U.S. patent application number 16/931986 was filed with the patent office on 2021-01-14 for lentiviral vectors for regulated expression of a chimeric antigen receptor molecule.
The applicant listed for this patent is CENTRE NATIONAL DE LA RECHERCHE SCIENTIFIQUE, INSTITUT CURIE, INSTITUT NATIONAL DE LA SANTE ET DE LA RECHERCHE MEDICALE (INSERM), THERAVECTYS, UNIVERSITE PIERRE ET MARIE CURIE. Invention is credited to Sophie AGAUGUE, Sebastian AMIGORENA, Cecile BAUCHE, Klervi EVEN-DESRUMAUX, Franck PEREZ, Lorenzo TIBALDI, Dmitry TRUBETSKOY.
Application Number | 20210009653 16/931986 |
Document ID | / |
Family ID | 1000005109525 |
Filed Date | 2021-01-14 |
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United States Patent
Application |
20210009653 |
Kind Code |
A1 |
AGAUGUE; Sophie ; et
al. |
January 14, 2021 |
LENTIVIRAL VECTORS FOR REGULATED EXPRESSION OF A CHIMERIC ANTIGEN
RECEPTOR MOLECULE
Abstract
The invention relates to the regulated expression of a chimeric
antigen receptor (CAR) within a lentiviral vector. The CAR
comprises a hook-binding domain that interacts with a hook,
preferably encoded by the same lentiviral vector, which prevents
proper processing and release of the CAR to the cell membrane. The
invention encompasses vectors, methods of making the vectors, and
methods of using them, including medicinal uses. The vectors can be
used for administration to humans to induce immune responses and to
treat cancers and tumors.
Inventors: |
AGAUGUE; Sophie; (Paris,
FR) ; TIBALDI; Lorenzo; (Paris, FR) ;
EVEN-DESRUMAUX; Klervi; (Antony, FR) ; TRUBETSKOY;
Dmitry; (Arcueil, FR) ; PEREZ; Franck; (Paris,
FR) ; AMIGORENA; Sebastian; (Paris, FR) ;
BAUCHE; Cecile; (Paris, FR) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
THERAVECTYS
INSTITUT CURIE
CENTRE NATIONAL DE LA RECHERCHE SCIENTIFIQUE
UNIVERSITE PIERRE ET MARIE CURIE
INSTITUT NATIONAL DE LA SANTE ET DE LA RECHERCHE MEDICALE
(INSERM) |
Paris
Paris Cedex 05
Paris Cedex 16
Paris Cedex 05
Paris |
|
FR
FR
FR
FR
FR |
|
|
Family ID: |
1000005109525 |
Appl. No.: |
16/931986 |
Filed: |
July 17, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15327517 |
Jan 19, 2017 |
10752668 |
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PCT/EP2015/067090 |
Jul 24, 2015 |
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16931986 |
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62155811 |
May 1, 2015 |
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62028889 |
Jul 25, 2014 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N 2799/027 20130101;
C07K 14/70503 20130101; A61K 35/17 20130101; C12N 5/0646 20130101;
C12N 5/0636 20130101; C07K 2319/03 20130101; C12N 2510/00 20130101;
C07K 16/00 20130101; A61K 38/00 20130101; C07K 2319/00 20130101;
C07K 2317/622 20130101; C07K 2319/22 20130101 |
International
Class: |
C07K 14/705 20060101
C07K014/705; C12N 5/0783 20060101 C12N005/0783; A61K 35/17 20060101
A61K035/17; C07K 16/00 20060101 C07K016/00 |
Foreign Application Data
Date |
Code |
Application Number |
Jun 29, 2015 |
EP |
15306036.3 |
Claims
1-18. (canceled)
19. An isolated cell comprising a nucleic acid molecule, a nucleic
acid vector or a lentiviral vector encoding a chimeric antigen
receptor, the said chimeric antigen receptor comprising: a binding
domain; a transmembrane domain; a hook-binding domain comprising a
streptavidin-binding peptide; and an activation domain comprising a
T cell activating fragment of at least 100 amino acids of SEQ ID
NO:3; SEQ ID NO:4; SEQ ID NO:5; SEQ ID NO:6, SEQ ID NO:7, or SEQ ID
NO:8, SEQ ID NO:33, or SEQ ID NO:34.
20. The isolated cell according to claim 19, wherein the isolated
cell is a cell of the immune system.
21. The isolated cell according to claim 19, wherein the isolated
cell is (i) a T cell, or (ii) a NK cell.
22. The isolated cell according to claim 19, wherein the isolated
cell is a mammalian cell, particularly a human cell.
23. The isolated cell according to claim 19, wherein the binding
domain comprises a single-chain Fv antibody or a nanobody.
24. The isolated cell according to claim 19, wherein the hook
comprises the amino acid sequence of SEQ ID NO:31 or SEQ ID
NO:32.
25. The isolated cell according to claim 19, wherein the nucleic
acid vector or the lentiviral vector comprises a
.beta.2-micro.sub.globulin, ubiquitin, MHCI or MHCII promoter.
26. The isolated cell according to claim 19, wherein the chimeric
antigen receptor comprises the amino acid sequence of any of SEQ ID
NO:46, SEQ ID NO:48, or SEQ ID NO:50.
27. An isolated cell comprising a vector or a lentiviral vector
particle expressing a chimeric antigen receptor, the said chimeric
antigen receptor comprising: a binding domain; a transmembrane
domain; a hook-binding domain comprising a streptavidin-binding
peptide; and an activation domain comprising a T cell activating
fragment of at least 100 amino acids of SEQ ID NO:3; SEQ ID NO:4;
SEQ ID NO:5; SEQ ID NO:6, SEQ ID NO:7, or SEQ ID NO:8, SEQ ID
NO:33, or SEQ ID NO:34.
28. The isolated cell according to claim 27, wherein the isolated
cell is a cell of the immune system.
29. The isolated cell according to claim 27, wherein the isolated
cell is (i) a T cell, or (ii) a NK cell.
30. The isolated cell according to claim 27, wherein the isolated
cell is a human cell.
31. The isolated cell according to claim 27, wherein the binding
domain comprises a single-chain Fv antibody or a nanobody.
32. The isolated cell according to claim 27, wherein the hook
comprises the amino acid sequence of SEQ ID NO:31 or SEQ ID
NO:32.
33. The isolated cell according to any one of claims 27, wherein
the vector or the lentiviral vector particle comprises a
.beta.2-microglobulin, ubiquitin, MHCI, or MHCII promoter.
34. The isolated cell according to claim 9, wherein the chimeric
antigen receptor comprises the amino acid sequence of any of SEQ ID
NO:46, SEQ ID NO:48, or SEQ ID NO:50.
35. A method for the induction of an immune response in a human
comprising the administration to said human of an isolated cell
according to claim 19.
36. The method according to claim 35, wherein the isolated cell is
intramuscularly, intravenously, intra-articularly or intra-tumoraly
administered to the human.
37. The method according to claim 35, wherein biotin is
administered to the human subsequently to the administration of the
cell.
38. A method for the treatment of cancers and tumors in a human
comprising the administration to said human of an isolated cell
according to claim 19.
39. The method according to claim 38, wherein the isolated cell is
intramuscularly, intravenously, intra-articularly or intra-tumoraly
administered to the human.
40. The method according to claim 38, wherein biotin is
administered to the human subsequently to the administration of the
cell.
41. A method for the treatment of auto-immune diseases in a human
comprising the administration to said human of an isolated cell
according to claim 19.
42. The method according to claim 41, wherein the isolated cell is
intramuscularly, intravenously, intra-articularly or intra-tumoraly
administered to the human.
43. The method according to claim 41, wherein biotin is
administered to the human subsequently to the administration of the
cell.
44. A method for expressing a chimeric antigen receptor in a cell
comprising: expressing a nucleic acid molecule, a nucleic acid
vector or a lentiviral vector encoding the chimeric antigen
receptor, or a vector expressing the chimeric antigen receptor in
the cell, or transducing the cell with a lentiviral vector particle
expressing the chimeric antigen receptor, under conditions that
allow the expression of the chimeric antigen receptor; said
chimeric antigen receptor comprising: a binding domain; a
transmembrane domain; a hook-binding domain comprising a
streptavidin-binding peptide; and an activation domain comprising a
T cell activating fragment of at least 100 amino acids of SEQ ID
NO:3; SEQ ID NO:4; SEQ ID NO:5; SEQ ID NO:6, SEQ ID NO:7, or SEQ ID
NO:8, SEQ ID NO:33, or SEQ ID NO:34.
45. The method according to claim 44, wherein it further comprises
harvesting or isolating the chimeric antigen receptor.
46. The method according to claim 44, wherein the cells are treated
with biotin.
47. The method according to claim 46, wherein the cells are treated
with biotin at an initial concentration of at least 0.2, 0.4, 0.8,
1.6, 2.5, 5, 10, 20, 40 or 80 .mu.M.
Description
TECHNICAL FIELD
[0001] The present invention is in the field of recombinant vaccine
technology and relates to improvements of lentiviral vectors, which
can be used as therapeutic and prophylactic vaccines. The vectors
provide improved induction of immune responses over other
vectors.
BACKGROUND
[0002] Recombinant vaccines have been developed with the progress
of recombinant DNA technology, allowing the modification of viral
genomes to produce modified viruses. In this manner, it has been
possible to introduce genetic sequences into non-pathogenic
viruses, so that they encode immunogenic proteins to be expressed
in target cells upon infection or transduction, in order to develop
a specific immune response in their host.
[0003] Such vaccines constitute a major advance in vaccine
technology (Kutzler et al., Nat Rev Genet, 9(10): 776-788, 2008).
In particular, they have the advantage over traditional vaccines of
avoiding live (attenuated) virus and eliminating risks associated
with the manufacture of inactivated vaccines.
[0004] Gene delivery using modified retroviruses (retroviral
vectors) was introduced in the early 1980s by Mann et al. (Cell,
33(1):153-9, 1983). The most commonly used oncogenic retroviral
vectors are based on the Moloney murine leukemia virus (MLV). They
have a simple genome from which the polyproteins Gag, Pol and Env
are produced and are required in trans for viral replication
(Breckpot et al., 2007, Gene Ther, 14(11):847-62; He et al. 2007,
Expert Rev vaccines, 6(6):913-24). Sequences generally required in
cis are the long terminal repeats (LTRs) and its vicinity: the
inverted repeats (IR or att sites) required for integration, the
packaging sequence Y, the transport RNA-binding site (primer
binding site, PBS), and some additional sequences involved in
reverse transcription (the repeat R within the LTRs, and the
polypurine tracts, PPT, necessary for plus strand initiation). To
generate replication-defective retroviral vectors, the gag, pol,
and env genes are generally entirely deleted and replaced with an
expression cassette.
[0005] Retroviral vectors deriving from lentivirus genomes (i.e.
lentiviral vectors) have emerged as promising tools for both gene
therapy and immunotherapy purposes, because they exhibit several
advantages over other viral systems. In particular, lentiviral
vectors themselves are not toxic and, unlike other retroviruses,
lentiviruses are capable of transducing non-dividing cells, in
particular dendritic cells (He et al. 2007, Expert Rev vaccines,
6(6):913-24), allowing antigen presentation through the endogenous
pathway.
[0006] Lentiviruses are linked by similarities in genetic
composition, molecular mechanisms of replication and biological
interactions with their hosts. They are best known as agents of
slow disease syndromes that begin insidiously after prolonged
periods of subclinical infection and progress slowly; thus, they
are referred to as the "slow" viruses (Narayan et al., 1989, J Gen
Virol, 70(7):1617-39). They have the same basic organization as all
retroviruses but are more complex due to the presence of accessory
genes (e.g., vif, vpr, vpu, nef, tat, and rev), which play key
roles in lentiviral replication in vivo.
[0007] Lentiviruses represent a genus of slow viruses of the
Retroviridae family, which includes the human immunodeficiency
viruses (HIV), the simian immunodeficiency virus (SIV), the equine
infectious encephalitis virus (EIAV), the caprine arthritis
encephalitis virus (CAEV), the bovine immunodeficiency virus (BIV),
and the feline immunodeficiency virus (FIV). Lentiviruses can
persist indefinitely in their hosts and replicate continuously at
variable rates during the course of the lifelong infection.
Persistent replication of the viruses in their hosts depends on
their ability to circumvent host defenses.
[0008] The design of recombinant integrating lentiviral vectors is
based on the separation of the cis- and trans-acting sequences of
the lentivirus. Efficient transduction in non-dividing cells
requires the presence of two cis-acting sequences in the lentiviral
genome, the central polypurine tract (cPPT) and the central
termination sequence (CTS). These lead to the formation of a
triple-stranded DNA structure called the central DNA "flap", which
maximizes the efficiency of gene import into the nuclei of
non-dividing cells, including dendritic cells (DCs) (Zennou et al.,
2000, Cell, 101(2) 173-85; Arhel et al., 2007, EMBO J,
26(12):3025-37).
[0009] Dendritic cells are of primary importance for antigen
presentation because they constitute the main class of antigen
presenting cells (APCs) whose primary function is to present
antigens and initiate an immune response.
[0010] To generate an immune response, antigenic proteins must be
processed by cells into peptides that are displayed on the cell
surface by major histocompatibility complex proteins (MHCs).
Circulating APCs present the peptide-MHC complexes to T cells in
the draining lymph nodes, where they interact with T cell
receptors, and, in conjunction with co-stimulatory signals,
activate the T cells.
[0011] A variety of studies have shown that inoculation with
lentiviral vectors leads to antigen presentation by DCs and strong
activation of antigen specific cytotoxic T lymphocytes (CTLs;
CD8.sup.+ T cells). Therefore, lentiviral vectors have been
engineered for the last 10 years for gene transfer and
immunotherapy applications.
[0012] The vectors routinely contain strong constitutive promoters
containing enhancers, such as the CMV promoter. Michelini et al.,
Vaccine 27(34):4622-29 (2009); Karwacz et al., J. Virol.
83(7):30943103 (2009); Negri et al., Molecular Therapy
15(9):1716-23 (2007); and Buffa et al., J. General Virology
87:1625-1634 (2006).
[0013] Lentiviral vectors have been improved in their safety by
removal of the LTR U3 sequence, resulting in "self-inactivating"
vectors that are entirely devoid of viral promoter and enhancer
sequences originally present within the LTRs.
[0014] The lentiviral particles, which contain lentiviral vectors,
can be produced by recombinant technology upon transient
transfection of cells, for example HEK 293T human cultured cells,
by different DNA plasmids:
[0015] (i) a packaging plasmid, which expresses at least the Gag,
Pol Rev, Tat and, in some cases, structural and enzymatic proteins
necessary for the packaging of the transfer construct;
[0016] (ii) a proviral transfer plasmid, containing an expression
cassette and HIV cis-acting factors necessary for packaging,
reverse transcription, and integration; and
[0017] (iii) an envelope-encoding plasmid, in most cases the
glycoprotein of vesicular stomatitis virus (VSV.G), a protein that
allows the formation of mixed particles (pseudotypes) that can
target a wide variety of cells, especially major histocompatibility
(MHC) antigen-presenting cells (APCs), including DCs.
[0018] This procedure allows obtaining transient production of
lentiviral particle vectors by the transfected cells. However, the
lentiviral particle vectors may also be continuously produced by
cells by stably inserting the packaging genes, the proviral coding
DNA, and the envelope gene into the cellular genome. This allows
the continuous production of lentiviral particle vectors by the
cells without the need for transient transfection. Of course, a
combination of these procedures can be used, with some of the
DNAs/plasmids integrated into the cellular genome and others
provided by transient transfection.
[0019] Non-integrating lentiviral vectors have been designed to
mitigate the risks of potential oncogenesis linked to insertional
mutagenesis events, particularly for vaccination purposes. Examples
of non-integrating lentiviral vectors are provided in Coutant et
al., PLOS ONE 7(11):e48644 (2102), Karwacz et al., J. Virol.
83(7):3094-3103 (2009), Negri et al., Molecular Therapy
15(9):1716-1723 (2007); Hu et al., Vaccine 28:6675-6683 (2010).
Consequently, it has been reported that a non-integrating
lentiviral vector system can mitigate the potential risk of
insertional mutagenesis as compared to an integrating system. Hu et
al., Vaccine 28:6675-6683 (2010).
[0020] In addition, deletion in the U3 region of the 3' LTR of the
viral promoter and enhancer sequences in self-inactivating
lentiviral vectors limits the likelihood of endogenous promoter
activation. These concerns with safety directly address the
experiences gained from the SCID-X1 gene therapy trial carried out
in 1998-1999, performed with Moloney virus-based retroviral vectors
on children suffering from a rare form of X-linked (SCID-X1 gene)
severe immunodeficiency disease (Cavazzana-Calvo et al., 2000,
Science., 288(5466):669-72). During this trial, four of nine
children developed leukemia as a result of the integration of the
Moloney-derived retroviral vector at close proximity to the human
LMO2 proto-oncogene (Hacein-Bey-Abina et al., 2008, J. Clin.
Invest., 118(9):3132-3142). It was demonstrated that malignancy was
the consequence of the proximity of the viral U3 promoter/enhancer
to the LM02 proto-oncogene. As a result, safety is a major concern
for the administration of lentivectors to humans.
[0021] Enhancers are cis-acting sequences, which can act as
transcriptional activators at a distance. They have been widely
employed in viral derived vectors because they appear to be the
most efficient for obtaining transgene strong expression in a
variety of cell types, in particular DCs (Chinnasamy et aL, 2000,
Hum Gene Ther 11(13):1901-9; Rouas et al., 2008, Cancer Gene Ther
9(9):715-24; Kimura et al., 2007, Mol Ther 15(7):1390-9; Gruh et
al., 2008, J Gene Med 10(1) 21-32). However, given the safety issue
of insertional mutagenesis, such transcriptional enhancer sequences
should be deleted from the lentiviral vector constructs to abolish
the risk of insertional mutagenesis by enhancer proximity effect.
This enhancer proximity effect is by far the most frequent
mechanism of insertional mutagenesis and is the only effect
described in human or animal cases of tumorigenic events after gene
transfer.
[0022] Thus, there is a need to develop retroviral, particularly
lentiviral vectors, which do not include viral enhancers, but still
allow sufficient expression of transgenes encoding immunogenic
peptides, if possible, as much expression as that observed when
using the CMV promoter.
[0023] Recent studies has reported on the replacement of viral
promoters by DC-specific promoters deriving from major
histocompatibility complex class II genes (MHC class II) (Kimura et
al., 2007, Mol Ther 15(7):1390-9) and dectin-2 genes (Lopes et al.,
2008, J Virol 82(1):86-95). The dectin-2 gene promoter used in
Lopes et al. contains a putative enhancer and an adenoviral
conserved sequence (inverted terminal repeats in adenovirus
promoter) (Bonkabara et al., 2001, J. Immunology, 167:6893-6900).
The MHC class II gene promoter used by Kimura et al. does not
contain any known enhancer.
[0024] Yet, without an enhancer, the MHC class II promoter was
found not to provide sufficient transgene expression in DCs, when
administered intravenously. In particular, lentiviral vectors
including MHC class II promoters did not provoke an immune reaction
in immunocompetent C57BL/6 mice, in contrast to the immune
responses observed with CMV promoters/enhancers. Although
integration and persistent transgene expression were observed after
injection in mice, the lentiviral vectors transcribed through MHC
class II promoters failed to stimulate an antigen-specific CD8+
cytotoxic T-lymphocyte response, even after vaccination boost. The
authors of these studies therefore concluded that the use of MHC
class II promoters was of interest only for applications where
persistence of expression is sought as in gene replacement therapy,
but not in the context of immunotherapy. Of note, MHC class II
promoters are expressed poorly in most cell types.
[0025] Thus, the MHC class II promoter is not an adequate promoter
for lentiviral vectors for induction of an immune response against
an antigen via IV injection. Moreover, the dectin-2 promoter is
expressed poorly in most cell types and appears to contain an
enhancer. Thus, the dectin-2 promoter is not a good promoter for
lentiviral vectors for safety reasons.
[0026] Preferably, in immunotherapy, lentiviral vectors provide
effective expression of the transgene that elicits a desired
specific immune response. This requires that the expression is at a
high level in APCs, such as dendritic cells.
[0027] It is also preferable that the cells transduced by the
lentiviral vectors are eliminated by the immune response to provide
a higher degree of safety. That is, the immune response generated
against the transgene can elicit an immune response in the host
sufficient to eliminate the cells that are transduced by the
lentiviral vectors. The elimination of transduced cells eliminates
the persistence of the lentiviral vector in the host, and possible
secondary effects of the vector. In order for the transduced cells
to be eliminated, expression is required in non-dendritic cells at
a level that allows elimination by the immune response. Thus,
appropriate expression of an antigen is desirable.
[0028] At the same time, the promoter should maximize immune
stimulation through the key cells (i.e., dendritic cells) involved
in the activation of naive and memory T cells, and should minimize
the risk of insertional mutagenesis and genotoxicity in stem cells,
leading to malignancies. Thus, the promoter should have
sufficiently high activity in dendritic and other cells, but not
contain an enhancer. Based on these criteria, viral promoters, such
as the CMV promoter, are not ideal because of the presence of
strong enhancers. These criteria are summarized as follows:
[0029] high expression in antigen presenting cells, including
dendritic cells, to induce maximal immune responses;
[0030] expression in other transduced cell types sufficient for
elimination by the induced immune response; and
[0031] lack of an enhancer element to avoid insertional
effects.
[0032] Chimeric antigen receptors (CARs) are recombinant receptors
for antigens. Sadelain et al., Cancer Discov. 2013 April; 3(4):
388-398. They are recombinant receptors that typically target
native cell surface antigens. Id. CARs engage molecules that do not
require peptide processing or HLA expression to be recognized. Id.
CARs have been shown to have T cell-proliferating and activating
potential on their own, including chimeric molecules between
CD3-.zeta. or Fc receptor .gamma. and CD8, CD4, CD25 or CD16. The
antigen-binding parts of the CARs generally are either scFv's
derived from antibodies, Fab's selected from libraries, or natural
receptor ligands. Han EQ et al.; J Hematol Oncol, 6:47, 2013.
