Dual specific TAG 72 CAR/WT-1 TCR T cells incubated with
CAOV4 ovarian cancer cells. D, E, F: Aqua amine levels on gated
CD3-ve cells (i.e., CAOVA4). Phase contrast images of G: Cancer
cells alone, H: non-CAR transfected WT-1 TCR cells with cancer
cells, and I: TAG-72 transfected WT-1 TCR T cells and cancer cells.
40.times. magnification. WT-1 TCR T cells caused approximately 10%
killing (above background); TAG72 CAR-T cells caused an additional
10% killing (i.e., approximately 20% above background). Dual
anti-cancer killing mechanisms are additive.
[0080] FIGS. 21A-21B. CAR transduction of iPS. Day 5 of growth on
MEF feeder layers, 4 days after incubation with CAR lentivirus.
CAR+ transduction (green) of TAG72, CD19 and GFP virus were
overlayed on bright field images at 20.times. magnification.
Non-transduced controls did not display any GFP signal. Images of
iPSC colonies at 4.times. magnification demonstrate the presence of
iPSC colonies on MEF feeder layers. In each system it is noted that
some of the iPSC colonies appeared to have begun to spontaneously
differentiate. Transduced fibroblast-derived iPSC are depicted in
FIG. 21A. FIG. 21B demonstrates the successful transduction of WT-1
T cell derived iPSCs with TAG72 CAR. These iPSCs were therefore
successfully imprinted for both WT-1 TCR and TAG 72
specificity.
[0081] FIG. 22. Flow cytometric analysis of Chimeric Antigen
Receptor transduction of iPSC. These iPSC are derived from adult
fibroblasts but can be from any origin including non-selected T
cells, CD8+ T cells or cancer antigen specific (e.g., WT-1) T
cells. There is clearly a population of fluorescent iPSC
successfully transduced by TAG 72 or CD19. Overlay of the
transduced cells compared to non-transduced controls is shown in
FIG. 23.
[0082] FIG. 23. Overlay of dot plots comparing non-transduced
control cells (blue) to transduced iPSC cultures (green). Events
within the GFP+ gate demonstrate successful transduction and are
presented as percent frequency of non-debris events.
[0083] FIG. 24. Reformation of CAR-Transduced iPSC colonies after
FACS sorting. CAR-transduced iPSC can be isolated by flow cytometry
(GFP positive fluorescence) and replated to form stable
colonies.
DETAILED DESCRIPTION OF THE INVENTION
[0084] The present invention is predicated, in part, on the
determination that dual TCR/CAR expressing T cells, directed to two
distinct antigenic determinants, can be consistently and stably
generated by, for example, transfecting a CAR cassette into an iPSC
derived from a T cell exhibiting TCR specificity directed to an
antigenic determinant of interest. By virtue of the actions of
epigenetic memory, a T cell differentiated from this iPSC has been
found to stably express both the TCR specificity of the somatic T
cell from which the iPSC was derived, and a CAR directed to a
distinct antigenic determinant. Specificity to additional antigenic
determinants can be achieved by introducing into cells additional
nucleic acid(s) encoding a molecule(s) that bind(s) to such
additional antigenic determinant(s). Such multi-specificity cell
thereby provides a more effective therapeutic outcome than
currently available. These determinations have therefore now
enabled the development of an ongoing source of stably transformed
dual antigen specific T cells, in particular cytotoxic
CD8+.alpha..beta. TCR T cells, for use in the context of any
disease condition which is characterised by an unwanted cellular
population, such as a neoplastic condition, a viral infection,
bacterial infection or an autoimmune condition. This finding, and
the generation of cells based thereon, have now facilitated the
improvement of therapeutic treatment regimes directed to treating
such conditions, in particular neoplastic conditions such as solid
tumours or blood cancers (e.g., leukaemias), including metastatic
disease.
[0085] Accordingly, one aspect of the present invention is directed
to a genetically modified mammalian stem cell, or a T cell
differentiated therefrom, which cell is capable of differentiating
to a T cell expressing a TCR directed to a first antigenic
determinant, and comprises a nucleic acid molecule encoding a
chimeric antigen receptor, wherein said receptor comprises an
antigen recognition moiety directed to a second antigenic
determinant, which antigen recognition moiety is operably linked to
a T cell activation moiety. In some embodiments, the genetically
modified mammalian stem cell expresses at least one homozygous HLA
haplotype.
[0086] Reference to a "T cell" should be understood as a reference
to any cell comprising a T cell receptor. In this regard, the T
cell receptor may comprise any one or more of the .alpha., .beta.,
.gamma. or .gamma. chains. As would be understood by the person of
skill in the art, NKT cells also express a T cell receptor and
therefore dual specific NKT cells can also be generated according
to the present invention. The present invention is not intended to
be limited to any particular sub-class of T cell, although in a
preferred embodiment the subject T cell expresses an .alpha./.beta.
TCR dimer. Still more preferably, said T cell is a CD4.sup.+ helper
T cell, a CD8.sup.+ killer T cell, or an NKT cell. Without limiting
the present invention to any one theory or mode of action,
CD8.sup.+ T cells are also known as cytotoxic cells. As a major
part of the adaptive immune system, CD8.sup.+ T cells scan the
intracellular environment in order to target and destroy,
primarily, infected cells. Small peptide fragments, derived from
intracellular content, are processed and transported to the cell
surface where they are presented in the context of MHC class I
molecules. However, beyond just responding to viral infections,
CD8+ T cells also provide an additional level of immune
surveillance by monitoring for and removing damaged or abnormal
cells, including cancers. CD8.sup.+ T cell recognition of an MHC I
presented peptide usually leads to either the release of cytotoxic
granules or lymphokines or the activation of apoptotic pathways via
the FAS/FASL interaction to destroy the subject cell. CD4.sup.+ T
cell, on the other hand, generally recognise peptide presented by
antigen presenting cells in the context of MHC class II, leading to
the release of cytokines designed to regulate the B cell and/or
CD8+ T cell immune responses. Accordingly, unlike cytotoxic T
cells, T helper cells do not directly kill unwanted cells, such as
cancer cells, although they can augment such a response, to the
extent that it is effected by cytotoxic T cells and/or antibody
based clearance mechanisms.
[0087] Natural killer T (NKT) cells are a specialised population of
T cells that express a semi-invariant T cell receptor (TCR
.alpha..beta.) and surface antigens typically associated with
natural killer cells. The TCR on NKT cells is unique in that it
recognizes glycolipid antigens presented by the MHC I-like molecule
CD1d. Most NKT cells express an invariant TCR alpha chain and one
of a small number of TCR beta chains. The TCRs present on type I
NKT cells recognise the antigen alpha-galactosylceramide
(alpha-GalCer). Within this group, distinguishable subpopulations
have been identified, including CD4.sup.+CD8.sup.- cells,
CD4.sup.-CD8.sup.+ cells and CD4''/CD8'' cells. Type II NKT cells
(or noninvariant NKT cells) express a wider range of TCR .alpha.
chains and do not recognise the alpha-GalCer antigen. NKT cells
produce cytokines with multiple, often opposing, effects, for
example either promoting inflammation or inducing immune
suppression including tolerance. As a result, they can contribute
to antibacterial and antiviral immune responses, promote
tumour-related immunosurveillance, and inhibit or promote the
development of autoimmune diseases. Like natural killer cells, NKT
cells can also induce perforin-, Fas-, and TNF-related cytoxicity.
Accordingly, reference to the genetically modified T cells of the
present invention should be understood to include reference to NKT
cells.
[0088] Since thymus-based T cell production is characterised by
random generation of the T cell receptor (TCR) repertoire,
thymopoiesis must also include very strict selection processes that
eliminate or functionally silence those developing thymus T cells
with the potential to attack self. This "self tolerance" therefore
reduces the potential for autoimmune disease. However, by
necessity, this very process compromises the immune surveillance
against cancers--given that non-viral induced cancers are by
definition diseases of "self". This means that many T cells arising
in the thymus, which could potentially have been reactive with
tumour-associated antigens, may be eliminated before entry into the
blood. At the very least they will be numerically deficient and
perhaps express a low affinity TCR.
[0089] In one embodiment there is provided a genetically modified
mammalian stem cell, or a T cell differentiated therefrom, which
cell is capable of differentiating to a CD4.sup.+ T cell expressing
a TCR directed to a first antigenic determinant, and comprises a
nucleic acid molecule encoding a chimeric antigen receptor, wherein
said receptor comprises an antigen recognition moiety directed to a
second antigenic determinant, which antigen recognition moiety is
operably linked to a T cell activation moiety. In one embodiment,
the genetically modified mammalian stem cell expresses at least one
homozygous HLA haplotype.
[0090] In another embodiment there is provided a genetically
modified mammalian stem cell, or a T cell differentiated therefrom,
which cell is capable of differentiating to a CD8.sup.+ T cell
expressing a TCR directed to a first antigenic determinant, and
comprises a nucleic acid molecule encoding a chimeric antigen
receptor, wherein said receptor comprises an antigen recognition
moiety directed to a second antigenic determinant, which antigen
recognition moiety is operably linked to a T cell activation
moiety. In one embodiment, the genetically modified mammalian stem
cell expresses at least one homozygous HLA haplotype.
[0091] In some embodiments, the genetically modified cell of the
present invention, e.g., the genetically modified stem cell (such
as an iPSC or an HSC) or T cell, is homozygous for at least one HLA
haplotype. Without limiting the present invention to any one theory
or mode of action, the major histocompatibility complex (MHC)
represents a set of cell surface molecules, the major function of
which is to bind peptide fragments derived from antigens and to
present them to T cells. The MEW gene family is divided into three
subgroups: class I, class II and class III. Class I MHC molecules
express (32 subunits and therefore can only be recognised by CD8
co-receptors. Class II MHC molecules express no (32 subunits and
can therefore be recognised by CD4 co-receptors. In this way, MHC
molecules regulate which type of lymphocytes may bind to a given
antigen with high affinity, since different lymphocytes express
different TCR co-receptors. Diversity of antigen presentation,
mediated by MHC classes I and II, is attained in at least three
ways:
[0092] (1) an organism's MEW repertoire is usually polygenic (via
multiple, interacting genes);
[0093] (2) MHC expression is codominant (from both sets of
inherited alleles); and
[0094] (3) MHC gene variants are highly polymorphic (diversely
varying from organism to organism within a species).
[0095] MHC molecules bind to both the T cell receptor and a CD4/CD8
co-receptor on T lymphocytes. The antigen epitope held in the
peptide-binding groove of the MHC molecule interacts with the
variable Ig-Like domain of the TCR to trigger T-cell activation.
However, the MEW molecules can also themselves act as antigens and
can provoke an immune response in the recipient of a tissue or
cells which express a foreign MHC, thus causing transplant
rejection. Still further, the transplantation of immunocompetent
cells can actually result in rejection of host tissue, also known
as graft vs host disease. In this regard, each human cell expresses
six MHC class I alleles (one HLA-A, -B, and -C allele from each
parent) and six to eight MHC class II alleles (one HLA-DP and -DQ,
and one or two HLA-DR from each parent, and combinations of these).
The MHC variation in the human population is high, with at least
350 alleles for HLA-A genes, 620 alleles for HLA-B, 400 alleles for
DR, and 90 alleles for DQ. Any two individuals who are not
identical twins will express differing MHC molecules.
[0096] All MHC molecules can mediate transplant rejection, but
HLA-C and HLA-DP, which show low polymorphism, are less important.
Transplant rejection can be minimised by attempting to match as
much of the cell surface HLA repertoire as possible between a donor
and a recipient. A complete match is only possible as between
identical twins. However, selecting donors based on minimising
incompatibility at one or more of the range of HLA antigens
expressed on a cell is highly desirable and can significantly
minimise rejection problems. This is a particular issue addressed
by the present invention since the usual method of managing
tissue/cell rejection is the administration of immunosuppressive
treatment regimes, this not being desirable in the context of a
treatment regime based on the administration of genetically
modified immune cells which are required to function at an optimum
level of functionality. In accordance with the present invention,
this can be achieved by utilizing cells, such as iPSCs, or cells
such as T cells from which iPSCs are derived, which are homozygous
for one or more MHC haplotypes, the HLA allele of interest being
one which is a major transplantation antigen and which is
preferably expressed by a significant proportion of the population,
such as at least 5%, at least 10%, at least 15%, at least 17%, at
least 20%, or more of the population. Where the homozygous HLA
haplotype corresponds to a dominant MHC I or MHC II HLA type (in
terms of tissue rejection), the use of such a cell will result in
significantly reduced problems with tissue rejection in the wider
population who receive the cells of the present invention in the
context of a treatment regime. In terms of the present invention,
the genetically modified cells may be homozygous in relation to one
cellular HLA antigen or they may be homozygous in relation to more
than one HLA antigen, e.g., 2, 3, or more HLA antigens. In some
embodiments, the genetically modified cells are homozygous in
relation to one HLA antigen selected from those listed in Table 1,
including e.g., HLA A1, B8, C7, DR17, DQ2, or HLA A2, B44, C5, DR4,
DQ8, or HLA A3, B7, C7, DR15, DQ6. In some embodiments, the
genetically modified cells are homozygous in relation to two or
more HLA antigens selected from those listed in Table 1, including
e.g., HLA A1, B8, C7, DR17, DQ2, or HLA A2, B44, C5, DR4, DQ8, or
HLA A3, B7, C7, DR15, DQ6.
[0097] The term "HLA-type" should therefore be understood to refer
to the complement of HLA antigens present on the cells of an
individual.
[0098] Obtaining a suitable homozygous HLA T cell for use in
generating an iPSC can be achieved by any suitable method
including, for example, screening a population (such as via a blood
bank) to identify individuals expressing HLA homozygocity and then
screening for T cells from that individual which exhibit the TCR
specificity of interest. These normally very rare T cells can be
selectively stimulated by the specific antigenic peptide that their
TCR recognises and vastly increased in frequency (e.g., from
<0.0001 to 0.2).
[0099] It would be appreciated by the person skilled in the art
that significant information is widely available in the public
literature which describes the identification and utility of
homozygous haplotypes in terms of minimizing donor-recipient HLA
mismatch across a given population of interest, thereby enabling
the generation of donor banks. See for example Pappas et al (2015).
In one example, Table 1 identifies the 15 highest ranked homozygous
HLA haplotypes relative to the proportion of the UK population to
which this provides minimal mismatch. The first 8 listed homozygous
HLA haplotypes are compatible with 49% of the population. A further
example is outlined in Table 2 which details the first 10 ranked
haplotypes compatible with the ethnically diverse Californian
population. Table 2 includes match frequencies for subpopulations,
including, black or African American, Asian and Pacific Islander,
white, Hispanic and American Indian and Alaska natives. Further
still, Table 3 outlines the 50 most frequent haplotypes for
HLA-A-B-DR, A-B, A-DR and B-DR in the North China population. It
would be appreciated that a person skilled in the art would
understand that the data depicted in Table 3 can be used to define
a set of homozygous haplotypes which would provide minimal mismatch
for the North Chinese population.
TABLE-US-00001 TABLE 1 Utility of 15 highest ranked homozygous
HLA-A, -B, -DR types identified to provide a zero HLA mismatch for
the UK population. Recipients Recipients matched matched Rank HLA-A
HLA-B HLA-DR (%) (cumulative %) 1 A1 B8 DR17(3) 16.87 16.87 2 A2
B44(12) DR4 9.51 26.38 3 A3 B7 DR15(2) 7.45 33.83 4 A2 B7 DR15(2)
4.28 38.11 5 A2 B44(12) DR7 3.41 41.52 6 A2 B62(15) DR4 2.85 44.37
7 A1 B57(17) DR7 2.54 46.91 8 A3 B35 DR1 2.10 49.01 9 A29(19)
B44(12) DR7 2.04 51.05 10 A2 B60(40) DR4 1.75 52.80 11 A2 B8
DR17(3) 1.60 54.40 12 A2 B27 DR1 1.28 55.68 13 A2 B44(12) DR13(6)
1.23 56.91 14 A3 B7 DR4 1.20 58.11 15 A1 B8 DR4 0.94 59.05
TABLE-US-00002 TABLE 2 Top cis and trans matched haplolines of the
California population. CIS TRANS Haplotype HLA- Match Match
Expected CIS match frequency A~B~DRB1 % (K.sub.i) SD % (K.sub.i) SD
f.sub.CAU f.sub.HIS f.sub.API f.sub.AFA f.sub.NAM f.sub.exp
01:01g~08:01g~03:01 6.32 0.24 6.64 0.26 11.63 3.57 0.47 2.181 8.484
5.59 03:01g~07:02g~15:01 3.47 0.18 1.06 0.19 5.967 2.37 0.4 1.198
4.618 3.06 29:02g~44:03~07:01 2.57 0.15 2.71 0.15 3.731 1.17 0.11
0.68 2.88 1.8 02:01g~07:02g~15:01 2.03 0.15 3.6 0.2 3.565 0.76 0.06
0.927 3.206 1.64 02:01g~44:02g~04:01 1.85 0.13 2.22 0.15 2.851 3.63
0.07 0.779 2.55 2.23 01:01g~57:01g~07:01 1.68 0.13 1.95 0.14 2.356
0.84 0.35 0.465 1.617 1.2 03:01g~35:01g~01:01 1.35 0.11 1.61 0.11
2.521 0.47 0.05 0.435 1.877 1.11 02:01g~15:01g~04:01 1.24 0.12 1.57
0.14 2.124 0.89 2.89 0.43 1.973 1.47 30:01g~13:02g~07:01 1.24 0.12
1.3 0.12 1.663 0.3 0.04 0.282 1.203 0.73 33:01g~14:02~01:02 0.99
0.1 1.02 0.1 1.532 0.62 0.08 0.304 1.21 0.79 Abbreviations AFA,
black or African American; API, Asian and Pacific Islander; CAU,
white (non-Hispanic); CIS, cis match benefit; f.sub.exp, expected
cis match frequency; HIS, Hispanic; K.sub.i, number of matches as a
count or percentages of the total number of subjects; NAM, American
Indian and Alaska native; TRANS, trans match benefit.
TABLE-US-00003 TABLE 3 50 most frequent haplotypes for HLA-A-B-DR,
A-B, A-DR and B-DR (at 10.sup.-5) HF = haplotype frequency per
100,000 HLA-A-B-DR HLA-A-A-B HLA-A-DR HLA-A-B-DR Haplotype HF R.L.D
Haplotype HF R.L.D Haplotype HF R.L.D Haplotype HF R.L.D A30-B13-
4446 0.58 A30-B13 5538 0.81 A2-DR9 5882 0.23 B13-DR7 5617 0.44 DR7
A2-B46- 2388 0.16 A2-B46 5090 0.60 A2-DR15 4703 -0.04 B46-DR9 3225
0.37 DR9 A33-B58- 1436 0.29 A33-B58 3201 0.74 A30-DR7 4532 0.64
B13-DR12 2303 0.12 DR17 A2-B13- 1088 0.04 A2-B61 2592 0.17 A2-DR12
4118 0.13 B52-DR15 2285 0.55 DR12 A2-B46- 1046 0.07 A2-B51 2411 0.6
A11-DR15 3426 0.03 B62-DR4 2070 0.18 DR8 A33-B58- 1010 0.18 A2-B62
2198 0.00 A24-DR15 3143 0.03 B61-DR9 2045 0.22 DR13 A33-B44- 936
0.14 A11-B60 2197 0.18 A2-DR4 2987 -0.11 B62-DR15 1921 0.11 DR13
A2-B61- 904 0.01 A2-B13 2136 -0.35 A11-DR12 2979 0.12 B44-DR7 1869
0.28 DR9 A1-B37- 860 0.46 A11-B62 2106 0.13 A11-DR4 2704 0.07
B7-DR15 1867 0.29 DR10 A11-B75- 848 0.12 A2-B60 1879 -0.3 A2-DR8
2660 0.20 B58-DR17 1858 0.42 DR12 A11-B62- 814 0.04 A24-B61 1802
0.15 A24-DR4 2584 0.08 B51-DR9 1727 0.12 DR4 A24-B54- 697 0.11
A24-B62 1798 0.10 A11-DR9 2378 0.01 B54-DR4 1497 0.38 DR4 A2-B62-
676 0.02 A24-B60 1765 0.13 A24-DR9 2375 0.03 B44-DR13 1480 0.25
DR15 A3-B7- 658 0.10 A33-B44 1752 0.29 A2-DR14 2169 0.08 B46-DR8
1453 0.18 DR15 A1-B57- 647 0.38 A11-B13 1689 -0.16 A33-DR13 2057
0.34 B75-DR12 1405 0.26 DR7 A11-B7- 647 0.11 A2-B75 1679 0.18
A2-DR11 2012 -0.01 B60-DR15 1325 0.04 DR1 A24-B61- 607 0.03 A11-B75
1632 0.28 A24-DR12 1860 0.02 B58-DR13 1269 0.26 DR9 A2-B51- 597
0.15 A24-B54 1468 0.33 A2-DR7 1553 -0.53 B13-DR15 1204 -0.35 DR9
A2-B61- 586 0.02 A2-B35 1446 -0.19 A24-DR11 1524 0.07 B37-DR10 1188
0.67 DR12 A24-B62- 579 0.02 A24-B51 1445 0.4 A33-DR17 1502 0.31
B35-DR15 1122 0.02 DR4 A32-B52- 575 0.23 A11-B51 1399 0.1 A24-DR14
1410 0.08 B7-DR1 1102 0.24 DR15 A11-B13- 572 0.03 A2-B48 1365 0.18
A11-DR14 1348 0.04 B61-DR12 1094 0.07 DR15 A11-B62- 556 0.01 A1-B37
1325 0.69 A11-DR8 1211 0.02 B8-DR17 1058 0.82 DR15 A33-B44- 532
0.05 A2-B38 1264 0.19 A3-DR15 1173 0.07 B61-DR15 1001 -0.07 DR7
A11-B13- 532 0.00 A24-B35 1079 0.3 A11-DR11 1070 -0.15 B57-DR7 986
0.65 DR12 A11-B52- 512 0.01 A11-B52 1060 0.13 A1-DR7 1011 0.08
B75-DR15 986 0.09 DR15 A32-B44- 493 0.19 A24-B48 1003 0.17 A2-DR16
958 0.19 B51-DR15 982 -0.21 DR7 A2-B75- 484 0.03 A3-B7 1001 0.18
A24-DR8 954 -0.03 B60-DR9 967 0.02 DR9 A11-B51- 452 0.02 A1-B57 984
0.67 A11-DR1 921 0.06 B35-DR11 949 0.10 DR9 A2-B13- 431 0.67 A3-B35
960 0.13 A1-DR10 887 0.48 B60-DR4 943 0.03 DR7 A2-B46- 427 0.01
A11-B46 906 -0.30 A31-DR15 879 0.08 B60-DR11 929 0.08 DR14 A11-B75-
425 0.02 A24-B13 885 -0.50 A33-DR7 863 0.01 B62-DR12 850 0.01 DR15
A24-B60- 420 0.01 A2-B54 874 -0.08 A3-DR1 785 0.15 B51-DR4 837
-0.01 DR15 A2-B60- 417 0.01 A11-B7 848 0.1 A1-DR15 781 -0.13
B62-DR9 821 -0.15 DR15 A24-B51- 414 0.01 A11-B35 787 -0.27 A2-DR17
691 -0.43 B51-DR11 794 0.04 DR9 A2-B62- 408 0.30 A11-B61 776 -0.32
A3-DR7 691 0.02 B60-DR12 786 0.01 DR4 A2-B75- 397 0.21 A32-B44 763
0.34 A32-DR15 683 0.20 B60-DR8 771 0.06 DR12 A2-B46- 389 0.28
A31-B51 757 0.14 A3-DR4 678 0.02 B75-DR9 750 0.06 DR12 A11-B46- 387
0.28 A29-B7 726 0.62 A11-DR7 652 -0.68 B51-DR12 701 -0.11 DR9
A11-B60- 379 0.01 A3-B44 705 0.9 A2-DR13 624 -0.59 B27-DR4 693 0.25
DR9 A11-B60- 378 0.03 A2-B71 697 0.23 A26-DR15 616 0.02 B48-DR15
665 0.05 DR8 A24-B13- 377 0.01 A24-B7 679 -0.5 A2-DR1 609 -0.49
B71-DR4 663 0.35 DR12 A2-B54- 377 0.09 A32-B52 677 0.31 A24-DR7 596
-0.67 B51-DR14 644 0.03 DR4 A2-B75- 362 0.00 A11-B55 674 0.19
A32-DR7 579 0.19 B50-DR7 640 0.68 DR15 A24-B7- 362 0.01 A2-B55 670
0.7 A1-DR13 572 0.06 B35-DR9 632 -0.18 DR15 A2-B71- 350 0.10 A2-B39
654 0.7 A33-DR15 550 -0.54 B35-DR4 625 -0.09 DR4 A11-B60- 350 0.19
A11-B54 649 0.2 A3-DR13 484 0.04 B46-DR14 612 0.03 DR15 A2-B61- 345
0.15 A2-B67 620 0.56 A31-DR9 480 -0.01 B62-DR14 599 0.02 DR15
A2-B50- 332 0.21 A24-B46 604 -0.46 A33-DR4 476 -0.42 B48-DR9 556
0.05 DR7 A2-B48- 332 0.02 A31-B62 570 0.8 A26-DR4 468 0.02 B35-DR1
542 0.08 DR9
[0100] As detailed hereinbefore, the present invention is
predicated on the determination that a stem cell can be
consistently and stably engineered to express dual T cell and
chimeric antigen receptors directed to multiple distinct antigens,
thereby providing an ongoing source of T cells which are more
therapeutically effective than the cells used in currently
available therapeutic cellular treatment regimes. In this regard,
reference to a "stem cell" should be understood as a reference to
any cell which exhibits the potentiality to develop in the
direction of multiple lineages, given its particular genetic
constitution, and thus to form a new organism or to regenerate a
tissue or cellular population of an organism. The stem cells which
are utilised in accordance with the present invention may be of any
suitable type capable of differentiating along two or more lineages
and include, but are not limited to, embryonic stem cells, adult
stem cells, umbilical cord stem cells, haemopoietic stem cells
(HSCs), totipotent cells, progenitor cells, precursor cells,
pluripotent cells, multipotent cells or de-differentiated somatic
cells (such as an induced pluripotent stem cell). By "totipotent"
is meant that the subject stem cell can self renew. By
"pluripotent" is meant that the subject stem cell can differentiate
to form, inter alia, cells of any one of the three germ layers,
these being the ectoderm, endoderm and mesoderm.
[0101] In one particular embodiment, the subject stem cell is an
induced pluripotent stem cell (iPSC). Without limiting the present
invention to any one theory or mode of action, adult stem cell
expansion is not necessarily based on the occurrence of
asymmetrical stem cell division in order to effect both stem cell
renewal and differentiation along a specific somatic cell lineage.
In particular, pluripotent stem cells can be sourced from T cells
which are induced to transition to a state of multilineage
potential. The development of technology to enable the
de-differentiation of adult cells is of significant importance due
to the difficulty of otherwise inducing stem cell renewal and
expansion in vitro.
[0102] According to this embodiment there is therefore provided a
genetically modified mammalian stem cell, or a T cell
differentiated therefrom, which stem cell is an iPSC, is capable of
differentiating to a T cell expressing a TCR directed to a first
antigenic determinant, and comprises a nucleic acid molecule
encoding a chimeric antigen receptor, wherein said receptor
comprises an antigen recognition moiety directed to a second
antigenic determinant, which antigen recognition moiety is operably
linked to a T cell activation moiety. In one embodiment, the
genetically modified mammalian iPSC expresses at least one
homozygous HLA haplotype.
[0103] iPSCs are usually generated directly from somatic cells,
although it should be understood that the present invention is not
limited in this regard. That is, the subject iPSC may be generated
from a cell which is not terminally differentiated; indeed iPSC can
be induced in principle from any nucleated cell including, for
example, mononucleocytes from blood and skin cells. For example, in
the context of one embodiment of the present invention, the subject
iPSCs may be generated from fully differentiated T cells or they
may be generated from precursor T cells, such as thymocytes. To the
extent that the subject thymocyte has re-arranged its TCR and
exhibits an antigen specificity of interest in the context of the
present invention, one may seek to generate the iPSC from this
cell. This may be relevant, for example, where the particular TCR
rearrangement in issue is one which might be expected to be
selected against during thymopoiesis. It would be appreciated by
the skilled person that one of the complicating factors with
respect to immunoresponsiveness to tumour cells or autoreactive
cells is that in this situation the immune system is required to
direct an immune response to a self cell and, therefore, a
self-antigen. Such immune cells are usually selected against during
T lymphocyte differentiation in the thymus in order to minimize the
prospect of the onset of an autoimmune disease. In the context of
neoplastic and autoimmune conditions, however, the unwanted cell is
a self cell and, accordingly, the cell surface antigens which one
may seek to target will be self antigens. Without limiting the
present invention in any way, and as discussed in more detail
hereafter, one of the advantages of using an iPSC from which to
generate a TCR/CAR expressing T cell directed to multiple distinct
antigenic determinants is that it has been determined that the
actions of epigenetic memory may potentiate the differentiation of
an iPSC to a functional T cell which expresses a TCR directed to
the same antigen as the T cell from which the iPSC has been
derived. However, in terms of selecting a specific TCR expressing
cell from which to derive an iPSC, it may be difficult to identify
a suitable fully differentiated T cell since a T cell expressing a
functional TCR directed to a self antigen may have been selected
against during thymopoiesis. It may therefore be more feasible to
screen for a thymocyte which expresses the TCR re-arrangement of
interest, which thymocyte has not yet undergone negative selection
to remove potentially self reactive cells.
[0104] In another embodiment, an iPSC is transfected with one or
more nucleic acid molecules coding for a TCR (such as rearranged
TCR genes) directed to a first antigenic determinant (e.g., a
tumour antigenic determinant).
[0105] In still another embodiment, the subject stem cell is a
haemopoietic stem cell (HSC). Haemopoietic stem cells (HSCs) refer
to stem cells that give rise to all the blood cells of the lymphoid
and myeloid lineages through the process of haematopoiesis. HSCs
are derived from mesoderm, and can be found in adult bone marrow,
peripheral blood, and umbilical cord blood. HSCs can be collected
from bone marrow, peripheral blood, and umbilical cord blood by
established techniques, and are commonly associated with CD34+
expression. In some embodiments, human HSCs can be defined as being
CD34+CD38-CD90+CD45RA- (see Reinisch et al (2015)). An HSC can be
genetically modified, e.g., transfected, with one or more nucleic
acids encoding a TCR directed to a first antigenic determinant,
then subsequently directed to differentiate into a T cell. Nucleic
acids encoding one or more CARs, and optionally nucleic acids
encoding one or more docking antigen-binding receptors, can also be
introduced into an HSC, before or after differentiation of the HSC
into a T cell.
[0106] Reference to a "T cell receptor" (TCR) should therefore be
understood as a reference to the heterodimer found on the surface
of T cells or NKT cells which recognise peptides presented by MHC.
Specifically, CD8+ T cells recognise peptide presented in the
context of MHC class I while CD4+ T cells recognise peptide
presented in the context of WIC class II. Without limiting the
present invention to any one theory or mode of action, in the
majority of human T cells, the TCR comprises an .alpha. and .beta.
chain, while a minor population of cells express a TCR comprising a
.gamma..delta. heterodimer. The TCR is a disulfide-linked
membrane-anchored heterodimeric protein. The .gamma., .delta.,
.alpha. and .beta. chains are composed of two extracellular
domains: a variable (V) region and a constant (C) region, which
both form part of the immunoglobulin superfamily and which fold to
form antiparallel .beta.-sheets. The constant region is proximal to
the cell membrane, followed by a transmembrane region and a short
cytoplasmic tail, while the variable region binds to the
peptide/WIC complex.
[0107] The variable domains of both the TCR .alpha.-chain and
.beta.-chain each express three hypervariable or complementarity
determining regions (CDRs), whereas the variable region of the
.beta.-chain has an additional area of hypervariability (HV4) that
does not normally contact antigen and, therefore, is not considered
a CDR. The processes for the generation of TCR diversity are based
mainly on genetic recombination of the DNA encoded segments in
precursor T cells--either somatic V(D)J recombination using RAG1
and RAG2 recombinases or gene conversion using cytidine deaminases.
Each recombined TCR possesses unique antigen specificity,
determined by the structure of the antigen-binding site formed by
the .alpha. and .beta. chains, in the case of .alpha..beta. T
cells, or .gamma. and .delta. chains in the case of .gamma..delta.
T cells. The TCR .alpha. chain is generated by VJ recombination,
whereas the .beta. chain is generated by VDJ recombination.
Likewise, generation of the TCR .gamma. chain involves VJ
recombination, whereas generation of the TCR .delta. chain occurs
by VDJ recombination. The intersection of these specific regions (V
and J for the .alpha. or .gamma. chain; V, D, and J for the .beta.
and .delta. chain) corresponds to the CDR3 region that is important
for peptide/MHC recognition. It is the unique combination of the
segments at this region, along with palindromic and random
nucleotide additions, which account for the even greater diversity
of T cell receptor specificity for processed antigenic
peptides.
[0108] Accordingly, reference to a TCR "directed" to an antigenic
determinant should be understood as a reference to a TCR which has
undergone rearrangement and which exhibits specificity for an
antigenic determinant, preferably a self (particularly a self
cancer) antigenic determinant.
[0109] In one embodiment, an iPSC is derived from a cell which
expresses a rearranged TCR, preferably a rearranged .alpha..beta.
TCR. Examples of cells suitable for use in generating the iPSCs of
the present invention include, but are not limited to CD4.sup.+ T
cells, CD8.sup.+ T cells, NKT cells, thymocytes or other form of
precursor T cells. In another embodiment, said cell expresses a
rearranged .gamma..delta. TCR.
[0110] There is therefore provided a genetically modified mammalian
iPSC or HSC, or a T cell differentiated therefrom, which iPSC or
HSC is capable of differentiating to a T cell expressing a TCR
directed to a first antigenic determinant, is derived from a cell
in which the TCR genes have undergone re-arrangement, or has been
transduced with said rearranged genes, and comprises a nucleic acid
molecule encoding a chimeric antigen receptor, wherein said
receptor comprises an antigen recognition moiety directed to a
second antigenic determinant, which antigen recognition moiety is
operably linked to a T cell activation moiety. In some embodiments,
the genetically modified mammalian iPSC or HSC expresses at least
one homozygous HLA haplotype.
[0111] In one embodiment, said iPSC is derived from a T cell or a
thymocyte.
[0112] In another embodiment, said iPSC is derived from a T cell or
thymocyte expressing an .alpha..beta. TCR.
[0113] In still another embodiment, said iPSC is derived from a T
cell or thymocyte expressing a .gamma..delta. TCR.
[0114] The subject stem cells may have been freshly isolated from
an individual who is the subject of treatment or they may have been
sourced from a non-fresh source, such as from a culture (for
example, where cell numbers were expanded and/or the cells were
cultured so as to render them receptive to differentiation signals)
or a frozen stock of cells, which had been isolated at some earlier
time point either from an individual or from another source. It
should also be understood that the subject cells, prior to
undergoing differentiation, may have undergone some other form of
treatment or manipulation, such as but not limited to purification,
modification of cell cycle status or the formation of a cell line
such as an embryonic stem cell line. Accordingly, the subject cell
may be a primary cell or a secondary cell. A primary cell is one
which has been isolated from an individual. A secondary cell is one
which, following its isolation, has undergone some form of in vitro
manipulation such as the preparation of an embryonic stem cell
line, prior to the application of the method of the invention.
[0115] To the extent that the stem cells of the present invention
are iPSCs, methods for generating iPSCs are well known to the
person of skill in the art. In this regard, and as detailed
hereinbefore, iPSCs are cells which are derived from a more mature
cell type, such as a somatic cell, which has been
transitioned/de-differentiated back to a pluripotent state.
[0116] Without limiting the present invention to any one theory or
mode of action, iPSCs can be derived by introducing a specific set
of pluripotency-associated genes, or "reprogramming factors", into
a somatic cell type. The most commonly used set of reprogramming
factors (also know as the Yamanaka factors) are the genes Oct4
(Pou5f1), Sox2, cMyc, and Klf4. The transfection of these four
specific genes encoding transcription factors were shown by
Yamanaka in 2006 to convert adult human cells into pluripotent
cells. While this combination is the most conventional combination
used for producing iPSCs, each of the factors can be functionally
replaced by related transcription factors, miRNAs, small molecules,
or even non-related genes such as lineage specifiers. For example,
the induction of iPSCs following transfection of Oct 3/4, Sox2,
Klf4 and c-Myc using a retroviral system has been achieved, as it
has also been via the transfection of Oct4, Sox2, Nanog and Lin28
using a lentiviral system. The former set of transcription factors
are known as the Yamanaka factors while the latter are commonly
known as the Thomson factors. As would be appreciated by the person
of skill in the art, a wide range of modifications to the basic
reprogramming factor expression vectors have been made and new
modes of delivery have been designed in order to increase
efficiency and minimise or remove vector sequences that might
otherwise be integrated into the reprogrammed iPSC genome. These
methods would be well known to the skilled person and include, but
are not limited to:
[0117] single cassette reprogramming vectors with Cre-Lox mediated
transgene excision;
[0118] (ii) reprogramming by non-integrating viruses such as
adenovirus or sendai virus.
[0119] Alternatively, expression of reprogramming factors as
proteins provides a means of generating iPSCs which have not
undergone integration of the introduced vector DNA into the
germline.
[0120] Non-viral reprograming methods have also been developed.
These include, but are not limited to:
[0121] mRNA Transfection--The ability to express reprogramming
factors as mRNA offers a method to make iPSCs into which
chromosomal integration of viral vectors does not occur. Warren et
al. transcribes mRNAs to efficiently express reprogramming factors
(Warren et al (2010)). By adding Lin28 to the Yamanaka
reprogramming factor protocol, culturing at 5% 02, and including
valproic acid in the cell culture medium, the efficiency can be
increased. Reprogramming factor mRNAs are commercially
available.
[0122] (ii) miRNA Infection/Transfection--Several miRNA clusters
are strongly expressed in embryonic stem cells. When synthetic
mimics of the mature miR-302b and/or miR-372 plus the four
lentiviral Yamanaka factors are added to MRCS and BJ-1 fibroblasts
there is a 10- to 15-fold increase in reprogramming efficiency in
comparison with the four lentiviral factors alone (Subramanyam et
al (2011)). It has also been found that certain miRNAs can
reprogram cells at high efficiency without the presence of the
Yamanaka factors.
[0123] (iii) PiggyBac--PiggyBac is a mobile genetic element
(transposon) that in the presence of a transposase can be
integrated into chromosomal TTAA sites and subsequently excised
from the genome upon re-expression of the transposase. When cloned
into a piggyBac vector and co-transfected into MEFs the Yamanaka
factors can reprogram cells 14-25 days post-transfection (Kaji et
al (2009); Woltj en et al (2009)). The piggyBac vector can be
excised from the iPSCs upon re-expression of the transposase.
[0124] (iv) Minicircle Vectors--Minicircle vectors are minimal
vectors containing only the eukaryotic promoter and cDNA(s) that
will be expressed. A Lin28, GFP, Nanog, Sox2, and Oct4 minicircle
vector expressed in human adipose stromal cells is able to
reprogram cells (Narsinh et al (2011)).
[0125] (v) Episomal Plasmids--Transient expression of reprogramming
factors as episomal plasmids allows for the generation of iPSCs.
For example oriP/EBNA vectors can be constructed with the Yamanaka
factors plus Lin28 in one cassette and another oriP/EBNA vector
containing SV40 large T antigen (Chuo et al (2011)). These vectors
have been shown to be expressed in CD34+ cord blood, peripheral
blood, and bone mononuclear cells in media supplemented with sodium
butyrate, resulting in iPSC colonies in 14 days. The transfected
plasmids are ultimately lost.
[0126] In another aspect, the skilled person would also be familiar
with adjunct methods which are known to enhance the programming
efficiency of cells. For example, even when using the same method
there can be variability in iPSC efficiency between cells. Various
small molecules have been shown to enhance reprogramming efficiency
(Table 4).
TABLE-US-00004 TABLE 4 Compounds increasing iPSC reprogramming
efficiency Treatment Process affected Valproic acid Histone
deacetylase inhibition Sodium butyrate Histone deacetylase
inhibition PD0325901 MEK inhibition A-83-01 TGF.beta.-inhibition
SB43152 TGF.beta.-inhibition Vitamin C Enhances epigenetic
modifiers, promotes survival of antioxidant effects Thiazovivin
ROCK inhibitor, promotes cell survival PS48 P13K/Akit activation,
promotes glycolysis 5% Oxygen Promotes glycolysis
[0127] Several known mechanisms enable these molecules to
facilitate reprogramming including inhibition of histone
deacetylation (Mali et al (2010); Huangfu et al (2008)) blockade of
the TGF.beta. and MEK signalling pathways (Lin et al (2009); Ichida
et al (2009)), enhancement of function of epigenetic modifiers
(Esteban et al (2010)), inhibition of the ROCK pathway (Noggle et
al (2011)) and induction of glycolysis (Zhu et al (2010)). Amongst
these small molecules, the histone deacetylatase inhibitors
valproic acid and sodium butyrate are the most commonly used in
reprogramming protocols. It should also be noted that culture of
cells in 5% oxygen during the reprogramming process can also
increase efficiency of iPSC derivation (Yoshida et al (2009)). For
cells that are particularly difficult to reprogram, the addition of
a small molecule and culture in hypoxic conditions can yield
improvements. Another option is to use embryonic stem
cell-conditioned medium (ESCM) to induce expression of endogenous
reprogramming factors (Balasubramanian et al (2009)). The
efficiency can be improved further with the addition of valproic
acid. Such a strategy can also be used to enhance the ability of
exogenously introduced reprogramming factors to increase
reprogramming efficiency.
[0128] To the extent that the stem cells of the present invention
are HSCs, methods for generating or preparing HSCs are well known
to the person of skill in the art. HSCs can be obtained by direct
extraction from the bone marrow or from the blood after the HSCs
are released from the bone marrow following e.g., treatment with
specific molecules such as GM-CSF. The HSCs can then be purified
through their plasma membrane expression of CD34 by for example
magnetic beads coated with anti-CD34 or cell sorting by flow
cytometry after labelling with fluorescent anti CD34. These so
purified HSCs can be induced to T cell differentiation using the OP
9/OP9 DL-L1 system outlined in Example 3 and FIGS. 3-10
inclusive.
[0129] Reference to the subject stem cell, in particular iPSC or
HSC, being "capable of" differentiating to a T cell expressing a
TCR directed to an antigenic determinant should be understood as a
reference to a cell which either does, or has the capacity to,
transcribe and translate the subject TCR genes and then assemble
the TCR heterodimer as a functional receptor on the cell surface.
As would be appreciated by the skilled person, in most situations a
stem cell such as an iPSC will not, in its undifferentiated form,
express a TCR. TCR expression is generally expected to occur once
directed differentiation along the T cell lineage has been induced.
In one embodiment, the cell is one which, with or without a CAR
genetic modification, can be induced to differentiate to a T cell
expressing a functional TCR. It should be understood that the
capacity of the cell to express a TCR of a particular specificity
may be enabled by any suitable means. For example, the cell may
have been transfected with genes encoding the two TCR chains (eg.
.alpha. and .beta. chain) which, when expressed, will associate to
form the TCR heterodimer. Alternatively, and in the context of a
preferred embodiment of the present invention, the stem cell of the
present invention is one which has been generated from a T cell,
thymocyte or other cell in which the TCR genes have been
rearranged. It has been determined that an iPSC which has been
generated from such a cell, if directed to differentiate to a
CD4.sup.+ or CD8.sup.+ T cell under appropriate cell culture
conditions, will express the same TCR antigen specificity as the
somatic T cell from which the iPSC was derived. Of still further
significance, and as discussed in more detail hereinafter, is that
it has been determined that with or without transfection of the
iPSC or HSC with one or more nucleic acids encoding one or more
CARs, or the .alpha. and .beta. chains of an antigen/MHC class I
specific TCR, the T cell differentiated therefrom is capable of
stably expressing both a functional TCR and one or more CARs (and
optionally one or more antigen-binding receptors), and is therefore
directed to two or more distinct antigenic determinants.
Accordingly, such a stem cell is deemed "capable of"
differentiating to a T cell and expressing the requisite TCR on the
basis that if the iPSC or HSC is provided with the appropriate
differentiative signal, this will occur. In this regard, since the
rearrangement of the TCR genes is an entirely independent genomic
event, the choice of T cell sub-population from which to generate
the iPSC need not necessarily be the same as the T cell
sub-population which it is sought to ultimately be produced via the
directed differentiation of the iPSC. For example, one may select a
CD4.sup.+ T cell which exhibits an appropriate TCR specificity in
order to generate an iPSC. However, once that iPSC has been
generated, the skilled person may seek to direct the
differentiation of the iPSC to a CD8.sup.+ T cell. In this case, by
virtue of epigenetic memory, the newly generated CD8.sup.+ T cell
will exhibit the functionality of a CD8.sup.+ T cell but the TCR
specificity will be that of the CD4.sup.+ T cell from which the
iPSC was derived. The converse is also true.
[0130] Reference to inducing the "transition" of a somatic cell,
such as a T cell, to a multilineage potential phenotype, such as an
iPSC, should be understood as a reference to inducing the genetic,
morphologic and/or functional changes which are required to change
a somatic phenotype to a multilineage (pluripotent) phenotype of
the type defined herein.
[0131] To the extent that one may elect to render an iPSC capable
of producing a TCR via the transfection of the cell with DNA
encoding a TCR, it would be appreciated that this transfection may
occur at any time point, such as prior to the generation of the
iPSC of the present invention, subsequently to the generation of
the iPSC, or it may occur simultaneously with the CAR
transfection.
[0132] As detailed hereinbefore, a somatic cell, in particular a T
cell or thymocyte, can be induced to transition into a stem cell,
that is a functional state of multilineage differentiation
potential. Accordingly, reference to a cell exhibiting
"multilineage differentiation potential" or "multilineage
potential" should be understood as a reference to a cell which
exhibits the potentiality to develop along more than one somatic
differentiative path. For example, the cell may be capable of
generating a limited range of somatic cell types, such cells
usually being referred to as pluripotent or multipotent. These
cells exhibit the potential to commit to a more limited range of
lineages than a totipotent cell, the latter being a cell which can
develop in any of the differentiation directions inherently
possible including all the somatic lineages and the gametes.
[0133] Cells that are classically termed "progenitor" cells or
"precursor" cells fall within the scope of the definition of
"multilineage differentiation potential" on the basis that, under
appropriate stimulatory conditions, they can give rise to cells of
more than one somatic lineage. To the extent that reference to
"stem cell" is made herein in terms of the cells generated by the
method of the invention, this should be understood as a reference
to a cell exhibiting multilineage differentiative potential as
herein defined.
[0134] In terms of the present invention, it should be understood
that the important feature of the subject stem cell is that the
multilineage differentiative potential which the cell exhibits
includes the capacity to differentiate to a T cell and to express a
TCR exhibiting specificity for an antigen of interest. Whether the
TCR specificity is induced before or after the stem cell is
generated (such as via the transfection of the stem cell with DNA
encoding the TCR of interest) is irrelevant. It should be
understood that the stem cells claimed herein encompass all stem
cells exhibiting the requisite differentiative potential,
irrespective of when or how that capability has been introduced.
Still further, it should also be understood that the subject stem
cells need not be totipotent. Provided that they exhibit the
capacity to differentiate along more than one somatic cell lineage
and provided that one of these lineages is a T cell lineage, said
cells fall within the scope of the present invention.
[0135] As detailed hereinbefore, the stem cells provided by the
present invention are genetically modified. By "genetically
modified" is meant that the subject cell results from some form of
molecular manipulation relative to that which is observed in the
context of a corresponding unmodified cell. In the context of the
present invention, the subject stem cell comprises a nucleic acid
molecule encoding a chimeric antigen receptor, and optionally
further comprises a nucleic acid molecule encoding an
antigen-binding receptor. As disclosed herein, a nucleic acid
encoding a receptor, whether a chimeric antigen receptor or an
antigen-binding receptor, can be introduced to a stem cell such as
an iPSC or an HSC, or to a cell (e.g., a T cell) from which a stem
cell is derived; and in both instances, the resulting stem cell
which comprises the receptor-encoding nucleic acid is considered
herein to be a genetically modified stem cell. A T cell
differentiated from a genetically modified stem cell, and a T cell
engineered to contain a nucleic acid encoding a genetically
engineered CAR or antigen-binding receptor, are also considered
herein genetically modified T cells.
[0136] Reference to a "nucleic acid molecule" should be understood
as a reference to both deoxyribonucleic acid and ribonucleic acid
thereof. The subject nucleic acid molecule may be any suitable form
of nucleic acid molecule including, for example, a genomic, cDNA or
ribonucleic acid molecule. To this end, the term "expression"
refers to the transcription and translation of DNA or the
translation of RNA resulting in the synthesis of a peptide,
polypeptide or protein. A DNA construct, for example, corresponds
to the construct which one may seek to transfect into a cell for
subsequent expression while an example of an RNA construct is the
RNA molecule transcribed from a DNA construct, which RNA construct
merely requires translation to generate the protein of interest.
Reference to "expression product" is a reference to the product
produced from the transcription and translation of a nucleic acid
molecule.
[0137] Reference to "chimeric antigen receptor" (also known as an
"artificial T cell receptor", "chimeric T cell receptor" and
"chimeric immunoreceptors") should be understood as a reference to
engineered receptors which graft an antigen binding moiety onto an
immune effector cell. Typically, these receptors are used to graft
the specificity of a monoclonal antibody onto a T cell; with
transfection of their coding sequence facilitated by retroviral
vectors. More specifically, and without limiting the invention in
any way, the most common form of these molecules are fusions of
single-chain variable fragments (scFv) derived from monoclonal
antibodies, fused to a CD3-zeta chain transmembrane and endodomain.
Such molecules result in the transmission of a CD3-zeta chain
signal in response to recognition by the scFv of its target. When T
cells express this chimeric molecule, they recognize and kill
target cells that express the antigen to which the scFv is
directed. For example, to target malignant B cells, the specificity
of T cells has been redirected using a chimeric immunoreceptor
specific for the B-lineage molecule, CD19.