[0033] CARs have been generated against many different cell surface
molecules, including CD19, HER2, GD2, PSMA, and many are in
clinical trials. Davila et al., Oncolmmunology 1:9, 1577-1583;
2012. The most promising clinical outcomes of CARs have been in
patients treated with autologous CAR-modified T cells targeting
CD19. Maus M V et al.; 123(17): 2625-35, 2014.
[0034] The in vivo elimination of T cells expressing CARs has been
investigated. Human T cells have been genetically modified with a
lentiviral vector to express a CD20-CAR containing a suicide gene
relying on inducible activation of caspase 9. Budde et al., PLoS
ONE 8(12): e82742 (2013). This lentivector used the human
EF1.alpha. promoter to obtain expression in T cells. Activation of
the suicide fuse resulted in efficient removal of transduced T
cells both in vitro and in vivo. Id.
[0035] It would be better to be able quickly turn off the
expression of the CAR at the surface of the cells to prevent
adverse events and be able reactivate the system after it has been
turned off. Thus, a need exists in the art for improved vectors and
methods for treatment of humans. The present invention fulfills
these needs in the art.
SUMMARY OF THE INVENTION
[0036] The invention encompasses nucleic acid molecules and vectors
encoding CARs and methods of making and using the nucleic acid
molecules and vectors. In one embodiment, the invention encompasses
a chimeric antigen receptor comprising a binding domain; a
transmembrane domain; a hook-binding domain, preferably comprising
a streptavidin-binding peptide; and an activation domain comprising
a T cell activating fragment of at least 100 amino acids of SEQ ID
NO:3; SEQ ID NO:4; SEQ ID NO:5; SEQ ID NO:6, SEQ ID NO:7, or SEQ ID
NO:8, SEQ ID NO:33, or SEQ ID NO:34.
[0037] In one embodiment, the invention encompasses a lentiviral
vector expressing a chimeric antigen receptor comprising a binding
domain; a transmembrane domain; a hook-binding domain comprising a
streptavidin-binding peptide; and an activation domain comprising a
T cell activating fragment of at least 100 amino acids of SEQ ID
NO:3; SEQ ID NO:4; SEQ ID NO:5; SEQ ID NO:6, SEQ ID NO:7, or SEQ ID
NO:8, SEQ ID NO:33, or SEQ ID NO:34.
[0038] In one embodiment, the invention encompasses a nucleic acid
vector encoding a chimeric antigen receptor comprising a binding
domain; a transmembrane domain; a hook-binding domain, preferably
comprising a streptavidin-binding peptide; and an activation domain
comprising a T cell activating fragment of at least 100 amino acids
of SEQ ID NO:3; SEQ ID NO:4; SEQ ID NO:5; SEQ ID NO:6, SEQ ID NO:7,
or SEQ ID NO:8, SEQ ID NO:33, or SEQ ID NO:34.
[0039] In one embodiment, invention encompasses a lentiviral vector
particle encoding a chimeric antigen receptor comprising a binding
domain; a transmembrane domain; a hook-binding domain, preferably
comprising a streptavidin-binding peptide; and an activation domain
comprising a T cell activating fragment of at least 100 amino acids
of SEQ ID NO:3; SEQ ID NO:4; SEQ ID NO:5; SEQ ID NO:6, SEQ ID NO:7,
or SEQ ID NO:8, SEQ ID NO:33, or SEQ ID NO:34.
[0040] In one embodiment, the chimeric antigen receptor or the
lentiviral vector or the nucleic acid vector or the lentiviral
vector particle comprises a single-chain Fv antibody or a
single-domain antibodies (i.e., nanobody).
[0041] In one embodiment, the vector encodes a hook comprising the
amino acid sequence of SEQ ID NO:31 or SEQ ID NO:32.
[0042] In one embodiment, the vector comprises a
.beta.2-microglobulin, Ubi, EF1.alpha., MHCI, or MHCII promoter. In
one embodiment, the vector is a DNA.
[0043] In one embodiment, the lentiviral vector particle comprises
a vesicular stomatitis virus glycoprotein. In one embodiment, the
lentiviral vector particle comprises HIV-1 subtype D Gag and Pol
proteins.
[0044] In one embodiment, the invention encompasses an isolated
cell comprising a vector of the invention.
[0045] The invention encompasses the use of the chimeric antigen
receptor or the lentiviral vector or the nucleic acid vector or the
lentiviral vector particle of any of the invention for inducing an
immune response in a human.
[0046] The invention encompasses a method for inducing an immune
response in a human comprising administering the lentiviral vector
particle or cells transduced by the lentiviral vector particle
encoding a chimeric antigen receptor to a human and, optionally,
subsequently administering biotin to the human.
[0047] In some embodiments, the chimeric antigen receptor or the
lentiviral vector or the nucleic acid vector comprises the
nucleotide sequence of any of SEQ ID NO:45, SEQ ID NO:47, or SEQ ID
NO:49. In some embodiments, the chimeric antigen receptor receptor
comprises the amino acid sequence of any of SEQ ID NO:46, SEQ ID
NO:48, or SEQ ID NO:50.
[0048] In some embodiments, the lentiviral vector or the nucleic
acid vector further encodes the amino acid sequence of SEQ ID NO:42
or comprises the nucleic acid sequence of SEQ ID NO:43 and/or SEQ
ID NO:44.
BRIEF DESCRIPTION OF THE DRAWINGS
[0049] FIG. 1 depicts GFP expression in HEK 293T and BDCM cells
from lentivectors with the indicated promoters.
[0050] FIG. 2 depicts cell-specific expression of various promoters
analyzed using the data sets at biogps.org. The expression levels
were normalized to expression in skeletal muscle(=1.0).
[0051] FIG. 3A-C depict expression of a CAR in T cells from various
lentivectors with human ubiquitin, MHC class I, MHC class II, and
.beta.2 microglobulin (.beta.2m) promoters.
[0052] FIG. 4 depicts titers with a pseudotyping vector encoding a
codon-optimized VSV-G protein fused to Hook-streptavidin with a
dose escalation of hook-streptavidin encoding vector and
+/-biotin.
[0053] FIG. 5A-B depict structure and expression of CAR-RUSH. A)
Scheme of the three CAR-RUSH constructions designed. CAR-RUSH are
second generation CARs displaying 4-1 BB and CD3zeta intracellular
domains. The peculiarity of CAR-RUSH resides in the presence of a
SBP (Streptavidin Binding Protein) region allowing the interaction
with a HOOK protein coupled to streptavidin and, consequently, the
retention in the endoplasmic reticulum. The difference between
SBP1, SBP2 and SBP3 consists in the position of the SBP region,
which has been placed between the transmembrane domain and 4-1 BB,
or between 4-1 BB and CD3zeta, or at the C-terminus, respectively.
B) MFI showing median fluorescence intensity of CAR-positive T
lymphocytes analysed by FACS. Cells were transduced at a MOI of 10
and analysed at day 3 post-transduction. MFI values reported in the
histogram are normalized by substracting the mfi from untransduced
cells, corresponding to the background. All CAR-positive T cells
showed comparable MFI, with second generation CAR-CD19 (positive
control) displaying MFI three-fold higher than the other constructs
tested. All three CAR-RUSH (SBP1, SBP2 and SBP3) proteins showed
very similar MFI and were therefore expressed at similar levels at
the cellular surface.
[0054] FIG. 6A-B depict expression and behavior of CAR-RUSH
constructs following co-transduction with a lentivector encoding a
HOOK-streptavidin, and biotin treatment. Percentage (A) and MFI
showing median fluorescence intensity (B) of CAR-positive T
lymphocytes analysed by FACS. Cells were co-transduced with a
lentivector encoding a HOOK-streptavidin (MOI 5) and a lentivector
encoding a CAR-SBP (MOI 5), corresponding to a final total MOI of
10. Biotin was added at the time of co-transduction and cells were
analysed at day 3 post-transduction. The percentage and the MFI
values reported in the histograms were obtained by subtracting the
value from untransduced cells, corresponding to the background,.
CAR-RUSH constructs were expressed at low percentages specifically
following biotin addition into the medium, suggesting that in a
co-transduction context, the CAR-RUSH was efficiently retained by
the HOOK protein and it was able to be presented at the cellular
membrane only upon biotin-mediated induction.
[0055] FIG. 7A-B depict expression and behavior of CAR-RUSH
bicistronic constructs. Percentage and MFI showing median
fluorescence intensity of CAR-positive T lymphocytes analysed by
FACS. Cells were transduced at a MOI of 10 (A) or 30 (B) and
analysed at day 3 post-transduction. Biotin was added at the time
of transduction. The percentage and the MFI values reported in the
histograms were obtained by substracting values from untransduced
cells, corresponding to the background. SBP1-CD19 and SBP3-CD19
CAR-RUSH constructs showed expression at the cellular surface only
upon addition of biotin in the culture medium, suggesting that
CAR-RUSH were specifically retained by the HOOK-streptavidin
protein in absence of biotin and that, following biotin delivery,
the RUSH regulation system induced their trafficking towards and
their expression on the plasma membrane. SBP1-CD19 and SBP3-CD19
CAR-RUSH constructs showed expression at the cellular surface upon
addition of biotin in the culture medium. At high MOI (MOI=30) and
even in absence of biotin, SBP1-CD19 showed slight expression at
the cellular surface. In view of these results, CAR-RUSH were
considered as largely retained by the HOOK streptavidin protein in
the absence of biotin. Moreover, as expected, the biotin delivery
induced their trafficking towards, and their expression on the
plasma membrane.
[0056] FIG. 8A-B depict CAR-RUSH system switch evaluation (OFF/ON).
Purified T cells were transduced at day 0 with lentivectors
encoding the CAR-RUSH system and biotin was added or not at the
same time. At day 3, T cells were analyzed by flow cytometry for
the expression of CARs at the T cell surface. Percentage (A) and
MFI showing median fluorescence intensity (B) of CAR-positive T
lymphocytes from one representative donor analyzed by FACS.
CAR-RUSH constructs were transduced at a MOI of 10 or 20, biotin
was added at the same time and T cells were analysed for CAR
expression at the cellular surface at day 3 post-transduction. The
rationale behind this experiment was to study the kinetics of
induction of CAR-RUSH constructs upon biotin delivery and the
efficiency of CAR-RUSH retention in absence of the biotin inducer.
As shown in the top panel, a low percentage of CAR-positive T cells
was detected and, depending on the CAR-RUSH construct, the effect
was dependent or independent on biotin addition. Depending on the
CAR-RUSH construct analyzed, a low percentage of CAR-positive T
cells was detected. The ability of biotin to induce CAR-RUSH
expression at the cellular surface was detected for CAR-SBP1 and
CAR-SBP2. CAR-SBP3 showed a slight CAR expression in both the
presence and absence of biotin.
[0057] FIG. 9A-D depict CAR-RUSH system switch evaluation
(OFF/ON/OFF). Purified T cells were transduced at day 0 with
lentivectors encoding the CAR-RUSH system and biotin was added or
not at the same time. At day 3 a set of wells was analysed by flow
cytometry for the expression of CARs in both the presence and
absence of biotin. Other sets of wells were washed and put back in
culture with or without biotin, in order to monitor the ability of
CAR-positive cells to switch the expression of CAR off. T cells
were then analysed for CAR expression at the T cell surface at day
7. Percentage (A) and MFI showing median fluorescence intensity (B)
of CAR-positive T lymphocytes analysed by FACS and derived from
healthy donor 32. CAR-RUSH constructs were transduced at a MOI of
10 and analysed for CAR expression at the cellular surface at day 7
post-transduction following the re-addition or not of biotin at day
3. As shown in A), a low percentage of CAR-positive T cells was
detected. Biotin induced CAR-RUSH expression at the extracellular
surface for both CAR-SBP2 and CAR-SBP3 and, in particular for
CAR-SBP3, CAR expression was also re-induced following washing and
biotin re-addition at day 3. Percentage (C) and MFI showing median
fluorescence intensity (D) of CAR-positive T lymphocytes analysed
by FACS and derived from healthy donor 31. CAR-RUSH constructs were
transduced at a MOI of 20 and analysed for CAR expression at the
cellular surface at day 7 post-transduction following the
re-addition or not of biotin at day 3. All three CAR-RUSH tested
showed a low percentage of CAR-positive cells. In addition, at
exception of CAR-SBP1 which showed CAR expression both in the
presence and absence of biotin, CAR-RUSH were exclusively expressed
in the conditions in which biotin was added at day 0 and/or at day
7.
[0058] FIG. 10A-C depicts the amino acid and nucleotide sequences
of CAR-CD19 2nd generation and its constituents. A) nucleotide
sequence (SEQ ID NO:39), B) amino acid sequence (SEQ ID NO:51), and
C) key indicating signal sequence, CD19 ScFv, CD8 hinge,
transmembrane domain, 4-1 BB, and CD3z sequences.
[0059] FIG. 11 depicts the structure of the bicistronic HOOK
streptavidin-IRES-CAR-SBP-CD19 2nd generation constructs.
DETAILED DESCRIPTION OF THE INVENTION
[0060] Various lentivector constructs encoding chimeric antigen
receptors (CARs) were generated for the expression of CARs in T
cells. It was unknown what promoter might be most suitable for
expression in human T cells. To compare promoters, a kidney cell
line and a dendritic cell line were transduced with lentiviral
vectors expressing green fluorescent protein (GFP) from various
promoters. It was found that the human EF1.alpha. promoter was the
strongest promoter in the dendritic cell line BDCM (FIG. 1). This
promoter was also very strong in the kidney cell line 293T.
Cell-specific expression of various promoters was analyzed using
the data sets at biogps.org (FIG. 2). From this data set,
expression of the EF1.alpha. promoter in T cells is similar to
expression in BDCA+ cells. In fact, the human EF1.alpha. promoter
was used previously to express a CAR in activated human T cells.
Budde et al., PLoS ONE 8(12): e82742 (2013.
[0061] Based on differences in expression in different cell types,
it was not evident which promoters might be used successfully for
expression of CARs in human T cells. Human ubiquitin MHC I, MHC II,
and .beta.2 microglobulin (.beta.2m) promoters were assessed for
their suitability for expression of CARs in human T cells.
Unexpectedly, it was found that all of these promoters worked in
human T cells (FIG. 3A-C).
[0062] In order find the best conditions to generate CAR T-Cells,
several parameters that could affect T cells transduction and CAR
expression (e.g. MOI, incubation time, promoters, T cell activation
and purification strategies) were tested.
[0063] Following subtraction of background (% of CAR+ cells in
untransduced (UTD) control) CAR+ cells were detected at 24 hrs (UBC
vector at MOI=10 and 30), at 96 hrs (.beta.2M vector at MOI=3 and
UBC vector MOI=3 and 10) and at 8 days (.beta.2M vector at MOI=30
and UBC vector at every MOI tested). At 96 hrs and at 8 days, a
high percentage (about 30%) of CAR-CD19 positive cells in
comparison to untransduced lymphocytes was found. At day 7, a high
percentage (about 70%) of CAR-CD19 positive cells in comparison to
untransduced lymphocytes was found when the viable CD3+ population
was analyzed.
[0064] Strong expression of CAR-CD19 in several donors (up to 70%
of CD3 positive lymphocytes) was found. CAR expression was found
even at MOI=3; however, a high percentage of CAR-positive cells was
achieved at late time points (day 7 in this experiment).
Interestingly, the CAR expression between donors was variable. For
CAR-CD19, expression was restricted to the CD3 positive population,
as CD3 negative cells failed to express CAR-CD19.
[0065] The Hook-streptavidin sequence was cloned into a
pseudotyping vector encoding a codon-optimized VSV-G protein as a
fusion with VSV-G. This vector was evaluated by cotransfection with
a lentivector encoding GFP and a dose escalation of
hook-streptavidin encoding vector. No reduction in titers due to
retention of the VSV protein in the ER was seen (FIG. 4).
Immunofluorescence studies showed a lack of retention of the VSV
protein in the ER. This contrasts with the results seen for the VSV
protein alone. Nature Methods 9(5): 493-500 (2102). Thus, it was
unclear whether CAR expression might be regulated with the
Hook-Steptavidin system.
[0066] The expression at the surface of T-cells of CAR-CD19 or
CAR-CD19 with a streptavidin binding protein (SBP) at various
positions between the intracellular domains was evaluated to check
whether the presence of the SBP sequence was modifying the CAR
expression. CAR-CD19+ with the SBP at various positions were cloned
into lentiviral vectors under the control of the .beta.2m
promoter.
[0067] Human T-cells were transduced with the lentiviral vectors
and the expression of the CD19 was evaluated at the surface of the
cells. The CAR-CD19-SBP are expressed at the surface of the
T-cells, with a slight reduction of the MFI compared to the
CAR-CD19 (FIG. 5).
[0068] CAR-CD19 lentivectors with the SBP at various positions
between the intracellular domains were evaluated in presence of the
Hook-Streptavidin. Human T-cells were co-transduced with a
lentivector expressing the CAR-CD19-SBP and with a lentivector
expressing the Hook-streptavidin and the percentage of transduced
cells and MFI were evaluated before or after addition of biotin. It
was found that the Hook-streptavidin and the CAR-CD19-SBP can be
co-expressed in human T-cells. The presence of the
Hook-streptavidin could retain the CAR-CD19-SBP in the endoplasmic
reticulum of the T-cells and the addition of biotin in the media
induced the release of the CAR-CD19 and its expression at the
surface of the cells (FIG. 6).
[0069] Several lentivectors were then constructed containing both
the Hook and the CAR containing the SBP at various locations (FIG.
11). The constructs contained the .beta.2m promoter linked to the
Hook streptavidin, followed by an IRES linked to the CAR-CD19 with
the SBP at 3 different locations.
[0070] Human T-cells from various donors were transduced with the
lentiviral vectors and the expression of the CAR-CD19-SBP was
evaluated at the surface of the cells in the presence and absence
of biotin. The Hook-IRES-CAR-CD19-SBP was expressed in human
T-cells. The expression of the Hook-streptavidin could retain the
CAR-CD19-SBP in the endoplasmic reticulum of the T-cells,
regardless of its position. Addition of biotin in the media induced
the release of the CAR-CD19 and its expression at the surface of
the cells, regardless of its position (FIG. 7).
[0071] The present invention encompasses lentiviral vectors
encoding a CAR, and their use for the induction of immune responses
in a host, especially a human.
[0072] Before the present proteins, compositions, methods, and
other embodiments are disclosed and described, it is to be
understood that the terminology used herein is for the purpose of
describing particular embodiments only and is not intended to be
limiting. It must be noted that, as used in the specification and
the appended claims, the singular forms "a," "an" and "the" include
plural referents unless the context clearly dictates otherwise.
[0073] The term "comprising" as used herein is synonymous with
"including" or "containing", and is inclusive or open-ended and
does not exclude additional, unrecited members, elements or method
steps.
[0074] The full name of amino acids is used interchangeably with
the standard three letter and one letter abbreviations for each in
this disclosure. For the avoidance of doubt, those are: Alanine
(Ala, A), Arginine (Arg, R), Asparagine (Asn, N), Aspartic acid
(Asp, D), Cysteine (Cys, C), Glutamic Acid (Glu, E), Glutamine
(Gln, Q), Glycine (Gly, G), Histidine (His, H), Isoleucine (Ile,
I), Leucine (Leu, L), Lysine (Lys, K), Methionine (Met, M),
Phenylalanine (Phe, F), Proline (Pro, P), Serine (Ser, S),
Threonine (Thr, T), Tryptophan (Trp, W), Tyrosine (Tyr, Y), Valine
(Val, V).
[0075] As used herein, the term "in vitro" refers to events that
occur in an artificial environment, e.g., in a test tube or
reaction vessel, in cell culture, in a Petri dish, etc., rather
than within an organism (e.g., animal, plant, or microbe).
[0076] As used herein, the term "in vivo" refers to events that
occur within an organism (e.g., animal, plant, or microbe).
[0077] As used herein, the term "isolated" refers to a substance or
entity that has been (1) separated from at least some of the
components with which it was associated when initially produced
(whether in nature or in an experimental setting), and (2)
produced, prepared, and/or manufactured by the hand of man.
Isolated substances and/or entities may be separated from at least
about 10%, about 20%, about 30%, about 40%, about 50%, about 60%,
about 70%, about 80%, about 90%, or more of the other components
with which they were initially associated. In some embodiments,
isolated agents are more than about 80%, about 85%, about 90%,
about 91%, about 92%, about 93%, about 94%, about 95%, about 96%,
about 97%, about 98%, about 99%, or more than about 99% pure. As
used herein, a substance is "pure" if it is substantially free of
other components.
[0078] The "isolated" products of this invention, including
isolated nucleic acids, proteins, polypeptides, and antibodies are
not products of nature (i.e., "non-naturally occurring"). Rather,
the "isolated" nucleic acids, proteins, polypeptides, and
antibodies of this invention are "man-made" products. The
"isolated" products of this invention can be "markedly different"
or "significantly different" from products of nature. By way of
non-limiting example, the isolated nucleic acids may be purified,
recombinant, synthetic, labeled, and/or attached to a solid
substrate. Such nucleic acids can be markedly different or
significantly different than nucleic acids that occur in nature. By
way of further non-limiting example, the "isolated" proteins,
polypeptides, and antibodies of this invention may be purified,
recombinant, synthetic, labeled, and/or attached to a solid
substrate. Such proteins, polypeptides, and antibodies can be
markedly different or significantly different from proteins,
polypeptides, and antibodies that occur in nature.
[0079] The term "peptide" as used herein refers to a short
polypeptide, e.g., one that typically contains less than about 50
amino acids and more typically less than about 30 amino acids. The
term as used herein encompasses analogs and mimetics that mimic
structural and thus biological function.
[0080] The term "polypeptide" encompasses both naturally-occurring
and non-naturally occurring proteins, and fragments, mutants,
derivatives and analogs thereof. A polypeptide may be monomeric or
polymeric. Further, a polypeptide may comprise a number of
different domains each of which has one or more distinct
activities. For the avoidance of doubt, a "polypeptide" may be any
length greater two amino acids.
[0081] The term "isolated protein" or "isolated polypeptide" is a
protein or polypeptide that by virtue of its origin or source of
derivation (1) is not associated with naturally associated
components that accompany it in its native state, (2) exists in a
purity not found in nature, where purity can be adjudged with
respect to the presence of other cellular material (e.g., is free
of other proteins from the same species) (3) is expressed by a cell
from a different species, or (4) does not occur in nature (e.g., it
is a fragment of a polypeptide found in nature or it includes amino
acid analogs or derivatives not found in nature or linkages other
than standard peptide bonds). Thus, a polypeptide that is
chemically synthesized or synthesized in a cellular system
different from the cell from which it naturally originates will be
"isolated" from its naturally associated components. A polypeptide
or protein may also be rendered substantially free of naturally
associated components by isolation, using protein purification
techniques well known in the art. As thus defined, "isolated" does
not necessarily require that the protein, polypeptide, peptide or
oligopeptide so described has been physically removed from a cell
in which it was synthesized.
[0082] The protein or polypeptide can be purified. Preferably, the
purified protein or polypeptide is more than 50%, 75%, 85%, 90%,
95%, 97%, 98%, or 99% pure. Within the context of this invention, a
purified protein that is more than 50% (etc.) pure means a purified
protein sample containing less than 50% (etc.) other proteins. For
example, a sample of a protein comprising can be 99% pure if it
contains less than 1% contaminating host cell proteins.
[0083] The term "polypeptide fragment" as used herein refers to a
polypeptide that has a deletion, e.g., an amino-terminal and/or
carboxy-terminal deletion compared to a full-length polypeptide,
such as a naturally occurring protein. In an embodiment, the
polypeptide fragment is a contiguous sequence in which the amino
acid sequence of the fragment is identical to the corresponding
positions in the naturally-occurring sequence. Fragments typically
are at least 5, 6, 7, 8, 9 or 10 amino acids long, or at least 12,
14, 16 or 18 amino acids long, or at least 20 amino acids long, or
at least 25, 30, 35, 40 or 45, amino acids, or at least 50 or 60
amino acids long, or at least 70 amino acids long, or at least 100
amino acids long.
[0084] The term "fusion protein" refers to a polypeptide comprising
a polypeptide or fragment coupled to heterologous amino acid
sequences. Fusion proteins are useful because they can be
constructed to contain two or more desired functional elements that
can be from two or more different proteins. A fusion protein
comprises at least 10 contiguous amino acids from a polypeptide of
interest, or at least 20 or 30 amino acids, or at least 40, 50 or
60 amino acids, or at least 75, 100 or 125 amino acids. The
heterologous polypeptide included within the fusion protein is
usually at least 6 amino acids in length, or at least 8 amino acids
in length, or at least 15, 20, or 25 amino acids in length. Fusions
that include larger polypeptides, such as an IgG Fc region, and
even entire proteins, such as the green fluorescent protein ("GFP")
chromophore-containing proteins, have particular utility. Fusion
proteins can be produced recombinantly by constructing a nucleic
acid sequence which encodes the polypeptide or a fragment thereof
in frame with a nucleic acid sequence encoding a different protein
or peptide and then expressing the fusion protein. Alternatively, a
fusion protein can be produced chemically by crosslinking the
polypeptide or a fragment thereof to another protein.
[0085] As used herein, "recombinant" may refer to a biomolecule,
e.g., a gene or protein, or to an organism. The term "recombinant"
may be used in reference to cloned DNA isolates, chemically
synthesized polynucleotides, or polynucleotides that are
biologically synthesized by heterologous systems, as well as
proteins or polypeptides and/or RNAs encoded by such nucleic acids.
A "recombinant" nucleic acid is a nucleic acid linked to a
nucleotide or polynucleotide to which it is not linked in nature. A
"recombinant" protein or polypeptide may be (1) a protein or
polypeptide linked to an amino acid or polypeptide to which it is
not linked in nature; and/or (2) a protein or polypeptide made by
transcription and/or translation of a recombinant nucleic acid.
Thus, a protein synthesized by a microorganism is recombinant, for
example, if it is synthesized from an mRNA synthesized from a
recombinant nucleic acid present in the cell. A "recombinant" cell
is a cell comprising a "recombinant" biomolecule. For example, a T
cell that comprises a "recombinant" nucleic acid is a "recombinant"
cell.
[0086] The term "polynucleotide", "nucleic acid molecule", "nucleic
acid", or "nucleic acid sequence" refers to a polymeric form of
nucleotides of at least 10 bases in length. The term includes DNA
molecules (e.g., cDNA or genomic or synthetic DNA) and RNA
molecules (e.g., mRNA or synthetic RNA), as well as analogs of DNA
or RNA containing non-natural nucleotide analogs, non-native
internucleoside bonds, or both. The nucleic acid can be in any
topological conformation. For instance, the nucleic acid can be
single-stranded, double-stranded, triple-stranded, quadruplexed,
partially double-stranded, branched, hairpinned, circular, or in a
padlocked conformation. The nucleic acid (also referred to as
polynucleotides) may include both sense and antisense strands of
RNA, cDNA, genomic DNA, and synthetic forms and mixed polymers of
the above. They may be modified chemically or biochemically or may
contain non-natural or derivatized nucleotide bases, as will be
readily appreciated by those of skill in the art. Such
modifications include, for example, labels, methylation,
substitution of one or more of the naturally occurring nucleotides
with an analog, internucleotide modifications such as uncharged
linkages (e.g., methyl phosphonates, phosphotriesters,
phosphoramidates, carbamates, etc.), charged linkages (e.g.,
phosphorothioates, phosphorodithioates, etc.), pendent moieties
(e.g., polypeptides), intercalators (e.g., acridine, psoralen,
etc.), chelators, alkylators, and modified linkages (e.g., alpha
anomeric nucleic acids, etc.) Also included are synthetic molecules
that mimic polynucleotides in their ability to bind to a designated
sequence via hydrogen bonding and other chemical interactions. Such
molecules are known in the art and include, for example, those in
which peptide linkages substitute for phosphate linkages in the
backbone of the molecule. Other modifications can include, for
example, analogs in which the ribose ring contains a bridging
moiety or other structure such as the modifications found in
"locked" nucleic acids.
[0087] A "synthetic" RNA, DNA or a mixed polymer is one created
outside of a cell, for example one synthesized chemically.
[0088] The term "nucleic acid fragment" as used herein refers to a
nucleic acid sequence that has a deletion, e.g., a 5'-terminal or
3'-terminal deletion compared to a full-length reference nucleotide
sequence. In an embodiment, the nucleic acid fragment is a
contiguous sequence in which the nucleotide sequence of the
fragment is identical to the corresponding positions in the
naturally-occurring sequence. In some embodiments, fragments are at
least 10, 15, 20, or 25 nucleotides long, or at least 20, 30, 40,
50, 60, 70, 80, 90, 100, 110, 120, 130, 140, or 150 nucleotides
long. In some embodiments a fragment of a nucleic acid sequence is
a fragment of an open reading frame sequence.
[0089] In some embodiments such a fragment encodes a polypeptide
fragment (as defined herein) of the protein encoded by the open
reading frame nucleotide sequence.
[0090] The nucleic acid can be purified. Preferably, the purified
nucleic acid is more than 50%, 75%, 85%, 90%, 95%, 97%, 98%, or 99%
pure. Within the context of this invention, a purified nucleic acid
that is at least 50% pure means a purified nucleic acid sample
containing less than 50% other nucleic acids. For example, a sample
of a plasmid can be at least 99% pure if it contains less than 1%
contaminating bacterial DNA.
[0091] As used herein, an endogenous nucleic acid sequence in the
genome of an organism (or the encoded protein product of that
sequence) is deemed "recombinant" herein if a heterologous sequence
is placed adjacent to the endogenous nucleic acid sequence. In this
context, a heterologous sequence is a sequence that is not
naturally adjacent to the endogenous nucleic acid sequence, whether
or not the heterologous sequence is itself endogenous (originating
from the same host cell or progeny thereof) or exogenous
(originating from a different host cell or progeny thereof). By way
of example, a promoter sequence can be substituted (e.g., by
homologous recombination) for the native promoter of a gene in the
genome of a host cell, such that this gene has an altered
expression pattern. This gene would now become "recombinant"
because it is separated from at least some of the sequences that
naturally flank it.
[0092] A nucleic acid is also considered "recombinant" if it
contains any modifications that do not naturally occur to the
corresponding nucleic acid in a genome. For instance, an endogenous
coding sequence is considered "recombinant" if it contains an
insertion, deletion or a point mutation introduced artificially,
e.g., by human intervention. A "recombinant nucleic acid" also
includes a nucleic acid integrated into a host cell chromosome at a
heterologous site and a nucleic acid construct present as an
episome.
[0093] As used herein, the phrase "degenerate variant" of a
reference nucleic acid sequence encompasses nucleic acid sequences
that can be translated, according to the standard genetic code, to
provide an amino acid sequence identical to that translated from
the reference nucleic acid sequence. The term "degenerate
oligonucleotide" or "degenerate primer" is used to signify an
oligonucleotide capable of hybridizing with target nucleic acid
sequences that are not necessarily identical in sequence but that
are homologous to one another within one or more particular
segments.
[0094] The term "percent sequence identity" or "identical" in the
context of nucleic acid sequences refers to the residues in the two
sequences which are the same when aligned for maximum
correspondence. The length of sequence identity comparison may be
over a stretch of at least about nine nucleotides, usually at least
about 20 nucleotides, more usually at least about 24 nucleotides,
typically at least about 28 nucleotides, more typically at least
about 32, and even more typically at least about 36 or more
nucleotides. There are a number of different algorithms known in
the art which can be used to measure nucleotide sequence identity.
For instance, polynucleotide sequences can be compared using FASTA,
Gap or Bestfit, which are programs in Wisconsin Package Version
10.0, Genetics Computer Group (GCG), Madison, Wis. FASTA provides
alignments and percent sequence identity of the regions of the best
overlap between the query and search sequences. Pearson, Methods
Enzymol. 183:63-98 (1990). For instance, percent sequence identity
between nucleic acid sequences can be determined using FASTA with
its default parameters (a word size of 6 and the NOPAM factor for
the scoring matrix) or using Gap with its default parameters as
provided in GCG Version 6.1, herein incorporated by reference.
Alternatively, sequences can be compared using the computer
program, BLAST (Altschul et al., J. Mol. Biol. 215:403-410 (1990);
Gish and States, Nature Genet. 3:266-272 (1993); Madden et al.,
Meth. Enzymol. 266:131-141 (1996); Altschul et al., Nucleic Acids
Res. 25:3389-3402 (1997); Zhang and Madden, Genome Res. 7:649-656
(1997)), especially blastp or tblastn (Altschul et al., Nucleic
Acids Res. 25:3389-3402 (1997)).
[0095] As used herein, an "expression control sequence" refers to
polynucleotide sequences which affect the expression of coding
sequences to which they are operatively linked. Expression control
sequences are sequences which control the transcription,
post-transcriptional events and translation of nucleic acid
sequences. Expression control sequences include appropriate
transcription initiation, termination, promoter and enhancer
sequences; efficient RNA processing signals such as splicing and
polyadenylation signals; sequences that stabilize cytoplasmic mRNA;
sequences that enhance translation efficiency (e.g., ribosome
binding sites); sequences that enhance protein stability; and when
desired, sequences that enhance protein secretion. The nature of
such control sequences differs depending upon the host organism; in
prokaryotes, such control sequences generally include promoter,
ribosomal binding site, and transcription termination sequence. The
term "control sequences" is intended to encompass, at a minimum,
any component whose presence is essential for expression, and can
also encompass an additional component whose presence is
advantageous, for example, leader sequences and fusion partner
sequences.
[0096] As used herein, "operatively linked" or "operably linked"
expression control sequences refers to a linkage in which the
expression control sequence is contiguous with the gene of interest
to control the gene of interest, as well as expression control
sequences that act in trans or at a distance to control the gene of
interest.
[0097] As used herein, a "vector" is intended to refer to a nucleic
acid molecule capable of transporting another nucleic acid to which
it has been linked. One type of vector is a "plasmid," which
generally refers to a circular double stranded DNA loop into which
additional DNA segments may be ligated, but also includes linear
double-stranded molecules such as those resulting from
amplification by the polymerase chain reaction (PCR) or from
treatment of a circular plasmid with a restriction enzyme. Other
vectors include cosmids, bacterial artificial chromosomes (BAC) and
yeast artificial chromosomes (YAC). Another type of vector is a
viral vector, wherein additional DNA segments may be ligated into
the viral genome (discussed in more detail below). Certain vectors
are capable of autonomous replication in a host cell into which
they are introduced (e.g., vectors having an origin of replication
which functions in the host cell). Other vectors can be integrated
into the genome of a host cell upon introduction into the host
cell, and are thereby replicated along with the host genome.
Moreover, certain vectors are capable of directing the expression
of genes to which they are operatively linked. Such vectors are
referred to herein as "recombinant expression vectors" (or simply
"expression vectors"). The integrating cosmid vector pYUB412 is an
example of a "vector".
[0098] The term "recombinant host cell" (or simply "recombinant
cell" or "host cell"), as used herein, is intended to refer to a
cell into which a recombinant nucleic acid such as a recombinant
vector has been introduced. In some instances the word "cell" is
replaced by a name specifying a type of cell. For example, a
"recombinant microorganism" is a recombinant host cell that is a
microorganism host cell. It should be understood that such terms
are intended to refer not only to the particular subject cell but
to the progeny of such a cell. Because certain modifications may
occur in succeeding generations due to either mutation or
environmental influences, such progeny may not, in fact, be
identical to the parent cell, but are still included within the
scope of the term "recombinant host cell," "recombinant cell," and
"host cell", as used herein. A recombinant host cell may be an
isolated cell or cell line grown in culture or may be a cell which
resides in a living tissue or organism.
[0099] As used herein, the term "mammal" refers to any member of
the taxonomic class mammalia, including placental mammals and
marsupial mammals. Thus, "mammal" includes humans, primates,
livestock, and laboratory mammals. Exemplary mammals include a
rodent, a mouse, a rat, a rabbit, a dog, a cat, a sheep, a horse, a
goat, a llama, cattle, a primate, a pig, and any other mammal. In
some embodiments, the mammal is at least one of a transgenic
mammal, a genetically-engineered mammal, and a cloned mammal.
Chimeric Antigen Receptors
[0100] The invention encompasses a chimeric antigen receptor (CAR)
comprising an extracellular antigen-binding domain (binding
domain), a hinge and transmembrane domain (transmembrane domain); a
hook-binding domain; and an intracellular signaling domain
(activation domain). The CAR can contain one, two, three, or more
of each of these domains.
[0101] The invention encompasses individually all possible
combinations of the specific polypeptides and fragments thereof
recited herein.
[0102] The invention encompasses CARs comprising a hook-binding
domain. A "hook-binding domain" is a domain that reversibly binds
directly or indirectly to a "hook" protein inside of the cell,
which binding prevents the CAR from exiting the endoplasmic
reticulum (ER) or Golgi under appropriate conditions.
[0103] In one embodiment, the hook-binding domain comprises a
streptavidin-binding peptide (SBP), which can bind to a hook
protein that bears the core streptavidin. Biotin causes the release
of the CAR containing the hook-binding domain from the hook by
out-competing the SBP. The CAR can then move to the cell
membrane.
[0104] Preferably, a system referred to as RUSH (retention using
selective hooks) system can be employed, Boncompain et al., Nat.
Methods 9:493-498, 2012, which is hereby incorporated by
reference.
[0105] Preferably, the hook-binding domain comprises the amino acid
sequence: MDEKTTGWRGGHVVEGLAGELEQLRARLEHHPQGQREP (SEQ ID NO:1) or
is encoded by the nucleic acid sequence:
ATGGACGAGAAAACCACCGGCTGGCGGGGAGGCCACGTGGTGGAAGGACTGGC
CGGCGAGCTGGAACAGCTGCGGGCCAGACTGGAACACCACCCCCAGGGCCAGA GAGAGCCC (SEQ
ID NO:2).
[0106] Shorter SBP fragments, deleted at their N-terminus and
C-terminus may be used with identical efficacy. See Barrette-Ng, I.
H., S. C. Wu, W. M. Tjia, S. L. Wong, and K. K. Ng. 2013, The
structure of the SBP-Tag-streptavidin complex reveals a novel
helical scaffold bridging binding pockets on separate subunits,
Acta crystallographica. Section D, Biological crystallography
69:879-887.
[0107] One embodiment of a hook-binding domain is set forth in
WO2010/142785, which is hereby incorporated by reference. The
FKBPF-K506 binding domain 12 (FKBPI2) can be used with
FKBP-rapamycin associated protein (FRAP) as the hook. In this
embodiment, the interaction occurs only in the presence of
rapamycin or analogues thereof that are able to mediate the
interaction between FKBP12 and FRAP and can be, in particular,
selected from the group consisting of FKI012, FK-CsA, and
rapamycin.
[0108] In one embodiment, the hook-binding domain is located in the
intracytoplasmic region of the CAR. In other embodiments, the
hook-binding domain is located in other positions, i.e., in all the
junctions between the different intracytoplamsic elements (e.g.,
between the transmembrane region and the first co-stimulation
element) or between the different co-stimulation elements.
[0109] The invention comprises CARs containing a binding domain
that comprises an antibody that binds specifically to a human
polypeptide. The term "antibody" is meant to include polyclonal
antibodies, monoclonal antibodies, fragments thereof, such as
F(ab')2 and Fab fragments, single-chain variable fragments (scFvs),
single-domain antibody fragments (VHHs or Nanobodies, preferably
camelid), and bivalent and trivalent ntibody fragments (diabodies
and triabodies).
[0110] Preferably, the antibody is a single-chain Fv antibody or a
nanobody.
[0111] In one embodiment, the antibody is monospecific. In one
embodiment, the antibody is multispecific for 2, 3, or 4
polypeptides. Preferably, the antibody is bispecific.
[0112] Antibodies can be synthetic, monoclonal, or polyclonal and
can be made by techniques well known in the art. Such antibodies
specifically bind to human proteins via the antigen-binding sites
of the antibody (as opposed to non-specific binding). Human
proteins, polypeptide fragments, and peptides can be employed as
immunogens in producing antibodies immunoreactive therewith. The
human proteins, polypeptides, and peptides contain antigenic
determinants or epitopes that elicit the formation of
antibodies.
[0113] These antigenic determinants or epitopes can be either
linear or conformational (discontinuous). Linear epitopes are
composed of a single section of amino acids of the polypeptide,
while conformational or discontinuous epitopes are composed of
amino acids sections from different regions of the polypeptide
chain that are brought into close proximity upon protein folding
(C. A. Janeway, Jr. and P. Travers, Immuno Biology 3:9 (Garland
Publishing Inc., 2nd ed. 1996)). Because folded proteins have
complex surfaces, the number of epitopes available is quite
numerous; however, due to the conformation of the protein and
steric hindrance, the number of antibodies that actually bind to
the epitopes is less than the number of available epitopes (C. A.
Janeway, Jr. and P. Travers, Immuno Biology 2:14 (Garland
Publishing Inc., 2nd ed. 1996)). Epitopes can be identified by any
of the methods known in the art.
[0114] Thus, one aspect of the present invention relates to the
antigenic epitopes of human proteins. Such epitopes are useful for
raising antibodies, in particular monoclonal antibodies, as
described in detail below.
[0115] Antibodies are defined to be specifically binding if they
bind human proteins or polypeptides with a Ka of greater than or
equal to about 10.sup.7 M.sup.-1. Affinities of binding partners or
antibodies can be readily determined using conventional techniques,
for example those described by Scatchard et al., Ann. N.Y. Acad.
Sci., 51:660 (1949).
[0116] Polyclonal antibodies can be readily generated from a
variety of sources, for example, horses, cows, goats, sheep, dogs,
chickens, rabbits, mice, or rats, using procedures that are well
known in the art. In general, a purified human protein or
polypeptide that is appropriately conjugated is administered to the
host animal typically through parenteral injection. The
immunogenicity can be enhanced through the use of an adjuvant, for
example, Freund's complete or incomplete adjuvant. Following
booster immunizations, small samples of serum are collected and
tested for reactivity to human proteins or polypeptides. Examples
of various assays useful for such determination include those
described in Antibodies: A Laboratory Manual, Harlow and Lane
(eds.), Cold Spring Harbor Laboratory Press, 1988; as well as
procedures, such as countercurrent immuno-electrophoresis (CIEP),
radioimmunoassay, radio-immunoprecipitation, enzyme-linked
immunosorbent assays (ELISA), dot blot assays, and sandwich assays.
See U.S. Pat. Nos. 4,376,110 and 4,486,530.
[0117] Monoclonal antibodies can be readily prepared using well
known procedures. See, for example, the procedures described in
U.S. Pat. Nos. RE 32,011, 4,902,614, 4,543,439, and 4,411,993;
Monoclonal Antibodies, Hybridomas: A New Dimension in Biological
Analyses, Plenum Press, Kennett, McKeam, and Bechtol (eds.),
1980.
[0118] For example, the host animals, such as mice, can be injected
intraperitoneally at least once and preferably at least twice at
about 3 week intervals with isolated and purified human proteins or
conjugated human polypeptides, for example a peptide comprising or
consisting of the specific amino acids set forth above. Mouse sera
are then assayed by conventional dot blot technique or antibody
capture (ABC) to determine which animal is best to fuse.
Approximately two to three weeks later, the mice are given an
intravenous boost of the human protein or polypeptide. Mice are
later sacrificed and spleen cells fused with commercially available
myeloma cells, such as Ag8.653 (ATCC), following established
protocols. Briefly, the myeloma cells are washed several times in
media and fused to mouse spleen cells at a ratio of about three
spleen cells to one myeloma cell. The fusing agent can be any
suitable agent used in the art, for example, polyethylene glycol
(PEG). Fusion is plated out into plates containing media that
allows for the selective growth of the fused cells. The fused cells
can then be allowed to grow for approximately eight days.
Supernatants from resultant hybridomas are collected and added to a
plate that is first coated with goat anti-mouse Ig. Following
washes, a label, such as a labeled human protein or polypeptide, is
added to each well followed by incubation. Positive wells can be
subsequently detected. Positive clones can be grown in bulk culture
and supernatants are subsequently purified over a Protein A column
(Pharmacia).