[0138] The variable portions of an immunoglobulin heavy and light
chain are generally fused by a flexible linker to form a scFv. This
scFv is usually preceded by a signal peptide to direct the nascent
protein to the endoplasmic reticulum and subsequent surface
expression, which the signal peptide ultimately being cleaved. A
flexible spacer allows the scFv to orient in different directions
to enable antigen binding. The transmembrane domain is generally a
typical hydrophobic alpha helix usually derived from the original
molecule of the signalling endodomain which protrudes into the cell
and transmits the desired signal. Accordingly, reference to an
"antigen recognition moiety" should be understood as a reference to
an extracellular portion of the receptor which recognises and binds
to an antigenic determinant of interest, that is, a target specific
binding element. The antigen recognition domain is usually an scFv.
There are, however, many other alternatives. For example, an
antigen recognition moiety from native T-cell receptor (TCR) alpha
and beta single chains have also been used, as have simple
ectodomains (e.g., CD4 ectodomain to recognize HIV infected cells)
and other recognition components such as a linked cytokine (which
leads to recognition of cells bearing the cytokine receptor). In
fact any moiety that binds a given target with sufficiently high
affinity can be used as an antigen recognition domain. Such
molecules are well known to the person of skill in the art and
selecting an appropriate molecule for use would be well within the
skill of the person in the art. In terms of designing a chimeric
antigen receptor, in particular the extracellular domain, the
skilled person may include additional moieties which are useful in
terms of effecting efficient expression or functioning. For
example, and as detailed earlier, the nucleic acid molecule
expressing a CAR may be designed to express a signal peptide at the
N-terminal end of the antigen recognition moiety. Without limiting
the present invention to any one theory or mode of action, a signal
peptide directs the nascent protein into the endoplasmic reticulum.
This is necessary if the receptor is to be glycosylated and
anchored in the cell membrane. Any eukaryotic signal peptide
sequence may be used. Generally, a signal peptide natively attached
to the amino-terminal is used (e.g., in a scFv with orientation
light chain-linker-heavy chain, the native signal of the
light-chain is used). In another example the extracellular domain
may also comprise a spacer region which may be used to link the
antigen recognition domain to the transmembrane domain. It should
be flexible enough to allow the antigen recognition domain to
orient in different directions to facilitate antigen recognition
and binding. The simplest form of a spacer region is the hinge
region from IgG1. Alternatives include the CH.sub.2CH.sub.3 region
of immunoglobulin and portions of CD3. For most scFv based
constructs, the IgG1 hinge suffices. Accordingly, the term "spacer"
refers to any oligo- or polypeptide that functions to link the
transmembrane domain to either the extracellular domain or, the
cytoplasmic domain in the polypeptide chain. A spacer domain may
comprise up to 300 amino acids, preferably 10 to 100 amino acids
and most preferably 25 to 50 amino acids. In yet another example,
one may modify the hinge region to change its length and thereby
achieve additional functional benefits. For example, in a
traditional CAR which comprises a CD8 or CD28 hinge, a single
Cysteine (Cys) can be left in the hinge to stabilize dimerization
on the T-cell surface. Thus two scFv are usually displayed
(bivalent). In another example, one may substitute the Cys (for
Ser) so that the stabilizing disulphide bond cannot form thereby
preventing dimerization and hence premature activation. The Cys may
also be removed entirely. Another design is to display just the VH
domain on one CAR and VL domain on another, thus the Cys pairing
will align the VH/VL to form a functional monovalent Fv, targeting
the antigen of interest.
[0139] The antigen recognition moiety of the subject chimeric
antigen receptor is operably linked to a T cell activation moiety.
By "T cell activation moiety" is meant the sub-region of the
receptor which, after antigen recognition and binding, is
responsible for transmitting the signal into the T cell to enable
its activation and effector mechanism induction. The T cell
activation moiety of a CAR is generally located within the
intracellular domain (or "endodomain") of the CAR; hence, the
intracellular domain of a CAR molecule also typically comprises, or
is, its "intracellular signalling domain". A commonly used
endodomain component is the intracellular domain of CD3-zeta which
contains 3 ITAMs. This transmits an activation signal to the T cell
after antigen is bound. CD3-zeta may not provide a fully competent
activation signal and additional co-stimulatory signalling is
desirable. For example, chimeric CD28 and OX40 can be used with
CD3-Zeta to transmit a proliferative/survival signal, or all three
can be used together. It should be understood that this
intracellular signalling domain of the CAR is responsible for
activation of at least one of the normal effector functions of the
immune cell, preferably a T cell in which the CAR has been
expressed. The term "intracellular signalling domain" refers to the
portion of the protein which transduces the effector function
signal and directs the cell to perform a specialized function.
While usually the entire intracellular signalling domain can be
employed, in many cases it is not necessary to use the entire
domain. To the extent that a truncated portion of the intracellular
signalling domain is used, such truncated portion may be used in
place of the intact chain as long as it transduces the effector
function signal. The term "intracellular signalling domain" is thus
meant to include any truncated portion of the intracellular domain
sufficient to transduce the effector function signal.
[0140] Preferred examples of intracellular signalling domains for
use in a CAR include the cytoplasmic sequences of the T cell
receptor (TCR) and co-receptors that act in concert to initiate
signal transduction following antigen receptor engagement, as well
as any derivative or variant of these sequences and any synthetic
sequence that has the same functional capability.
[0141] It is known that signals generated through the TCR alone are
insufficient for full activation of the T cell and that a secondary
or co-stimulatory signal is also required. Thus, T cell activation
can be said to be mediated by two distinct classes of cytoplasmic
signalling sequence: those that initiate antigen-dependent primary
activation through the TCR (primary cytoplasmic signalling
sequences) and those that act in an antigen-independent manner to
provide a secondary or co-stimulatory signal (secondary cytoplasmic
signalling sequences). Primary cytoplasmic signalling sequences
regulate primary activation of the TCR complex either in a
stimulatory way, or in an inhibitory way. Primary cytoplasmic
signalling sequences that act in a stimulatory manner may contain
signalling motifs which are known as immunoreceptor tyrosine-based
activation motifs or ITAMs. Examples of ITAM containing primary
cytoplasmic signalling sequences that are of particular use include
those derived from TCR zeta, FcR gamma, FcR beta, CD3 gamma, CD3
delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66d. It is
particularly preferred that cytoplasmic signalling molecule in the
CAR comprise a cytoplasmic signalling sequence derived from
CD3-zeta.
[0142] In a preferred embodiment, the cytoplasmic domain of the CAR
can be designed to comprise the CD3-zeta signalling domain by
itself or combined with any other desired cytoplasmic domain(s)
useful in the context of the CAR of the invention. For example, the
cytoplasmic domain of the CAR can comprise a CD3 zeta chain portion
and a costimulatory signalling region. The costimulatory signalling
region refers to a portion of the CAR comprising the intracellular
domain of a costimulatory molecule. A costimulatory molecule is a
cell surface molecule other than an antigen receptor or its ligands
that is required for an efficient response of lymphocytes to an
antigen. Examples of such molecules include CD27, CD28, 4-1BB
(CD137), OX40, CD30, CD40, PD-1, ICOS, lymphocyte
function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C,
B7-H3, and a ligand that specifically binds with CD83, and the
like. The cytoplasmic signalling sequences within the cytoplasmic
signalling portion of the CAR of the invention may be linked to
each other in a random or specified order. Optionally, a short
oligo- or polypeptide linker, preferably between 2 and 10 amino
acids in length may form the linkage. A glycine-serine doublet
provides a particularly suitable linker. In one embodiment, the
cytoplasmic domain is designed to comprise the signalling domain of
CD3-zeta and the signalling domain of CD28.
[0143] As detailed hereinbefore, the antigen recognition moiety is
operably linked to the T cell activation moiety. By "operably
linked" is meant that the antigen recognition moiety is linked,
bound or otherwise associated with the T cell activation moiety,
such that upon binding of the antigen recognition moiety to the
antigenic determinant, a signal is induced via the T cell
activation moiety to activate the subject T cell and enable its
effector functions to be activated. This is achieved, for example,
via the design of a transmembrane domain.
[0144] In one embodiment, the transmembrane domain that is
naturally associated with one of the domains in the CAR is used. In
some instances, the transmembrane domain can be selected or
modified by amino acid substitution to avoid binding of such
domains to the transmembrane domains of the same or different
surface membrane proteins to minimize interactions with other
members of the receptor complex. The transmembrane domain may be
derived either from a natural or from a synthetic source. Where the
source is natural, the domain may be derived from any
membrane-bound or transmembrane protein. For example, transmembrane
regions may be derived from (ie. comprise at least the
transmembrane region(s) of) the alpha, beta or zeta chain of the
T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16,
CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, or from an
immunoglobulin such as IgG4. Alternatively, the transmembrane
domain may be synthetic, in which case it will comprise
predominantly hydrophobic residues such as leucine and valine.
Preferably a triplet of phenylalanine, tryptophan and valine will
be found at each end of a synthetic transmembrane domain.
Optionally, a short oligo- or polypeptide linker, preferably
between 2 and 10 amino acids in length may form the linkage between
the transmembrane domain and the cytoplasmic signalling domain of
the CAR. A glycine-serine doublet provides a particularly suitable
linker. Typically, the transmembrane domain is a hydrophobic alpha
helix that spans the membrane. Generally, the transmembrane domain
from the most membrane proximal component of the endodomain is
used.
[0145] Reference to an "antigen-binding receptor" should be
understood as a reference to engineered receptors which are
anchored to the cell surface and bind to an antigen. Similar to
chimeric antigen receptors disclosed herein, antigen-binding
receptors disclosed herein also comprise an antigen recognition
moiety directed to an antigenic determinant. The antigen
recognition moiety in an antigen-binding receptor can take the same
form and designed in the same way as the antigen recognition moiety
of a chimeric antigen receptor, as described herein. Also similar
to chimeric antigen receptors disclosed herein, the antigenic
recognition moiety in an antigen-binding receptor is operably
linked (e.g., through a spacer sequence such as a hinge region) to
a transmembrane domain, such that the antigen-binding receptor is
anchored to the cell surface. The spacer sequence and the
transmembrane domain in an antigen-binding receptor can also be
designed in the same way as the spacer sequence and the
transmembrane domain of a chimeric antigen receptor, as described
above. However, unlike chimeric antigen receptors, the
antigen-binding receptor as defined herein is generally
non-signalling, and may include an intracellular sequence that
lacks a T-cell activation domain. Such a non-signalling
antigen-binding receptor can bind to an antigen but does not
trigger any signal transduction in T cells, and therefore is also
referred to as a "docking receptor" or "anchoring receptor".
Certain embodiments of antigen-binding receptors, such as a
non-signalling CD47-binding receptor, are further described herein
below.
[0146] Examples of nucleic acid constructs encoding a CAR and/or an
antigen-binding receptor are depicted in FIG. 11, and exemplary
sequences for CAR and antigen-binding receptor as well as various
domains suitable for use in CARs and/or an antigen-binding
receptors are provided in SEQ ID NOS: 1-20.
[0147] It would be appreciated by the person of skill in the art
that the mechanism by which these genetic modifications are
introduced into the cell may take any suitable form which would be
well known and understood by those of skill in the art. For
example, genetic material is generally conveniently introduced to
cells via the use of an expression construct.
[0148] In one embodiment, a cell capable of differentiating into a
T cell expressing a TCR (i.e., a stem cell such as an iPSC or HSC)
or a cell that expresses a TCR from which a stem cell such as an
iPSC can be derived, is transfected with a CAR-encoding expression
construct. The expression construct can comprise one or more DNA
regions comprising a promoter operably linked to a nucleotide
sequence encoding a CAR and, optionally, a second DNA region
encoding a selectable marker and, optionally, a third DNA region
encoding a suicide protein. In this regard, it should be
appreciated that one may design the construct with any one or more
additional components, such as a suicide gene, which the person of
skill in the art would deem useful, as a matter of routine
procedure. In the context of the cells of the present invention,
which are proposed to be used in vivo to treat patients, the
ability to control the killing of the genetically modified cells of
the invention, and therefore effect their elimination from the in
vivo environment, is highly desirable. Without limiting the present
invention to any one theory or mode of action, the adoptive
transfer of the cells of the present invention, particularly to the
extent that they may be directed to "self" antigens such as tumour
antigens or antigens expressed on autoreactive cells, or antigens
to which cross-reactivity with self antigens may occur, is not
without risk. In this situation, outcomes similar to graft versus
host disease may occur, where these cells attack healthy
(non-diseased) cells. In the overall therapeutic scheme, these
side-effects may still be more desirable than the non-specific
systemic killing of healthy tissue which is characteristic of a
treatment such as chemotherapy or the uncontrolled killing of
healthy tissue in an autoimmune disorder. Nevertheless, killing the
cancer cells is paramount but the ability to control the
elimination of the cells of the present invention is highly
desirable and can be routinely achieved by the very well known and
widely used technique of building an inducible suicide gene into
the gene construct which is introduced into the stem/T cells of the
present invention.
[0149] The subject promoter may be constitutive or inducible. Where
the subject construct expresses more than one protein of interest,
these may be under the control of separate promoters or they may be
under the control of a single promoter, such as occurs in the
context of a bicistronic vector which makes use of an IRES sequence
to facilitate the translation of more than one protein product, in
an unfused form, from a single RNA transcript. The subject
construct may additionally be designed to facilitate use of the Cre
recombinase mediated splicing inducible gene expression system.
[0150] Reference to a nucleic acid "expression construct" should be
understood as a reference to a nucleic acid molecule which is
transmissible to a cell and designed to undergo transcription. The
RNA molecule is then transcribed therefrom. In general, expression
constructs are also referred to by a number of alternative terms,
which terms are widely utilised interchangeably, including
"expression cassette" and "vector".
[0151] For purposes of introducing nucleic acids encoding multiple
receptors, whether the receptor is a CAR, an antigen-binding
receptor, or a combination thereof, the multiple receptor-encoding
nucleic acids can be placed in one construct which is transfected
into a cell. In one embodiment, the multiple receptor-encoding
nucleic acids can be included in a multicistronic vector which
makes use of an IRES sequence to facilitate the translation of the
multiple receptor proteins. In another embodiment, the multiple
receptor-encoding nucleic acids can be linked to each other within
one expression unit and reading frame, for example, by utilizing a
self-cleaving peptide (e.g., P2A) such that one single polypeptide
comprising multiple receptor sequences is initially produced and
subsequently processed to produce multiple receptors. In another
embodiment, the multiple receptor-encoding nucleic acids are placed
in separate constructs which are used in transfection.
[0152] The expression construct of the present invention may be
generated by any suitable method including recombinant or synthetic
techniques. To this end, the subject construct may be constructed
from first principles, as would occur where an entirely synthetic
approach is utilised, or it may be constructed by appropriately
modifying an existing vector. Where one adopts the latter approach,
the range of vectors which could be utilised as a starting point
are extensive and include, but are not limited to: [0153] (i)
Plasmids: Plasmids are small independently replicating pieces of
cytoplasmic DNA, generally found in prokaryotic cells, which are
capable of autonomous replication. Plasmids are commonly used in
the context of molecular cloning due to their capacity to be
transferred from one organism to another. Without limiting the
present invention to any one theory or mode of action, plasmids can
remain episomal or they can become incorporated into the genome of
a host. Examples of plasmids which one might utilise include the
bacterial derived pBR322 and pUC. [0154] (ii) Bacteriophage:
Bacteriophages are viruses which infect and replicate in bacteria.
They generally consist of a core of nucleic acid enclosed within a
protein coat (termed the capsid). Depending on the type of phage,
the nucleic acid may be either DNA (single or double stranded) or
RNA (single stranded) and they may be either linear or circular.
Phages may be filamentous, polyhedral or polyhedral and tailed, the
tubular tails to which one or more tubular tail fibres are
attached. Phages can generally accommodate larger fragments of
foreign DNA than, for example, plasmids. Examples of phages
include, but are not limited to the E. coli lambda phages, P1
bacteriophage and the T-even phages (eg. T4). [0155] (iii)
Baculovirus: These are any of a group of DNA viruses which multiply
only in invertebrates and are generally classified in the family
Baculoviridae. Their genome consists of double-stranded circular
DNA. [0156] (iv) Mammalian virus: Examples of such viruses which
infect mammals, include lentivirus, sendai virus, retrovirus, and
vaccinia virus. [0157] (v) Artificial Chromosomes: Artificial
chromosomes such as yeast artificial chromosomes or bacterial
artificial chromosomes. [0158] (vi) Hybrid vectors such as cosmids,
phagemids and phasmids: Cosmids are generally derived from plasmids
but also comprise cos sites for lambda phage while phagemids
represent a chimeric phage-plasmid vector. Phasmids generally also
represent a plasmid-phage chimaera but are defined by virtue of the
fact that they contain functional origins of replication of both.
Phasmids can therefore be propagated either as a plasmid or a phage
in an appropriate host strain. [0159] (vii) Commercially available
vectors which are themselves entirely synthetically generated or
are modified versions of naturally occurring vectors, such as viral
vectors.
[0160] It would be understood by the person of skill in the art
that the selection of an appropriate vector for modification, to
the extent that one chooses to do this rather than synthetically
generate a construct, will depend on a number of factors including
the ultimate use to which the genetically modified cell will be
put. For example, where the cell is to be administered in vivo into
a human, it may be less desirable to utilise certain types of
vectors, such as viral vectors. Further, it is necessary to
consider the amount of DNA which is sought to be introduced to the
construct. It is generally understood that certain vectors are more
readily transfected into certain cell types. For example, the range
of cell types which can act as a host for a given plasmid may vary
from one plasmid type to another. In still yet another example, the
larger the DNA insert which is required to be inserted, the more
limited the choice of vector from which the expression construct of
the present invention is generated. To this end, the size of the
inserted DNA can vary depending on factors such as the size of the
DNA sequence encoding the protein of interest, the number of
proteins which are sought to be expressed, the number of selection
markers which are utilised and the incorporation of features such
as linearisation polylinker regions and the like.
[0161] The expression construct which is used in the present
invention may be of any form including circular or linear. In this
context, a "circular" nucleotide sequence should be understood as a
reference to the circular nucleotide sequence portion of any
nucleotide molecule. For example, the nucleotide sequence may be
completely circular, such as a plasmid, or it may be partly
circular, such as the circular portion of a nucleotide molecule
generated during rolling circle replication (this may be relevant,
for example, where a construct is being initially replicated, prior
to its introduction to a cell population, by this type of method
rather than via a cellular based cloning system). In this context,
the "circular" nucleotide sequence corresponds to the circular
portion of this molecule. A "linear" nucleotide sequence should be
understood as a reference to any nucleotide sequence which is in
essentially linear form. The linear sequence may be a linear
nucleotide molecule or it may be a linear portion of a nucleotide
molecule which also comprises a non-linear portion such as a
circular portion. An example of a linear nucleotide sequence
includes, but is not limited to, a plasmid derived construct which
has been linearised in order to facilitate its integration into the
chromosomes of a host cell or a construct which has been
synthetically generated in linear form. To this end, it should also
be understood that the configuration of the construct of the
present invention may or may not remain constant. For example, a
circular plasmid-derived construct may be transfected into a cell
where it remains a stable circular episome which undergoes
replication and transcription in this form. However, in another
example, the subject construct may be one which is transfected into
a cell in circular form but undergoes intracellular linearisation
prior to chromosomal integration. This is not necessarily an ideal
situation since such linearisation may occur in a random fashion
and potentially cleave the construct in a crucial region thereby
rendering it ineffective.
[0162] The nucleic acid molecules which are utilised in the method
of the present invention are derivable from any human or non-human
source. Non-human sources contemplated by the present invention
include primates, livestock animals (e.g., sheep, pigs, cows,
goats, horses, donkeys), laboratory test animal (e.g., mice,
hamsters, rabbits, rats, guinea pigs), domestic companion animal
(e.g., dogs, cats), birds (e.g., chicken, geese, ducks and other
poultry birds, game birds, emus, ostriches) captive wild or tamed
animals (e.g., oxes, kangaroos, dingoes), reptiles, fish, insects,
prokaryotic organisms or synthetic nucleic acids.
[0163] It should be understood that the receptor-encoding
constructs of the present invention may comprise nucleic acid
material from more than one source. For example, whereas the
construct may originate from a particular microorganism, in
modifying that construct to introduce the features defined herein,
nucleic acid material from other microorganism sources may be
introduced. These sources may include, for example, viral or
bacterial DNA (eg. IRES DNA), mammalian DNA (e.g., the DNA encoding
a CAR) or synthetic DNA (e.g., to introduce specific restriction
endonuclease sites). Still further, the cell type in which it is
proposed to express the subject construct may be different again in
that it does not correspond to the same organism as all or part of
the nucleic acid material of the construct. For example, a
construct consisting of essentially bacterial and viral derived DNA
may nevertheless be expressed in the mammalian stem cells
contemplated herein.
[0164] Without limiting the present invention in any way, the
present invention preferably uses a DNA construct comprising
sequences of a CAR, wherein the sequence comprises the nucleic acid
sequence of an antigen binding moiety operably linked to the
nucleic acid sequence of an intracellular domain. For example, an
intracellular domain that can be used in the subject CAR includes
but is not limited to the intracellular domain of CD3-zeta. In
another embodiment, the intracellular domain of a CAR includes the
intracellular domain of CD3-zeta in operable linkage to the
intracellular domain of CD28; and in a further embodiment, the
intracellular domain of a CAR includes the intracellular domains of
CD3-zeta, CD28 and OX40, in operable linkage with each other.
[0165] Vectors derived from retroviruses such as the lentivirus are
one example of vectors suitable to achieve long-term gene transfer
since they allow long-term, stable integration of a transgene and
its propagation in daughter cells. Other suitable viruses include
Sendai virus and Vaccinia virus. The vector should be suitable for
replication and integration into eukaryotes. Typical cloning
vectors contain transcription and translation terminators,
initiation sequences, and promoters useful for regulation of the
expression of the desired nucleic acid sequence. Viral vector
technology is well known in the art and is described, for example,
in Sambrook et al (2001, Molecular Cloning: A Laboratory Manual,
Cold Spring Harbor Laboratory, New York), and in other virology and
molecular biology manuals. Viruses, which are useful as vectors
include, but are not limited to, retroviruses, adenoviruses,
adeno-associated viruses, herpes viruses and lentiviruses. In
general, a suitable vector contains an origin of replication
functional in at least one organism, a promoter sequence,
convenient restriction endonuclease sites, and one or more
selectable markers, (eg. WO 01/96584; WO 01/29058; and U.S. Pat.
No. 6,326,193).
[0166] A number of viral based systems have been developed for gene
transfer into mammalian cells. For example, retroviruses provide a
convenient platform for gene delivery systems. A selected gene can
be inserted into a vector and packaged in retroviral particles
using techniques known in the art. The recombinant virus can then
be isolated and delivered to the subject stem cells. A number of
retroviral systems are known in the art.
[0167] Additional promoter elements, eg. enhancers, regulate the
frequency of transcriptional initiation. Typically, these are
located in the region 30-110 bp upstream of the start site,
although a number of promoters have recently been shown to contain
functional elements downstream of the start site as well. The
spacing between promoter elements frequently is flexible, so that
promoter function is preserved when elements are inverted or moved
relative to one another. In the thymidine kinase (tk) promoter, the
spacing between promoter elements can be increased to 50 bp apart
before activity begins to decline. Depending on the promoter, the
individual elements can function either cooperatively or
independently to activate transcription.
[0168] One example of a suitable promoter is the immediate early
cytomegalovirus (CMV) promoter sequence. This promoter sequence is
a strong constitutive promoter sequence capable of driving high
levels of expression of any polynucleotide sequence operatively
linked thereto. Another example of a suitable promoter is
Elongation Growth Factor-1a (EF-1a). However, other constitutive
promoter sequences may also be used, including, but not limited to
the simian virus 40 (SV40) early promoter, mouse mammary tumor
virus (MMTV), human immunodeficiency virus (HIV) long terminal
repeat (LTR) promoter, MoMuLV promoter, an avian leukemia virus
promoter, an Epstein-Barr virus immediate early promoter, a Rous
sarcoma virus promoter, as well as human gene promoters such as,
but not limited to, the actin promoter, the myosin promoter, the
hemoglobin promoter, and the creatine kinase promoter. Further, the
construct should not be limited to the use of constitutive
promoters. Inducible promoters are also contemplated to be used.
The use of an inducible promoter provides a molecular switch
capable of turning on expression of the CAR polynucleotide sequence
to which it is operatively linked when such expression is desired,
or turning off the expression when expression is not desired.
Examples of inducible promoters include, but are not limited to a
metallothionine promoter, a glucocorticoid promoter, a progesterone
promoter, and a tetracycline promoter.
[0169] In order to assess the expression of a CAR polypeptide or
portions thereof, the expression vector to be introduced into a
cell can also contain either a selectable marker gene or a reporter
gene or both to facilitate identification and selection of
expressing cells from the population of cells sought to be
transfected or infected through viral vectors. In other aspects,
the selectable marker may be carried on a separate piece of DNA and
used in a co-transfection procedure. Both selectable markers and
reporter genes may be flanked with appropriate regulatory sequences
to enable expression in the host cells. Useful selectable markers
include, for example, antibiotic-resistance genes, such as neo and
the like. An epitope tag can also be included in the extracellular
domain of a CAR molecule, such as the commonly used short
polypeptide c-myc or FLAG, preferably placed within the hinge
region, to identify CAR expression by epitope specific targeting
agents such as antibodies used in combination for example with flow
cytometry.