[0119] The monoclonal antibodies of the invention can be produced
using alternative techniques, such as those described by
Alting-Mees et al., "Monoclonal Antibody Expression Libraries: A
Rapid Alternative to Hybridomas", Strategies in Molecular Biology
3:1-9 (1990), which is incorporated herein by reference. Similarly,
binding partners can be constructed using recombinant DNA
techniques to incorporate the variable regions of a gene that
encodes a specific binding antibody. Such a technique is described
in Larrick et al., Biotechnology, 7:394 (1989).
[0120] Antigen-binding fragments of such antibodies, which can be
produced by conventional techniques, are also encompassed by the
present invention. Examples of such fragments include, but are not
limited to, Fab and F(ab')2 fragments. Antibody fragments and
derivatives produced by genetic engineering techniques are also
provided.
[0121] The monoclonal antibodies of the present invention include
chimeric antibodies, e.g., humanized versions of murine monoclonal
antibodies. Such humanized antibodies can be prepared by known
techniques, and offer the advantage of reduced immunogenicity when
the antibodies are administered to humans. In one embodiment, a
humanized monoclonal antibody comprises the variable region of a
murine antibody (or just the antigen binding site thereof) and a
constant region derived from a human antibody. Alternatively, a
humanized antibody fragment can comprise the antigen binding site
of a murine monoclonal antibody and a variable region fragment
(lacking the antigen-binding site) derived from a human antibody.
Procedures for the production of chimeric and further engineered
monoclonal antibodies include those described in Riechmann et al.
(Nature 332:323, 1988), Liu et al. (PNAS 84:3439, 1987), Larrick et
al. (Bio/Technology 7:934, 1989), and Winter and Harris (TIPS
14:139, May, 1993). Procedures to generate antibodies
transgenically can be found in GB 2,272,440, U.S. Pat. Nos.
5,569,825 and 5,545,806.
[0122] Antibodies produced by genetic engineering methods, such as
chimeric and humanized monoclonal antibodies, comprising both human
and non-human portions, which can be made using standard
recombinant DNA techniques, can be used. Such chimeric and
humanized monoclonal antibodies can be produced by genetic
engineering using standard DNA techniques known in the art, for
example using methods described in Robinson et al. International
Publication No. WO 87/02671; Akira, et al. European Patent
Application 0184187; Taniguchi, M., European Patent Application
0171496; Morrison et al. European Patent Application 0173494;
Neuberger et al. PCT International Publication No. WO 86/01533;
Cabilly et al. U.S. Pat. No. 4,816,567; Cabilly et al. European
Patent Application 0125023; Better et al., Science 240:1041 1043,
1988; Liu et al., PNAS 84:3439 3443, 1987; Liu et al., J. Immunol.
139:3521 3526, 1987; Sun et al. PNAS 84:214 218, 1987; Nishimura et
al., Canc. Res. 47:999 1005, 1987; Wood et al., Nature 314:446 449,
1985; and Shaw et al., J. Natl. Cancer Inst. 80:1553 1559, 1988);
Morrison, S. L., Science 229:1202 1207, 1985; Oi et al.,
BioTechniques 4:214, 1986; Winter U.S. Pat. No. 5,225,539; Jones et
al., Nature 321:552 525, 1986; Verhoeyan et al., Science 239:1534,
1988; and Beidler et al., J. Immunol. 141:4053 4060, 1988.
[0123] An immunoglobulin library can be expressed by a population
of display packages, preferably derived from filamentous phage, to
form an antibody display library. Examples of methods and reagents
particularly amenable for use in generating antibody display
library can be found in, for example, Ladner et al. U.S. Pat. No.
5,223,409; Kang et al. PCT publication WO 92/18619; Dower et al.
PCT publication WO 91/17271; Winter et al. PCT publication WO
92/20791; Markland et al. PCT publication WO 92/15679; Breitling et
al. PCT publication WO 93/01288; McCafferty et al. PCT publication
WO 92/01047; Garrard et al. PCT publication WO 92/09690; Ladner et
al. PCT publication WO 90/02809; Fuchs et al. (1991) Bio/Technology
9:1370 1372; Hay et al. (1992) Hum Antibod Hybridomas 3:81 85; Huse
et al. (1989) Science 246:1275 1281; Griffths et al. (1993) supra;
Hawkins et al. (1992) J Mol Biol 226:889 896; Clackson et al.
(1991) Nature 352:624 628; Gram et al. (1992) PNAS 89:3576 3580;
Garrad et al. (1991) Bio/Technology 9:1373 1377; Hoogenboom et al.
(1991) Nuc Acid Res 19:4133 4137; and Barbas et al. (1991) PNAS
88:7978 7982. Once displayed on the surface of a display package
(e.g., filamentous phage), the antibody library is screened to
identify and isolate packages that express an antibody that binds a
human protein or polypeptide. In a preferred embodiment, the
primary screening of the library involves panning with an
immobilized human protein or polypeptide and display packages
expressing antibodies that bind immobilized human protein or
polypeptide are selected.
[0124] In connection with synthetic and semi-synthetic antibodies,
such terms are intended to cover but are not limited to antibody
fragments, isotype switched antibodies, humanized antibodies (e.g.,
mouse-human, human-mouse), hybrids, antibodies having plural
specificities, and fully synthetic antibody-like molecules.
[0125] The invention encompasses a CAR comprising an activation
domain comprising CD3-.zeta. or Fc receptor .gamma. amino acid
sequences. Sadelain et al., Cancer Discov. 2013 April; 3(4):
388-398, which is hereby incorporated by reference. The invention
further encompasses a CAR comprising an activation domain
comprising a CD3-.zeta. chain and the cytoplasmic domain of a
costimulatory receptor such as CD28, 4-1 BB (CD137), DAP10, OX40
(CD134), ICOS, CD27, or CD40L. Id.
[0126] Preferably, the CAR comprises a fragment of at least 50, 60,
70, 80, 90,100, 110, 120, 150, or 200 amino acids of at least one
of the following amino acid sequences having T-cell activating
activity.
TABLE-US-00001 CD3-.zeta. (SEQ ID NO: 3) MKWKALFTAA ILQAQLPITE
AQSFGLLDPK LCYLLDGILF IYGVILTALF LRVKFSRSAD APAYQQGQNQ LYNELNLGRR
EEYDVLDKRR GRDPEMGGKP QRRKNPQEGL YNELQKDKMA EAYSEIGMKG ERRRGKGHDG
LYQGLSTATK DTYDALHMQA LPPR CD28: (SEQ ID NO: 4) MLRLLLALNL
FPSIQVTGNK ILVKQSPMLV AYDNAVNLSC KYSYNLFSRE FRASLHKGLD SAVEVCVVYG
NYSQQLQVYS KTGFNCDGKL GNESVTFYLQ NLYVNQTDIY FCKIEVMYPP PYLDNEKSNG
TIIHVKGKHL CPSPLFPGPS KPFWVLVVVG GVLACYSLLV TVAFIIFWVR SKRSRLLHSD
YMNMTPRRPG PTRKHYQPYA PPRDFAAYRS 4-1BB (CD137): (SEQ ID NO: 5)
MGNSCYNIVA TLLLVLNFER TRSLQDPCSN CPAGTFCDNN RNQICSPCPP NSFSSAGGQR
TCDICRQCKG VFRTRKECSS TSNAECDCTP GFHCLGAGCS MCEQDCKQGQ ELTKKGCKDC
CFGTFNDQKR GICRPWTNCS LDGKSVLVNG TKERDVVCGP SPADLSPGAS SVTPPAPARE
PGHSPQIISF FLALTSTALL FLLFFLTLRF SVVKRGRKKL LYIFKQPFMR PVQTTQEEDG
CSCRFPEEEE GGCEL DAP10: (SEQ ID NO: 6) MIHLGHILFL LLLPVAAAQT
TPGERSSLPA FYPGTSGSCS GCGSLSLPLL AGLVAADAVA SLLIVGAVFL CARPRRSPAQ
EDGKVYINMP GRG OX40 (CD134): (SEQ ID NO: 7) MCVGARRLGR GPCAALLLLG
LGLSTVTGLH CVGDTYPSND RCCHECRPGN GMVSRCSRSQ NTVCRPCGPG FYNDVVSSKP
CKPCTWCNLR SGSERKQLCT ATQDTVCRCR AGTQPLDSYK PGVDCAPCPP GHFSPGDNQA
CKPWTNCTLA GKHTLQPASN SSDAICEDRD PPATQPQETQ GPPARPITVQ PTEAWPRTSQ
GPSTRPVEVP GGRAVAAILG LGLVLGLLGP LAILLALYLL RRDQRLPPDA HKPPGGGSFR
TPIQEEQADA HSTLAKI ICOS: (SEQ ID NO: 8)
MKSGLWYFFLFCLRIKVLTGEINGSANYEMFIFHNGGVQILCKYPDIVQQFKMQLLKGG
QILCDLTKTKGSGNTVSIKSLKFCHSQLSNNSVSFFLYNLDHSHANYYFCNLSIFDPPPF
KVTLTGGYLHIYESQLCCQLKFWLPIGCAAFVVVCILGCILICWLTKKKYSSSVHDPNGE
YMFMRAVNTAKKSRLTDVTL CD27: (SEQ ID NO: 33)
MARPHPWWLCVLGTLVGLSATPAPKSCPERHYWAQGKLCCQMCEPGTFLVKDCDQ
HRKAAQCDPCIPGVSFSPDHHTRPHCESCRHCNSGLLVRNCTITANAECACRNGWQ
CRDKECTECDPLPNPSLTARSSQALSPHPQPTHLPYVSEMLEARTAGHMQTLADFRQ
LPARTLSTHWPPQRSLCSSDFIRILVIFSGMFLVFTLAGALFLHQRRKYRSNKGESPVE
PAEPCHYSCPREEEGSTIPIQEDYRKPEPACSP CD40L (CD154): (SEQ ID NO: 34)
MIETYNQTSPRSAATGLPISMKIFMYLLTVFLITQMIGSALFAVYLHRRLDKIEDERNLHE
DFVFMKTIQRCNTGERSLSLLNCEEIKSQFEGFVKDIMLNKEETKKENSFEMQKGDQN
PQIAAHVISEASSKTTSVLQWAEKGYYTMSNNLVTLENGKQLTVKRQGLYYIYAQVTFC
SNREASSQAPFIASLCLKSPGRFERILLRAANTHSSAKPCGQQSIHLGGVFELQPGASV
FVNVTDPSQVSHGTGFTSFGLLKL
[0127] In various embodiments, CAR comprises a fragment of at least
20, 30, 40, 50, 60, 70, 80, 90,100, 110, 120, 150, or 200 amino
acids that shares at least than 90%, preferably more than 95%, more
preferably more than 99% identity with the amino acid sequence of
SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7,
SEQ ID NO:8, SEQ ID NO:33, or SEQ ID NO:34.
[0128] In various embodiments, the activation domain of the CAR
comprises one, two, three, or more fragments of at least 20, 30,
40, 50, 60, 70, 80, 90,100, 110, 120, 150, or 200 amino acids that
share at least than 90%, preferably more than 95%, more preferably
more than 99% identity with the amino acid sequence of SEQ ID NO:3,
SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8,
SEQ ID NO:33, or SEQ ID NO:34.
[0129] The invention encompasses a CAR comprising a transmembrane
(TM) domain, preferably a fragment of at least 20, 30, 40, 50, 60,
70, 80, 90,100, 110, 120, 150, or 200 amino acids, most preferably
of at least one of CD28, CD3z, CD8, CD4, FcR.gamma., TM
regions.
[0130] The CAR can be purified. Preferably, the purified CAR is
more than 50%, 75%, 85%, 90%, 95%, 97%, 98%, or 99% pure. Within
the context of this invention, a purified CAR that is more than 50%
(etc.) pure means a purified CAR sample containing less than 50%
(etc.) other proteins. For example, a sample of a recombinant CAR
purified from a host cell can be 99% pure if it contains less than
1% contaminating host cell proteins.
[0131] In a preferred embodiment, the CAR encodes the amino acid
sequence of any of SEQ ID NO:46, SEQ ID NO:48, or SEQ ID NO:50.
[0132] Particularly preferred CARs include those encoding any of
the constituents of the CAR depicted in FIG. 11, including signal
sequence, CD19 ScFv, CD8 hinge, transmembrane domain, 4-1BB, and/or
CD3z sequences. Other preferred CARs include those encoding any of
the constituents of the CARs of SEQ ID NOs:44-50. Particularly
preferred CARs have the CD19 ScFv domain replaced by another
binding region.
Nucleic Acids
[0133] The invention encompasses nucleic acids encoding a CAR. The
nucleic acid can be single-stranded or double-stranded. The nucleic
acid can be an RNA or DNA molecule. Preferred nucleic acids encode
an amino acid sequence of at least one of the SEQ ID NOs detailed
herein. The invention encompasses an isolated nucleic acid of the
invention inserted into a vector.
[0134] In one embodiment, the nucleic acid sequence comprises the
nucleic acid sequence of SEQ ID NO:2. In one embodiment the nucleic
acid sequence comprises one or more of the following nucleic acid
sequences:
TABLE-US-00002 HOOK: (CORE + HA TAG + OTHER SEQUENCE) (SEQ ID NO:
35) ATGCACCGGAGGAGATCACGCTCTTGTAGGGAGGACCAGAAACCTGTCAC
CGGTGACCCTAGCAAAGACTCAAAAGCTCAGGTGTCCGCTGCCGAGGCTG
GCATTACTGGAACATGGTACAATCAGCTCGGGAGCACCTTTATTGTGACT
GCTGGAGCCGATGGAGCCCTCACCGGAACATACGAATCTGCTGTGGGAAA
CGCCGAATCACGGTACGTCCTCACTGGCCGATACGATAGTGCCCCTGCCA
CCGACGGATCTGGGACTGCCCTGGGATGGACTGTCGCTTGGAAAAACAAC
TACCGGAATGCTCATTCTGCCACAACATGGAGTGGACAGTACGTGGGAGG
CGCTGAGGCTAGAATCAATACACAGTGGCTGCTCACATCTGGCACAACCG
AGGCAAATGCTTGGAAATCCACCCTGGTGGGACATGACACATTCACCAAA
GTGAAACCCTCCGCCGCTTCAATCGATGCCGCCAAAAAAGCCGGAGTCAA
CAACGGCAATCCTCTGGATGCCGTCCAGCAGGTCGACTATCCGTACGACG
TACCAGACTACGCAGTCGGACCGATGGACGATCAGAGGGACCTCATTAGC
AACAACGAACAGCTGCCTATGCTGGGACGGCGACCTGGAGCCCCTGAATC
CAAATGCTCTAGGGGAGCACTGTACACTGGCTTCTCCATTCTCGTGACAC
TGCTGCTGGCCGGGCAGGCTACTACTGCTTACTTCCTGTACCAGCAGCAG
GGGCGGCTGGACAAACTCACTGTGACATCTCAGAACCTCCAGCTGGAAAA
TCTGAGGATGAAACTGCCCAAACCCCCTAAACCCGTGTCCAAAATGAGGA
TGGCCACACCTCTGCTCATGCAGGCACTGCCAATGGGAGCCCTGCCCCAG
GGGCCCATGCAGAATGCCACCAAGTATGGCAACATGACAGAGGACCATGT
GATGCACCTGCTCCAGAATGCTGACCCCCTGAAGGTGTACCCGCCACTGA
AGGGGAGCTTCCCGGAGAACCTGAGACACCTTAAGAACACCATGGAGACC
ATAGACTGGAAGGTCTTTGAGAGCTGGATGCACCATTGGCTCCTGTTTGA
AATGAGCAGGCACTCCTTGGAGCAAAAGCCCACTGACGCTCCACCGAAAG
AGTCACTGGAACTGGAGGACCCGTCTTCTGGGCTGGGTGTGACCAAGCAG
GATCTGGGCCCAGTCCCCATGTGA. CORE STREPTAVIDIN: (SEQ ID NO: 36)
GACCCTAGCAAAGACTCAAAAGCTCAGGTGTCCGCTGCCGAGGCTGGCAT
TACTGGAACATGGTACAATCAGCTCGGGAGCACCTTTATTGTGACTGCTG
GAGCCGATGGAGCCCTCACCGGAACATACGAATCTGCTGTGGGAAACGCC
GAATCACGGTACGTCCTCACTGGCCGATACGATAGTGCCCCTGCCACCGA
CGGATCTGGGACTGCCCTGGGATGGACTGTCGCTTGGAAAAACAACTACC
GGAATGCTCATTCTGCCACAACATGGAGTGGACAGTACGTGGGAGGCGCT
GAGGCTAGAATCAATACACAGTGGCTGCTCACATCTGGCACAACCGAGGC
AAATGCTTGGAAATCCACCCTGGTGGGACATGACACATTCACCAAAGTGA
AACCCTCCGCCGCTTCAATCGATGCCGCCAAAAAAGCCGGAGTCAACAAC
GGCAATCCTCTGGATGCCGTCCAGCAG. HA TAG: (SEQ ID NO: 37)
TATCCGTACGACGTACCAGACTACGCA.
[0135] Preferred nucleic acids are of at least 50, 60, 70, 80,
90,100, 110, 120, 150, 200, 300, 400, 500, or 600 nucleotides.
[0136] The nucleic acid can be purified. Preferably, the purified
nucleic acid is more than 50%, 75%, 85%, 90%, 95%, 97%, 98%, or 99%
pure. Within the context of this invention, a purified nucleic acid
that is more than 50% pure means a purified nucleic acid sample
containing less than 50% other nucleic acids. For example, a sample
of a plasmid purified from a host bacteria can be 99% pure if it
contains less than 1% contaminating bacterial DNA.
[0137] Particularly preferred nucleic acids include the
following:
TABLE-US-00003 CAR_CD19 2.sup.Nd generation: 1518 bp (SEQ ID NO:
39) ATGGCCCTGCCTGTGACAGCCCTGCTGCTGCCCCTGGCTCTCCTGCTGCA
TGCCGCCAGACCCGCTAGCGACATCCAGATGACCCAGACCACCAGCAGCC
TGAGCGCCAGCCTGGGCGACAGAGTGACCATCAGCTGCCGGGCCAGCCAG
GACATCAGCAAGTACCTGAACTGGTATCAGCAGAAACCCGACGGCACCGT
GAAGCTGCTGATCTACCACACCAGCCGGCTCCACAGCGGCGTGCCCAGCA
GATTTTCTGGCAGCGGCAGCGGCACCGACTACAGCCTGACCATCTCCAAC
CTGGAACAGGAAGATATCGCTACCTACTTCTGTCAGCAAGGCAACACCCT
GCCCTACACCTTCGGCGGAGGCACCAAGCTGGAAATCACCGGCGGAGGCG
GAAGTGGAGGTGGAGGATCTGGCGGCGGAGGCTCCGAAGTGAAGCTGCAG
GAAAGCGGCCCTGGCCTCGTGGCCCCTAGCCAGAGCCTGTCCGTGACCTG
TACCGTGTCCGGCGTGTCCCTGCCCGACTACGGCGTGTCCTGGATCAGAC
AGCCTCCCAGAAAGGGCCTGGAATGGCTGGGCGTGATCTGGGGCAGCGAG
ACAACCTACTACAACAGCGCCCTGAAGTCCCGGCTGACCATCATCAAGGA
CAACAGCAAGAGCCAGGTGTTCCTGAAGATGAACAGCCTGCAGACCGACG
ACACCGCCATCTACTACTGCGCCAAGCACTACTACTACGGCGGCAGCTAC
GCCATGGACTACTGGGGCCAGGGCACCAGCGTGACCGTGTCCAGCCATAT
GGCCCTGAGCAACAGCATCATGTACTTCAGCCACTTCGTGCCCGTGTTTC
TGCCCGCCAAGCCCACCACCACCCCTGCCCCTAGACCTCCCACCCCAGCC
CCAACAATCGCCAGCCAGCCTCTGTCCCTGCGGCCCGAAGCCTGTAGACC
TGCTGCCGGCGGAGCCGTGCACACCAGAGGCCTGGATATCTACATCTGGG
CCCCTCTGGCCGGCACCTGTGGCGTGCTGCTGCTGAGCCTGGTGATCACA
AAGCGGGGCAGAAAGAAGCTGCTGTACATCTTCAAGCAGCCATTCATGCG
GCCCGTGCAGACCACCCAGGAAGAGGACGGCTGCAGCTGCCGGTTCCCCG
AGGAAGAGGAAGGCGGCTGCGAACTGCCCAAGCTGTGCTACCTGCTGGAC
GGCATCCTGTTCATCTATGGCGTGATCCTGACCGCCCTGTTCCTGAGAGT
GAAGTTCAGCAGAAGCGCCGACGCCCCTGCCTACCAGCAGGGCCAGAACC
AGCTGTACAACGAGCTGAACCTGGGCAGACGGGAAGAGTACGACGTGCTG
GACAAGCGGAGAGGCCGGGACCCTGAGATGGGCGGCAAGCCCCAGCGGCG
GAAGAACCCTCAGGAAGGCCTGTATAACGAACTGCAGAAAGACAAGATGG
CCGAGGCCTACAGCGAGATCGGCATGAAGGGCGAGCGGCGGAGAGGCAAG
GGCCACGATGGCCTGTAC CAR_CD19 3.sup.rd generation: 1641 bp (SEQ ID
NO: 40) ATGGCCCTGCCTGTGACAGCCCTGCTGCTGCCCCTCGCTCTGCTGCTGCA
TGCCGCCAGACCCGCTAGCGACATCCAGATGACCCAGACCACCAGCAGCC
TGAGCGCCAGCCTGGGCGACAGAGTGACCATCAGCTGCCGGGCCAGCCAG
GACATCAGCAAGTACCTGAACTGGTATCAGCAGAAACCCGACGGCACCGT
GAAGCTGCTGATCTACCACACCAGCCGGCTCCACAGCGGCGTGCCCAGCA
GATTTTCTGGCAGCGGCAGCGGCACCGACTACAGCCTGACCATCTCCAAC
CTGGAACAGGAAGATATCGCTACCTACTTCTGTCAGCAAGGCAACACCCT
GCCCTACACCTTCGGCGGAGGCACCAAGCTGGAAATCACCGGCGGAGGCG
GAAGTGGAGGGGGAGGATCTGGCGGCGGAGGCTCCGAAGTGAAGCTGCAG
GAAAGCGGCCCTGGCCTGGTGGCCCCTAGCCAGAGCCTGTCCGTGACCTG
TACCGTGTCCGGCGTGTCCCTGCCCGACTACGGCGTGTCCTGGATCAGAC
AGCCCCCCAGAAAGGGCCTGGAATGGCTGGGCGTGATCTGGGGCAGCGAG
ACAACCTACTACAACAGCGCCCTGAAGTCCCGGCTGACCATCATCAAGGA
CAACAGCAAGAGCCAGGTGTTCCTGAAGATGAACAGCCTGCAGACCGACG
ACACCGCCATCTACTACTGCGCCAAGCACTACTACTACGGCGGCAGCTAC
GCCATGGACTACTGGGGCCAGGGCACCAGCGTGACCGTGTCCAGCCATAT
GGCCCTGAGCAACAGCATCATGTACTTCAGCCACTTCGTGCCCGTGTTTC
TGCCCGCCAAGCCCACCACCACCCCTGCCCCTAGACCTCCCACCCCAGCC
CCAACAATCGCCAGCCAGCCTCTGTCCCTGAGGCCCGAAGCCTGTAGACC
TGCTGCCGGCGGAGCCGTGCACACCAGAGGCCTGGATATCTACATCTGGG
CCCCTCTGGCCGGCACCTGTGGCGTGCTGCTGCTGAGCCTGGTGATCACC
CGGTCCAAGCGGAGCAGACTGCTGCACTCCGACTACATGAACATGACCCC
CAGACGGCCTGGCCCCACCCGGAAGCACTACCAGCCTTACGCCCCTCCCC
GGGACTTCGCCGCCTACAGAAGCAAGCGGGGCAGAAAGAAGCTGCTGTAC
ATCTTCAAGCAGCCCTTCATGCGGCCCGTGCAGACCACCCAGGAAGAGGA
CGGCTGCAGCTGCCGGTTCCCCGAGGAAGAGGAAGGCGGCTGCGAACTGC
CCAAGCTGTGCTACCTGCTGGACGGCATCCTGTTCATCTATGGCGTGATC
CTGACCGCCCTGTTCCTGAGAGTGAAGTTCAGCAGAAGCGCCGACGCCCC
TGCCTACCAGCAGGGCCAGAACCAGCTGTACAACGAGCTGAACCTGGGCA
GACGGGAAGAGTACGACGTGCTGGACAAGCGCAGAGGCCGGGACCCTGAG
ATGGGCGGCAAGCCTCAGCGGCGGAAGAACCCTCAGGAAGGCCTGTATAA
CGAACTGCAGAAAGACAAGATGGCCGAGGCCTACAGCGAGATCGGCATGA
AGGGCGAGCGGCGGAGAGGCAAGGGCCACGATGGCCTGTAC.