[0170] Reporter genes are used for identifying potentially
transfected cells and for evaluating the functionality of
regulatory sequences. In general, a reporter gene is a gene that is
not present in or expressed by the recipient organism or tissue and
encodes a polypeptide whose expression is manifested by some easily
detectable property, e.g., enzymatic activity. Expression of the
reporter gene is assayed at a suitable time after the DNA has been
introduced into the recipient cells. Suitable reporter genes may
include genes encoding luciferase, beta-galactosidase,
chloramphenicol acetyl transferase, secreted alkaline phosphatase,
or the green fluorescent protein gene (e.g., Ui-Tei et al, 2000
FEBS Letters 479: 79-82). Suitable expression systems are well
known and may be prepared using known techniques or obtained
commercially. In general, the construct with the minimal 5'
flanking region showing the highest level of expression of reporter
gene is identified as the promoter. Such promoter regions may be
linked to a reporter gene and used to evaluate agents for the
ability to modulate promoter-driven transcription. It will be
appreciated by those skilled in the art that a reporter such as
eGFP (enhanced green fluorescent protein) can be incorporated as a
C-terminal polypeptide extension to a CAR, separated by a
self-cleaving peptide such as P2A, which will release the reporter
such as eGFP intracellularly.
[0171] Methods of introducing and expressing genes in a cell are
known in the art. In the context of an expression vector, the
vector can be readily introduced into a host cell by physical,
chemical, or biological means.
[0172] Physical methods for introducing a polynucleotide into a
host cell include calcium phosphate precipitation, lipofection,
particle bombardment, microinjection, electroporation, and the
like. Methods for producing cells comprising vectors and/or
exogenous nucleic acids are well-known in the art. See, for
example, Sambrook et al (2001, Molecular Cloning: A Laboratory
Manual, Cold Spring Harbor Laboratory, New York). A preferred
method for the introduction of a polynucleotide into a host cell is
calcium phosphate transfection.
[0173] Biological methods for introducing a polynucleotide of
interest into a host cell include the use of DNA and RNA vectors.
Viral vectors, and especially retroviral vectors, have become the
most widely used method for inserting genes into mammalian, eg.,
human cells. Other viral vectors can be derived from lentivirus,
poxviruses, herpes simplex virus I, adenoviruses and
adeno-associated viruses, and the like. See, for example, U.S. Pat.
Nos. 5,350,674 and 5,585,362.
[0174] Chemical means for introducing a polynucleotide into a host
cell include colloidal dispersion systems, such as macromolecule
complexes, nanocapsules, microspheres, beads, and lipid-based
systems including oil-in-water emulsions, micelles, mixed micelles,
and liposomes. An exemplary colloidal system for use as a delivery
vehicle is a liposome (eg., an artificial membrane vesicle).
[0175] In the case where a non-viral delivery system is sought to
be utilized, an exemplary delivery vehicle is a liposome. The use
of lipid formulations is contemplated for the introduction of the
nucleic acids into a host cell. In another aspect, the nucleic acid
may be associated with a lipid. The nucleic acid associated with a
lipid may be encapsulated in the aqueous interior of a liposome,
interspersed within the lipid bilayer of a liposome, attached to a
liposome via a linking molecule that is associated with both the
liposome and the oligonucleotide, entrapped in a liposome,
complexed with a liposome, dispersed in a solution containing a
lipid, mixed with a lipid, combined with a lipid, contained as a
suspension in a lipid, contained or complexed with a micelle, or
otherwise associated with a lipid. Lipid, lipid/DNA or
lipid/expression vector associated compositions are not limited to
any particular structure in solution. For example, they may be
present in a bilayer structure, as micelles, or with a "collapsed"
structure. They may also simply be interspersed in a solution,
possibly forming aggregates that are not uniform in size or shape.
Lipids are fatty substances which may be naturally occurring or
synthetic lipids. For example, lipids include the fatty droplets
that naturally occur in the cytoplasm as well as the class of
compounds which contain long-chain aliphatic hydrocarbons and their
derivatives, such as fatty acids, alcohols, amines, amino alcohols,
and aldehydes.
[0176] Lipids suitable for use can be obtained from commercial
sources. For example, dimyristyl phosphatidylcholine ("DMPC") can
be obtained from Sigma, St. Louis, Mo.; dicetyl phosphate ("DCP")
can be obtained from K & K Laboratories (Plainview, N.Y.);
cholesterol ("Choi") can be obtained from Calbiochem-Behring;
dimyristyl phosphatidylglycerol ("DMPG") and other lipids may be
obtained from Avanti Polar Lipids, Inc. (Birmingham, Ala.). Stock
solutions of lipids in chloroform or chloroform/methanol can be
stored at about -20.degree. C. Chloroform is used as the only
solvent since it is more readily evaporated than methanol.
"Liposome" is a generic term encompassing a variety of single and
multilamellar lipid vehicles formed by the generation of enclosed
lipid bilayers or aggregates. Liposomes can be characterized as
having vesicular structures with a phospholipid bilayer membrane
and an inner aqueous medium. Multilamellar liposomes have multiple
lipid layers separated by aqueous medium. They form spontaneously
when phospholipids are suspended in an excess of aqueous solution.
The lipid components undergo self-rearrangement before the
formation of closed structures and entrap water and dissolved
solutes between the lipid bilayers (Ghosh et al (1991)). However,
compositions that have different structures in solution than the
normal vesicular structure are also encompassed. For example, the
lipids may assume a micellar structure or merely exist as
nonuniform aggregates of lipid molecules. Also contemplated are
lipofectamine-nucleic acid complexes.
[0177] Regardless of the method used to introduce exogenous nucleic
acids into a host cell, in order to confirm the presence of the
recombinant DNA sequence in the host cell, a variety of assays may
be performed. Such assays include, for example, Southern and
Northern blotting, RT-PCR and PCR or by detecting the presence or
absence of a particular peptide, eg., by immunological means
(ELISAs and Western blots).
[0178] The TCR and CAR, and antigen-binding receptors in some
embodiments, of the present cells are each directed to an antigenic
determinant. Reference to "antigenic determinant" should be
understood as a reference to any proteinaceous or non-proteinaceous
molecule expressed by a cell which is sought to be targeted by the
receptor-expressing T cells of the present invention. It would be
appreciated that these are molecules which may be "self" molecules
in that they are normally expressed in the body of a patient (such
as would be expected on some tumour cells or an autoreactive cells)
or they may be non-self molecules such as would be expected where a
cell is infected with a microorganism (eg. viral proteins). It
should also be understood that the subject antigen is not limited
to antigens (whether self or not) which are naturally able to
elicit a T or B cell immune response. Rather, in the context of the
present invention, reference to "antigen" or "antigenic
determinant" is a reference to any proteinaceous or
non-proteinaceous molecule which is sought to be targeted. As
detailed hereinbefore, the target molecule may be one to which the
immune system is naturally tolerant, such as a tumour antigen or
auto-reactive immune cell antigen. However, it may be desirable
(even in light of potential collateral damage) to nevertheless
target this antigen, for example to minimize the potentially even
more severe side effects which might be observed with a highly
non-specific and systemic treatment, such as chemotherapy or
immunosuppression, or to reduce the duration of treatment via a
highly targeted treatment and/or to maximise the prospect of
killing all unwanted cells. Preferably, said molecule is expressed
on the cell surface.
[0179] It would be understood by the skilled person that in the
context of TCR binding, the subject antigenic determinant will take
the form of a peptide derived from an antigen, which peptide is
expressed in the context of either MHC I or MHC II. In the context
of the CAR, since the design of this receptor is based on the use
of an immunoglobulin variable region binding domain, the receptor
will recognise an epitope present on the native form of the
antigen. The subject epitope may be either linear or
conformational. It should be understood that the subject antigenic
determinant may be any molecule expressed by the cell which is
sought to be targeted. That is, the molecule which is targeted may
be exclusively expressed by the target cell or it may also be
expressed by non-target cells too. Preferably, the subject
antigenic determinant is a non-self antigenic determinant or an
antigenic determinant which is otherwise expressed exclusively, or
at a significantly higher level than by normal cells, by the cells
which are sought to be targeted. However, as discussed
hereinbefore, depending on the disease condition to be treated, it
may not always be possible to identify and target a non-self
antigenic determinant.
[0180] Reference herein to TCR/CAR receptors which are directed to
a "first" antigenic determinant and to a "second" antigenic
determinant should be understood as a reference to the fact that
the subject receptors are directed to two different epitopic
regions. In this regard, however, it should be understood that the
receptors may be directed to epitopes on two entirely different
cell surface molecules or the receptors may be directed to two
different regions/epitopes of the same cell surface molecule. In
embodiments where reference is made to a TCR together with multiple
CARs, or where reference is made to a TCR with one or more CARs and
one or more antigen-binding receptors, it should be understood that
each receptor is directed to an antigenic determinant, and the
antigenic determinants are preferably different from one another,
i.e., the antigenic determinants corresponding to different
epitopic regions of the same or different molecules.
[0181] Accordingly in one embodiment there is provided a
genetically modified mammalian stem cell, or T cell differentiated
therefrom, which cell expresses at least one homozygous HLA
haplotype, is capable of differentiating to a T cell expressing a
TCR directed to a first antigenic determinant, and comprises at
least one (i.e., one or more) nucleic acid molecule encoding a
chimeric antigen receptor, wherein said receptor comprises an
antigen recognition moiety directed to a second antigenic
determinant, which antigen recognition moiety is operably linked to
a T cell activation moiety, and optionally further comprises a
nucleic acid encoding an antigen-binding receptor directed to a
third antigenic determinant, and wherein said antigenic
determinants are selected from tumour antigens, microorganism
antigens or autoreactive immune cell antigens.
[0182] In one embodiment, said stem cell is an iPSC. In another
embodiment, the stem cell is an HSC.
[0183] In still another embodiment, said stem cell is capable of
differentiating to a CD4.sup.+ T cell or a CD8.sup.+ T cell.
[0184] In still another embodiment, said TCR is an .alpha..beta.
TCR.
[0185] In yet still another embodiment, said stem cell such as iPSC
derived from a T cell or thymocyte, preferably a CD8.sup.+ T cell
or thymocyte.
[0186] As would be appreciated by the skilled person, the
identification of antigens which are exclusive to tumours is a
significant area of research, but in respect of which there has
been limited progress. Since tumour cells are usually self cells,
(as opposed to, for example, tumours arising from transplant
tissues), it is the case that the antigens which they express are
not only self antigens, but are likely to also be expressed by the
non-neoplastic cells of the tissue from which the tumour is
derived. This is clearly a less than ideal situation due to the
side-effects (in terms of destruction of non-neoplastic tissue)
which can arise when an anti-neoplastic treatment regime is
targeted to such an antigen, but is unavoidable. Nevertheless, some
progress has been made in terms of identifying target tumour
antigens which, even if not expressed exclusively by tumour cells,
are expressed at lower levels or otherwise less frequently on
non-neoplastic cells.
[0187] The selection of the antigen binding moiety of the invention
will depend on the particular type of cancer to be treated. Tumor
antigens are well known in the art and include, for example, MAGE,
LMP-2, CD19, CD20, WT1, MART-1 glioma-associated antigen,
carcinoembryonic antigen (CEA), .beta.-human chorionic
gonadotropin, tumour associated glycoprotein 72 (TAG 72),
alphafetoprotein (AFP), lectin-reactive AFP, thyroglobulin, RAGE-1,
MN-CA IX, human telomerase reverse transcriptase, RU1, RU2 (AS),
intestinal carboxyl esterase, mut hsp70-2, M-CSF, prostase,
prostate-specific antigen (PSA), PAP, NY-ESO-1, LAGE-1a, p53,
prostein, PSMA, Her2/neu, survivin and telomerase,
prostate-carcinoma tumor antigen-1 (PCTA-1), ELF2M, neutrophil
elastase, ephrinB2, CD22, insulin growth factor (IGF)-I, IGF-II,
IGF-I receptor and mesothelin. CD47 ("don't eat me" receptor) is
also a tumour target because it is often highly expressed in cancer
cells, as compared to normal cells, and prevents these cancer cells
from being attacked by cells of the immune system including, and in
particular, scavenger macrophages.
[0188] In one embodiment, the tumor antigen comprises one or more
epitopes associated with a malignant tumor. Malignant tumors
express a number of proteins that can serve as target antigens for
an immune attack. These molecules include but are not limited to
tissue-specific antigens such as MART-1, WT-1, tyrosinase and GP
100 in melanoma and prostatic acid phosphatase (PAP) and
prostate-specific antigen (PSA) in prostate cancer. Other target
molecules belong to the group of transformation-related molecules
such as the oncogene HER-2/Neu/ErbB-2. Yet another group of target
antigens are onco-fetal antigens such as carcinoembryonic antigen
(CEA). In B-cell lymphoma, the tumor-specific idiotype
immunoglobulin constitutes a truly tumor-specific immunoglobulin
antigen that is unique to the individual tumor. B-cell
differentiation antigens such as CD 19, CD20 and CD37 are other
candidates for target antigens in B-cell lymphoma.
[0189] Non-limiting examples of antigens include the following:
Differentiation antigens such as MART-1/MelanA (MART-I), gp1OO
(Pmel 17), tyrosinase, TRP-1, TRP-2 and tumor-specific multilineage
antigens such as MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, p15;
overexpressed embryonic antigens such as CEA; overexpressed
oncogenes and mutated tumor-suppressor genes such as p53, Ras,
HER-2/neu; unique tumor antigens resulting from chromosomal
translocations; such as BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR;
and viral antigens, such as the Epstein Barr virus antigens EBVA
and the human papillomavirus (HPV) antigens E6 and E7. Other large,
protein-based antigens include CD47, TSP-180, MAGE-4, MAGE-5,
MAGE-6, RAGE, NY-ESO, pl 85erbB2, p180erbB-3, cMet, nm-23H1, PSA,
TAG-72, CA 19-9, CA 72-4, CAM 17.1, NuMa, K-ras, beta-Catenin,
CDK4, Mum-1, p 15, p 16, 43-9F, 5T4, 791Tgp72, alpha-fetoprotein,
beta-HCG, BCA225, BTAA, CA 125, CA 15-3\CA 27. 29\BCAA, CA 195, CA
242, CA-50, CAM43, CD68\P1, CO-029, FGF-5, G250, Ga733\EpCAM,
HTgp-175, M344, MA-50, MG7-Ag, MOV 18, NB/70K, NY-CO-1, RCAS 1,
SDCCAG16, TA-90\Mac-2 binding protein\cyclophilin C-associated
protein, TAAL6, TAG72, TLP, and TPS.
[0190] The cells of the present invention are designed to be
directed to multiple, i.e., two or more, antigenic determinants. As
detailed herein, the multiple antigenic determinants may be, or
include, in some embodiments, multiple epitopes of one molecule,
or, in other embodiments, epitopes of multiple entirely distinct
molecules. The selection of which multiple antigenic determinants
should be targeted and, further, whether they should be targeted by
the TCR or the CAR is well within the skill of the person in the
art. In one embodiment, the cells of the present invention are
designed to clear tumour cells and said TCR/CAR are directed to
tumour antigens, in particular TAG 72, MAGE and WT1. In another
embodiment, said cells are designed to clear autoreactive immune
cells and said TCR/CAR are directed to idiotypic T cell or B cell
receptors.
[0191] Accordingly, in one embodiment there is provided a
genetically modified mammalian stem cell, or T cell differentiated
therefrom, which cell is capable of differentiating to a T cell
expressing a TCR directed to a first tumour antigenic determinant,
and comprises one or more nucleic acid molecules encoding one or
more chimeric antigen receptors, wherein each chimeric antigen
receptor comprises an antigen recognition moiety directed to a
tumour antigenic determinant, which antigen recognition moiety
operably linked to a T cell activation moiety and wherein said
antigenic determinants are selected from TAG 72, CD47, CD19, WT-1,
MAGE and EBVLMP2.
[0192] Preferably, said genetically modified cell is directed to
TAG72 and WT-1. Still more preferably, said CAR is directed to
TAG72 and CD47, and said TCR is directed to WT-1.
[0193] In one embodiment, said stem cell is an iPSC. In another
embodiment, the stem cell is an HSC.
[0194] In still another embodiment, said stem cell is capable of
differentiating to a CD4.sup.+ T cell or a CD8.sup.+ T cell.
[0195] In still another embodiment, said TCR is an .alpha..beta.
TCR.
[0196] In yet still another embodiment, said stem cell (such as
iPSC) is derived from a T cell or thymocyte, preferably a CD8.sup.+
T cell or thymocyte.
[0197] To the extent that the cells of the present invention, in
one embodiment, are directed to treating neoplasias, a wide range
of CARs have been developed to target known tumour antigens. A
non-limiting summary exemplifying some of these CARs, together with
the structure of the receptor, is provided in Table 5, below:
TABLE-US-00005 TABLE 5 Target antigen Associated malignancy
Receptor type .alpha.-Folate receptor Ovarian cancer
ScFv-Fc.epsilon.RI.gamma.CAIX CAIX Renal cell carcinoma
ScFv-Fc.epsilon.RI.gamma. CAIX Renal cell carcinoma
ScFv-Fc.epsilon.RI.gamma. CD19 B-cell malignancies ScFv-CD3.zeta.
(EBV) CD19 B-cell malignancies, CLL ScFv-CD3.zeta. CD19 B-ALL
ScFv-CD28-CD3.zeta. CD19 ALL CD3.zeta.(EBV) CD19 ALL post-HSCT
ScFv-CD28-CD3.zeta. CD19 Leukemia, lymphoma, CLL
ScFv-CD28-CD3.zeta. vs. CD3.zeta. CD19 B-cell malignancies
ScFv-CD28-CD3.zeta. CD19 B-cell malignancies post-HSCT
ScFv-CD28-CD3.zeta. CD19 Refractory Follicular Lymphoma
ScFv-CD3.zeta. CD19 B-NHL ScFv-CD3.zeta. CD19 B-lineage lymphoid
malignancies post-UCBT ScFv-CD28-CD3.zeta. CD19 CLL, B-NHL
ScFv-CD28-CD3.zeta. CD19 B-cell malignancies, CLL, B-NHL
ScFv-CD28-CD3.zeta. CD19 ALL, lymphoma ScFv-41BB-CD3.zeta. vs
CD3.zeta. CD19 ALL ScFv-41BB-CD3.zeta. CD19 B-cell malignancies
ScFv-CD3.zeta. (Influenza MP-1) CD19 B-cell malignancies
ScFv-CD3.zeta. (VZV) CD20 Lymphomas ScFv-CD28-CD3.zeta. CD20 B-cell
malignancies ScFv-CD4-CD3.zeta. CD20 B-cell lymphomas
ScFv-CD3.zeta. CD20 Mantle cell lymphoma ScFv-CD3.zeta. CD20 Mantle
cell lymphoma, indolent B-NHL CD3 .zeta./CD137/CD28 CD20 indolent B
cell lymphomas ScFv-CD28-CD3.zeta. CD20 Indolent B cell lymphomas
ScFv-CD28-41BB-CD3.zeta. CD22 B-cell malignancies
ScFV-CD4-CD3.zeta. CD30 Lymphomas ScFv-Fc.epsilon.RI.gamma. CD30
Hodgkin lymphoma ScFv-CD3.zeta. (EBV) CD33 AML ScFv-CD28-CD3.zeta.
CD33 AML ScFv-41BB-CD3.zeta. CD44v7/8 Cervical carcinoma
ScFv-CD8-CD3.zeta. CEA Breast cancer ScFv-CD28-CD3.zeta. CEA
Colorectal cancer ScFv-CD3.zeta. CEA Colorectal cancer
ScFv-FceRI.gamma. CEA Colorectal cancer ScFv-CD3.zeta. CEA
Colorectal cancer ScFv-CD28-CD3.zeta. CEA Colorectal cancer
ScFv-CD28-CD3.zeta. EGP-2 Multiple malignancies scFv-CD3.zeta.
EGP-2 Multiple malignancies scFv-Fc.epsilon.RI.gamma. EGP-40
Colorectal cancer scFv-Fc.epsilon.RI.gamma. erb-B2 Colorectal
cancer CD28/4-1BB-CD3.zeta. erb-B2 Breast and others
ScFv-CD28-CD3.zeta. erb-B2 Breast and others ScFv-CD28-CD3.zeta.
(Influenza) erb-B2 Breast and others ScFv-CD28mut-CD3.zeta. erb-B2
Prostate cancer ScFv-Fc.epsilon.RI.gamma. erb-B 2, 3, 4 Breast and
others Heregulin-CD3.zeta. erb-B 2, 3, 4 Breast and others
ScFv-CD3.zeta. FBP Ovarian cancer ScFv-Fc.epsilon.RI.gamma. FBP
Ovarian cancer ScFv-Fc.epsilon.RI.gamma. (alloantigen) Fetal
acetylcholine receptor Rhabdomyosarcoma ScFv-CD3.zeta. GD2
Neuroblastoma ScFv-CD28 GD2 Neuroblastoma ScFv-CD3.zeta. GD2
Neuroblastoma ScFv-CD3.zeta. GD2 Neuroblastoma
ScFv-CD28-OX40-CD3.zeta. GD2 Neuroblastoma ScFv-CD3.zeta. (VZV) GD3
Melanoma ScFv-CD3.zeta. GD3 Melanoma ScFv-CD3.zeta. Her2/neu
Medulloblastoma ScFv-CD3.zeta. Her2/neu Lung malignancy
ScFv-CD28-CD3.zeta. Her2/neu Advanced osteosarcoma
ScFv-CD28-CD3.zeta. Her2/neu Glioblastoma ScFv-CD28-CD3.zeta.
IL-13R-a2 Glioma IL-13-CD28-4-1BB-CD3.zeta. IL-13R-a2 Glioblastoma
IL-13-CD3.zeta. IL-13R-a2 Medulloblastoma IL-13-CD3.zeta. KDR Tumor
neovasculature ScFv-Fc.epsilon.RI.gamma. k-light chain B-cell
malignancies ScFv-CD3.zeta. k-light chain (B-NHL, CLL)
ScFv-CD28-CD3.zeta. vs CD3.zeta. LeY Carcinomas
ScFv-Fc.epsilon.RI.gamma. LeY Epithelial derived tumors
ScFv-CD28-CD3.zeta. L1 cell adhesion molecule Neuroblastoma
ScFv-CD3.zeta. MAGE-A1 Melanoma ScFV-CD4-Fc.epsilon.RI.gamma.
MAGE-A1 Melanoma ScFV-CD28-Fc.epsilon.RI.gamma. Mesothelin Various
tumors ScFv-CD28-CD3.zeta. Mesothelin Various tumors
ScFv-41BB-CD3.zeta. Mesothelin Various tumors
ScFv-CD28-41BB-CD3.zeta. Murine CMV infected cells Murine CMV
Ly49H-CD3.zeta. MUC1 Breast, Ovary ScFV-CD28-OX40-CD3.zeta. NKG2D
ligands Various tumors NKG2D-CD3.zeta. Oncofetal antigen (h5T4)
Various tumors ScFV-CD3.zeta. (vaccination) PSCA Prostate carcinoma
ScFv-b2c-CD3.zeta. PSMA Prostate/tumor vasculature ScFv-CD3.zeta.
PSMA Prostate/tumor vasculature ScFv-CD28-CD3.zeta. PSMA
Prostate/tumor vasculature ScFv-CD3.zeta. TAA targeted by mAb IgE
Various tumors FceRI-CD28-CD3.zeta. (+a-TAA IgE mAb) TAG-72
Adenocarcinomas scFv-CD3.zeta. VEGF-R2 Tumor neovasculature
scFv-CD3.zeta.
[0198] In some embodiments, a CAR comprises an antigen recognition
domain which is comprised of an scFv directed to CD19 or TAG-72,
and a hinge (Stalk) region and a transmembrane region both of which
are derived from CD28 or CD8, and a cytoplasmic endodomain which is
also derived from CD28 or CD8 and comprises a T cell activation
moiety. The CAR can include a reporter protein (such as EGFP) as a
C-terminal polypeptide extension, joined together by a P2A
self-cleaving polypeptide to release EGFP after translation. See,
e.g., FIGS. 11 and 14.
[0199] In a related aspect, it has been further determined that the
cells of the present invention are rendered particularly effective
if they are engineered to express a non-signalling antigen-binding
receptor, for example, a CD47 binding molecule which is unable to
effect signal transduction. The expression of a CD47 binding
molecule on the cell surface anchors the cell of the present
invention to the neoplastic cell to which it is directed, thereby
facilitating improved interaction of the TCR and the CAR with their
respective ligands. In terms of the treatment of solid tumours, in
particular, the increased stability and binding affinity of the
interaction of the subject cell enables improved functional
outcomes, in terms of neoplastic cell killing, relative to a cell
which does not express the subject CD47 binding molecule.
[0200] Accordingly, in a related aspect of the present invention
there is provided a genetically modified mammalian stem cell, or T
cell differentiated therefrom, which cell is capable of
differentiating to a T cell expressing a TCR directed to a first
antigenic determinant, and comprises (i) a nucleic acid molecule
encoding a chimeric antigen receptor, wherein said receptor
comprises an antigen recognition moiety directed to a second
antigenic determinant, which antigen recognition moiety is operably
linked to a T cell activation moiety and (ii) a nucleic acid
molecule encoding a non-signalling antigen-binding receptor, such
as a non-signalling CD47 binding receptor. In some embodiments, the
genetically modified mammalian stem cell expresses at least one
homozygous HLA haplotype.
[0201] Without limiting the present invention to any one theory or
mode of action, CD47 (also known as integrin associated protein) is
a transmembrane protein that in humans is encoded by the CD47 gene.
CD47 belongs to the immunoglobulin superfamily. CD47 is involved in
a range of cellular processes, including apoptosis, proliferation,
adhesion, and migration. Furthermore, it plays a key role in immune
and angiogenic responses. CD47 is ubiquitously expressed in human
cells and has been found to be overexpressed in many different
tumor cells.
[0202] CD47 is a 50 kDa membrane receptor that comprises an
extracellular N-terminal IgV domain, five transmembrane domains,
and a short C-terminal intracellular tail. There are four
alternatively spliced isoforms of CD47 that differ only in the
length of their cytoplasmic tail. Form 2 is the most widely
expressed form that is found in all circulating and immune cells.
The second most abundant isoform is form 4, which is predominantly
expressed in the brain and in the peripheral nervous system. Only
keratinocytes express significant amounts of form 1. These isoforms
are highly conserved between mouse and man, suggesting an important
role for the cytoplasmic domains in CD47 function.
[0203] CD47 is a receptor for thrombospondin-1 (TSP-1), a secreted
glycoprotein that plays a role in vascular development and
angiogenesis. Binding of TSP-1 to CD47 influences several
fundamental cellular functions including cell migration and
adhesion, cell proliferation or apoptosis, and plays a role in the
regulation of angiogenesis and inflammation. CD47 also interacts
with signal-regulatory protein alpha (SIRP.alpha.), an inhibitory
transmembrane receptor present on myeloid cells. The
CD47/SIRP.alpha. interaction leads to bidirectional signalling,
resulting in different cell-to-cell responses including inhibition
of phagocytosis (facilitating cancer cell escape), stimulation of
cell-cell fusion, and T-cell activation. Still further, CD47
interacts with several membrane integrins, most commonly integrin
avb3. These interactions result in CD47/integrin complexes that
affect a range of cell functions including adhesion, spreading and
migration.
[0204] However, although CD47 is ubiquitously expressed, it has
been determined that the increased level of expression of CD47 on
neoplastic cells is sufficient to facilitate improved
responsiveness to, and clearing of, said neoplastic cells by
molecules targeting CD47, prior to any substantive adverse impact
on non-neoplastic cells.
[0205] Reference to a "binding receptor" directed to CD47 should be
understood as a reference to any receptor which interacts with
CD47. This may take the form of a CD47-binding receptor such as a
surface-displayed antibody fragment, for example, and preferably
lacking a signalling function.
[0206] Accordingly to this embodiment, there is provided a
genetically modified mammalian stem cell, or T cell differentiated
therefrom, which cell is capable of differentiating to a T cell
expressing a TCR directed to a first antigenic determinant, and
comprises (i) a nucleic acid molecule encoding a chimeric antigen
receptor, wherein said receptor comprises an antigen recognition
moiety directed to a second antigenic determinant, which antigen
recognition moiety is operably linked to a T cell activation moiety
and (ii) a nucleic acid molecule encoding a non-signalling
antigen-binding receptor, wherein said receptor comprises an
antigen recognition moiety directed to CD47. In some embodiments,
the genetically modified mammalian stem cell expresses at least one
homozygous HLA haplotype.
[0207] As detailed hereinbefore, the subject CD47 binding receptor
is a non-signalling receptor. By "non-signalling" is meant that
subsequently to binding of the subject receptor to CD47 on a target
cell, there is no signal transmitted which would effect a change to
the functionality of the cell of the present invention. Rather, the
purpose of the CD47 binding receptor is to provide improved
anchoring of the subject cell to a target cell, thereby improving
the effectiveness of binding of the TCR and the CAR which is
directed to the target antigen moiety, such as a tumour antigen
moiety.
[0208] For example, in one design of a non-signalling
antigen-binding receptor, the extracellular domain of the receptor
comprises an antigen recognition moiety with binding specificity to
CD47, a hinge (stalk) domain, a transmembrane domain, and an
intracellular domain which completely lacks a cytoplasmic
signalling function. Such non-signalling CD47 binding receptor can
be used simply for attachment, not for signalling, so it can drive
the docking of T-cells to cancer cells via CD47 binding and without
the unwanted activation and kill if engaging to normal
CD47-expressing cells.
[0209] In some embodiments, the antigen recognition moiety of a
non-signalling CD47-binding receptor includes antibody-like domains
such as scFv, Fv, Fab etc and any CD47 targeted V-domain, including
single human and mammalian V-domains and their equivalent (VhH or
vNAR) domains, or may include "alternative protein-based targeting
scaffolds" that are well known in the field including, but not
restricted to, darpins, anticalins, knottins, ImmE7s, affibodies,
Fn3 fibronectin domains etc. The antigen recognition moiety may
also include one or more of the V-like domains of SIRPa (the
natural ligand of CD47). In one embodiment, the antigen recognition
moiety may include one natural V-like domain of SIRP.alpha.. In
another embodiment, the antigen recognition moiety may include all
three of the natural V-like domain of SIRP.alpha.. In other
embodiments, a molecule suitable for use to provide an antigen
recognition moiety in a non-signalling CD47 binding receptor is
Hu5F9-G4 scFv molecule (described in U.S. patent application Ser.
No. 14/656,431). Hu5F9 has been designed with 3 different versions
of VH (1,2,3) and 3 different versions of VL (11, 12, 13), shown in
FIGS. 12A, 12B of U.S. patent application Ser. No. 14/656,431,
published as US 20150183874 A1. Liu et al (PLOS One (2015)
September 21;10(9):e0137345) describes Hu5F9-G4 where the selected
V-domains were heavy VH-2 comprising 4 unique residue changes in
the framework (that differentiate VH-2 from VH-1,3) and light VL-12
comprising 2 unique residue changes in the framework (that
differentiate VL-12 from VL-11,13).
[0210] In some embodiments, the hinge region of a non-signalling
CD47-binding receptor can be the natural SIRP.alpha. hinge
sequence, or the CD8 or CD28 hinges as typically used in CARs, or
alternative hinges well known in the field such as CD4 domains or
Mucin peptide hinges. The hinge region can be designed to include
one or more Cysteine (Cys) residues in order to allow for
dimerization of the receptors. CD28 is a natural dimeric structure
linked via a single Cys in the stalk region. Thus, where the stalk
region of CD28 is used as the hinge of non-signalling CD47-binding
receptor, introduction of an additional Cys may not be necessary,
but may provide additional stabilization for dimers.
[0211] It will be understood by those skilled in the art that the
introduction of nucleic acids encoding a CAR and a non-signalling
antigen-binding receptor (such as a non-signalling CD47-binding
receptor) into a cell (e.g., a T cell or an iPSC) can be achieved
using two separate transfection vectors, or a single bicistronic
vector, or a single gene encoding an internal cleavage signal to
separate the CAR from the antigen-binding receptor. In one
embodiment the internal cleavage signal is P2A, a peptide sequence
that directs self-cleavage to separate CAR from the antigen-binding
receptor. In a specific embodiment, a non-signalling CD47 binding
receptor is expressed as a C-terminal extension of a CAR and
separated by a P2A self-cleaving peptide to separate the CAR and
the CD47-binding receptor after translation.
[0212] Means for modifying the stem cell of the present invention,
such that it also expresses a non-signalling CD47 binding molecule,
have been described in significant detail hereinbefore in terms of
effecting the expression of a chimeric antigen receptor directed to
a tumour antigen moiety. The transfection and other methods of
achieving receptor expression which are described herein would be
understood by the skilled person to be equally applicable in the
context of the subject CD47 binding molecule.
[0213] In one embodiment, said stem cell is an iPSC. In another
embodiment, said stem cell is an HSC.
[0214] In another embodiment, said stem cell is capable of
differentiating to a CD4.sup.+ T cell or a CD8.sup.+ T cell.
[0215] In still another embodiment, said TCR is an .alpha..beta.
TCR.
[0216] In yet still another embodiment, said stem cell such as iPSC
is derived from a T cell or thymocyte, preferably a CD8.sup.+ T
cell or thymocyte, and in some embodiments, a CD8.sup.+ T cell or
thymocyte with an endogenous TCR directed to a tumour antigen.
[0217] In still yet another embodiment, said stem cell is directed
to TAG 72 and WT 1. Still more preferably, said CAR is directed to
TAG 72 and said TCR is directed to WT 1.
[0218] In a further aspect there is provided a method of making a
genetically modified mammalian stem cell. The various means for
making a genetically modified mammalian stem cell, particularly an
iPSC have been described hereinabove.
[0219] In a further aspect there is provided a T cell that
expresses a TCR directed to a first antigenic determinant, and a
chimeric antigen receptor, wherein said receptor comprises an
antigen recognition moiety directed to a second antigenic
determinant, which antigen recognition moiety is operably linked to
a T cell activation moiety. In some embodiments, the T cell
expresses at least one homozygous HLA haplotype.
[0220] In one embodiment, the T cell expresses multiple chimeric
antigen receptors, wherein each chimeric antigen receptor comprises
an antigen recognition moiety directed to an antigenic determinant,
which antigen recognition moiety is operably linked to a T cell
activation moiety.
[0221] In one embodiment, the multiple antigenic determinants which
the multiple chimeric antigen receptors are directed to are each
distinct from said first antigenic determinant to which the TCR
expressed on the subject T cell is directed. In another embodiment,
the multiple antigenic determinants which the multiple chimeric
antigen receptors are directed to, are distinct one from another,
and are also distinct from said first antigenic determinant to
which the TCR expressed on the subject T cell is directed.
[0222] In one embodiment, the multiple CARs are encoded by one
contiguous nucleic acid fragment. For example, the multiple CARs
are encoded by multiple nucleic acids placed in one vector, which
is transfected into a cell to ultimately generate the subject T
cell. In a specific embodiment, the multiple CAR encoding nucleic
acids can be linked to each other within one expression unit and
reading frame (for example, by utilizing a self-cleaving peptide
such as P2A), such that one single polypeptide comprising multiple
CAR polypeptide sequences is initially produced and subsequently
processed to provide multiple CARs. In another embodiment, the
multiple CAR-encoding nucleic acids are placed in separate vectors,
which are used in transfection to generate the subject T cell.
[0223] In another embodiment, the T cell, which expresses one or
more CARs, further expresses at least one (i.e., one or more)
antigen-binding receptor which comprises an antigen recognition
moiety directed to a third antigenic determinant.
[0224] In one embodiment, the antigen-binding receptor is a
non-signalling antigen-binding receptor; namely, the receptor is
anchored to the cell surface of the subject T cell and binds to the
third antigenic determinant, but does not transduce signal into the
cytoplasmic part of the T cell that would affect the function of
the T cell (hence also referred to as a non-T cell signalling
antigen-binding receptor). In one embodiment, the antigen-binding
receptor comprises an antigen recognition moiety directed to a
third antigenic determinant, operably linked to a transmembrane
domain, but lacks a T cell activation moiety.
[0225] In a specific embodiment, the antigen-binding receptor is a
non-signalling antigen-binding receptor directed to CD47. For
example, the antigen-binding receptor is a non-signalling
CD47-binding molecule.
[0226] In some embodiments, the T cell provided herein is CD4+. In
other embodiments, the T cell is CD8+.
[0227] In some embodiments, the T cell provided herein expresses an
.alpha..beta. TCR. In other embodiments, the T cell provided herein
expresses a .gamma..delta. TCR.
[0228] In some embodiments, the multiple antigenic determinants to
which the subject T cell is directed, i.e., the first antigenic
determinant to which the TCR is directed, the antigenic
determinant(s) to which the chimeric antigen receptor(s) is(are)
directed, and the antigenic determinant(s) to which the
antigen-binding receptor(s) is(are) directed if such
antigen-binding receptor(s) is(are) present, can be selected from
tumour antigens, microorganism antigens, or autoreactive immune
cell antigens. In certain embodiments, the antigenic determinants
are selected from tumour antigens. In specific embodiments, the
antigenic determinant to which the TCR is directed, is selected
from TCR recognized peptides such as WT-1 or EbvLMP2. In other
specific embodiments, the antigenic determinants to which a
chimeric antigen receptor and an antigen-binding receptor are
directed, can be selected from for example, TAG-72, CD19, MAGE, or
CD47.
[0229] In some embodiments, the subject T cell, which expresses a
TCR directed to a first antigenic determinant, and expresses a
chimeric antigen receptor which comprises an antigen recognition
moiety directed to a second antigenic determinant, operably linked
to a T cell activation moiety, is derived from an iPSC or an
HSC.
[0230] In one embodiment, the iPSC or HSC from which the subject T
cell is derived, is a genetically modified iPSC or HSC which is
capable of differentiating into a T cell which expresses a TCR
directed to said first antigenic determinant, and comprises one or
more nucleic acid(s) encoding one or more chimeric antigen
receptor, and optionally comprises one or more nucleic acid
encoding an antigen-binding receptor(s). In another embodiment, the
iPSC or HSC from which the subject T cell is derived, is capable of
differentiating into a T cell which expresses a TCR directed to
said first antigenic determinant; and one or more nucleic acid(s)
encoding one or more chimeric antigen receptor, and optionally one
or more nucleic acid encoding an antigen-binding receptor(s), are
introduced after the iPSC or HSC has differentiated into a T cell.
In some embodiments, the iPSC or HSC from which the subject T cell
is derived, expresses at least one HLA haplotype, and the T cell
derived from such iPSC or HSC also expresses said at least one HLA
haplotype.
[0231] In one embodiment, the iPSC from which the subject T cell is
derived, is itself derived from a T cell or thymocyte. In one
embodiment, the iPSC is derived from a CD8+ T cell or thymocyte. In
one embodiment, the iPSC is derived from a T cell or thymocyte,
which expresses a TCR directed to the first antigenic determinant,
i.e., the same antigenic determinant to which the TCR of the
subject T cell derived from the iPSC is directed.
[0232] The value of the cells of the present invention is
predicated on directing the differentiation of the subject stem
cell to a CD4.sup.+ or CD8.sup.+ T cell. In this regard, reference
to "directing" the differentiation of a stem cell to a T cell
should be understood to mean that a cell culture system is applied
which induces commitment of a stem cell to the T cell lineage and
differentiation along that lineage to a mature T cell. Means for
effecting the directed differentiation of a stem cell along the T
cell lineage are well known to those of skill in the art. For
example, and as exemplified herein, the introduction of
Notch-dependent signalling into the culture system is known to
effect the directed differentiation of stem cells along the T cell
lineage. Still further, if this signalling is provided to stem
cells in the context of their co-culture over the OP-9 feeder cell
layer, particularly efficient differentiation is achieved. Examples
of Notch ligands which are suitable for use include, but are not
limited to, Delta-like 1, and Delta-4. In this regard, OP-9 cells
have been engineered to express Delta-like 1 (OP9-DL1), thereby
providing a highly convenient means of generating T cells from stem
cells. In another example, and as exemplified herein, the subject
stem cells are first cultured in feeder-free conditions to generate
mesoderm, followed by co-culture on the OP9-DL1 cell line. A
particularly preferred method of achieving the directed
differentiation to CD8.sup.+ T cells is exemplified herein.
[0233] In another aspect there is provided a method for making a T
cell that expresses a TCR directed to a first antigenic
determinant, and expresses one or more CARs, and optionally one or
more antigen-binding receptors. In some embodiments, the T cell
also expresses at least one homozygous HLA haplotype.
[0234] In one embodiment, the method comprises obtaining a
genetically modified stem cell (such as a genetically modified iPSC
or HSC) which is capable of differentiating into a T cell which
expresses a TCR directed to a first antigenic determinant, and
comprises one or more nucleic acid(s) encoding one or more chimeric
antigen receptor each directed to an antigenic determinant
(preferably distinct from the first antigenic determinant), and
optionally further comprises one or more nucleic acid encoding one
or more antigen-binding receptor(s) each directed to an antigenic
determinant (preferably distinct from the first antigenic
determinant); and differentiating such genetically modified stem
cell into a T cell. In some embodiments, the genetically modified
stem cell also expresses at least one homozygous HLA haplotype.
[0235] In another embodiment, the method comprises obtaining a stem
cell (such as an iPSC or HSC) which is capable of differentiating
into a T cell which expresses a TCR directed to a first antigenic
determinant; differentiating the stem cell into a T cell;
introducing into the T cell one or more nucleic acid(s) encoding
one or more chimeric antigen receptor, each directed to an
antigenic determinant (preferably distinct from the first antigenic
determinant), and optionally also one or more nucleic acid encoding
one or more antigen-binding receptor(s) each directed to an
antigenic determinant (preferably distinct from the first antigenic
determinant). In some embodiments, the genetically modified stem
cell (such as an iPSC or HSC) also expresses at least one
homozygous HLA haplotype.
[0236] Irrespective of whether a CAR-encoding nucleic acid is
introduced into a stem cell before differentiation into a T cell,
or introduced into a T cell after differentiation from a stem cell,
the stem cell (such as an iPSC) can be itself derived from a T cell
or thymocyte. Such T cell and thymocyte can have a TCR specific for
a nominal antigen, e.g., a tumour antigen. In one embodiment, the
stem cell is an iPSC. In one embodiment, the iPSC is derived from a
CD8+ T cell or thymocyte. In another embodiment, the iPSC is
derived from a T cell or thymocyte expressing a TCR directed to the
same antigenic determinant to which the TCR expressed on the T cell
derived from the iPSC is directed.
[0237] Reference to "mammal" should be understood to include
reference to a mammal such as but not limited to human, primate,
livestock animal (e.g., sheep, cow, horse, donkey, pig), companion
animal (e.g., dog, coat), laboratory test animal (e.g., mouse,
rabbit, rat, guinea pig, hamster), captive wild animal (e.g., fox,
deer). Preferably the mammal is a human or primate. Most preferably
the mammal is a human.
[0238] The development of the present invention has now facilitated
the development of means for treating disease conditions
characterised by the presence of an unwanted cellular population
such as a neoplastic population of cells, virally infected cells,
autoreactive immune cells or infection with microorganisms such as
antibiotic resistant bacteria. More specifically, the cells of the
present invention provide a means of clearing these cells in a more
targeted fashion than current highly non-specific methods such as
chemotherapy to treat a neoplastic condition, anti-inflammatory
therapy to treat the symptoms of autoimmune disease or
immunosuppression to manage autoimmunity. In this regard, reference
to a disease condition "characterised by the presence of an
unwanted cellular population" should be understood as a reference
to any condition, a symptom or cause of which is the presence or
functioning of a population of cells which can be targeted by
virtue of an expressed cell surface antigen and the elimination of
some or all of which cells would be beneficial to the patient.
Treatment of the subject condition is achieved by administering T
cells differentiated from the stem cells of the present invention,
the dual TCR/CAR of which T cells are directed to two or more
antigenic determinants expressed by the cells which are sought to
be cleared.
[0239] It should be understood that the "cells" which are sought to
be cleared by the T cells of the present invention may be any cell,
whether self or non-self. For example, to the extent that the T
cells of the present invention are designed to treat a disease
condition such as a neoplasia, viral infection or autoimmune
disease, the target population of cells which are sought to be
cleared are self cells. However, to the extent that the condition
which is sought to be treated is, for example, infection by a
microorganism, such as antibiotic resistant bacteria or a parasite,
the "cell" to be cleared is a foreign cell. In this regard, the
cell may be in suspension (such as leukaemic cells which are
present in the circulation) or they may be part of a mass (such as
a tumour or tissue). To the extent that the condition being treated
is a microorganism infection, the cells may correspond to a
unicellular microorganism (such as many bacteria) or they may be
part of a multicellular organism. The T cells of the present
invention are useful for targeting any type of cell which presents
in any type of formation.
[0240] Accordingly, another aspect of the present invention is
directed to a method of treating a condition characterised by the
presence of an unwanted population of cells in a mammal, said
method comprising administering to said mammal an effective number
of stem cells or T cells differentiated therefrom, as hereinbefore
defined.
[0241] In one embodiment, said condition is a neoplastic condition,
a microorganism infection (such as HIV, STD or antibiotic resistant
bacteria), or an autoimmune condition.
[0242] In another embodiment, said stem cell is an iPSC or an
HSC.
[0243] In still another embodiment, said stem cell is capable of
differentiating to a CD4.sup.+ T cell or a CD8.sup.+ T cell.
[0244] In still another embodiment, said TCR is an .alpha..beta.
TCR.
[0245] In yet still another embodiment, said stem cell such as iPSC
is derived from a T cell or thymocyte.
[0246] In still another embodiment, the cell further comprises a
nucleic acid molecule encoding a non-signalling antigen-binding
receptor, wherein said receptor comprises an antigen recognition
moiety directed to CD47.
[0247] According to these embodiments, in one particular aspect
there is provided a method of treating a neoplastic condition, said
method comprising administering to said mammal an effective number
of stem cells, or T cells differentiated therefrom, as hereinbefore
defined wherein said TCR is directed to a first tumour antigenic
determinant and said CAR is directed to one or more additional
tumour antigenic determinant(s).
[0248] In one embodiment, said first tumour antigenic determinant
is WT 1.
[0249] In another embodiment, said second tumour antigenic
determinant is TAG72.
[0250] In another embodiment, the cell further comprises a nucleic
acid molecule encoding a non-signalling antigen-binding receptor,
wherein said receptor comprises an antigen recognition moiety
directed to CD47.
[0251] In another embodiment the genetically modified stem cell
also expresses at least one homozygous HLA haplotype.
[0252] Reference to a "neoplastic condition" should be understood
as a reference to a condition characterised by the presence or
development of encapsulated or unencapsulated growths or aggregates
of neoplastic cells. Reference to a "neoplastic cell" should be
understood as a reference to a cell exhibiting abnormal growth. The
term "growth" should be understood in its broadest sense and
includes reference to enlargement of neoplastic cell size as well
as proliferation.
[0253] The phrase "abnormal growth" in this context is intended as
a reference to cell growth which, relative to normal cell growth,
exhibits one or more of an increase in individual cell size and
nuclear/cytoplasmic ratio, an increase in the rate of cell
division, an increase in the number of cell divisions, a decrease
in the length of the period of cell division, an increase in the
frequency of periods of cell division or uncontrolled proliferation
and evasion of apoptosis. Without limiting the present invention in
any way, the common medical meaning of the term "neoplasia" refers
to "new cell growth" that results as a loss of responsiveness to
normal growth controls, eg. to neoplastic cell growth. Neoplasias
include "tumours" which may be benign, pre-malignant or malignant.
The term "neoplasm" should be understood as a reference to a
lesion, tumour or other encapsulated or unencapsulated mass or
other form of growth or cellular aggregate which comprises
neoplastic cells.
[0254] The term "neoplasm", in the context of the present invention
should be understood to include reference to all types of cancerous
growths or oncogenic processes, metastatic tissues or malignantly
transformed cells, tissues or organs irrespective of
histopathologic type or state of invasiveness.
[0255] The term "carcinoma" is recognised by those skilled in the
art and refers to malignancies of epithelial or endocrine tissues
including respiratory system carcinomas, gastrointestinal system
carcinomas, genitourinary system carcinomas, testicular carcinomas,
breast carcinomas, prostate carcinomas, endocrine system carcinomas
and melanomas. The term also includes carcinosarcomas, e.g. which
include malignant tumours composed of carcinomatous and sarcomatous
tissues. An "adenocarcinoma" refers to a carcinoma derived from
glandular tissue or in which the tumour cells form recognisable
glandular structures.
[0256] The neoplastic cells comprising the neoplasm may be any cell
type, derived from any tissue, such as an epithelial or
non-epithelial cell. Reference to the terms "malignant neoplasm"
and "cancer" and "carcinoma" herein should be understood as
interchangeable.
[0257] The term "neoplasm" should be understood as a reference to a
lesion, tumour or other encapsulated or unencapsulated mass or
other form of growth or cellular aggregate which comprises
neoplastic cells. The neoplastic cells comprising the neoplasm may
be any cell type, derived from any tissue, such as an epithelial or
non-epithelial cell. Examples of neoplasms and neoplastic cells
encompassed by the present invention include, but are not limited
to central nervous system tumours, retinoblastoma, neuroblastoma,
paediatric tumours, head and neck cancers (e.g. squamous cell
cancers), breast and prostate cancers, lung cancer (both small and
non-small cell lung cancer), kidney cancers (e.g. renal cell
adenocarcinoma), oesophagogastric cancers, hepatocellular
carcinoma, pancreaticobiliary neoplasias (e.g. adenocarcinomas and
islet cell tumours), colorectal cancer, cervical and anal cancers,
uterine and other reproductive tract cancers, urinary tract cancers
(e.g. of ureter and bladder), germ cell tumours (e.g. testicular
germ cell tumours or ovarian germ cell tumours), ovarian cancer
(e.g. ovarian epithelial cancers), carcinomas of unknown primary,
human immunodeficiency associated malignancies (e.g. Kaposi's
sarcoma), lymphomas, leukemias, malignant melanomas, sarcomas,
endocrine tumours (e.g. of thyroid gland), mesothelioma and other
pleural or peritoneal tumours, neuroendocrine tumours and carcinoid
tumours.