[0138] Particularly preferred nucleic acids include those
comprising or encoding any of the constituents of the CAR depicted
in FIG. 9, including signal sequence, CD19 ScFv, CD8 hinge,
transmembrane domain, 4-1 BB, and/or CD3z sequences. Other
preferred nucleic acids include those comprising or encoding any of
the constituents of the CARs of SEQ ID NOs:45-50.
[0139] In some embodiments, the nucleic acid comprises a hook
operably-linked to a promoter, preferably UBC or .beta.2M, and a
CAR comprising a hook-binding protein, operably-linked to an IRES.
In a preferred embodiment, the hook is a streptavidin protein,
preferably core Streptavidin, and the hook-binding protein is a
streptavidin-binding protein.
Vectors
[0140] The invention encompasses vectors encoding a CAR and a
hook-binding domain. Preferred vectors comprise a nucleic acid
sequence or encode an amino acid sequence of at least one of the
SEQ ID NOs detailed herein.
[0141] The vector can further encode a "hook." A "hook" is a
protein that prevents a CAR containing a hook-binding domain from
exiting the endoplasmic reticulum (ER) or Golgi by reversibly
binding, directly or indirectly, the hook-binding domain within the
CAR.
[0142] The retention can take place in the lumen of the ER or at
its cytoplasmic face, depending on the topology of the protein and
the orientation of tagging with the interaction domains. Boncompain
et al., Current Protocols in Cell Biology 15.19.1-15.19.16,
December 2012, which is hereby incorporated by reference.
[0143] In some embodiments, the hook for the ER comprises a mutant
of stromal interaction molecule 1 (STIM1-NN; a type I protein) that
localizes in the ER but that cannot bind microtubules, an isoform
of the human invariant chain of the major histocompatibility
complex (Ii; a type II protein) that has an N-terminal
arginine-based motif; or a C-terminal ER retention signal
(Lys-Asp-Glu-Leu; KDEL). Boncompain et al., Nat. Methods 9:493-498,
2012, which is hereby incorporated by reference. The hook can be
fused to a core Streptavidin in their luminal or cytoplasmic domain
depending on the hook-binding protein. Id. at FIG. 1.
[0144] In an alternative embodiment, the hook for the Golgi can be
Golgin-84 to be used as a cytoplasmic Golgi hook. Id.
[0145] Preferably, the hook comprises a Streptavidin protein
sequence, most preferably core Streptavidin. U.S. Pat. No.
5,672,691, which is hereby incorporated by reference.
[0146] Preferably, the hook comprises one of the following
Streptavidin protein sequences:
TABLE-US-00004 (SEQ ID NO: 31)
MDPSKDSKAQVSAAEAGITGTWYNQLGSTFIVTAGADGALTGTYESAVGN
AESRYVLTGRYDSAPATDGSGTALGWTVAWKNNYRNAHSATTWSGQYVGG
AEARINTQWLLTSGTTEANAWKSTLVGHDTFTKVKPSAASIDAAKKAGVN NGNPLDAVQQ or
(SEQ ID NO: 32) MDPSKDSKAQVSAAEAGITGTWYNQLGSTFIVTAGADGALTGTYESAVGN
AESRYTLTGRYDSAPATDGSGTALGWRVAWKNNYRNAHSATTWSGQYVGG
AEARINTQWTLTSGTTEANAWKSTLRGHDTFTKVKPSAASIDAAKKAGVN NGNPLDAVQQ or
(SEQ ID NO: 42) MHRRRSRSCREDQKPVTGDPSKDSKAQVSAAEAGITGTWYNQLGSTFIVT
AGADGALTGTYESAVGNAESRYVLTGRYDSAPATDGSGTALGWTVAWKNN
YRNAHSATTWSGQYVGGAEARINTQWLLTSGTTEANAWKSTLVGHDTFTK
VKPSAASIDAAKKAGVNNGNPLDAVQQVDYPYDVPDYAVGPMDDQRDLIS
NNEQLPMLGRRPGAPESKCSRGALYTGFSILVTLLLAGQATTAYFLYQQQ
GRLDKLTVTSQNLQLENLRMKLPKPPKPVSKMRMATPLLMQALPMGALPQ
GPMQNATKYGNMTEDHVMHLLQNADPLKVYPPLKGSFPENLRHLKNTMET
IDWKVFESWMHHWLLFEMSRHSLEQKPTDAPPKESLELEDPSSGLGVTKQ DLGPVPM.
Preferably, the hook is encoded by the following nucleotide
sequence: (SEQ ID NO: 43)
ATGCACCGGAGGAGATCACGCTCTTGTAGGGAGGACCAGAAACCTGTCAC
CGGTGACCCTAGCAAAGACTCAAAAGCTCAGGTGTCCGCTGCCGAGGCTG
GCATTACTGGAACATGGTACAATCAGCTCGGGAGCACCTTTATTGTGACT
GCTGGAGCCGATGGAGCCCTCACCGGAACATACGAATCTGCTGTGGGAAA
CGCCGAATCACGGTACGTCCTCACTGGCCGATACGATAGTGCCCCTGCCA
CCGACGGATCTGGGACTGCCCTGGGATGGACTGTCGCTTGGAAAAACAAC
TACCGGAATGCTCATTCTGCCACAACATGGAGTGGACAGTACGTGGGAGG
CGCTGAGGCTAGAATCAATACACAGTGGCTGCTCACATCTGGCACAACCG
AGGCAAATGCTTGGAAATCCACCCTGGTGGGACATGACACATTCACCAAA
GTGAAACCCTCCGCCGCTTCAATCGATGCCGCCAAAAAAGCCGGAGTCAA
CAACGGCAATCCTCTGGATGCCGTCCAGCAGGTCGACTATCCGTACGACG
TACCAGACTACGCAGTCGGACCGATGGACGATCAGAGGGACCTCATTAGC
AACAACGAACAGCTGCCTATGCTGGGACGGCGACCTGGAGCCCCTGAATC
CAAATGCTCTAGGGGAGCACTGTACACTGGCTTCTCCATTCTCGTGACAC
TGCTGCTGGCCGGGCAGGCTACTACTGCTTACTTCCTGTACCAGCAGCAG
GGGCGGCTGGACAAACTCACTGTGACATCTCAGAACCTCCAGCTGGAAAA
TCTGAGGATGAAACTGCCCAAACCCCCTAAACCCGTGTCCAAAATGAGGA
TGGCCACACCTCTGCTCATGCAGGCACTGCCAATGGGAGCCCTGCCCCAG
GGGCCCATGCAGAATGCCACCAAGTATGGCAACATGACAGAGGACCATGT
GATGCACCTGCTCCAGAATGCTGACCCCCTGAAGGTGTACCCGCCACTGA
AGGGGAGCTTCCCGGAGAACCTGAGACACCTTAAGAACACCATGGAGACC
ATAGACTGGAAGGTCTTTGAGAGCTGGATGCACCATTGGCTCCTGTTTGA
AATGAGCAGGCACTCCTTGGAGCAAAAGCCCACTGACGCTCCACCGAAAG
AGTCACTGGAACTGGAGGACCCGTCTTCTGGGCTGGGTGTGACCAAGCAG
GATCTGGGCCCAGTCCCCATGTGA.
[0147] In some embodiments, the glycine at aa 49 of SEQ ID NO:31 or
SEQ ID NO:32 is replaced with a bulkier residue (e.g., threonine)
to reduces the biotin binding affinity without affecting the SBP
binding affinity. Wu et al., PLoS ONE 8(7): e69530 (2013), which is
hereby incorporated by reference. Another mutation can also be
introduced to further favor SBP binding over biotin (mutation
S27A).
[0148] In some embodiments, the vector comprises a hook
operably-linked to a promoter, preferably UBC or .beta.2M, and a
CAR comprising a hook-binding protein, operably-linked to an
IRES.
TABLE-US-00005 A preferred RES nucleotide sequence is: (SEQ ID NO:
44) GCCCCTCTCCCTCCCCCCCCCCTAACGTTACTGGCCGAAGCCGCTTGGAA
TAAGGCCGGTGTGCGTTTGTCTATATGTTATTTTCCACCATATTGCCGTC
TTTTGGCAATGTGAGGGCCCGGAAACCTGGCCCTGTCTTCTTGACGAGCA
TTCCTAGGGGTCTTTCCCCTCTCGCCAAAGGAATGCAAGGTCTGTTGAAT
GTCGTGAAGGAAGCAGTTCCTCTGGAAGCTTCTTGAAGACAAACAACGTC
TGTAGCGACCCTTTGCAGGCAGCGGAACCCCCCACCTGGCGACAGGTGCC
TCTGCGGCCAAAAGCCACGTGTATAAGATACACCTGCAAAGGCGGCACAA
CCCCAGTGCCACGTTGTGAGTTGGATAGTTGTGGAAAGAGTCAAATGGCT
CTCCTCAAGCGTATTCAACAAGGGGCTGAAGGATGCCCAGAAGGTACGCC
ATTGTATGGGATCTGATCTGGGGCCTCGGTGCACATGCTTTACATGTGTT
TAGTCGAGGTTAAAAAACGTCTAGGCCCCCCGAACCACGGGGACGTGGTT
TTCCTTTGAAAAACACGATGATAA.
[0149] In a preferred embodiment, the hook is a streptavidin
protein, preferably core Streptavidin, and the hook-binding protein
is a streptavidin-binding protein. Preferred vectors are
lentivectors comprising any of the following nucleotide or amino
acid sequences:
TABLE-US-00006 CAR-CD19 2nd generation-SBP1 (nt): (SEQ ID NO: 45)
ATGGCCCTGCCTGTGACAGCCCTGCTGCTGCCCCTGGCTCTCCTGCTGCA
TGCCGCCAGACCCGCTAGCGACATCCAGATGACCCAGACCACCAGCAGCC
TGAGCGCCAGCCTGGGCGACAGAGTGACCATCAGCTGCCGGGCCAGCCAG
GACATCAGCAAGTACCTGAACTGGTATCAGCAGAAACCCGACGGCACCGT
GAAGCTGCTGATCTACCACACCAGCCGGCTCCACAGCGGCGTGCCCAGCA
GATTTTCTGGCAGCGGCAGCGGCACCGACTACAGCCTGACCATCTCCAAC
CTGGAACAGGAAGATATCGCTACCTACTTCTGTCAGCAAGGCAACACCCT
GCCCTACACCTTCGGCGGAGGCACCAAGCTGGAAATCACCGGCGGAGGCG
GAAGTGGAGGTGGAGGATCTGGCGGCGGAGGCTCCGAAGTGAAGCTGCAG
GAAAGCGGCCCTGGCCTCGTGGCCCCTAGCCAGAGCCTGTCCGTGACCTG
TACCGTGTCCGGCGTGTCCCTGCCCGACTACGGCGTGTCCTGGATCAGAC
AGCCTCCCAGAAAGGGCCTGGAATGGCTGGGCGTGATCTGGGGCAGCGAG
ACAACCTACTACAACAGCGCCCTGAAGTCCCGGCTGACCATCATCAAGGA
CAACAGCAAGAGCCAGGTGTTCCTGAAGATGAACAGCCTGCAGACCGACG
ACACCGCCATCTACTACTGCGCCAAGCACTACTACTACGGCGGCAGCTAC
GCCATGGACTACTGGGGCCAGGGCACCAGCGTGACCGTGTCCAGCCATAT
GGCCCTGAGCAACAGCATCATGTACTTCAGCCACTTCGTGCCCGTGTTTC
TGCCCGCCAAGCCCACCACCACCCCTGCCCCTAGACCTCCCACCCCAGCC
CCAACAATCGCCAGCCAGCCTCTGTCCCTGCGGCCCGAAGCCTGTAGACC
TGCTGCCGGCGGAGCCGTGCACACCAGAGGCCTGGATATCTACATCTGGG
CCCCTCTGGCCGGCACCTGTGGCGTGCTGCTGCTGAGCCTGGTGATCACC
ACCGGTATGGACGAGAAAACCACCGGCTGGCGGGGAGGCCACGTGGTGGA
AGGACTGGCCGGCGAGCTGGAACAGCTGCGGGCCAGACTGGAACACCACC
CCCAGGGCCAGAGAGAGCCCAAGCGGGGCAGAAAGAAGCTGCTGTACATC
TTCAAGCAGCCCTTCATGCGGCCCGTGCAGACCACCCAGGAAGAGGACGG
CTGCAGCTGCCGGTTCCCCGAGGAAGAGGAAGGCGGCTGCGAACTGCCCA
AGCTGTGCTACCTGCTGGACGGCATCCTGTTCATCTACGGCGTGATCCTG
ACCGCCCTGTTCCTGAGAGTGAAGTTCAGCAGAAGCGCCGACGCCCCTGC
CTACCAGCAGGGCCAGAACCAGCTGTACAACGAGCTGAACCTGGGCAGAC
GGGAAGAGTACGACGTGCTGGACAAGCGGAGAGGCCGGGACCCTGAGATG
GGCGGCAAGCCCCAGCGGCGGAAGAACCCCCAGGAAGGCCTGTATAACGA
ACTGCAGAAAGACAAGATGGCCGAGGCCTACAGCGAGATCGGCATGAAGG
GCGAGCGGCGGAGAGGCAAGGGCCACGATGGCCTGTAC. CAR-CD19 2nd
generation-SBP1 (aa): (SEQ ID NO: 46)
MALPVTALLLPLALLLHAARPASDIQMTQTTSSLSASLGDRVTISCRASQ
DISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISN
LEQEDIATYFCQQGNTLPYTFGGGTKLEITGGGGSGGGGSGGGGSEVKLQ
ESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSE
TTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSY
AMDYWGQGTSVTVSSHMALSNSIMYFSHFVPVFLPAKPTTTPAPRPPTPA
PTIASQPLSLRPEACRPAAGGAVHTRGLDIYIWAPLAGTCGVLLLSLVIT
TGMDEKTTGWRGGHVVEGLAGELEQLRARLEHHPQGQREPKRGRKKLLYI
FKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELPKLCYLLDGILFIYGVIL
TALFLRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEM
GGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLY. CAR-CD19 2nd
generation-SBP2(nt): (SEQ ID NO: 47)
ATGGCCCTGCCTGTGACAGCCCTGCTGCTGCCCCTGGCTCTCCTGCTGCA
TGCCGCCAGACCCGCTAGCGACATCCAGATGACCCAGACCACCAGCAGCC
TGAGCGCCAGCCTGGGCGACAGAGTGACCATCAGCTGCCGGGCCAGCCAG
GACATCAGCAAGTACCTGAACTGGTATCAGCAGAAACCCGACGGCACCGT
GAAGCTGCTGATCTACCACACCAGCCGGCTCCACAGCGGCGTGCCCAGCA
GATTTTCTGGCAGCGGCAGCGGCACCGACTACAGCCTGACCATCTCCAAC
CTGGAACAGGAAGATATCGCTACCTACTTCTGTCAGCAAGGCAACACCCT
GCCCTACACCTTCGGCGGAGGCACCAAGCTGGAAATCACCGGCGGAGGCG
GAAGTGGAGGTGGAGGATCTGGCGGCGGAGGCTCCGAAGTGAAGCTGCAG
GAAAGCGGCCCTGGCCTCGTGGCCCCTAGCCAGAGCCTGTCCGTGACCTG
TACCGTGTCCGGCGTGTCCCTGCCCGACTACGGCGTGTCCTGGATCAGAC
AGCCTCCCAGAAAGGGCCTGGAATGGCTGGGCGTGATCTGGGGCAGCGAG
ACAACCTACTACAACAGCGCCCTGAAGTCCCGGCTGACCATCATCAAGGA
CAACAGCAAGAGCCAGGTGTTCCTGAAGATGAACAGCCTGCAGACCGACG
ACACCGCCATCTACTACTGCGCCAAGCACTACTACTACGGCGGCAGCTAC
GCCATGGACTACTGGGGCCAGGGCACCAGCGTGACCGTGTCCAGCCATAT
GGCCCTGAGCAACAGCATCATGTACTTCAGCCACTTCGTGCCCGTGTTTC
TGCCCGCCAAGCCCACCACCACCCCTGCCCCTAGACCTCCCACCCCAGCC
CCAACAATCGCCAGCCAGCCTCTGTCCCTGCGGCCCGAAGCCTGTAGACC
TGCTGCCGGCGGAGCCGTGCACACCAGAGGCCTGGATATCTACATCTGGG
CCCCTCTGGCCGGCACCTGTGGCGTGCTGCTGCTGAGCCTGGTGATCACA
AAGCGGGGCAGAAAGAAGCTGCTGTACATCTTCAAGCAGCCCTTCATGCG
GCCCGTGCAGACCACCCAGGAAGAGGACGGCTGCAGCTGCCGGTTCCCCG
AGGAAGAGGAAGGCGGCTGCGAGCTGACCGGTATGGACGAGAAAACCACC
GGCTGGCGGGGAGGCCACGTGGTGGAAGGACTGGCCGGCGAGCTGGAACA
GCTGCGGGCCAGACTGGAACACCACCCCCAGGGCCAGAGGGAACCCCCCA
AGCTGTGCTACCTGCTGGACGGCATCCTGTTCATCTACGGCGTGATCCTG
ACCGCCCTGTTCCTGAGAGTGAAGTTCAGCAGAAGCGCCGACGCCCCTGC
CTACCAGCAGGGCCAGAACCAGCTGTACAACGAGCTGAACCTGGGCAGAC
GGGAAGAGTACGACGTGCTGGACAAGCGGAGAGGCCGGGACCCTGAGATG
GGCGGCAAGCCCCAGCGGCGGAAGAACCCCCAGGAAGGCCTGTATAACGA
ACTGCAGAAAGACAAGATGGCCGAGGCCTACAGCGAGATCGGCATGAAGG
GCGAGCGGCGGAGAGGCAAGGGCCACGATGGCCTGTAC. CAR-CD19 2nd
generation-SBP2(aa): (SEQ ID NO: 48)
MALPVTALLLPLALLLHAARPASDIQMTQTTSSLSASLGDRVTISCRASQ
DISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISN
LEQEDIATYFCQQGNTLPYTFGGGTKLEITGGGGSGGGGSGGGGSEVKLQ
ESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSE
TTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSY
AMDYWGQGTSVTVSSHMALSNSIMYFSHFVPVFLPAKPTTTPAPRPPTPA
PTIASQPLSLRPEACRPAAGGAVHTRGLDIYIWAPLAGTCGVLLLSLVIT
KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELTGMDEKTT
GWRGGHVVEGLAGELEQLRARLEHHPQGQREPPKLCYLLDGILFIYGVIL
TALFLRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEM
GGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLY. CAR-CD19 2nd
generation-SBP3(nt): (SEQ ID NO: 49)
ATGGCCCTGCCTGTGACAGCCCTGCTGCTGCCCCTGGCTCTCCTGCTGCA
TGCCGCCAGACCCGCTAGCGACATCCAGATGACCCAGACCACCAGCAGCC
TGAGCGCCAGCCTGGGCGACAGAGTGACCATCAGCTGCCGGGCCAGCCAG
GACATCAGCAAGTACCTGAACTGGTATCAGCAGAAACCCGACGGCACCGT
GAAGCTGCTGATCTACCACACCAGCCGGCTCCACAGCGGCGTGCCCAGCA
GATTTTCTGGCAGCGGCAGCGGCACCGACTACAGCCTGACCATCTCCAAC
CTGGAACAGGAAGATATCGCTACCTACTTCTGTCAGCAAGGCAACACCCT
GCCCTACACCTTCGGCGGAGGCACCAAGCTGGAAATCACCGGCGGAGGCG
GAAGTGGAGGTGGAGGATCTGGCGGCGGAGGCTCCGAAGTGAAGCTGCAG
GAAAGCGGCCCTGGCCTCGTGGCCCCTAGCCAGAGCCTGTCCGTGACCTG
TACCGTGTCCGGCGTGTCCCTGCCCGACTACGGCGTGTCCTGGATCAGAC
AGCCTCCCAGAAAGGGCCTGGAATGGCTGGGCGTGATCTGGGGCAGCGAG
ACAACCTACTACAACAGCGCCCTGAAGTCCCGGCTGACCATCATCAAGGA
CAACAGCAAGAGCCAGGTGTTCCTGAAGATGAACAGCCTGCAGACCGACG
ACACCGCCATCTACTACTGCGCCAAGCACTACTACTACGGCGGCAGCTAC
GCCATGGACTACTGGGGCCAGGGCACCAGCGTGACCGTGTCCAGCCATAT
GGCCCTGAGCAACAGCATCATGTACTTCAGCCACTTCGTGCCCGTGTTTC
TGCCCGCCAAGCCCACCACCACCCCTGCCCCTAGACCTCCCACCCCAGCC
CCAACAATCGCCAGCCAGCCTCTGTCCCTGCGGCCCGAAGCCTGTAGACC
TGCTGCCGGCGGAGCCGTGCACACCAGAGGCCTGGATATCTACATCTGGG
CCCCTCTGGCCGGCACCTGTGGCGTGCTGCTGCTGAGCCTGGTGATCACA
AAGCGGGGCAGAAAGAAGCTGCTGTACATCTTCAAGCAGCCCTTCATGCG
GCCCGTGCAGACCACCCAGGAAGAGGACGGCTGCAGCTGCCGGTTCCCCG
AGGAAGAGGAAGGCGGCTGCGAACTGCCCAAGCTGTGCTACCTGCTGGAC
GGCATCCTGTTCATCTACGGCGTGATCCTGACCGCCCTGTTCCTGAGAGT
GAAGTTCAGCAGAAGCGCCGACGCCCCTGCCTACCAGCAGGGCCAGAACC
AGCTGTACAACGAGCTGAACCTGGGCAGACGGGAAGAGTACGACGTGCTG
GACAAGCGGAGAGGCCGGGACCCTGAGATGGGCGGCAAGCCCCAGCGGCG
GAAGAACCCCCAGGAAGGCCTGTATAACGAACTGCAGAAAGACAAGATGG
CCGAGGCCTACAGCGAGATCGGCATGAAGGGCGAGCGGCGGAGAGGCAAG
GGCCACGATGGCCTGTACACCGGTATGGACGAGAAAACCACCGGCTGGCG
GGGAGGCCACGTGGTGGAAGGACTGGCCGGCGAGCTGGAACAGCTGCGGG
CCAGACTGGAACACCACCCCCAGGGCCAGAGGGAACCC. CAR-CD19 2nd
generation-SBP3(aa): (SEQ ID NO: 50)
MALPVTALLLPLALLLHAARPASDIQMTQTTSSLSASLGDRVTISCRASQ
DISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISN
LEQEDIATYFCQQGNTLPYTFGGGTKLEITGGGGSGGGGSGGGGSEVKLQ
ESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSE
TTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSY
AMDYWGQGTSVTVSSHMALSNSIMYFSHFVPVFLPAKPTTTPAPRPPTPA
PTIASQPLSLRPEACRPAAGGAVHTRGLDIYIWAPLAGTCGVLLLSLVIT
KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELPKLCYLLD
GILFIYGVILTALFLRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVL
DKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGK
GHDGLYTGMDEKTTGWRGGHVVEGLAGELEQLRARLEHHPQGQREP.