[0258] In one particular embodiment, said neoplastic condition is a
leukaemia or lymphoma.
[0259] In another embodiment, said neoplastic condition is
metastatic.
[0260] The subject undergoing treatment or prophylaxis may be any
human or animal in need of therapeutic or prophylactic treatment.
In this regard, reference herein to "treatment" and "prophylaxis"
is to be considered in its broadest context. The term "treatment"
does not necessarily imply that a mammal is treated until total
recovery. Similarly, "prophylaxis" does not necessarily mean that
the subject will not eventually contract a disease condition.
Accordingly, treatment and prophylaxis include amelioration of the
symptoms of a particular condition or preventing or otherwise
reducing the risk of developing a particular condition. The term
"prophylaxis" may be considered as reducing the severity of the
onset of a particular condition. "Treatment" may also reduce the
severity of an existing condition.
[0261] The present invention should therefore be understood to
encompass reducing or otherwise ameliorating a condition in a
mammal. This should be understood as a reference to the reduction
or amelioration of any one or more symptoms of disease. Although it
is always most desirable to achieve the cure of a disease, there is
nevertheless significant clinical value in slowing the progression
of a disease. For example, in the context of a viral infection such
as HIV or STD, even if complete cure cannot be achieved, a
reduction in the extent of viral load and spread may provide a
means of controlling the infection such that the severe
immunodeficiency of HIV, for example, which is ultimately fatal is
not experienced and a relatively normal life span can be achieved
without the severe side effects that are characteristic of the
current anti-viral drug cocktails which patients are required to
take. In the specific context of neoplastic conditions, the T cells
of the present invention, when administered to a patient,
down-regulate the growth of a neoplasm. Reference to "growth" of a
cell or neoplasm should be understood as a reference to the
proliferation, differentiation and/or maintenance of viability of
the subject cell, while "down-regulating the growth" of a cell or
neoplasm is a reference to the process of cellular senescence or to
reducing, preventing or inhibiting the proliferation,
differentiation and/or maintenance of viability of the subject
cell. In a preferred embodiment the subject growth is proliferation
and the subject down-regulation is CD8.sup.+ T cell mediated
killing. In this regard, the killing may be evidenced either by a
reduction in the size of the tumour mass or by the inhibition of
further growth of the tumour or by a slowing in the growth of the
tumour. In this regard, and without limiting the present invention
to any one theory or mode of action, the neoplastic cells may be
killed by any suitable mechanism such as direct lysis or apoptosis
induction or some other mechanism which can be facilitated by
CD4.sup.+ or CD8.sup.+ T cells, or T cells lacking these CD4 and
CD8 markers. The present invention should therefore be understood
to encompass reducing or otherwise ameliorating a neoplastic
condition in a mammal. This should be understood as a reference to
the prevention, reduction or amelioration of any one or more
symptoms of a neoplastic condition. Symptoms can include, but are
not limited to, pain at the site of tumour growth or impaired
metabolic or physiological bodily functions due to the neoplastic
condition. It should be understood that the method of the present
invention may either reduce the severity of any one or more
symptoms or eliminate the existence of any one or more symptoms.
The method of the present invention also extends to preventing the
onset of any one or more symptoms.
[0262] Accordingly, the method of the present invention is useful
both in terms of therapy and palliation. To this end, reference to
"treatment" should be understood to encompass both therapy and
palliative care. As would be understood by the person of skill in
the art, although it is always the most desirable outcome that a
neoplastic condition is cured, there is nevertheless significant
benefit in being able to slow down or halt the progression of the
neoplasm, even if it is not fully cured. Without limiting the
present invention in any way, there are some neoplastic conditions
which, provided they are sufficiently down-regulated in terms of
cell division, will not be fatal to a patient and with which the
patient can still have a reasonable quality of life. Still further,
it should be understood that the present method provides a useful
alternative to existing treatment regimes. For example, in some
situations the therapeutic outcome of the present method may be
equivalent to chemotherapy or radiation but the benefit to the
patient is a treatment regime which induces either fewer side
effects or a shortened period of side effects and will therefore be
tolerated by the patient much better. As detailed above, it should
also be understood that the term "treatment" does not necessarily
imply that a subject is treated until total recovery. Accordingly,
as detailed above, treatment includes reducing the severity of an
existing condition or amelioration of the symptoms of a particular
condition or palliation. In this regard, where the treatment of the
present invention is applied at the time that a primary tumour is
being treated it may effectively function as a prophylactic to
prevent the onset of metastatic cancer. For example, for certain
types of solid tumours, it may still be most desirable to
surgically excise the tumour. However, there is always a risk that
the entirety of the tumour may not be successfully removed or that
there may be escape of some neoplastic cells. In this case, by
applying the method of the present invention to lyse any such
neoplastic cells, the method is effectively being applied as a
prophylactic to prevent metastatic spread.
[0263] In accordance with this aspect of the invention, the subject
cells are preferably autologous cells which are isolated and
genetically modified ex vivo and transplanted back into the
individual from which they were originally harvested. However, it
should be understood that the present invention nevertheless
extends to the use of cells derived from any other suitable source
where the subject cells exhibit a similar histocompatability
profile as the individual who is the subject of treatment, so that
the transferred cells can perform their function of removing
unwanted cells, before being subjected to immune rejection by the
host. Accordingly, such cells are effectively autologous in that
they would not result in the histocompatability problems which are
normally associated with the transplanting of cells exhibiting a
foreign MHC profile. Such cells should be understood as falling
within the definition of being histocompatible. For example, under
certain circumstances it may be desirable, necessary or of
practical significance that the subject cells are isolated from a
genetically identical twin, or from an embryo generated using
gametes derived from the subject individual or cloned from the
subject individual (in this case the cells are likely to correspond
to stem cells which have undergone directed differentiation to an
appropriate somatic cell type). The cells may also have been
engineered to exhibit the desired major histocompatability profile.
The use of such cells overcomes the difficulties which are
inherently encountered in the context of tissue and organ
transplants.
[0264] However, where it is not possible or feasible to isolate or
generate autologous or histocompatible cells, it may be necessary
to utilise allogeneic cells. "Allogeneic" cells are those which are
isolated from the same species as the subject being treated but
which exhibit a different MHC profile. Although the use of such
cells in the context of therapeutics could result in graft vs host
problems, or graft rejection by the host, this problem can
nevertheless be minimised by use of cells which exhibit an MHC
profile exhibiting similarity to that of the subject being treated,
such as a cell population which has been isolated/generated from a
relative such as a sibling, parent or child or which has otherwise
been generated in accordance with the methods exemplified
herein.
[0265] It would be appreciated that in a preferred embodiment the
cells which are used are autologous. However, due to the
circumstances of a given situation, it may not always be possible
to generate an autologous stem cell population. This may be due to
issues such as the urgency of commencing treatment or the
availability of facilities to effect transformation and directed
differentiation. In this case, and as detailed hereinbefore, it may
be desirable or necessary to use syngeneic or allogeneic cells,
such as cells which have been previously transfected and are
available as frozen stock in a cell bank. Such cells, although
allogeneic, may have been selected for transformation based on the
expression of an MHC haplotype which exhibits less immunogenicity
than some haplotypes which are known to be highly immunogenic or
which has otherwise been generated in accordance with the methods
exemplified herein.
[0266] Reference to an "effective number" means that number of
cells necessary to at least partly attain the desired effect, or to
delay the onset of, inhibit the progression of, or halt altogether
the onset or progression of the particular condition being treated.
Such amounts will depend, of course, on the particular condition
being treated, the severity of the condition and individual patient
parameters including age, physical conditions, size, weight,
physiological status, concurrent treatment, medical history and
parameters related to the disorder in issue. One skilled in the art
would be able to determine the number of cells of the present
invention that would constitute an effective dose, and the optimal
mode of administration thereof without undue experimentation, this
latter issue being further discussed hereinafter. These factors are
well known to those of ordinary skill in the art and can be
addressed with no more than routine experimentation. It is
preferred generally that a maximal cell number be used, that is,
the highest safe number according to sound medical judgement. It
will be understood by those of ordinary skill in the art, however,
that a lower cell number may be administered for medical reasons,
psychological reasons or for any other reasons.
[0267] As hereinbefore discussed, it should also be understood that
although the method of the present invention is predicated on the
introduction of genetically modified cells to an individual
suffering a condition as herein defined, it may not necessarily be
the case that every cell of the population introduced to the
individual will have acquired or will maintain the subject
modification and differentiation. For example, where a transfected
and expanded cell population is administered in total (i.e. the
successfully modified or differentiated cells are not enriched
for), there may exist a proportion of cells which have not acquired
or retained the genetic modification and/or the desired T cell
differentiation. The present invention is therefore achieved
provided that the relevant portion of the cells thereby introduced
constitute the "effective number" as defined above. However, in a
particularly preferred embodiment the population of cells which
have undergone differentiation will be subjected to the
identification of successfully modified and differentiated cells,
their selective isolation.
[0268] In the context of this aspect of the present invention, the
subject cells require introduction into the subject individual. To
this end, the cells may be introduced by any suitable method. For
example, cell suspensions may be introduced by direct injection or
inside a blood clot whereby the cells are immobilised in the clot
thereby facilitating transplantation. The cells may also be
introduced by surgical implantation. This may be necessary, for
example, where the cells exist in the form of a tissue graft. The
site of transplant may be any suitable site, for example,
subcutaneously. Without limiting the present invention to any one
theory or mode of action, where cells are administered as an
encapsulated cell suspension, the cells will coalesce into a mass.
It should also be understood that the cells may continue to divide
following transplantation. In this regard, the introduction of a
suicide gene, as hereinbefore described, provides a convenient
means of controlling ongoing division.
[0269] The cells which are administered to the patient can be
administered as single or multiple doses by any suitable route.
Preferably, and where possible, a single administration is
utilised. Administration via injection can be directed to various
regions of a tissue or organ, depending on the type of treatment
required.
[0270] In accordance with the method of the present invention,
other proteinaceous or non-proteinaceous molecules may be
co-administered with the introduction of the transfected cells. By
"co-administered" is meant simultaneous administration in the same
formulation or in different formulations via the same or different
routes or sequential administration via the same or different
routes. By "sequential" administration is meant a time difference
of from seconds, minutes, hours or days between the transplantation
of these cells and the administration of the proteinaceous or
non-proteinaceous molecules. For example, depending on the nature
of the condition being treated, it may be necessary to maintain the
patient on a course of medication to alleviate the symptoms of the
condition until such time as the transplanted cells become
integrated and fully functional (for example, the administration of
anti-viral drugs in the case of an HIV patient). Alternatively, at
the time that the condition is treated, it may be necessary to
commence the long term use of medication to prevent re-occurrence
of the condition. For example, where the subject damage was caused
by an autoimmune condition, the ongoing use of a low level of
immunosuppressive drugs may be required once the autoreactive cells
have been destroyed.
[0271] It should also be understood that the method of the present
invention can either be performed in isolation to treat the
condition in issue or it can be performed together with one or more
additional techniques designed to facilitate or augment the subject
treatment. These additional techniques may take the form of the
co-administration of other proteinaceous or non-proteinaceous
molecules or surgery, as detailed hereinbefore.
[0272] Yet another aspect of the present invention is directed to
the use of stem cells or T cells differentiated therefrom, as
hereinbefore defined in the manufacture of a medicament for the
treatment of a condition characterised by the presence of an
unwanted population of cells in a mammal.
[0273] In another embodiment, said stem cell is an iPSC or an
HSC.
[0274] In still another embodiment, said stem cell is capable of
differentiating to a CD4.sup.+ T cell or a CD8.sup.+ T cell.
[0275] In still another embodiment, said TCR is an .alpha..beta.
TCR.
[0276] In yet still another embodiment, said stem cell such as iPSC
is derived from a T cell or thymocyte, preferably a CD8.sup.+ T
cell or thymocyte.
[0277] In still another embodiment, the cell further comprises a
nucleic acid molecule encoding a non-signalling antigen-binding
receptor, wherein said receptor comprises an antigen recognition
moiety directed to CD47.
[0278] References made herein to "a cell" should be understood as
referring to an isolated cell, or an isolated or substantially
purified population of cells. In reference to a cell population, by
"substantially pure" it is meant that a relevant cell type accounts
for at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or greater
percentage of all the cells in the cell population. For example, a
cell population is substantially pure for a relevant T cell if such
T cell accounts for at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%
or greater percentage of all the cells in the cell population.
[0279] The present invention is further described by reference to
the following non-limiting examples.
EXAMPLES
[0280] The present description is further illustrated by the
following Examples which demonstrate the development of certain
embodiments of the present invention, including dual anti-cancer
specific T cells, derived from iPSC cells or HSCs. These examples
should not be construed as limiting in any way.
Example 1: Enrichment of Cancer Peptide Antigen-Specific T Cells
from Blood WT-1 Specific TCR T-Cell Stimulation and Expansion
[0281] WT-1 specific T cells are very rare in normal human blood,
but can be expanded and enriched in order to be detected. In this
context, peripheral blood mononuclear cells (PBMCs) were isolated
using Ficoll-Hypaque density gradient centrifugation. Freshly
isolated PBMCs were resuspended in tissue culture medium
supplemented with human AB serum, L-glutamine and CD28 monoclonal
antibody added to act as a co-stimulant of the T cells when WT-1 is
present; anti-CD28 alone doesn't activate T cells. PBMCs were then
stimulated with Wilm's Tumor 1 (WT-1) peptides overnight at 0.6
nmol/ml for each of the four WT-1 peptides: WT-137 (VLDFAPPGA, SEQ
ID NO: 22), WT-1126 (RMFPNAPYL, SEQ ID NO: 23), WT-1187 (SLGEQQYSV,
SEQ ID NO: 24), and WT-1235 (CMTWNQMNL, SEQ ID NO: 25), which
represent the main HLA Class I binding motifs. Data presented in
the examples of this application used WT-1 peptide 1-37 as
representative of this family of WT-1 peptides. WT-1 specific T
cells can be identified with HLA-WT-1 specific tetramers or by the
early induction of the surface molecule CD137 on stimulated but not
resting T cells. CD137 is a member of the tumor necrosis factor
(TNF) receptor family. It is also known as 4-1BB. After 24-36
hours, CD137 positive cells (that is WT-1 stimulated T cells) were
magnetically separated using a magnetic cell separator. CD137
positive (WT-1 specific TCR) cells were cultured in T cell
expansion media consisting of X-Vivo-15 base medium supplemented
with human AB-serum, recombinant interleukin 7, interleukin 15 and
interleukin 21. The corresponding CD137 negative cells were further
subjected to CD3 magnetic separation. CD3 negative cells
(predominantly B cells) were subjected to mitomycin C treatment and
used as WT-1 peptide-loaded antigen presenting feeder cells to the
induced CD137 positive population, while the remaining CD3 positive
cells (non-WT-1 specific) were grown in culture to act as a control
T cell type for the down-stream functional assays. Media with the
recombinant cytokines were replenished every second day.
[0282] For flow cytometry analysis, cells were resuspended in FACs
Buffer: 30 .mu.l per 10.sup.6 cells. 10 .mu.l of FcR blocking
reagent was added to cells for 5 mins at room temperature. 10 .mu.l
of HLA-A02 WT-1 tetramer was added and cells incubated for 20 mins
at 4.degree. C. protected from light. 50 .mu.l of "T-Cell
Activation" Cocktail was added and cells incubated for 20 mins at
4.degree. C. protected from light. 100 .mu.l of FACs Buffer was
added plus 41 of Aqua Amine and cells incubated for 5 mins, and
subsequently centrifuged at 150.times.g for 5 mins. The supernatant
was aspirated or decanted and the pellet resuspended in 100 .mu.l
of BD Cytofix/Cytoperm solution per sample and cells incubated for
20 mins at 4.degree. C. Cells were washed in BD/Perm wash.
IFN-.gamma. antibody was diluted 1/100 in BD/Perm wash solution and
incubated with cells for 30 minutes in the dark at 4.degree. C.
Cells were washed in BD/Perm wash and resuspended in FACs buffer
prior to flow cytometric analysis. FACS data acquisition was done
on a Miltenyi Quant cytometer.
[0283] T cells with a TCR specific for WT-1 peptide are normally
very low in frequency (e.g., Schmeid et al (2015)) showed they are
as few 1 per 10.sup.-6 of CD8+ cells (range 3.times.10.sup.-7 to
3.times.10.sup.-6 cells). Following the stimulation protocol
described above, WT-1 TCR specific T cells increased .about.100
fold to .about.3.0% (WT-1 patient #1 1.5%; WT-1 patient #2 4.0%;
FIG. 1).
Functional Analysis of WT-1 TCR T-Cells
[0284] The in vitro expanded T cells were additionally stimulated
with autologous antigen presenting cells (B cells transformed with
EBV) and primed with the range of WT-1 peptides: WT-137
(VLDFAPPGA), WT-1126 (RMFPNAPYL), WT-1187 (SLGEQQYSV), and WT-1235
(CMTWNQMNL). The T cells were examined by flow cytometry for
interferon gamma (IFN.gamma.) production using the fluorescent bead
assay. Cells were double labelled for WT-1 peptide specificity via
binding to a WT-1 peptide-HLA tetramer (see FIG. 2).
[0285] The WT-1 stimulated T cells clearly expressed (80-90%)
interferon gamma (IFN.gamma.) (FIG. 2), a well recognised measure
of T cell function (e.g., Ghanekar et al (2001)). To potentially
increase the level of CD8 T cell activation (targeting WT-1 T
cells), use was made of the LAG 3 inhibitor IMP 321. LAG3 is
normally a "check point blockade", inhibiting the stimulatory
function of dendritic cells (DC) and the response to DC's as
antigen presenting cells, by CD8 T cells. When added to the WT-1
specific T cell activation assay, there was no effect of IMP 321 at
24 hours, but after 4 days there was a doubling of the rare
CD8+WT-1 specific TCR T cells (FIG. 2H).
Example 2: Generation of iPSC from Human Blood T-Cells
[0286] For derivation of iPSC from human blood T cells, there are a
number of approaches with varying levels of faithful retention of
the original T cell properties. iPSC have been produced from a
broad repertoire of peripheral blood T lymphocyte pool (T-iPSC)
from a normal healthy human. The T-cells were pre-activated, for
example, with the mitogen PHA or anti CD3 and anti CD28 antibodies.
Using dual retroviral vector cassettes each containing two of the
Yamanaka reprogramming factors (Oct4, Sox 2, KLF, cMyc), multiple
T-iPSC clones were generated which were validated at the cellular
and molecular level, including flow cytometry and qRT-PCR for a
range of markers including Nanog, Oct3/4, SSEA 3,4, TRA-1-60 and
TRA-1-81. Their pluripotency was confirmed by teratoma formation
after injection into NOD-SCID-IL common gamma chain -/- (NSGMice).
Confirmation of T cell origin was confirmed by showing that the TCR
genes were rearranged.
[0287] The production of iPSC from WT-1 specific blood T cells is
summarised in FIG. 3.
Example 3: Induction of Human T Cells from iPSC
[0288] This Example shows generation of genuine T cells from iPSC.
These T cells were shown to express the key features of typical T
cells as normally produced by the thymus. They were shown to
express the mainstream T cell .alpha..beta.TCR and CD8 with both
.beta. and .alpha. chains.
[0289] T cells have been induced from iPSC derived from whole adult
blood T cells or pre-selected CD8+ T cells, or antigen specific T
cells (e.g., those specific for WT-1) (T-iPSC), or adult
fibroblasts. There are two basic stages specialisation to
haemopoiesis (haemopoietic stem cells or "HSC") and partially
lymphoid lineage, by culture on OP9 cells; transfer of these
cultured cells to OP cell line genetically modified to express
Notch signalling molecule Delta-like Ligand 1 (OP9-DL-L1) for the
subsequent induction of T cell differentiation.
Phase 1--Preparation of OP9 Support Cells and iPSC Colonies
[0290] Day -8: Mitomycin treated Mouse embryonic fibroblast feeder
layers were plated onto 0.1% gelatin coated TC plates at
0.3.times.10.sup.6 (14,250 cells/cm.sup.2) in 3 mL of MEF media
(DMEM+15% FCS+1% pen/strep L-glutamine), and incubated overnight.
OP9 cells were pre-prepared by plating onto 0.1% gelatin coated 10
cm TC plates at 0.25.times.10.sup.6 cells in 11 mL OP9 media
(.alpha.MEM+20% FCS+1% pen/strep).
[0291] Day -7: iPS cells were thawed and plated onto the MEF cells,
and incubated at 37.degree. C. 5% CO.sub.2 for 7 days.
Phase 2--Conversion of iPSCs to Haemopoietic Cells
[0292] Day 0: Start of haemopoietic specialisation. The iPS
colonies were dissociated and plated onto OP9 for HSC
differentiation. The colony suspension was added dropwise for even
distribution onto the OP9 plate. Fresh differentiation medium was
added on days 1, 5 and 9.
[0293] Day 13 Harvest Induced HSC Precursors for T-Cell
Differentiation
[0294] Cells cultured on the OP9 cell line were gently removed by
Collagenase (working solution 100 .mu.g/mL collagenase/HBSS;
37.degree. C. for 1:15 hrs) and the colonies further disrupted into
single cells by trypsin/EDTA 0.05% at 37.degree. C. for 30 mins.
The cells were gently washed and examined by phase Contrast
microscopy (FIG. 4) and by flow cytometry (FIG. 5). The
haemopoietic nature of the cells was confirmed by flow cytometry
(FIG. 5).
Phase 3--Induction of iPSC-Derived HSC to T Cells
[0295] Day 13: Induction of T Cell Differentiation: Transfer of Day
13 OP9 Conditioned (Haemopoiesis Induced) Cells to OP9DL-L1
Cells.
[0296] In a preferred embodiment, to enhance the efficiency of
contact with the OP9DL-L1 cells, the OP9 conditioned cells were
purified for CD34.sup.+CD43+(HSC) and then plated onto the OP9
DLL-1 cells for the first stage of T cell differentiation. A
critical component of the process disclosed herein was to collect
the cells which initially grew underneath the OP9 DL-L1 cells.
[0297] Cells collected from the OP9 cultures were resuspended in T
cell differentiation culture medium (OP9 media, SCF 5 ng/mL, Flt3 5
ng/mL, IL-7 5 ng/mL & Vitamin C 100 .mu.M) and the suspension
was added dropwise to the OP9 DLL-1 cells and incubated at
37.degree. C. Cells were harvested after 2, 9, 16, 23 and 30 days
culture on the OP9 DL-L1 and subjected to flow cytometry analysis
(FIG. 6 and FIG. 7).
[0298] When these cultures were examined for T cell development
there was clear evidence of expression of the early markers CD7 and
CD9 and the next markers of T cell development with CD4 and CD8
expression (FIG. 7). Even at this early stage there was already
.about.10% of the cells expressing both CD4+ and CD8+; these
CD4+CD8+ cells are characteristic of T cells which develop normally
in the thymus cortex (Heng et al (2010)).
[0299] Flow cytometry showed progressive development of T cells
from the initial expression of CD5, CD7+ then CD8+. Most
importantly the induced T cells expressed the phenotype of
"optimal, thymus produced" CD8 T cells. They expressed the
CD8.beta. chain in addition to the CD8.alpha. chain (other reported
T cell induction systems do not induce the optimal, signalling
CD8.beta. chain; e.g., Themeli et al (2013)). As indications of
function they also expressed CD3 with the .alpha..beta.TCR.
Furthermore, these cells were present as early as Day 16 of culture
on OP9 DL-L1 cells compared to day 30 in other reported
systems.
Phase 4 Development of Mature T Cells
[0300] After a further 7 days (a total of 13 days on OP9 cells
followed by 16 days on OP DL-L1 cells), these developing T cells
made a critical transition to expression of the T cell receptor
complex with CD8+ T cells clearly positive for CD3 and the
.alpha..beta.TCR; in addition, these cells expressed the important
CD8.beta.--these are the desired cells for CAR-T. There was a
corresponding further reduction in CD34+CD43+ HSC (FIG. 8).
[0301] This induction system has thus successfully produced mature
CD8 T cells from iPSC after 13 days culture on OP9 cells followed
by 16 days on OP9DL-L1 cells.
[0302] Using the process described above, T cells expressing a TCR
specific for WT-1 were produced from iPSC which were themselves
derived from WT-1 TCR CD8+T cells (FIG. 9). These iPSC derived WT-1
T cells had a cytotoxic function equivalent to the original T cells
from which the iPSC were derived (FIG. 10).
Example 4: Development of CAR Constructs
[0303] A component of Chimeric Antigen Receptor (CAR)-T cells is
the antigen recognition component of the CAR mediated by the scFv
ectodomain, represented by a single-chain Fv (scFv) anchored by a
CD8 or CD28 hinge and including a transmembrane (TM) region and the
signal transduction of the CAR via the cytoplasmic
endodomain--represented by CD28, 4-1BB and the CD3 zeta (CD3.zeta.)
chain. There are also two suitable viral delivery
systems--retrovirus and lentivirus. Exemplary CAR and CD47-binding
receptor constructs are shown in FIG. 11.
Example 5: Chimeric Antigen Receptor Vector Cloning Strategies
[0304] Exemplary chimeric antigen receptor vector cloning
strategies are illustrated in FIGS. 12-13. FIG. 14 shows our
2.sup.nd generation CAR and the strategy for a non-signalling
anti-CD47 construct. Exemplary sequences of chimeric antigen
receptors, non-signalling antigen-binding receptors, and the
various domains thereof, are provided in SEQ ID NOS: 1-20.
Example 6: Chimeric Antigen Receptor Transduction of T Cells
Lentivirus Production
[0305] 293T cells were plated onto poly-L-lysine (Sigma) coated 175
cm.sup.2 flasks. Two hours prior to transfection, medium was
replaced with DMEM supplemented with 10% FCS. The lentiviral
transfer vector DNA, together with packaging and envelope plasmid
DNA were combined and mixed with Lipofectamine2000. The solution
was briefly vortexed and incubated at room temperature for 30 min.
Following this, the solution was mixed again and then added
dropwise to the cells. Flasks were returned to the incubator. Six
hours later, fresh growth medium added. Viral supernatant was
collected after 48 hrs and cleared by centrifugation at 1500 rpm
for 5 min at 4.degree. C. then passed through a 0.45 .mu.m pore
PVDF Millex-HV filter (Millipore). Concentration of lentivirus
using ultracentrifugation was performed with a Sorval Discovery 100
SE centrifuge using an AH-629 rotor. 30 mL of filtered virus
supernatant was added to 36 mL polyallomer conical tubes (Beckman).
Centrifugation was performed for 90 min at 20,000 g. Supernatant
was completely removed and virus pellets resuspended in 300 .mu.L
PBS and stored at -80.degree. C. until use.
Generation of CAR-T Cells
[0306] FIG. 11 and SEQ ID NOS: 1-6 show a panel of Chimeric Antigen
Receptor (CAR) and CD47-binding receptor constructs that have been
developed--with scFv specific for either TAG 72 or CD19 (as a
positive control). These constructs use either human CD8 or CD28 as
hinge region and CD28, CD3.zeta. chain or 4-1BB cytoplasmic
activation signalling domains. CAR and CD47-binding receptor
constructs are cloned into lentiviral vectors as described in the
previous paragraph.
[0307] Optimal lentiviral transduction of T cells involves their
activation at the TCR and co-stimulatory receptors. Accordingly, on
day 0, fresh PBMC were collected by apheresis from healthy donors,
were enriched for activated T cells with the use of anti-CD3 and
anti-CD28 antibodies bound to paramagnetic beads (Dynabeads
ClinExVivo CD3/CD28, Invitrogen, Camarillo, Calif., USA) at a ratio
of 3:1 (beads:cells). The cells and beads were co-incubated for 1 h
at room temperature, andCD3+ cell enrichment was performed with the
use of magnet (Invitrogen). Cells in the CD3+ fraction were
resuspended in initiation media at a concentration of
1.times.10.sup.6 cells/ml in T cell expansion medium with 100 IU/ml
IL-2. On day 1, RetroNectin was used to coat cell culture dishes at
a concentration of 2 mg/cm2 in a solution of 10 mg/mL in PBS
overnight at 4.degree. C. On day 2, the RetroNectin solution was
aspirated and the same volume of blocking solution, consisting of
0.5% human serum albumin in PBS, was added to each bag and
incubated at room temperature for 30 min. The blocking solution was
aspirated, and each bag washed with PBS. Lentiviral supernatant was
rapidly thawed and added to each dish with T cell expansion medium
with 300 IU/ml IL-2. The cultures were placed back into the
incubator and left for at least 24 h. On day 4, the transduction
was stopped; cells were resuspended in fresh T-cell expansion
medium at a concentration of 0.5-1.times.10.sup.6 cells/mL. The
cultures were maintained until day 14 and fed every other day with
fresh expansion media to maintain cell concentration at
1.times.10.sup.6 cells/mL.
[0308] Initially blood derived human T cells were subjected to CAR
transduction, the success of which was measured by flow cytometry,
depicting the eGFP+ cells (FIG. 15). This was also confirmed by
Western Blot analysis (FIG. 16).
Assessing the Functionality of the CAR-T Cells
[0309] The TAG-72 CAR-T cells (created from normal PBMC) were
examined for their ability to kill TAG72 expressing target cancer
cells in vitro. The real time cell monitoring system (xCELLigence)
was employed to determine the killing efficiency of CAR-T cells in
vitro. 10,000-2.times.10.sup.6 target cells/1004, (for example the
TAG72+ ovarian cancer cell line CaOV4) were deposited into RTCA
plates. In some instances, tethering of target cells by anti-hCD40
or by human fibronectin pre-coating of the plate may be required.
Target cells are maintained at 37.degree. C., 5% CO.sub.2 for 3-12
h to allow for cellular attachment. Following attachment of target
cells, CAR-T effector cells were added at variable effector:target
ratios (ranging from 1:1 to 10:1). In some experiments, CAR-T
effector cells were isolated based of GFP expression of CAR-T cells
via FACS prior to use. Co-cultures were maintained in optimal
growth conditions for at least 12 h. Cellular impedance was
monitored throughout; a decrease in impedance is indicative of cell
detachment and ultimately cell death.
[0310] FIG. 17 shows results from this experiment monitored over 40
hours. The ovarian cancer cell line CaOV4 grew consistently over
this time period (blue line). In contrast, cultures supplemented
with TAG-72 specific CAR T cells showed an initial growth phase
that was significantly less than that of target cells alone,
followed by gradual elimination of the target cells over time
(purple line). To overcome the non-specific killing due to CD3/CD28
activation, the TAG 72 CAR-T cells were isolated by flow cytometry
and compared to CD19 CAR-T cells and non-CAR-T cells--without prior
CD3/CD28 activation (FIG. 21). The data shown in FIG. 21 indicate
strong antigen specificity of TAG-72 CAR-T cells in the first 24
hours of culture with TAG-72 expressing cancer cells, since
negative controls of vector only transfected T cells and
non-transfected T cells showed no killing of the cancer cells in
this time frame.
[0311] The above studies were performed on polyclonal T cells
derived from peripheral blood. To demonstrate CAR-transduction of
mono-specific T cells expressing a TCR specific for a nominal
cancer peptide antigen, WT-1 TCR specific T cells derived from iPSC
formed from WT-1 specific TCR, were transduced by TAG 72 CAR
lentivirus. FIG. 22A shows successful CAR transduction of these
WT-1 specific TCR CD8+ T cells themselves derived from iPSC
produced from WT-1 specific T cells. The CAR contained the
specificity for TAG 72. Most importantly, FIG. 22B shows the
successful transduction of WT-1 specific TCR CD8+ T cells
themselves derived from iPSC produced from WT-1 specific T cells,
with a CAR construct for both TAG 72 and CD47. This indicates that
T cells can be produced with three specificities for cancer: WT-1
(TCR), TAG 72 (CAR) and CD47 (truncated, CD47-binding
receptor).
[0312] The results demonstrate the development of dual specific
CAR-transduced cancer specific TCR (WT-1) derived from iPSCs, which
were themselves derived from WT-1 specific TCR T cells from normal
adult blood.
[0313] FIG. 20 shows that both components of the dual specific T
cells (containing the WT-1 TCR and the TAG72 CAR) can contribute to
the killing of cancer cells. When corrected for spontaneous cell
death, even at low effector-target ratio (here it is 2 effectors to
1 target cell) the WT-1 cells caused approximately 10% cell killing
and then addition of the TAG72 CAR by transduction caused an
additional 10% killing.
Example 7: Chimeric Antigen Receptor Transduction of iPSC
[0314] The production of multi-specific CAR-T cells can be achieved
by multiple approaches including CAR transduction of existing blood
T cells (FIG. 15) or by transduction of iPSC which are then induced
to T cells (expressing cancer specific TCR and the CAR's) (e.g.,
SEQ ID NOS: 1-6). Multiple iPSC lines have been used to progress
with CAR-T transduction. These iPSC could be derived from non-T
cells, or from cancer antigen specific T cells (e.g., WT-1) which
would retain the TCR gene rearrangements. These iPSC were either
derived from adult fibroblasts or from T cells with an endogenous
TCR specific for a specific cancer antigen (WT-1 peptide).
[0315] The iPSC were stably transduced with a single cistron, as
shown in FIG. 14 in which the CAR ectodomain comprises an scFv
specific for TAG72 (or as control CD19). The hinge (Stalk) region
and the transmembrane region are derived from CD28 or CD8 and the
cytoplasmic endodomain, which comprises T cell signal transduction
domains, is derived from CD28 and TCR chain. The CAR has a
C-terminal extension encoding EGFP linked by a P2A self-cleaving
polypeptide to separate the CAR and reporter. Following viral
integration, the P2A was cleaved and the success of transduction
was quantified by measuring the fluorescent of the released EGFP
reporter. GFP fluorescence illuminates the success of transduction.
It can be used to show transduction in situ (FIG. 21) or to
identify and isolate CAR transduced iPSC via flow cytometry (FIG.
22, 23).
[0316] These studies clearly show the ability to transduce iPSC
with lenti-virus CAR constructs. FIG. 21A shows the successful
transduction of iPSC derived from human fibroblasts with a CAR
encoding TAG 72 or CD19 (FIG. 21A). FIG. 21B shows the successful
transduction of iPSC derived from WT-1 TCR specific T cells, with
TAG72. Any T cell derived from this line will thus express dual
anti-cancer specificity (WT-1 via TCR; TAG 72 via CAR).
[0317] It is also possible to isolate the transduced iPSC by
fluorescent-based cell sorting. The positive cells can be collected
and replated to successfully form (CAR transduced) iPSC colonies
(FIG. 24).
[0318] Those skilled in the art will appreciate that the invention
described herein is susceptible to variations and modifications
other than those specifically described. It is to be understood
that the invention includes all such variations and modifications.
The invention also includes all of the steps, features,
compositions and compounds referred to or indicated in this
specification, individually or collectively, and any and all
combinations of any two or more of said steps or features.
BIBLIOGRAPHY
[0319] Balasubramanian S, Babai N, Chaudhuri A, Qiu F, Bhattacharya
S, Dave B J, Parameswaran S, Carson S D, Thoreson W B, Sharp J G,
et al. (2009) Non cell-autonomous reprogramming of adult ocular
progenitors: generation of pluripotent stem cells without oxogenous
transcription factors. Stem Cells; 27:3053-3062 [PubMed: 19859985]
[0320] Brignone, C., C. Grygar, M. Marcu, K. Schakel, and F.
Triebel. 2007. A soluble form of lymphocyte activation gene-3
(IMP321) induces activation of a large range of human effector
cytotoxic cells. J Immunol 179:4202. [0321] Casucci M, Hawkins R E,
Dotti G, Bondanza A. (2015). Overcoming the toxicity hurdles of
genetically targeted T cells. Cancer Immunol Immunother.;
64(1):123-30 [0322] Chuo B K, Mali P, Huang X, Ye Z, Dowey S N,
Resar L M, Zou C, Zhang Y A, Tong J, Cheng L. (2011) Efficient
human iPS cell derivation by a non-integrating plasmid from blood
cells with unique epigenetic and gene expression signatures. Cell
Res.; 21: 518-529 [PubMed: 21243013] [0323] Corrigan-Curay J, Kiem
H P, Baltimore D, O'Reilly M, et al, Kohn D B. (2014). T-cell
immunology: looking forward. Mol Ther.; 22(9):1564-74 [0324] Curran
K J, Pegram H J, Brentj ens R J. (2012). Chimeric antigen receptors
for T cell immunotherapy: current understanding and future
directions. J Gene Med.; 14(6):405-15 [0325] Curran K J, Seinstra B
A, Nikhamin Y, Yeh R et al Brentj ens R J (2015) Enhancing
Antitumor Efficacy of Chimeric Antigen Receptor T Cells Through
Constitutive CD40L Expression. Mol Ther.; Jan. 13. doi: 10.
1038/mt. 2015. 4. [Epub ahead of print] [0326] Davila M L, Riviere
I, Wang X, Bartido S, et al Brentj ens R. (2014). Efficacy and
toxicity management of 19-28z CAR T cell therapy in B cell acute
lymphoblastic leukemia. Sci Transl Med.; 6(224):224 [0327] Dotti G,
Gottschalk S, Savoldo B, Brenner M K. (2014). Design and
development of therapies using chimeric antigen receptor-expressing
T cells. Immunol Rev.; 257(1):107-26 [0328] Esteban M A, Wang T,
Qin B, Yang J, Qin D, Cai J, Li W, Weng Z, Chen J, Ni S, et al.
(2010) Vitamin C enhances the generation of mouse and human induced
pluripotent stem cells. Cell Stem Cell.; 6:71-79 [PubMed: 20036631]
[0329] Fedorov V D, Sadelain M, Kloss C C. (2014). Novel approaches
to enhance the specificity and safety of engineered T cells. Cancer
J; 20(2):160-53. [0330] Fletcher A L, Calder A, Hince M N, Boyd R
L, Chidgey A P. (2011). The contribution of thymic stromal
abnormalities to autoimmune disease. Crit Rev Immunol; 7(12):954-63
[0331] Ghanekar S A, Nomura L E, Suni M A, Picker L J, Maecker H T,
Maino V C. (2001). Gamma interferon expression in CD8(+) T cells is
a marker for circulating cytotoxic T lymphocytes that recognize an
HLA A2-restricted epitope of human cytomegalovirus phosphoprotein
pp65. Clin Diagn Lab Immunol.8(3):628-31 [0332] Gargett, T, Brown M
P. (2014). He inducible caspase 9 suicide gene systems as a `safety
switch" to limit on-target, off-tumourtoxicitiesof chimeric antigen
receptorT cells. Front. Pharmacol 28:5:235 [0333] Ghosh et al.,
1991 Glycobiology 5: 505-10 [0334] Han E Q, Li X L, Wang C R, Li T
F, Han S Y. (2013). Chimeric antigen receptor-engineered T cells
for cancer immunotherapy: progress and challenges. J Hematol
Oncol.; 8; 6:47 [0335] Heng, T. S. P, Chidgey, A. P., and Boyd, R.
L., (2010) Getting back at nature: understanding thymic development
and overcoming its atrophy. Curr Opin Pharmacol, 10: 425-433 [0336]
Huangfu D, Osafune K, Maehr R, Guo W, Eijkelenboom A, Chen S,
Melton D A. (2008) Induction of pluripotent stem cells from primary
human fibroblasts with only Oct4 and Sox2. Nat Biotechnol.;
26:1269-1275 [PubMed: 18849973] [0337] Ichida J K, "Blanchard J,
Lam K, Son E Y, Chung J E, Egli D, Loh K M, Carter A C, DiGiorgio F
P, Kiszka K, et al. (2009) A small-molecule inhibitor of TGF-.beta.
signalling replaces Sox2 in reprogramming by inducing nanog. Cell
Stem Cell; 5:491-503 [PubMed: 19818703] [0338] Kaji K, Norrby K,
Paca A, Mileikovsky M, Mohseni P, Woltjen K. (2009) Virus-free
induction of pluripotency and subsequent excision of reprogramming
factors. Nature. 458:771-775 [0339] Lin T, Ambasudhan R, Yuan X, Li
W, Hilcove S, Aburjarour R, Lin X, Hahm H S, Hao E, Hayek, A, Ding
S. (2009) A chemical platform for improved induction of human
iPSCs. Nat Methods; 6:805-808 [PubMed: 19838168] [0340] Liu J et
al. (PLOS One (2015) September 21;10(9):e0137345) [0341] Mali P,
Chuo B K, Yen J, Ye Z, Zou J, Dowey S, Brodsky R A, Ohm J E, Yu W,
Baylin S B, et al. (2010) Butyrate greatly enhances derivation of
human induced pluripotent stem cells by promoting epigenetic
remodeling and the expression of pluripotency-associated genes.
Stem Cells; 28:713-720. [PubMed: 20201064] [0342] Narsihn K H, Jia
F, Robbins R C, Kay M A, Longaker M T, Wu J C. (2011) Generation of
adult human induced pluripotent stem cells sing nonviral minicircle
DNA vectors. Nature Protoc.; 6:78-88. [PubMed: 21212777] [0343]
Noggle S, Fung H-L, Gore A, Martinez H, Satriani K S, Prosser R,
Oum K, Paull D, Druckenmiller S, Freeby M, et al. (2011) Human
oocytes reprogram somatic cells to a pluripotent state. Nature;
478:70-75 [PubMed: 21979046] [0344] Pappas D J, Gourraud, R-A, Le
Gall C, Laurent J, Trounson A, DeWitt N and Talib S. (2015)
Proceedings: Human Leukocyte Antigen Haplo-Homozygous Induced
Pluripotent Stem Cell Haplobank Modeled After the California
Population: Evaluating Matching in a Multiethnic and Admixed
Population. Stem Cells Translational Medicine 4:413-418 [0345]
Perna S K, Savoldo B, Dotti G (2014). Genetic modification of
cytotoxic T lymphocytes to express cytokine receptors. Methods Mol
Biol.; 1139:189-200 [0346] "Sambrook et al. (2001, Molecular
Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New
York) [0347] Schmied, S, Gostsick E, Price D A, Abken H,
Assenmacher M, Richter A. (2015). Analysis of the functional WT1
specific T-cell repertoire in healthy donors reveals a discrepancy
between CD4+ and C D*+ memory formation. Immunology 145: 558-569
[0348] Subramanyam D, Lamouille S, Judson R L, Liu Jy, Bucay N,
Derynck R, Blelloc R. (2011)Multiple targets of miR-302 and miS-372
promote reprogramming of human fibroblasts to induced pluripotent
stem cells. Nature Biotechnol.; 29:443-448. [PubMed: 21490602]
[0349] Themeli M, Kloss C C, Ciriello G et al M, Sadelain M (2013)
Nat Biotechnol 31(10):928-33 [0350] Ui-Tei et al, 2000 FEBS Letters
479: 79-82 [0351] Warren L, Manos P D, Ahfeldt, Loh Y H, Li H, Lau
F, Ebina W, Mandal P K, Smith Z D, Meissner A, et al. (2010) Highly
efficient reprogramming to pluripotency and directed
differentiation of human cells with synthetic modified mRNA. Cell
Stem Cell; 7:618-630. [PubMed: 20888316] [0352] Woltjen K, Michael
I P, Mohseni P, Desai R, Mileikovsky M, Hamalainen R, Cowling R,
Wang W, Liu P, Gertsenstein M, et al. (2009) piggy Bac
transposition reprograms fibroblasts to induced pluripotent stem
cells. Nature; 458:766-770. [PubMed: 19252478] [0353] Yoshida Y,
Taskahashi K, Okita K, Ichisaka T, Yamanaka S. (2009) Hypoxia
enhances the generation of induced pluripotent stem cells. Cell
Stem Cell; 5:237-241 [PubMed: 19716359] [0354] Zhu S, Li w, Zhou H,
Wei W, Ambasudhan R, Lin T, Kim J, Zhang K, King S. (2010)
Reprogramming of human primary somatic cells by OCT4 and chemical
compounds. Cell Stem Cell; 7:651-655 [PubMed:21112560] [0355] U.S.