[0150] The vector can be an expression vector. The vector can be a
plasmid vector. Preferably, the vector is a lentiviral vector.
[0151] Within the context of this invention, a "lentiviral vector"
means a non-replicating vector for the transduction of a host cell
with a transgene comprising cis-acting lentiviral RNA or DNA
sequences, and requiring lentiviral proteins (e.g., Gag, Pol,
and/or Env) that are provided in trans. The lentiviral vector lacks
expression of functional Gag, Pol, and Env proteins. The lentiviral
vector may be present in the form of an RNA or DNA molecule,
depending on the stage of production or development of said
retroviral vectors.
[0152] The lentiviral vector can be in the form of a recombinant
DNA molecule, such as a plasmid. The lentiviral vector can be in
the form of a lentiviral vector particle, such as an RNA
molecule(s) within a complex of lentiviral and other proteins.
Typically, lentiviral particle vectors, which correspond to
modified or recombinant lentivirus particles, comprise a genome
which is composed of two copies of single-stranded RNA. These RNA
sequences can be obtained by transcription from a double-stranded
DNA sequence inserted into a host cell genome (proviral vector DNA)
or can be obtained from the transient expression of plasmid DNA
(plasmid vector DNA) in a transformed host cell.
[0153] Preferably the lentiviral vector particles have the capacity
for integration. As such, they contain a functional integrase
protein. Non-integrating vector particles have one or more
mutations that eliminate most or all of the integrating capacity of
the lentiviral vector particles. For, example, a non-integrating
vector particle can contain mutation(s) in the integrase encoded by
the lentiviral pol gene that cause a reduction in integrating
capacity. In contrast, an integrating vector particle comprises a
functional integrase protein that does not contain any mutations
that eliminate most or all of the integrating capacity of the
lentiviral vector particles.
[0154] Lentiviral vectors derive from lentiviruses, in particular
human immunodeficiency virus (HIV-1 or HIV-2), simian
immunodeficiency virus (SIV), equine infectious encephalitis virus
(EIAV), caprine arthritis encephalitis virus (CAEV), bovine
immunodeficiency virus (BIV) and feline immunodeficiency virus
(FIV), which are modified to remove genetic determinants involved
in pathogenicity and introduce new determinants useful for
obtaining therapeutic effects.
[0155] Such vectors are based on the separation of the cis- and
trans-acting sequences. In order to generate replication-defective
vectors, the trans-acting sequences (e.g., gag, pol, tat, rev, and
env genes) can be deleted and replaced by an expression cassette
encoding a transgene.
[0156] Efficient integration and replication in non-dividing cells
generally requires the presence of two cis-acting sequences at the
center of the lentiviral genome, the central polypurine tract
(cPPT) and the central termination sequence (CTS). These lead to
the formation of a triple-stranded DNA structure called the central
DNA "flap", which acts as a signal for uncoating of the
pre-integration complex at the nuclear pore and efficient
importation of the expression cassette into the nucleus of
non-dividing cells, such as dendritic cells.
[0157] In one embodiment, the invention encompasses a lentiviral
vector comprising a central polypurine tract and central
termination sequence referred to as cPPT/CTS sequence as described,
in particular, in the European patent application EP 2 169 073.
[0158] Further sequences are usually present in cis, such as the
long terminal repeats (LTRs) that are involved in integration of
the vector proviral DNA sequence into a host cell genome. Vectors
may be obtained by mutating the LTR sequences, for instance, in
domain U3 of said LTR (.DELTA.U3) (Miyoshi H et al, 1998, J Virol.
72(10):8150-7; Zufferey et al., 1998, J Virol 72(12):9873-80).
[0159] Preferably, the vector does not contain an enhancer. In one
embodiment, the invention encompasses a lentiviral vector
comprising LTR sequences, preferably with a mutated U3 region
(.DELTA.U3) removing promoter and enhancer sequences in the 3'
LTR.
[0160] The packaging sequence LP (psi) can also be incorporated to
help the encapsidation of the polynucleotide sequence into the
vector particles (Kessler et al., 2007, Leukemia, 21(9):1859-74;
Paschen et al., 2004, Cancer Immunol Immunother 12(6):196-203).
[0161] In one embodiment, the invention encompasses a lentiviral
vector comprising a lentiviral packaging sequence LP (psi).
[0162] Further additional functional sequences, such as a transport
RNA-binding site or primer binding site (PBS) or a Woodchuck
PostTranscriptional Regulatory Element (WPRE), can also be
advantageously included in the lentiviral vector polynucleotide
sequence of the present invention, to obtain a more stable
expression of the transgene in vivo.
[0163] In one embodiment, the invention encompasses a lentiviral
vector comprising a PBS. In one embodiment, the invention
encompasses a lentiviral vector comprising a WPRE and/or an
IRES.
[0164] Thus, in a preferred embodiment, the lentiviral vector
comprises at least one cPPT/CTS sequence, one LP sequence, one
(preferably 2) LTR sequence, and an expression cassette including a
transgene under the transcriptional control of a .beta.2m or class
I MHC promoter.
Promoter
[0165] The invention encompasses the use of promoters to drive high
expression of CARs from lentivectors in T cells, preferably human T
cells. Preferred promoters are human ubiquitin, MHC class I, MHC
class II, and .beta.2 microglobulin (.beta.2m) promoters.
[0166] In various embodiments, the promoter drives high expression
in antigen presenting cells, including dendritic cells. Preferably,
the promoter lacks an enhancer element to avoid insertional
effects.
[0167] Most preferably, the promoter is not a CMV
promoter/enhancer. Preferably, the promoter is not a dectin-2 or
MHCII promoter.
[0168] The sequences of various mammalian (human) MHC class I
promoters are shown below:
TABLE-US-00007 HLA-A2 (MHC I): (SEQ ID NO: 9)
attggggagtcccagccttggggattccccaactccgcagtttcttttct
ccctctcccaacctatgtagggtccttcttcctggatactcacgacgcgg
acccagttctcactcccattgggtgtcgggtttccagagaagccaatcag
tgtcgtcgcggtcgcggttctaaagtccgcacgcacccaccgggactcag
attctccccagacgccgagg HLA-B7 (MHC I): (SEQ ID NO: 10)
ggggaggcgcagcgttggggattccccactcccctgagtttcacttcttc
tcccaacttgtgtcgggtccttcttccaggatactcgtgacgcgtcccca
cttcccactcccattgggtattggatatctagagaagccaatcagcgtcg
ccgcggtcccagttctaaagtccccacgcacccacccggactcagag HLA-Cw5 (MHC I):
(SEQ ID NO: 11) cactggggaggcgccgcgttgaggattctccactcccctcagtttcactt
cttctcccaacctgcgtcgggtccttcttcctgaatactcatgacgcgtc
cccaattcccactcccattgggtgtcgggttctagagaagccaatcagcg
tctccgcagtcccggtctaaagtccccagtcacccacccggactcagatt ctccccagacgccgag
HLA-E (MHC I): (SEQ ID NO: 12)
taagaactgctgattgctgggaaactctgcagtttcccgttcctctcgta
acctggtcatgtgtccttcttcctggatactcatgacgcagactcagttc
tcattcccaatgggtgtcgggtttctagagaagccaatcagcgtcgccac
gactcccgactataaagtccccatccggactcaagaagttctcaggactc agagg HLA-F (MHC
I): (SEQ ID NO: 13)
aggccccgaggcggtgtctggggttggaaggctcagtattgagaattccc
catctccccagagtttctctttctctcccaacccgtgtcaggtccttcat
cctggatactcataacgcggccccatttctcactcccattgggcgtcgcg
tttctagagaagccaatcagtgtcgccgcagttcccaggttctaaagtcc
cacgcaccccgcgggactcatatttttcccagacgcggaggttggggtca tg
[0169] A sequence of the human .beta.2-microglobulin promoter is
shown below:
TABLE-US-00008 (SEQ ID NO: 14)
aacatcacgagactctaagaaaaggaaactgaaaacgggaaagtccctct
ctctaacctggcactgcgtcgctggcttggagacaggtgacggtccctgc
gggccttgtcctgattggctgggcacgcgtttaatataagtggaggcgtc
gcgctggcgggcattcctgaagctgacagcattcgggccgag.
[0170] A sequence of the human ubiquitin (Ubi) promoter is shown
below:
TABLE-US-00009 (SEQ ID NO: 38)
ggcctccgcgccgggttttgggcctcccgcgggcgcccccctcctcacg
gcgagcgctgccacgtcagacgaagggcgcagcgagcgtcctgatcctt
ccgcccggacgctcaggacagcggcccgctgctcataagactcggcctt
agaaccccagtatcagcagaaggacattttaggacgggacttgggtgac
tctagggcactggttttctttccagagagcggaacaggcgaggaaaagt
agtcccttctcggcgattctgcggagggatctccgtggggcggtgaacg
ccgatgattatataaggacgcgccgggtgtggcacagctagttccgtcg
cagccgggatttgggtcgcggttcttgtttgtggatcgctgtgatcgtc
acttggtgagtagcgggctgctgggctggccggggctttcgtggccgcc
gggccgctcggtgggacggaagcgtgtggagagaccgccaagggctgta
gtctgggtccgcgagcaaggttgccctgaactgggggttggggggagcg
cagcaaaatggcggctgttcccgagtcttgaatggaagacgcttgtgag
gcgggctgtgaggtcgttgaaacaaggtggggggcatggtgggcggcaa
gaacccaaggtcttgaggccttcgctaatgcgggaaagctcttattcgg
gtgagatgggctggggcaccatctggggaccctgacgtgaagtttgtca
ctgactggagaactcggtttgtcgtctgttgcgggggcggcagttatgg
cggtgccgttgggcagtgcacccgtacctttgggagcgcgcgccctcgt
cgtgtcgtgacgtcacccgttctgttggcttataatgcagggtggggcc
acctgccggtaggtgtgcggtaggcttttctccgtcgcaggacgcaggg
ttcgggcctagggtaggctctcctgaatcgacaggcgccggacctctgg
tgaggggagggataagtgaggcgtcagtttctttggtcggttttatgta
cctatcttcttaagtagctgaagctccggttttgaactatgcgctcggg
gttggcgagtgtgttttgtgaagttttttaggcaccttttgaaatgtaa
tcatttgggtcaatatgtaattttcagtgttagactagtaaattgtccg
ctaaattctggccgtttttggcttttttgttagaccgatc.
[0171] A sequence of the human HLA-DRa promoter is shown below:
TABLE-US-00010 (SEQ ID NO: 41)
gtctagaagtcagattggggttaaagagtctgtccgtgattgactaaca
gtcttaaatacttgatttgttgttgttgttgtcctgtttgtttaagaac
tttacttctttatccaatgaacggagtatcttgtgtcctggaccctttg
caagaacccttcccctagcaacagatgcgtcatctcaaaatatttttct
gattggccaaagagtaattgatttgcattttaatggtcagactctatta
caccccacattctcttttcttttattcttgtctgttctgcctcactccc gagctc.
[0172] In various embodiments, the lentiviral vector comprises a
.beta.2m, Ubi, MHCII, or MHC class I promoter. Preferably, the MHC
class I promoter is an HLA-A2 promoter, an HLA-B7 promoter, an
HLA-Cw5 promoter, an HLA-F, or an HLA-E promoter. In various
embodiments, the promoter sequence comprises a polynucleotide
sequence that shares more than 90%, preferably more than 95%, more
preferably more than 99% identity with the promoter sequence of SEQ
ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13,
SEQ ID NO:14, SEQ ID NO:38, or SEQ ID NO:41.
[0173] In some embodiments, the expression of the promoter in
BDCA+dendritic cells is at least 10, 12, 15, 20, 25, 30, 35, 40,
50, or 60 times the expression of that promoter in skeletal muscle
cells.
[0174] In one embodiment, the invention encompasses lentiviral
vector particles comprising a lentiviral vector that comprises a
dendritic cell-specific promoter directing expression of a
microbial or tumor antigen, wherein the lentiviral vector particles
exhibit higher expression of the antigen in BDCM cells than in HEK
293 T cells.
[0175] The invention encompasses lentiviral vectors containing a
promoter that does not contain an enhancer.
[0176] The invention encompasses the insertion of an MHC Class I
(MHCI), Ubi, EF1.alpha., or .beta.2 microglobulin promoter
(.beta.2m) promoter into a lentiviral vector. As used herein, an
"MHC Class I (MHCI) promoter" or ".beta.2 microglobulin promoter"
or "MHC Class II (MHCII)" or "human ubiquitin promoter" includes a
naturally occurring or synthetic MHC Class I promoter or .beta.2
microglobulin promoter or MHC Class II promoter or human ubiquitin
promoter. The term "MHC Class I promoter" does not include a
.beta.2m promoter.
[0177] In one embodiment, the lentiviral vector particles
comprising the promoter exhibit higher expression in BDCM cells
than in HEK 293 T cells.
[0178] The promoter can be a naturally occurring promoter. Examples
of naturally occurring promoters are the human .beta.2m, HLA-A2,
HLA-B7, HLA-Cw5, HLA-E, HLA-F, HLA-DR.alpha., and ubiquitin gene
promoters.
[0179] These naturally occurring MHCI promoters are generally
cloned or reproduced from the promoter region of a gene encoding
the MHC class I protein, or referred to as putatively encoding such
proteins in genome databases (ex: NCBI polynucleotide database
http://www.ncbi.nlm.nih.gov/guide/dna-rna). Both .beta.2m and class
I MHC proteins enter the Major Histocompatibility Complex
(MHC).
[0180] The proteins encoded by these genes are found in almost all
cell types. MHCI proteins are generally present at the surface of
the membrane of leucocytes, where they are associated with the
.beta.2-microglobulin (.beta.2m). The role of these associated
proteins is to present peptides from endogenous sources to CD8+ T
cells. They thus play a central role to the generation of the
antigen-specific immune response. Because MHC class I proteins have
been widely studied and described for many years, their genes are
well characterized and detectable using sequence comparison tools,
such as the BLAST method (Altschul, S. F. et al. (1990). Basic
local alignment search tool. J. Mol. Biol. 215(3):403-410).
[0181] MHC class I promoters share the ability to be strongly
activated in antigen presenting cells, including dendritic cells,
as well as, to lower intensity, in the majority of the other human
body tissues.
[0182] The promoters of the invention can contain further
regulatory elements, such as one or more Sp1 and ETs binding sites.
In a preferred embodiment, the MHC class I promoter contains 2 Sp1
binding sites and 1 Ets binding site. In other embodiments, Ap1
and/or Ap2 sites are further contained in the promoter.
[0183] Preferred promoters are naturally occurring human 132m,
HLA-A2, HLA-B7, HLA-Cw5, HLA-E and HLA-F promoters.
[0184] Promoters can also be synthetic. Synthetic promoters include
promoters that are synthesized using molecular biological
techniques to assemble the individual components of a promoter or
that are derived from naturally occurring promoters using molecular
biological techniques.
[0185] In various embodiments, the synthetic promoter comprises a
polynucleotide sequence that shares more than 90%, preferably more
than 95%, more preferably more than 99% identity, or 100% with the
promoter sequence of a .beta.2m, Ubi, MHC class II, or MHC class I
gene promoter (e.g., SEQ ID NOs: 9-14,38, or 41).
[0186] The transcription of MHC class genes are usually mediated by
two major regulatory elements: Interferon stimulated response
element (ISRE) and the SXY module (encompassing the W/S, X1X2/Site
.alpha. and Y/enhancer B regulatory elements). See Van den Elsen,
Immunogenetics (1998) 48:208-211.
[0187] These regulatory promoter elements are localized in a region
extending approximately from nucleotides -220 to -95 upstream of
the transcription initiation site. They mediate tissue-specific and
cytokine-induced transcription of MHC class I genes.
[0188] The ISRE of MHC class I gene promoters generally contains
binding sites for interferon regulatory factor (IRF) family
members. It is thus a property of MHC class I promoters to bind to
interferon regulatory factor (IRF) family members. This may be
verified, for example, by gel shift assays.
[0189] Another regulatory element, the enhancer A (containing
binding sites for nuclear transcription factor KB (NF-KB)) is
present in most cases. It is thus a property of MHC class I
promoters to bind to nuclear transcription factor KB (NF-KB). This
may be verified, for example, by gel shift assays.
[0190] In addition to ISRE, MHC class I promoters generally share
another set of conserved upstream sequence motifs, consisting of
three regulatory elements: the S or W box, the X1/CREX2 boxes or
site a, and the Y box or enhancer B, which together are termed the
SXY module. This SXY module is generally cooperatively bound by a
multiprotein complex containing regulatory factor X (RFX;
consisting of RFX5, RFXB/ANK and RFXAP), cAMP response element
binding protein (CREB)/activating transcription factor (ATF), and
nuclear factor Y (NFY), which acts as an enhanceosome driving
transactivation of these genes. It is thus a property of MHC class
I promoters to bind to these factors. This may be verified, for
example, by gel shift assays.
[0191] In contrast, MHC class II promoters do not display enhancer
A nor ISRE elements (Van den Elsen, P. J. et al, 1998,
Immunogenetics. 48:208-221). Furthermore, RFX and CIITA in MHC
class II gene regulation have been found of crucial importance as
illustrated by studies with cell lines established from patients
with the bare lymphocyte syndrome (BLS), a severe combined
immunodeficiency due to mutations in one of the RFX subunits or
CIITA (DeSandro, A. et al., 1999, Am J Hum Genet, 65:279-286).
Also, lack of either CIITA or one of the RFX subunits affects the
functioning and assembly of the MHC enhanceosome, respectively,
leading to a lack of MHC class II and reduced levels of MHC class I
transcription (Van den Elsen, P. J. et al. 2004, Current Opinion in
Immunology, 16:67-75).
[0192] In one embodiment, the invention encompasses a method
comprising inserting a promoter of the invention, particularly a
.beta.2m, Ubi, MHC class II,or MHC class I promoter, into a
lentiviral vector to direct expression of a CAR of the invention.
The method can further comprise inserting any of the other nucleic
acid elements mentioned herein, such as a DNA flap sequence.
Isolated Cells
[0193] The invention encompasses cells, particularly cells of the
immune system, comprising vectors and lentiviral vector particles
encoding a CAR of the invention. Preferably, the cells are T cells,
including T.alpha..beta. and T.delta. cells, or NK cells.
[0194] In one embodiment, the cell contains the vector integrated
into the cellular genome. In one embodiment, the cell contains the
vector transiently expressing the CAR. In one embodiment, the cell
produces lentiviral vector particles encoding the CAR.
[0195] In various embodiments, the invention encompasses a cell
line, a population of cells, or a cell culture comprising vectors
and lentiviral vector particles encoding the CAR.