Pat. No. 5,350,674 [0356] U.S. Pat. No. 5,585,362 [0357] WO
2001/96584 [0358] WO 2001/29058 [0359] U.S. Pat. No. 6,326,193
[0360] U.S. patent application Ser. No. 14/656,431, published as US
20150183874 A1.
Sequence CWU 1
1
2512670DNAArtificial SequenceDNA coding for TAG72 CAR-P2A-CD47
binding receptor 1atggagttcg gcctgcgctg ggtgttcctg gtggccatcc
tgaaggacgt gcagtgccag 60gtgcagctgc agcagagcga cgccgagctg gtgaagcccg
gcgccagcgt gaagatcagc 120tgcaaggcca gcggctacac cttcaccgac
cacgccatcc actgggtgaa gcagaacccc 180gagcagggcc tggagtggat
cggctacttc agccccggca acgacgactt caagtacaac 240gagcgcttca
agggcaaggc caccctgacc gccgacaaga gcagcagcac cgcctacctg
300cagctgaaca gcctgaccag cgaggacagc gccgtgtact tctgcacccg
cagcctgaac 360atggcctact ggggccaggg caccagcgtg accgtgagca
gcggcggcgg cggcagcggc 420ggcggcggca gcggcggcgg cggcagcgac
atcgtgatga cccagagccc cagcagcctg 480cccgtgagcg tgggcgagaa
ggtgaccctg agctgcaaga gcagccagag cctgctgtac 540agcggcaacc
agaagaacta cctggcctgg taccagcaga agcccggcca gagccccaag
600ctcctgatct actgggccag cacccgcgag agcggcgtgc ccgaccgctt
caccggcagc 660ggcagcggca ccgacttcac cctgagcatc agcagcgtgg
agaccgagga cctggccgtg 720tactactgcc agcagtacta cagctacccc
ctgaccttcg gcgccggcac caagctggtg 780ctgaagcgcg actacaagga
cgacgacgac aagctgagca actccatcat gtacttcagc 840cacttcgtgc
cggtcttcct gccagcgaag cccaccacga cgccagcgcc gcgaccacca
900acaccggcgc ccaccatcgc gtcgcagccc ctgtccctgc gcccagaggc
gtgccggcca 960gcggcggggg gcgcagtgca cacgaggggg ctgttttggg
tgctggtggt ggttggtgga 1020gtcctggctt gctatagctt gctagtaaca
gtggccttta ttattttctg ggtgaggagt 1080aagaggagca ggctcctgca
cagtgactac atgaacatga ctccccgccg ccccgggccc 1140acccgcaagc
attaccagcc ctatgcccca ccacgcgact tcgcagccta tcgctccaga
1200gtgaagttca gcaggagcgc agacgccccc gcgtaccagc agggccagaa
ccagctctat 1260aacgagctca atctaggacg aagagaggag tacgatgttt
tggacaagag acgtggccgg 1320gaccctgaga tggggggaaa gccgagaagg
aagaaccctc aggaaggcct gtacaatgaa 1380ctgcagaaag ataagatggc
ggaggcctac agtgagattg ggatgaaagg cgagcgccgg 1440aggggcaagg
ggcacgatgg cctttaccag ggtctcagta cagccaccaa ggacacctac
1500gacgcccttc acatgcaggc cctgccccct cgcgtcgacg gaagcggagc
tactaacttc 1560agcctgctga agcaggctgg agacgtggag gagaaccctg
gacctatggc cctgcccgtg 1620accgccctgc tgctgcccct ggccctgctg
ctgcacgccg cccgcgccca ggtgcagctg 1680gtgcagagcg gcgccgaggt
gaagaagccc ggcgccagcg tgaaggtgag ctgcaaggcc 1740agcggctaca
ccttcaccaa ctacaacatg cactgggtgc gccaggcccc cggccagcgc
1800ctggagtgga tgggcaccat ctaccccggc aacgacgaca ccagctacaa
ccagaagttc 1860aaggaccgcg tgaccatcac cgccgacacc agcgccagca
ccgcctacat ggagctgagc 1920agcctgcgca gcgaggacac cgccgtgtac
tactgcgccc gcggcggcta ccgcgccatg 1980gactactggg gccagggcac
cctggtgacc gtgagcagcg gcggaggcgg aagcggaggc 2040ggaggcagcg
ggggcggcgg aagcgacatc gtgatgaccc agagccccct gagcctgccc
2100gtgacccccg gcgagcccgc cagcatcagc tgccgcagca gccagagcat
cgtgtacagc 2160aacggcaaca cctacctggg ctggtacctg cagaagcccg
gccagagccc ccagctgctg 2220atctacaagg tgagcaaccg cttcagcggc
gtgcccgacc gcttcagcgg cagcggcagc 2280ggcaccgact tcaccctgaa
gatcagccgc gtggaggccg aggacgtggg cgtgtactac 2340tgcttccagg
gcagccacgt gccctacacc ttcggccagg gcaccaagct ggagatcaag
2400gccgccgagc agaagctgat cagcgaggag gacctgatcg aggtgatgta
cccccccccc 2460tacctggaca acgagaagag caacggcacc atcatccacg
tgaagggcaa gcacctgtgc 2520cccagccccc tgttccccgg ccccagcaag
cccttctggg tgctggtggt ggtgggcggc 2580gtgctggcct gctacagcct
gctggtgacc gtggccttca tcatcttctg ggtgcgcagc 2640aagcgcagcc
gcctgctgca cagcgactaa 26702889PRTArtificial SequenceProtein
sequence of TAG72 CAR-P2A-CD47 binding
receptormisc_feature(1)..(19)Kappa
leadermisc_feature(20)..(134)anti-TAG72
VHmisc_feature(135)..(149)GS
Linkermisc_feature(150)..(263)anti-TAG72
VLmisc_feature(264)..(272)FLAG epitopemisc_feature(273)..(331)CD8
hingemisc_feature(332)..(358)CD28 TMmisc_feature(359)..(399)CD28
Signalling domainmisc_feature(400)..(513)TCR3-zeta Signalling
domainmisc_feature(514)..(535)P2A self cleaving
peptidemisc_feature(536)..(556)CD8a
leadermisc_feature(557)..(673)anti-CD47
VHmisc_feature(674)..(688)GS
linkermisc_feature(689)..(800)anti-CD47
VLmisc_feature(801)..(812)c-Myc epitopemisc_feature(813)..(851)CD28
hingemisc_feature(852)..(878)CD28
TMmisc_feature(879)..(889)cytoplasmic tail derived from CD28 2Met
Glu Phe Gly Leu Arg Trp Val Phe Leu Val Ala Ile Leu Lys Asp1 5 10
15Val Gln Cys Gln Val Gln Leu Gln Gln Ser Asp Ala Glu Leu Val Lys
20 25 30Pro Gly Ala Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr
Phe 35 40 45Thr Asp His Ala Ile His Trp Val Lys Gln Asn Pro Glu Gln
Gly Leu 50 55 60Glu Trp Ile Gly Tyr Phe Ser Pro Gly Asn Asp Asp Phe
Lys Tyr Asn65 70 75 80Glu Arg Phe Lys Gly Lys Ala Thr Leu Thr Ala
Asp Lys Ser Ser Ser 85 90 95Thr Ala Tyr Leu Gln Leu Asn Ser Leu Thr
Ser Glu Asp Ser Ala Val 100 105 110Tyr Phe Cys Thr Arg Ser Leu Asn
Met Ala Tyr Trp Gly Gln Gly Thr 115 120 125Ser Val Thr Val Ser Ser
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 130 135 140Gly Gly Gly Gly
Ser Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu145 150 155 160Pro
Val Ser Val Gly Glu Lys Val Thr Leu Ser Cys Lys Ser Ser Gln 165 170
175Ser Leu Leu Tyr Ser Gly Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gln
180 185 190Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile Tyr Trp Ala
Ser Thr 195 200 205Arg Glu Ser Gly Val Pro Asp Arg Phe Thr Gly Ser
Gly Ser Gly Thr 210 215 220Asp Phe Thr Leu Ser Ile Ser Ser Val Glu
Thr Glu Asp Leu Ala Val225 230 235 240Tyr Tyr Cys Gln Gln Tyr Tyr
Ser Tyr Pro Leu Thr Phe Gly Ala Gly 245 250 255Thr Lys Leu Val Leu
Lys Arg Asp Tyr Lys Asp Asp Asp Asp Lys Leu 260 265 270Ser Asn Ser
Ile Met Tyr Phe Ser His Phe Val Pro Val Phe Leu Pro 275 280 285Ala
Lys Pro Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro 290 295
300Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg
Pro305 310 315 320Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Phe
Trp Val Leu Val 325 330 335Val Val Gly Gly Val Leu Ala Cys Tyr Ser
Leu Leu Val Thr Val Ala 340 345 350Phe Ile Ile Phe Trp Val Arg Ser
Lys Arg Ser Arg Leu Leu His Ser 355 360 365Asp Tyr Met Asn Met Thr
Pro Arg Arg Pro Gly Pro Thr Arg Lys His 370 375 380Tyr Gln Pro Tyr
Ala Pro Pro Arg Asp Phe Ala Ala Tyr Arg Ser Arg385 390 395 400Val
Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln 405 410
415Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp
420 425 430Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly
Lys Pro 435 440 445Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu
Leu Gln Lys Asp 450 455 460Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly
Met Lys Gly Glu Arg Arg465 470 475 480Arg Gly Lys Gly His Asp Gly
Leu Tyr Gln Gly Leu Ser Thr Ala Thr 485 490 495Lys Asp Thr Tyr Asp
Ala Leu His Met Gln Ala Leu Pro Pro Arg Val 500 505 510Asp Gly Ser
Gly Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp 515 520 525Val
Glu Glu Asn Pro Gly Pro Met Ala Leu Pro Val Thr Ala Leu Leu 530 535
540Leu Pro Leu Ala Leu Leu Leu His Ala Ala Arg Ala Gln Val Gln
Leu545 550 555 560Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
Ser Val Lys Val 565 570 575Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr
Asn Tyr Asn Met His Trp 580 585 590Val Arg Gln Ala Pro Gly Gln Arg
Leu Glu Trp Met Gly Thr Ile Tyr 595 600 605Pro Gly Asn Asp Asp Thr
Ser Tyr Asn Gln Lys Phe Lys Asp Arg Val 610 615 620Thr Ile Thr Ala
Asp Thr Ser Ala Ser Thr Ala Tyr Met Glu Leu Ser625 630 635 640Ser
Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Gly Gly 645 650
655Tyr Arg Ala Met Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser
660 665 670Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
Gly Ser 675 680 685Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro
Val Thr Pro Gly 690 695 700Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser
Gln Ser Ile Val Tyr Ser705 710 715 720Asn Gly Asn Thr Tyr Leu Gly
Trp Tyr Leu Gln Lys Pro Gly Gln Ser 725 730 735Pro Gln Leu Leu Ile
Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 740 745 750Asp Arg Phe
Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 755 760 765Ser
Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Phe Gln Gly 770 775
780Ser His Val Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile
Lys785 790 795 800Ala Ala Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu
Ile Glu Val Met 805 810 815Tyr Pro Pro Pro Tyr Leu Asp Asn Glu Lys
Ser Asn Gly Thr Ile Ile 820 825 830His Val Lys Gly Lys His Leu Cys
Pro Ser Pro Leu Phe Pro Gly Pro 835 840 845Ser Lys Pro Phe Trp Val
Leu Val Val Val Gly Gly Val Leu Ala Cys 850 855 860Tyr Ser Leu Leu
Val Thr Val Ala Phe Ile Ile Phe Trp Val Arg Ser865 870 875 880Lys
Arg Ser Arg Leu Leu His Ser Asp 88531062DNAArtificial SequenceDNA
coding for CD47-binding receptor 3atggccctgc ccgtgaccgc cctgctgctg
cccctggccc tgctgctgca cgccgcccgc 60gcccaggtgc agctggtgca gagcggcgcc
gaggtgaaga agcccggcgc cagcgtgaag 120gtgagctgca aggccagcgg
ctacaccttc accaactaca acatgcactg ggtgcgccag 180gcccccggcc
agcgcctgga gtggatgggc accatctacc ccggcaacga cgacaccagc
240tacaaccaga agttcaagga ccgcgtgacc atcaccgccg acaccagcgc
cagcaccgcc 300tacatggagc tgagcagcct gcgcagcgag gacaccgccg
tgtactactg cgcccgcggc 360ggctaccgcg ccatggacta ctggggccag
ggcaccctgg tgaccgtgag cagcggcgga 420ggcggaagcg gaggcggagg
cagcgggggc ggcggaagcg acatcgtgat gacccagagc 480cccctgagcc
tgcccgtgac ccccggcgag cccgccagca tcagctgccg cagcagccag
540agcatcgtgt acagcaacgg caacacctac ctgggctggt acctgcagaa
gcccggccag 600agcccccagc tgctgatcta caaggtgagc aaccgcttca
gcggcgtgcc cgaccgcttc 660agcggcagcg gcagcggcac cgacttcacc
ctgaagatca gccgcgtgga ggccgaggac 720gtgggcgtgt actactgctt
ccagggcagc cacgtgccct acaccttcgg ccagggcacc 780aagctggaga
tcaaggccgc cgagcagaag ctgatcagcg aggaggacct gatcgaggtg
840atgtaccccc ccccctacct ggacaacgag aagagcaacg gcaccatcat
ccacgtgaag 900ggcaagcacc tgtgccccag ccccctgttc cccggcccca
gcaagccctt ctgggtgctg 960gtggtggtgg gcggcgtgct ggcctgctac
agcctgctgg tgaccgtggc cttcatcatc 1020ttctgggtgc gcagcaagcg
cagccgcctg ctgcacagcg ac 10624354PRTArtificial SequenceProtein
sequence of CD47-binding receptormisc_feature(1)..(21)CD8a
leadermisc_feature(22)..(138)anti-CD47 VHmisc_feature(139)..(153)GS
linkermisc_feature(154)..(265)anti-CD47
VLmisc_feature(266)..(277)c-Myc epitopemisc_feature(278)..(316)CD28
hingemisc_feature(317)..(343)CD28
TMmisc_feature(344)..(354)cytoplasmic tail derived from CD28 4Met
Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu1 5 10
15His Ala Ala Arg Ala Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val
20 25 30Lys Lys Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly
Tyr 35 40 45Thr Phe Thr Asn Tyr Asn Met His Trp Val Arg Gln Ala Pro
Gly Gln 50 55 60Arg Leu Glu Trp Met Gly Thr Ile Tyr Pro Gly Asn Asp
Asp Thr Ser65 70 75 80Tyr Asn Gln Lys Phe Lys Asp Arg Val Thr Ile
Thr Ala Asp Thr Ser 85 90 95Ala Ser Thr Ala Tyr Met Glu Leu Ser Ser
Leu Arg Ser Glu Asp Thr 100 105 110Ala Val Tyr Tyr Cys Ala Arg Gly
Gly Tyr Arg Ala Met Asp Tyr Trp 115 120 125Gly Gln Gly Thr Leu Val
Thr Val Ser Ser Gly Gly Gly Gly Ser Gly 130 135 140Gly Gly Gly Ser
Gly Gly Gly Gly Ser Asp Ile Val Met Thr Gln Ser145 150 155 160Pro
Leu Ser Leu Pro Val Thr Pro Gly Glu Pro Ala Ser Ile Ser Cys 165 170
175Arg Ser Ser Gln Ser Ile Val Tyr Ser Asn Gly Asn Thr Tyr Leu Gly
180 185 190Trp Tyr Leu Gln Lys Pro Gly Gln Ser Pro Gln Leu Leu Ile
Tyr Lys 195 200 205Val Ser Asn Arg Phe Ser Gly Val Pro Asp Arg Phe
Ser Gly Ser Gly 210 215 220Ser Gly Thr Asp Phe Thr Leu Lys Ile Ser
Arg Val Glu Ala Glu Asp225 230 235 240Val Gly Val Tyr Tyr Cys Phe
Gln Gly Ser His Val Pro Tyr Thr Phe 245 250 255Gly Gln Gly Thr Lys
Leu Glu Ile Lys Ala Ala Glu Gln Lys Leu Ile 260 265 270Ser Glu Glu
Asp Leu Ile Glu Val Met Tyr Pro Pro Pro Tyr Leu Asp 275 280 285Asn
Glu Lys Ser Asn Gly Thr Ile Ile His Val Lys Gly Lys His Leu 290 295
300Cys Pro Ser Pro Leu Phe Pro Gly Pro Ser Lys Pro Phe Trp Val
Leu305 310 315 320Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu
Leu Val Thr Val 325 330 335Ala Phe Ile Ile Phe Trp Val Arg Ser Lys
Arg Ser Arg Leu Leu His 340 345 350Ser Asp51065DNAArtificial
SequenceDNA coding for CD47-binding receptor with C to S
substitution 5atggccctgc ccgtgaccgc cctgctgctg cccctggccc
tgctgctgca cgccgcccgc 60gcccaggtgc agctggtgca gagcggcgcc gaggtgaaga
agcccggcgc cagcgtgaag 120gtgagctgca aggccagcgg ctacaccttc
accaactaca acatgcactg ggtgcgccag 180gcccccggcc agcgcctgga
gtggatgggc accatctacc ccggcaacga cgacaccagc 240tacaaccaga
agttcaagga ccgcgtgacc atcaccgccg acaccagcgc cagcaccgcc
300tacatggagc tgagcagcct gcgcagcgag gacaccgccg tgtactactg
cgcccgcggc 360ggctaccgcg ccatggacta ctggggccag ggcaccctgg
tgaccgtgag cagcggcgga 420ggcggaagcg gaggcggagg cagcgggggc
ggcggaagcg acatcgtgat gacccagagc 480cccctgagcc tgcccgtgac
ccccggcgag cccgccagca tcagctgccg cagcagccag 540agcatcgtgt
acagcaacgg caacacctac ctgggctggt acctgcagaa gcccggccag
600agcccccagc tgctgatcta caaggtgagc aaccgcttca gcggcgtgcc
cgaccgcttc 660agcggcagcg gcagcggcac cgacttcacc ctgaagatca
gccgcgtgga ggccgaggac 720gtgggcgtgt actactgctt ccagggcagc
cacgtgccct acaccttcgg ccagggcacc 780aagctggaga tcaaggccgc
cgagcagaag ctgatcagcg aggaggacct gatcgaggtg 840atgtaccccc
ccccctacct ggacaacgag aagagcaacg gcaccatcat ccacgtgaag
900ggcaagcacc tgagccccag ccccctgttc cccggcccca gcaagccctt
ctgggtgctg 960gtggtggtgg gcggcgtgct ggccagctac agcctgctgg
tgaccgtggc cttcatcatc 1020ttctgggtgc gcagcaagcg cagccgcctg
ctgcacagcg actaa 10656354PRTArtificial SequenceProtein sequence of
CD47-binding receptor with C to S substitution 6Met Ala Leu Pro Val
Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu1 5 10 15His Ala Ala Arg
Ala Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val 20 25 30Lys Lys Pro
Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr 35 40 45Thr Phe
Thr Asn Tyr Asn Met His Trp Val Arg Gln Ala Pro Gly Gln 50 55 60Arg
Leu Glu Trp Met Gly Thr Ile Tyr Pro Gly Asn Asp Asp Thr Ser65 70 75
80Tyr Asn Gln Lys Phe Lys Asp Arg Val Thr Ile Thr Ala Asp Thr Ser
85 90 95Ala Ser Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp
Thr 100 105 110Ala Val Tyr Tyr Cys Ala Arg Gly Gly Tyr Arg Ala Met
Asp Tyr Trp 115 120 125Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly
Gly Gly Gly Ser Gly 130 135 140Gly Gly Gly Ser Gly Gly Gly Gly Ser
Asp Ile Val Met Thr Gln Ser145 150 155 160Pro Leu Ser Leu Pro Val
Thr Pro Gly Glu Pro Ala Ser Ile Ser Cys 165 170 175Arg Ser Ser Gln
Ser Ile Val Tyr Ser Asn Gly Asn Thr Tyr Leu Gly 180 185 190Trp Tyr
Leu Gln Lys Pro Gly Gln Ser Pro Gln Leu Leu Ile Tyr Lys 195 200
205Val Ser
Asn Arg Phe Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly 210 215
220Ser Gly Thr Asp Phe Thr Leu Lys Ile Ser Arg Val Glu Ala Glu
Asp225 230 235 240Val Gly Val Tyr Tyr Cys Phe Gln Gly Ser His Val
Pro Tyr Thr Phe 245 250 255Gly Gln Gly Thr Lys Leu Glu Ile Lys Ala
Ala Glu Gln Lys Leu Ile 260 265 270Ser Glu Glu Asp Leu Ile Glu Val
Met Tyr Pro Pro Pro Tyr Leu Asp 275 280 285Asn Glu Lys Ser Asn Gly
Thr Ile Ile His Val Lys Gly Lys His Leu 290 295 300Ser Pro Ser Pro
Leu Phe Pro Gly Pro Ser Lys Pro Phe Trp Val Leu305 310 315 320Val
Val Val Gly Gly Val Leu Ala Ser Tyr Ser Leu Leu Val Thr Val 325 330
335Ala Phe Ile Ile Phe Trp Val Arg Ser Lys Arg Ser Arg Leu Leu His
340 345 350Ser Asp7732DNAArtificial SequenceDNA coding for TAG72
scFV 7caggtgcagc tgcagcagag cgacgccgag ctggtgaagc ccggcgccag
cgtgaagatc 60agctgcaagg ccagcggcta caccttcacc gaccacgcca tccactgggt
gaagcagaac 120cccgagcagg gcctggagtg gatcggctac ttcagccccg
gcaacgacga cttcaagtac 180aacgagcgct tcaagggcaa ggccaccctg
accgccgaca agagcagcag caccgcctac 240ctgcagctga acagcctgac
cagcgaggac agcgccgtgt acttctgcac ccgcagcctg 300aacatggcct
actggggcca gggcaccagc gtgaccgtga gcagcggcgg cggcggcagc
360ggcggcggcg gcagcggcgg cggcggcagc gacatcgtga tgacccagag
ccccagcagc 420ctgcccgtga gcgtgggcga gaaggtgacc ctgagctgca
agagcagcca gagcctgctg 480tacagcggca accagaagaa ctacctggcc
tggtaccagc agaagcccgg ccagagcccc 540aagctcctga tctactgggc
cagcacccgc gagagcggcg tgcccgaccg cttcaccggc 600agcggcagcg
gcaccgactt caccctgagc atcagcagcg tggagaccga ggacctggcc
660gtgtactact gccagcagta ctacagctac cccctgacct tcggcgccgg
caccaagctg 720gtgctgaagc gc 7328244PRTArtificial SequenceProtein
sequence of TAG72 scFV 8Gln Val Gln Leu Gln Gln Ser Asp Ala Glu Leu
Val Lys Pro Gly Ala1 5 10 15Ser Val Lys Ile Ser Cys Lys Ala Ser Gly
Tyr Thr Phe Thr Asp His 20 25 30Ala Ile His Trp Val Lys Gln Asn Pro
Glu Gln Gly Leu Glu Trp Ile 35 40 45Gly Tyr Phe Ser Pro Gly Asn Asp
Asp Phe Lys Tyr Asn Glu Arg Phe 50 55 60Lys Gly Lys Ala Thr Leu Thr
Ala Asp Lys Ser Ser Ser Thr Ala Tyr65 70 75 80Leu Gln Leu Asn Ser
Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys 85 90 95Thr Arg Ser Leu
Asn Met Ala Tyr Trp Gly Gln Gly Thr Ser Val Thr 100 105 110Val Ser
Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly 115 120
125Gly Ser Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Pro Val Ser
130 135 140Val Gly Glu Lys Val Thr Leu Ser Cys Lys Ser Ser Gln Ser
Leu Leu145 150 155 160Tyr Ser Gly Asn Gln Lys Asn Tyr Leu Ala Trp
Tyr Gln Gln Lys Pro 165 170 175Gly Gln Ser Pro Lys Leu Leu Ile Tyr
Trp Ala Ser Thr Arg Glu Ser 180 185 190Gly Val Pro Asp Arg Phe Thr
Gly Ser Gly Ser Gly Thr Asp Phe Thr 195 200 205Leu Ser Ile Ser Ser
Val Glu Thr Glu Asp Leu Ala Val Tyr Tyr Cys 210 215 220Gln Gln Tyr
Tyr Ser Tyr Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu225 230 235
240Val Leu Lys Arg9117PRTArtificial SequenceProtein sequence of
Hu5F9 VH-2 (CD47) 9Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys
Lys Pro Gly Ala1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr
Thr Phe Thr Asn Tyr 20 25 30Asn Met His Trp Val Arg Gln Ala Pro Gly
Gln Arg Leu Glu Trp Met 35 40 45Gly Thr Ile Tyr Pro Gly Asn Asp Asp
Thr Ser Tyr Asn Gln Lys Phe 50 55 60Lys Asp Arg Val Thr Ile Thr Ala
Asp Thr Ser Ala Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu
Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Gly Gly Tyr
Arg Ala Met Asp Tyr Trp Gly Gln Gly Thr Leu 100 105 110Val Thr Val
Ser Ser 11510112PRTArtificial SequenceProtein sequence of Hu5F9
VL-12 10Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro
Gly1 5 10 15Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile Val
Tyr Ser 20 25 30Asn Gly Asn Thr Tyr Leu Gly Trp Tyr Leu Gln Lys Pro
Gly Gln Ser 35 40 45Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe
Ser Gly Val Pro 50 55 60Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp
Phe Thr Leu Lys Ile65 70 75 80Ser Arg Val Glu Ala Glu Asp Val Gly
Val Tyr Tyr Cys Phe Gln Gly 85 90 95Ser His Val Pro Tyr Thr Phe Gly
Gln Gly Thr Lys Leu Glu Ile Lys 100 105 11011244PRTArtificial
SequenceProtein sequence of scFv for CD47 Attachment stalk 11Gln
Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10
15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr
20 25 30Asn Met His Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp
Met 35 40 45Gly Thr Ile Tyr Pro Gly Asn Asp Asp Thr Ser Tyr Asn Gln
Lys Phe 50 55 60Lys Asp Arg Val Thr Ile Thr Ala Asp Thr Ser Ala Ser
Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr
Ala Val Tyr Tyr Cys 85 90 95Ala Arg Gly Gly Tyr Arg Ala Met Asp Tyr
Trp Gly Gln Gly Thr Leu 100 105 110Val Thr Val Ser Ser Gly Gly Gly
Gly Ser Gly Gly Gly Gly Ser Gly 115 120 125Gly Gly Gly Ser Asp Ile
Val Met Thr Gln Ser Pro Leu Ser Leu Pro 130 135 140Val Thr Pro Gly
Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser145 150 155 160Ile
Val Tyr Ser Asn Gly Asn Thr Tyr Leu Gly Trp Tyr Leu Gln Lys 165 170
175Pro Gly Gln Ser Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe
180 185 190Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr
Asp Phe 195 200 205Thr Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Val
Gly Val Tyr Tyr 210 215 220Cys Phe Gln Gly Ser His Val Pro Tyr Thr
Phe Gly Gln Gly Thr Lys225 230 235 240Leu Glu Ile
Lys1260PRTArtificial SequenceCD8 hinge protein sequence 12Leu Ser
Asn Ser Ile Met Tyr Phe Ser His Phe Val Pro Val Phe Leu1 5 10 15Pro
Ala Lys Pro Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala 20 25
30Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg
35 40 45Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu 50 55
601324PRTArtificial SequenceCD8 TM protein sequence 13Ile Tyr Ile
Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu1 5 10 15Ser Leu
Val Ile Thr Leu Tyr Cys 201439PRTArtificial SequenceCD28 hinge
protein sequence 14Ile Glu Val Met Tyr Pro Pro Pro Tyr Leu Asp Asn
Glu Lys Ser Asn1 5 10 15Gly Thr Ile Ile His Val Lys Gly Lys His Leu
Cys Pro Ser Pro Leu 20 25 30Phe Pro Gly Pro Ser Lys Pro
351539PRTArtificial SequenceCD28 hinge protein sequence with C to S
substitution 15Ile Glu Val Met Tyr Pro Pro Pro Tyr Leu Asp Asn Glu
Lys Ser Asn1 5 10 15Gly Thr Ile Ile His Val Lys Gly Lys His Leu Ser
Pro Ser Pro Leu 20 25 30Phe Pro Gly Pro Ser Lys Pro
351627PRTArtificial SequenceCD28 TM protein sequence 16Phe Trp Val
Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu1 5 10 15Leu Val
Thr Val Ala Phe Ile Ile Phe Trp Val 20 251727PRTArtificial
SequenceCD28 TM with C to S substitution 17Phe Trp Val Leu Val Val
Val Gly Gly Val Leu Ala Ser Tyr Ser Leu1 5 10 15Leu Val Thr Val Ala
Phe Ile Ile Phe Trp Val 20 251841PRTArtificial SequenceCD28
signalling domain protein sequence 18Arg Ser Lys Arg Ser Arg Leu
Leu His Ser Asp Tyr Met Asn Met Thr1 5 10 15Pro Arg Arg Pro Gly Pro
Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro 20 25 30Pro Arg Asp Phe Ala
Ala Tyr Arg Ser 35 401942PRTArtificial Sequence4-1BB signalling
domain protein sequence 19Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile
Phe Lys Gln Pro Phe Met1 5 10 15Arg Pro Val Gln Thr Thr Gln Glu Glu
Asp Gly Cys Ser Cys Arg Phe 20 25 30Pro Glu Glu Glu Glu Gly Gly Cys
Glu Leu 35 4020112PRTArtificial SequenceTCR zeta signalling domain
protein sequence 20Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala
Tyr Gln Gln Gly1 5 10 15Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly
Arg Arg Glu Glu Tyr 20 25 30Asp Val Leu Asp Lys Arg Arg Gly Arg Asp
Pro Glu Met Gly Gly Lys 35 40 45Pro Arg Arg Lys Asn Pro Gln Glu Gly
Leu Tyr Asn Glu Leu Gln Lys 50 55 60Asp Lys Met Ala Glu Ala Tyr Ser
Glu Ile Gly Met Lys Gly Glu Arg65 70 75 80Arg Arg Gly Lys Gly His
Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala 85 90 95Thr Lys Asp Thr Tyr
Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg 100 105
1102122PRTArtificial SequenceP2A protein sequence 21Gly Ser Gly Ala
Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val1 5 10 15Glu Glu Asn
Pro Gly Pro 20229PRTArtificial SequenceSynthetic peptide WT-1(37)
22Val Leu Asp Phe Ala Pro Pro Gly Ala1 5239PRTArtificial
SequenceSynthetic peptide WT-1(126) 23Arg Met Phe Pro Asn Ala Pro
Tyr Leu1 5249PRTArtificial SequenceSynthetic peptide WT-1(187)
24Ser Leu Gly Glu Gln Gln Tyr Ser Val1 5259PRTArtificial
SequenceSynthetic peptide WT-1(235) 25Cys Met Thr Trp Asn Gln Met
Asn Leu1 5
* * * * *