Lentiviral Vector Particles
[0196] The present invention provides a method for producing a
lentiviral vector particle. A lentiviral vector particle (or
lentiviral particle vector) comprises a lentiviral vector in
association with viral proteins. The vector is preferably an
integrating vector.
[0197] In one embodiment, the lentiviral vector particles encode a
CAR of the invention.
[0198] In one embodiment, the lentiviral vector particle comprises
HIV-1 Gag and Pol proteins. Preferably, the lentiviral vector
particle comprises subtype D, especially HIV-1.sub.NDK, Gag and Pol
proteins.
[0199] According to one embodiment of this method, the lentivector
particles are obtained in a host cell transformed with a DNA
plasmid.
[0200] Such a DNA plasmid can comprise:
[0201] bacterial origin of replication (ex: pUC ori);
[0202] antibiotic resistance gene (ex: KanR) for selection; and
more particularly:
[0203] a lentiviral vector comprising at least one nucleic acid
encoding a CAR transcriptionally linked to a .beta.2m, Ubi, MHC
class II, or MHC class I promoter.
[0204] Such a method allows producing a recombinant vector particle
according to the invention, comprising the following steps of:
[0205] i) transfecting a suitable host cell with a lentiviral
vector;
[0206] ii) transfecting said host cell with a packaging plasmid
vector, containing viral DNA sequences encoding at least structural
and polymerase(+integrase) activities of a retrovirus (preferably
lentivirus); Such packaging plasmids are described in the art (Dull
et al., 1998, J Virol, 72(11):8463-71; Zufferey et al., 1998, J
Virol 72(12):9873-80).
[0207] iii) culturing said transfected host cell in order to obtain
expression and packaging of said lentiviral vector into lentiviral
vector particles; and
[0208] iv) harvesting the lentiviral vector particles resulting
from the expression and packaging of step iii) in said cultured
host cells.
[0209] For different reasons, it may be helpful to pseudotype the
obtained retroviral particles, i.e. to add or replace specific
particle envelope proteins. For instance, this may be advantageous
to have different envelope proteins in order to distinguish the
recombinant particle from natural particles or from other
recombinant particles. In matter of vaccination strategy,
pseudotyped particle vectors are more likely to escape the immune
system, when this latter already developed immunity against
lentiviruses. This is particularly helpful when successive
injections of similar particle vectors are required for immunizing
a patient against a disease.
[0210] In order to pseudotype the retroviral particles of the
invention, the host cell can be further transfected with one or
several envelope DNA plasmid(s) encoding viral envelope protein(s),
preferably a VSV-G envelope protein.
[0211] An appropriate host cell is preferably a human cultured cell
line as, for example, a HEK cell line.
[0212] Alternatively, the method for producing the vector particle
is carried out in a host cell, which genome has been stably
transformed with one or more of the following components: a
lentiviral vector DNA sequence, the packaging genes, and the
envelope gene. Such a DNA sequence may be regarded as being similar
to a proviral vector according to the invention, comprising an
additional promoter to allow the transcription of the vector
sequence and improve the particle production rate.
[0213] In a preferred embodiment, the host cell is further modified
to be able to produce viral particle in a culture medium in a
continuous manner, without the entire cells swelling or dying. One
may refer to Strang et al., 2005, J Virol 79(3)1165-71; Relander et
al., 2005, Mol Ther 11(3):452-9; Stewart et al., 2009, Gene Ther,
16(6):805-14; and Stuart et al., 2011, Hum gene Ther, with respect
to such techniques for producing viral particles.
[0214] An object of the present invention consists of a host cell
transformed with a lentiviral particle vector.
[0215] The lentiviral particle vectors can comprise the following
elements, as previously defined:
[0216] cPPT/CTS polynucleotide sequence; and
[0217] a nucleic acid encoding a CAR under control of a .beta.2m,
Ubi, or MHCI promoter, and optionally one of the additional
elements described above.
[0218] Preferably, the lentivector particles are in a dose of
10.sup.6, 2.times.10.sup.6, 5.times.10.sup.6, 10.sup.7,
2.times.10.sup.7, 5.times.10.sup.7, 10.sup.8, 2.times.10.sup.8,
5.times.10.sup.8, or 10.sup.9 TU.
Methods for Expressing a CAR in a Cell
[0219] The present invention encompasses methods for expressing a
CAR in a cell, preferably in T cells, and preferably in expanded T
cells. The method comprises transducing a cell with a lentiviral
vector or lentiviral particle vector of the invention under
conditions that allow the expression of the CAR, and preferably
expanding the T cells.
[0220] The cells are preferably mammalian cells, particularly human
cells. Particularly preferred are human non-dividing cells.
[0221] Preferably, the cells are primary T cells or NK cells.
[0222] The method can further comprise harvesting or isolating the
CAR.
[0223] The lentiviral vector or lentiviral particle vector
preferably comprises a promoter of the invention.
[0224] In one embodiment, the method comprises treating the cells
with biotin to release the CAR from the hook. Preferably, the cells
are treated with biotin at an initial concentration of, at least,
0.2, 0.4, 0.8. 1.6, 2.5, 5, 10, 20, 40, or 80 .mu.M.
[0225] In one embodiment, the invention encompasses a method for
expressing a CAR comprising inserting a .beta.2m, Ubi, or MHCI
promoter into a lentiviral vector such that it direct the
expression of a nucleic acid encoding a CAR and transducing a cell,
preferably a T or NK cell, with the vector containing the promoter,
and optionally, treating the cell with biotin at an initial
concentration of, at least, 0.2, 0.4, 0.8. 1.6, 2.5, 5, 10, 20, 40,
or 80 .mu.M.
Therapeutic use of Lentiviral Vectors
[0226] The present invention further relates to the use of the
lentiviral vectors according to the invention, especially in the
form of lentiviral vector particles, for the preparation of
therapeutic compositions or vaccines which are capable of inducing
or contributing to the occurrence or improvement of an
immunological reaction with the CAR encoded by the vectors.
[0227] The invention encompasses methods of administration of a
lentiviral vector (or "lentivector") to a human. Preferably, the
lentivector particle is an integrating lentivector particle,
comprising a functional integrase protein.
[0228] Preferred modes of administration include reinfusion of the
modified T cells, preferably intravenously or intra-articular
administration, most preferably intra-tumoral administration.
[0229] In one embodiment, the invention comprises a method for
inducing an immune response in a human comprising administering
lentiviral vector particles comprising a functional integrase
protein and a lentiviral vector to T or NK cells and administering
the modified cells to a human; wherein the integrating lentiviral
vector comprises a promoter directing expression of a CAR; and
generating immunological reaction with the CAR.
[0230] The invention can also be used in treatment protocols
against tumors and cancers and especially could be used in
protocols for immunotherapy or vaccination therapy against cancers
and tumors.
[0231] The invention further relates to an immunogenic composition
comprising a lentiviral vector as previously defined.
[0232] The immunogenic compositions of the invention preferably
contain cPPT and CTS sequences in the vector and vector particles
to induce or to stimulate the nuclear import of the vector genome
in the target cells.
[0233] During reverse transcription, cPPT and CTS sequences induce
the formation of a three stranded DNA structure referred as DNA
triplex, which stimulates the nuclear import of DNA vector
sequence. Preferably, the vector comprises a CAR and regulatory
signals of retrotranscription, expression and encapsidation of
retroviral or retroviral-like origin.
[0234] The lentiviral vectors according to the invention have the
ability to redirect the specificity and function of T lymphocytes
and/or other immune cells. They can rapidly generate T cells
targeted to a specific tumor antigen or an antigen relevant in
other pathologies like auto-immune diseases.
[0235] The lentiviral vectors of the invention can be used in
methods of treatment and methods of inducing an immune response
comprising administering the lentiviral vector to a cell,
preferably a T or NK cell, administering the cell to a host, and
generating a specific immune response that redirects the
specificity and function of T lymphocytes and/or other immune
cells.
[0236] A particular advantage of the immunogenic compositions of
the invention is that they can be used to redirect the specificity
and function of T lymphocytes and other immune cells against
multiple antigens against which the CAR in the vector or vector
particles are directed.
[0237] As a result, the invention encompasses a composition that
could be used in therapeutic vaccination protocols.
[0238] In particular, it can be used in combination with adjuvants,
other immunogenic compositions, chemotherapy, or any other
therapeutic treatment.
[0239] The invention encompasses a composition for administration
to a human comprising lentiviral vector particles comprising a
functional integrase protein and a lentiviral vector; wherein the
DNA of the lentiviral vector comprises a promoter directing
expression of an amino acid comprising or consisting of a CAR.
[0240] In one embodiment, the invention encompasses administering,
preferably via intramuscular administration, a lentiviral vector,
or cells transduced by the lentiviral vector, encoding a chimeric
antigen receptor comprising a binding domain; a transmembrane
domain; a hook-binding domain, preferably comprising a
streptavidin-binding peptide; and an activation domain comprising a
T cell activating fragment of at least 100 amino acids of SEQ ID
NO:3; SEQ ID NO:4; SEQ ID NO:5; SEQ ID NO:6, SEQ ID NO:7, or SEQ ID
NO:8, SEQ ID NO:33, or SEQ ID NO:34 to a human. Preferably, the
lentiviral vector further comprises a hook, preferably comprising a
streptavidin protein, most preferably comprising the amino acid
sequence of SEQ ID NO:32, SEQ ID NO:33, or a mutant thereof having
a mutation of the Glycine at amino acid 49, preferably to
threonine. Preferably, the hook-binding domain comprises the amino
acid sequence of SEQ ID NO:1 or is encoded by the nucleic acid
sequence of SEQ ID NO:2.
[0241] The method can further comprise administering biotin to the
human to release the CAR from the ER or Golgi. Preferably, the
biotin is administered at an initial concentration of at least,
0.2, 0.4, 0.8. 1.6, 3.2, 5, 10, 20, 40, or 80 .mu.M.
[0242] Having thus described different embodiments of the present
invention, it should be noted by those skilled in the art that the
disclosures herein are exemplary only and that various other
alternatives, adaptations, and modifications may be made within the
scope of the present invention. Accordingly, the present invention
is not limited to the specific embodiments as illustrated
herein.
EXAMPLES
Example 1. Molecular Constructions
[0243] PCR amplification of the proviral region of the
pTRIP.DELTA.U3-CMV-GFP(15) was performed using direct
(5'-CTTACTAGTTGGAAGGGCTAATTCACTCCCAAC-3'; SEQ ID NO:15) and reverse
(5'-CATTCTAGAACTGCTAGAGATTTTCCACACTG-3'; SEQ ID NO:16)
oligonucleotides encompassing respectively the SpeI and XbaI
restriction sites. The resulting fragment was digested and cloned
between the SpeI and XbaI sites of the pVAX-1 plasmid (Invitrogen,
Lifetech) from which the MluI site have been deleted. The resulting
plasmid was named pFLAP-CMV-GFP. The SV40 sequence was amplified by
PCR from the pTRIP.DELTA.U3-CMV-GFP plasmid (using the
5'-TACCCCGGGCCATGGCCTCCAAAAAAGCCTCCTCACTACTTC-3' (SEQ ID NO:17) and
5'-ACTCCCGGGTAATTTTTTTTATTTATGCAGAGGCCGAGGCCGCC-3' (SEQ ID NO:18)
oligonucleotides), and cloned into the Pml1 site of the
pFLAP-CMV-GFP, the resulting plasmid being then named
pFLAP-CMV-GFP-SV. The CMV promoter was amplified with direct
(5'-TACACGCGTGGAGTTCCGCGTTACATAACTTACGG-3'; SEQ ID NO:19) and
reverse (5'-CGTGGATCCGATCGCGGTGTCTTCTATGGAGGTCAAAAC-3'; SEQ ID
NO:20) oligonucleotides encompassing the MluI and BamHI sites,
respectively. The resulting fragment was cloned back between the
MluI and BamHI sites of the pFlap-CMV-GFP-SV allowing the easy
replacement of the promoters inside the lentiviral vectors. The
promoter was then amplified by PCR from HEK 293T cells DNA with
5'-GCCGGCGCGCCGAGAAACCCTGCAGGGAATTCCC-3' (SEQ ID NO:21) and
5'-CGTGGATCCGATCGCTCGGCCCGAATGCTGTCAGCTTCAGG-3' (SEQ ID NO:22) for
the .beta.2m promoter and cloned between the MluI and BamH1 sites
of pFLAP-CMV-GFP-SV to create pFlap-.beta.2m-SV. The amplified
.beta.2m promoter sequence is the following:
GAGAAACCCTGCAGGGAATTCCCCAGCTGTAGTTATAAACAGAAGTTCTCCTTCTG
CTAGGTAGCATTCAAAGATCTTAATCTTCTGGGTTTCCGTTTTCTCGAATGAAAAAT
GCAGGTCCGAGCAGTTAACTGGCGGGGGCACCATTAGCAAGTCACTTAGCATCTC
TGGGGCCAGTCTGCAAAGCGAGGGGGCAGCCTTAATGTGCCTCCAGCCTGAAGT
CCTAGAATGAGCGCCCGGTGTCCCAAGCTGGGGCGCGCACCCCAGATCGGAGGG
CGCCGATGTACAGACAGCAAACTCACCCAGTCTAGTGCATGCCTTCTTAAACATCA
CGAGACTCTAAGAAAAGGAAACTGAAAACGGGAAAGTCCCTCTCTCTAACCTGGCA
CTGCGTCGCTGGCTTGGAGACAGGTGACGGTCCCTGCGGGCCTTGTCCTGATTG
GCTGGGCACGCGTTTAATATAAGTGGAGGCGTCGCGCTGGCGGGCATTCCTGAA
GCTGACAGCATTCGGGCCGAG (SEQ ID NO:23). The CAR can be synthetized
and cloned between the BamHI and XhoI sites of the
pFlap-.beta.2m-SV, in place of the GFP gene.
[0244] For example, pFlap-.beta.2m-GFP-SV can be digested by BamHI
and XhoI, and a DNA linker containing a Multiple Cloning Site (MCS,
carrying SalI, SadI, NdeI, AscI and NheI restriction sites) can be
cloned between those sites, in place of the GFP gene to allow
insertion of a nucleic acid sequence encoding the CAR.
[0245] The packaging plasmid pTHV-GP-N was constructed by
amplifying the HIV-NDK genome by PCR (using the following
oligonucleotides with
5'-atgcatgcgtcgacctcgagttaatcctcatcctgtctacttgccac-3' (SEQ ID
NO:24) and
5'-gcatgcatcggccggggcggcgactgGTgagagGCCACCatgggtgcgagagcgtcagtattaag-3'
(SEQ ID NO:25)). The resulting fragment has been digested by EagI
and SalI restriction enzymes and inserted in the p8.74 packaging
plasmid (15) from which the Eag1-SalI fragment had been previously
removed.
[0246] Pseudotyping plasmids were generated by synthesizing the
codon optimized genes corresponding to the vesicular stomatitis
virus Indiana (GenBank #CAX62728.1), New Jersey GenBank
#CAX62729.1) and Cocal (GenBank #CAX62731.1) strains. Those genes
were then digested with EcoR1 and BamH1 and cloned between the
corresponding restriction sites of the pVAX1 plasmid (Invitrogen,
Lifetech).
[0247] The plasmids can produced using Nucleobond Xtra Maxi EF
column according to manufacturer's instructions (Macherey
Nagel).
Example 2. Lentiviral Production
[0248] R&D productions: Vectors can be produced by transient
calcium-phosphate transfection of HEK 293T as previously described
(25). HEK 293T (human embryonic kidney cell line, ATCC CRL-11268,
(Graham et al. 1977)) cells were maintained in Dubelcco's modified
Eagle's medium (DMEM/High modified, Hyclone) supplemented with 10%
fetal bovine serum (FBS, FAA), 1% L-Glutamine (Eurobio), 1%
Penicillin-Streptomycin (Gibco by Life technologies) and 1% Sodium
Pyruvate (Gibco by Life technologies).). The cell line was kept in
an incubator with humidified atmosphere of 5% CO.sub.2 at
37.degree. C. The lentiviral vectors were produced by transient
transfection of HEK 293T cells using a standard calcium phosphate
precipitation protocol. HEK 293T cells were seeded at
7.times.10.sup.6 cells in 10 cm.sup.2 Tissue Culture Dish (BD
Falcon) in 10 mL of complete culture medium and maintained 24 h in
an incubator with humidified atmosphere of 5% CO.sub.2 at
37.degree. C. to adhere. For each vector produced, one tissue
culture dish is transfected as following: the lentiviral backbone
plasmid pFlap-promoter-CAR_CD19 (10 .mu.g), the pThV-Env1 encoding
envelope plasmid (2 .mu.g), and the pThV-GP packaging plasmid (10
.mu.) were mixed with 353 .mu.L of sterile distilled water (Gibco
by Life Technologies) and 125 .mu.L of CaCl.sub.2 (Fluka). The DNA
mix is then added drop to drop to 500 .mu.L of 37.degree. C.
prewarmed HBS 2.times. pH=7.3 and the 1 mL of precipitate obtained
was added to the culture medium of the cells. The transfected cells
were then incubated at 37.degree. C., 5% CO.sub.2. The medium was
replaced 24 h after transfection by 7 mL of harvest medium without
serum and the viral supernatant was harvested after an additional
24 h, and clarified by centrifugation 5 min. at 2500 rpm. The
harvest clarified bulk (210 mL) is then treated 30 min. with DNase
(Roche) in the presence of MgCl.sub.2 (Sigma Aldrich) to avoid
residual transfection DNA, and ultraconcentrated by centrifugation
1 h at 22000 rpm, 4.degree. C. Each vector pellets are resuspended
in 70 .mu.l PBS-Lactose (40 mg/L), pooled, 30 .mu.L aliquoted and
stored at -70.degree. C.+-.10.degree. C.
[0249] For product characterization and pharmaceutical release,
quality tests can be performed according to regulatory texts on
vaccines: the quality control required for vaccines as per the
European Pharmacopeia (section 6.16), the "guideline on quality,
non-clinical and clinical aspects of live recombinant viral
vectored vaccines" (EMA/CHMP/141697/2009), the "guideline on
development and manufacture of lentiviral vectors"
(CHMP/BWP/2458/03); regulatory text on gene therapy medicinal
products: the quality controls required for gene transfer medicinal
products for human use as per the European Pharmacopeia (section
5.14), the quality controls specific to gene therapy products as
defined in the "note for guidance on the quality, preclinical and
clinical aspects of gene transfer medicinal products"
(CHMP/BWP/3088/99); regulatory texts on biotechnological products
(ICH Q5A to ICH Q5E); regulatory texts on specifications (ICH Q6A
and ICH Q6B) and the quality controls required for parenteral
preparations as per the European Pharmacopeia (section 7.0).
Example 3. Lentiviral Vector Titration
[0250] qPCR reactions: HEK 293T cells are seeded in 6-well plates
(BD Falcon) in culture medium and incubated for 4 h at 37.degree.
C., 5% CO.sub.2 in moist atmosphere. Cells are transduced with 3
successive dilutions of lentiviral vector. 72 h post-incubation,
cells are harvested and transduced HEK 293T cell pellets are
produced. Total genomic DNA from transduced cell-pellets is
extracted using a method based on QIAGEN QIAamp DNA mini kit
handbook. Proviral quantification is performed using Taqman qPCR.
The amplification is performed with the Master Mix (Fermentas
Thermo Scientific), the Flap A (CCCAAGAACCCAAGGAACA; SEQ ID NO:26)
and Flap S (AGACAA GATAGAGGAAGAGCAAAAC; SEQ ID NO:27) primers and
LENTI TM probe (6FAM-AACCATTAGGAGTAGCACCCACCAAGG-BBQ; SEQ ID
NO:52). Normalization is performed with the quantification of the
actin gene (same Mix, Actine A--CGGTGAGGATCTTCATGAGGTAGT- (SEQ ID
NO:28), Actine S--AACACCCCAGCCATGTACGT- (SEQ ID NO:29) primers and
HUMURA ACT TM probe--6FAM-CCAGCCAGGTCCAGACGCAGGA-BBQ- (SEQ ID
NO:30). Both reactions are achieved on MasterCycler Ep Realplex S
(Eppendorf, 2 min at 50.degree. C., 10 min at 95.degree. C. and 40
cycles of 15 seconds at 95.degree. C. and 1 min at 63.degree. C.).
The analysis is performed on MasterCycler Ep Realplex Software.
Example 4. Regulated CARs
[0251] Lentiviral vectors were generated encoding CARs. The
CAR_CD19 2nd and 3rd generation sequences (SEQ ID NO:39 and SEQ ID
NO:40) were purchased from GeneArt (Lifetech), and cloned replacing
the GFP gene between BamHI and XhoI restrictions sites of the
pFlap-.DELTA.U3-.beta.2m-GFP, pFlap-.DELTA.U3-HLA-A2-GFP,
pFlap-.DELTA.U3-HLA-DR.alpha.-GFP or pFlap-.DELTA.U3-UBC-GFP,
depending of the required promoter.
[0252] CARs were generated as a fusion protein with SBP at 3
different positions. The lentiviral vectors were further modified
to contain a hook for the ER fused to a core Streptavidin protein.
The promoter was an .beta.2m promoter.
[0253] These lentiviral vectors will be used in in vitro and animal
models of cancer. Biotin will be administered at 40 .mu.M (with a
titration of higher and lower concentrations) initial concentration
to test for release from the ER. First, migration of the CAR to the
surface of the cell (GFP in place of the binding domain or between
the signal sequence and the binding domain) will be analyzed in
vitro. Next, the same will be performed in animal models (mice and
rats) by injection of CAR-T cells and evaluation of the migration
at the surface of cells (GFP) from the animal.
[0254] Both "second generation" CAR (2 intra cytoplasmic activating
domains) and "third generation" CAR (3 intra cytoplasmic activating
domains) constructs will be generated containing a hook-binding
domain (SBP). Initially, the binding domains evaluated will be
anti-CD19, anti-PDL-1, anti-PD1, anti-hedgehog, anti-CD123, and
anti-CD123/CD33.
[0255] CARMIN 1.0: development of lentiviral vectors coding for CAR
of the second (containing the CD3_ and the 4-1 BB cosignaling
domains) and third generations (containing the CD3_, the CD28 and
the 4-1BB domains) directed against CD19 (for CD19+ leukemias and
lymphomas), LMP-1 and -2 (for EBV-induced leukemias). The
lentiviral vectors allow optimal expression of CAR in T cells and
the impact on the efficacy of CAR-T cells is under investigation.
Hematological malignancies can be used as a benchmark.
[0256] CARMIN 2.0: development of a switchable on/off system which
is based on a W protein anchored to the membrane of the endoplasmic
reticulum (ER) through a hook, and its binding partner Y introduced
into the CAR structure. The interaction between the X-hook (e.g.,
Streptavidin) and the Y (e.g., Streptavidin-Binding Protein)-CAR
allows the CAR retention inside the ER. The addition of a Z protein
(e.g., Biotin) displaces the equilibrium of binding of X towards Z
instead of Y, thus leading to the release of the CAR from the ER
and its expression to the cytoplasmic membrane. The release of the
CAR will stop with Z exhaustion (or antagonist) and remaining cells
can be easily reactivated through reintroduction of the Z
inducer.
[0257] Hook and CAR are vectorized in one lentivector and can be
used in clinics (b2m-HOOK-IRES-CAR). Evaluations can be performed
in vitro both on immortalized cells (HEK293T, Jurkat, HeLa) and on
primary cells (T-cells). This system will increase safety of CAR-T
cells. A switchable CD19-CAR system can be evaluated in vitro for
expression and in vivo for efficacy.
[0258] Most of the scFv used to date are of murine origin.
Neutralizing antibodies against these murine scFv can limit the
efficacy of CAR. As an alternative, we will develop camelid
nanobodies to be used as binding domains since they are highly
homologous to the human VH domain of antibodies and they display
high antigen binding capacities. The proof-of-concept will be made
with a second generation CAR containing a nanobody directed against
Her2 as a binding domain. These technological platforms allows
flexibility and reactivity in the CAR design, production and
evaluation, thus leading to the generation of optimal CAR-T cells.
This differentiated inductible and reversible (ON/OFF) CAR T-cell
technology is aimed to be delivered at the patient's bedside
(automated process).
Example 5. Expression in Human T Cells
[0259] Peripheral blood mononuclear cells (PBMC) were purified from
peripheral blood by gradient density centrifugation on Ficoll.
After PBMC washing, CD3+ T cells were purified by negative magnetic
selection (ie unwanted cells were magnetically labelled while T
cells were left untouched) using the Pan T Cell Isolation Kit
(Miltenyi). This step is required to obtain a highly purified T
cell population. According to the yield obtained after this step of
isolation, 10.sup.7 to 10.sup.8 T cells were cultured at
2.5.times.10.sup.6/ml in an optimized serum-free cell culture
medium developed for the cultivation and expansion of human T cells
(TexMACS medium, Miltenyi). These T cells are activated by the T
cell Activation/Expansion kit from Miltenyi. The kit consists of
anti-biotin MACSiBead Particles and biotinylated antibodies against
human CD2, CD3 and CD28. Anti-biotin MACSiBead Particles loaded
with biotinylated antibodies are used to mimic antigen-presenting
cells and to activate T cells. An optimal activation of T cells is
accomplished by using one loaded anti-biotin MACSiBead Particle per
two cells. T cells are activated for 3 days. Transduction of
activated T cells was then performed at a MOI of 4 which means that
1 T cell is incubated with 4 transduction units of lentiviral
particles. CAR expression was assessed by flow cytometry 48 or 72 h
lentiviral particle addition, by staining the murine CD19-binding
domain with a biotinylated goat anti-mouse IgG followed by
streptavidin conjugated to phycoerythrin. T cell subpopulations
were characterized by CD3, CD4 and CD8 staining. This allowed
specific detection of the CAR on the surface of T cells. The whole
process was performed with the TexMACS medium allowing survival and
expansion of T cells.
Example 6. Structure and Expression of CAR-RUSH
[0260] Qualified blood was obtained from the Etablissement Frangais
du Sang (Rungis, France). Peripheral Blood Mononuclear Cells (PBMC)
were purified by Ficoll (Lymphocyte Separation Medium, Eurobio)
gradient density separation. T cells were then purified from PBMC
by magnetic isolation using the Pan T cell isolation kit
(Miltenyi). T cells were separated according to the manufacturer's
instructions. T cells were put in culture at a concentration of
2.5.times.106 cells/ml in TexMACS medium (Miltenyi) at 37.degree.
C./5%CO2 and activated 3 days by the T cell activation/expansion
kit (anti-CD2/-CD3/-CD28 nanoparticles prepared according the
manufacturer's instructions) from Miltenyi at a bead:T cell ratio
of 1:2. After activation, T cells were harvested, counted and put
in culture in TexMACS medium in 24 well-plates at 37.degree. C./5%
CO2. Transduction was performed by adding directly into wells
lentiviral vectors at different MOI. The different lentiviral
vectors tested were: (i) the 2nd generation anti-CD19 CAR
containing the 4-1 BB and the CD3zeta intracellular domains; (ii)
the same vector containing the streptavidin binding protein at
three different positions (CAR-SBP1, CAR-SBP2, CAR-SBP3).
[0261] The volume of vector to add to each well according to the
MOI was calculated as follows: volume to be added
(.mu.l)=(MOI.times.number of cells (in millions)/concentration of
vector (transduction unit/ml)).times.1000.
[0262] 200 000 cells were transduced at a MOI of 10 with a vector
titer at 3.10.sup.9 TU/ml.fwdarw.volume of vector to be added
(.mu.I)=(10.times.0.2.times.10.sup.6/3.times.10.sup.9).times.1000=0.51
.mu.l. Human recombinant IL-2 (Miltenyi) was added the day of the
transduction at 50 IU/ml. At day 3, transduced T cells were
harvested, washed extensively with DPBS 1.times. and immunostaining
of molecules of interest was performed in 96 well-plates. T cells
were first incubated with a viability dye (Fixable Viability Dye,
eBiosciences) conjugated to eFluor 780 and incubated 30 minutes at
4.degree. C. The incubation was performed in azide-free and
protein-free DPBS1.times.. Cells were then washed with DPBS
1.times. and then incubated with a biotinylated Goat anti-mouse IgG
(Fab')2 (Jackson ImmunoResearch) for 30 minutes a 4.degree. C.
After incubation, cells were washed in autoMACS running buffer
(Miltenyi) and the third incubation was performed with a mix of
streptavidin conjugated to phycoerythrin (Jackson ImmunoResearch)
and mouse anti-human CD3 conjugated to PE-Cy7 (BD Biosciences).
Incubation was done for 30 minutes at 4.degree. C. After this third
incubation, cells were washed in autoMACS running buffer and fixed
in CellFIX (BD Biosciences) before acquisition on a MACSQuant
analyzer (Miltenyi). Flow cytometry data were analyzed using the
FlowJo software.
Example 7. Expression and Behaviour of CAR-RUSH Constructs
Following Co-Transduction with a Lentivector Encoding a
HOOK-Streptavidin, and Biotin Treatment
[0263] Qualified blood was obtained from the Etablissement Frangais
du Sang (Rungis, France). Peripheral Blood Mononuclear Cells (PBMC)
were purified by Ficoll (Lymphocyte Separation Medium, Eurobio)
gradient density separation. T cells were then purified from PBMC
by magnetic isolation using the Pan T cell isolation kit
(Miltenyi). T cells were separated according to the manufacturer's
instructions. T cells were put in culture at a concentration of
2.5.times.10.sup.6 cells/ml in TexMACS medium (Miltenyi) at
37.degree. C./5%CO2 and activated 3 days by the T cell
activation/expansion kit (anti-CD2/-CD3/-CD28 nanoparticles
prepared according the manufacturer's instructions) from Miltenyi
at a bead:T cell ratio of 1:2.
[0264] After activation, T cells were harvested, counted and put in
culture in TexMACS medium in 24 well-plates at 37.degree. C./5%
CO2. Co-transduction was performed by adding directly into wells
lentiviral vectors at different MOI. The two lentiviral vectors
tested were: (i) a hook-streptavidin vector and (ii) the 2nd
generation anti-CD19 CAR containing the 4-1 BB and the CD3zeta
intracellular domains with the streptavidin binding protein at
three different positions (CAR-SBP1, CAR-SBP2 and CAR-SBP3).
[0265] The volume of each vector to add to each well according to
the MOI was calculated as follows: volume to be added
(.mu.l)=(MOI.times.number of cells (in millions)/concentration of
vector (transduction unit/ml)).times.1000
[0266] Human recombinant IL-2 (50 IU/ml; Miltenyi) and biotin (40
.mu.M; Sigma-Aldrich) were added the day of the transduction.
[0267] At day 3, transduced T cells were harvested, washed
extensively with DPBS 1X and immunostaining of molecules of
interest was performed in 96 well-plates. T cells were first
incubated with a viability dye (Fixable Viability Dye,
eBiosciences) conjugated to eFluor 780 and incubated 30 minutes at
4.degree. C. The incubation was performed in azide-free and
protein-free DPBS1.times.. Cells were then washed with DPBS
1.times. and then incubated with a biotinylated Goat anti-mouse IgG
(Fab')2 (Jackson ImmunoResearch) for 30 minutes a 4.degree. C.
After incubation, cells were washed in autoMACS running buffer
(Miltenyi) and the third incubation was performed with a mix of
streptavidin conjugated to phycoerythrin (Jackson ImmunoResearch)
and mouse anti-human CD3 conjugated to PE-Cy7 (BD Biosciences).
Incubation was done for 30 minutes at 4.degree. C. After this third
incubation, cells were washed in autoMACS running buffer and fixed
in CellFIX (BD Biosciences) before acquisition on a MACSQuant
analyzer (Miltenyi). Flow cytometry data were analyzed using the
FlowJo software.
Example 8. Expression and Behavior of CAR-RUSH Bicistronic
Constructs
[0268] Qualified blood was obtained from the Etablissement Frangais
du Sang (Rungis, France). Peripheral Blood Mononuclear Cells (PBMC)
were purified by Ficoll (Lymphocyte Separation Medium, Eurobio)
gradient density separation. T cells were then purified from PBMC
by magnetic isolation using the Pan T cell isolation kit
(Miltenyi). T cells were separated according to the manufacturer's
instructions. T cells were put in culture at a concentration of
2.5.times.106 cells/ml in TexMACS medium (Miltenyi) at 37.degree.
C./5%CO2 and activated 3 days by the T cell activation/expansion
kit (anti-CD2/-CD3/-CD28 nanoparticles prepared according the
manufacturer's instructions) from Miltenyi at a bead:T cell ratio
of 1:2.
[0269] After activation, T cells were harvested, counted and put in
culture in TexMACS medium in 24 well-plates at 37.degree. C./5%
CO2. Transduction was performed by adding directly into wells
lentiviral vectors at different MOI. The different lentiviral
vectors tested were ; (i) the "classical" 2nd generation anti-CD19
CAR; (ii) three constructions containing the hook-streptavidin, an
RES and the 2nd generation anti-CD19 CAR containing the 4-1 BB and
the CD3zeta intracellular domains with the streptavidin binding
protein at three different positions (hook-IRES-CAR-SBP1,
hook-IRES-CAR-SBP2, hook-IRES-CAR-SBP3).
[0270] The volume of vector to add to each well according to the
MOI was calculated as follows: volume to be added
(.mu.l)=(MOI.times.number of cells (in millions)/concentration of
vector (transduction unit/ml)).times.1000.
[0271] The different MOI tested were 10, 20 or 30 depending on the
experiment.
[0272] Human recombinant IL-2 (50 IU/ml; Miltenyi) and biotin (40
.mu.M; Sigma-Aldrich) were added the day of the transduction.
[0273] At days 3 and 7, transduced T cells were harvested, washed
extensively with DPBS 1X and immunostaining of molecules of
interest was performed in 96 well-plates. T cells were first
incubated with a viability dye (Fixable Viability Dye,
eBiosciences) conjugated to eFluor 780 and incubated 30 minutes at
4.degree. C. The incubation was performed in azide-free and
protein-free DPBS1.times.. Cells were then washed with DPBS
1.times. and then incubated with a biotinylated Goat anti-mouse IgG
(Fab')2 (Jackson ImmunoResearch) for 30 minutes at 4.degree. C.
After incubation, cells were washed in autoMACS running buffer
(Miltenyi) and the third incubation was performed with a mix of
streptavidin conjugated to phycoerythrin (Jackson ImmunoResearch)
and mouse anti-human CD3 conjugated to PE-Cy7 (BD Biosciences).
Incubation was done for 30 minutes at 4.degree. C. After this third
incubation, cells were washed in autoMACS running buffer and fixed
in CellFIX (BD Biosciences) before acquisition on a MACSQuant
analyzer (Miltenyi). Flow cytometry data were analyzed using the
FlowJo software.
Example 9. CAR-RUSH System Switch Evaluation (OFF/ON/OFF)
[0274] Qualified blood was obtained from the Etablissement Frangais
du Sang (Rungis, France). Peripheral Blood Mononuclear Cells (PBMC)
were purified by Ficoll (Lymphocyte Separation Medium, Eurobio)
gradient density separation. T cells were then purified from PBMC
by magnetic isolation using the Pan T cell isolation kit
(Miltenyi). T cells were separated according to the manufacturer's
instructions. T cells were put in culture at a concentration of
2.5.times.10.sup.6 cells/ml in TexMACS medium (Miltenyi) at
37.degree. C./5%CO2 and activated 3 days by the T cell
activation/expansion kit (anti-CD2/-CD3/-CD28 nanoparticles
prepared according the manufacturer's instructions) from Miltenyi
at a bead:T cell ratio of 1:2.
[0275] After activation, T cells were harvested, counted and put in
culture in TexMACS medium in 24 well-plates at 37.degree. C./5%
CO.sub.2. Transduction was performed by adding directly into wells
lentiviral vectors at different MOI. The different lentiviral
vectors tested were; (i) the "classical" 2nd generation anti-CD19
CAR; (ii) three constructions containing the hook-streptavidin, an
RES and the 2nd generation anti-CD19 CAR containing the 4-1 BB and
the CD3zeta intracellular domains with the streptavidin binding
protein at three different positions (hook-IRES-CAR-SBP1,
hook-IRES-CAR-SBP2, hook-IRES-CAR-SBP3).
[0276] The volume of vector to add to each well according to the
MOI was calculated as follows: volume to be added
(.mu.l)=(MOI.times.number of cells (in millions)/concentration of
vector (transduction unit/ml)).times.1000
[0277] The different MOI tested were 10 and 20.
[0278] Human recombinant IL-2 (50 IU/ml; Miltenyi) and biotin (40
.mu.M; Sigma-Aldrich) were added the day of the transduction.
[0279] At day 3, cells were washed and biotin was added or not at
400 to re-induce CAR-SBP expression.
[0280] At day 7, transduced T cells were harvested, washed
extensively with DPBS 1.times. and immunostaining of molecules of
interest was performed in 96 well-plates. T cells were first
incubated with a viability dye (Fixable Viability Dye,
eBiosciences) conjugated to eFluor 780 and incubated 30 minutes at
4.degree. C. The incubation was performed in azide-free and
protein-free DPBS1.times.. Cells were then washed with DPBS
1.times. and then incubated with a biotinylated Goat anti-mouse IgG
(Fab')2 (Jackson ImmunoResearch) for 30 minutes at 4.degree. C.
After incubation, cells were washed in autoMACS running buffer
(Miltenyi) and the third incubation was performed with a mix of
streptavidin conjugated to phycoerythrin (Jackson ImmunoResearch)
and mouse anti-human CD3 conjugated to PE-Cy7 (BD Biosciences).
Incubation was done for 30 minutes at 4.degree. C. After this third
incubation, cells were washed in autoMACS running buffer and fixed
in CelIFIX (BD Biosciences) before acquisition on a MACSQuant
analyzer (Miltenyi).
Sequence CWU 1
1
52138PRTArtificial SequenceSynthetic hook-binding domain 1Met Asp
Glu Lys Thr Thr Gly Trp Arg Gly Gly His Val Val Glu Gly1 5 10 15Leu
Ala Gly Glu Leu Glu Gln Leu Arg Ala Arg Leu Glu His His Pro 20 25
30Gln Gly Gln Arg Glu Pro 352114DNAArtificial SequenceSynthetic
hook-binding domain 2atggacgaga aaaccaccgg ctggcgggga ggccacgtgg
tggaaggact ggccggcgag 60ctggaacagc tgcgggccag actggaacac cacccccagg
gccagagaga gccc 1143163PRTHomo sapiens 3Lys Trp Lys Ala Leu Phe Thr
Ala Ala Ile Leu Gln Ala Gln Leu Pro1 5 10 15Ile Thr Glu Ala Gln Ser
Phe Gly Leu Leu Asp Pro Lys Leu Cys Tyr 20 25 30Leu Leu Asp Gly Ile
Leu Phe Ile Tyr Gly Val Ile Leu Thr Ala Leu 35 40 45Phe Leu Arg Val
Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln 50 55 60Gln Gly Gln
Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu65 70 75 80Glu
Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly 85 90
95Gly Lys Pro Gln Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu
100 105 110Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly
Met Lys 115 120 125Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu
Tyr Gln Gly Leu 130 135 140Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala
Leu His Met Gln Ala Leu145 150 155 160Pro Pro Arg4220PRTHomo
sapiens 4Met Leu Arg Leu Leu Leu Ala Leu Asn Leu Phe Pro Ser Ile
Gln Val1 5 10 15Thr Gly Asn Lys Ile Leu Val Lys Gln Ser Pro Met Leu
Val Ala Tyr 20 25 30Asp Asn Ala Val Asn Leu Ser Cys Lys Tyr Ser Tyr
Asn Leu Phe Ser 35 40 45Arg Glu Phe Arg Ala Ser Leu His Lys Gly Leu
Asp Ser Ala Val Glu 50 55 60Val Cys Val Val Tyr Gly Asn Tyr Ser Gln
Gln Leu Gln Val Tyr Ser65 70 75 80Lys Thr Gly Phe Asn Cys Asp Gly
Lys Leu Gly Asn Glu Ser Val Thr 85 90 95Phe Tyr Leu Gln Asn Leu Tyr
Val Asn Gln Thr Asp Ile Tyr Phe Cys 100 105 110Lys Ile Glu Val Met
Tyr Pro Pro Pro Tyr Leu Asp Asn Glu Lys Ser 115 120 125Asn Gly Thr
Ile Ile His Val Lys Gly Lys His Leu Cys Pro Ser Pro 130 135 140Leu
Phe Pro Gly Pro Ser Lys Pro Phe Trp Val Leu Val Val Val Gly145 150
155 160Gly Val Leu Ala Cys Tyr Ser Leu Leu Val Thr Val Ala Phe Ile
Ile 165 170 175Phe Trp Val Arg Ser Lys Arg Ser Arg Leu Leu His Ser
Asp Tyr Met 180 185 190Asn Met Thr Pro Arg Arg Pro Gly Pro Thr Arg
Lys His Tyr Gln Pro 195 200 205Tyr Ala Pro Pro Arg Asp Phe Ala Ala
Tyr Arg Ser 210 215 2205255PRTHomo sapiens 5Met Gly Asn Ser Cys Tyr
Asn Ile Val Ala Thr Leu Leu Leu Val Leu1 5 10 15Asn Phe Glu Arg Thr
Arg Ser Leu Gln Asp Pro Cys Ser Asn Cys Pro 20 25 30Ala Gly Thr Phe
Cys Asp Asn Asn Arg Asn Gln Ile Cys Ser Pro Cys 35 40 45Pro Pro Asn
Ser Phe Ser Ser Ala Gly Gly Gln Arg Thr Cys Asp Ile 50 55 60Cys Arg
Gln Cys Lys Gly Val Phe Arg Thr Arg Lys Glu Cys Ser Ser65 70 75
80Thr Ser Asn Ala Glu Cys Asp Cys Thr Pro Gly Phe His Cys Leu Gly
85 90 95Ala Gly Cys Ser Met Cys Glu Gln Asp Cys Lys Gln Gly Gln Glu
Leu 100 105 110Thr Lys Lys Gly Cys Lys Asp Cys Cys Phe Gly Thr Phe
Asn Asp Gln 115 120 125Lys Arg Gly Ile Cys Arg Pro Trp Thr Asn Cys
Ser Leu Asp Gly Lys 130 135 140Ser Val Leu Val Asn Gly Thr Lys Glu
Arg Asp Val Val Cys Gly Pro145 150 155 160Ser Pro Ala Asp Leu Ser
Pro Gly Ala Ser Ser Val Thr Pro Pro Ala 165 170 175Pro Ala Arg Glu
Pro Gly His Ser Pro Gln Ile Ile Ser Phe Phe Leu 180 185 190Ala Leu
Thr Ser Thr Ala Leu Leu Phe Leu Leu Phe Phe Leu Thr Leu 195 200
205Arg Phe Ser Val Val Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe
210 215 220Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu
Asp Gly225 230 235 240Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly
Gly Cys Glu Leu 245 250 255693PRTHomo sapiens 6Met Ile His Leu Gly
His Ile Leu Phe Leu Leu Leu Leu Pro Val Ala1 5 10 15Ala Ala Gln Thr
Thr Pro Gly Glu Arg Ser Ser Leu Pro Ala Phe Tyr 20 25 30Pro Gly Thr
Ser Gly Ser Cys Ser Gly Cys Gly Ser Leu Ser Leu Pro 35 40 45Leu Leu
Ala Gly Leu Val Ala Ala Asp Ala Val Ala Ser Leu Leu Ile 50 55 60Val
Gly Ala Val Phe Leu Cys Ala Arg Pro Arg Arg Ser Pro Ala Gln65 70 75
80Glu Asp Gly Lys Val Tyr Ile Asn Met Pro Gly Arg Gly 85
907277PRTHomo sapiens 7Met Cys Val Gly Ala Arg Arg Leu Gly Arg Gly
Pro Cys Ala Ala Leu1 5 10 15Leu Leu Leu Gly Leu Gly Leu Ser Thr Val
Thr Gly Leu His Cys Val 20 25 30Gly Asp Thr Tyr Pro Ser Asn Asp Arg
Cys Cys His Glu Cys Arg Pro 35 40 45Gly Asn Gly Met Val Ser Arg Cys
Ser Arg Ser Gln Asn Thr Val Cys 50 55 60Arg Pro Cys Gly Pro Gly Phe
Tyr Asn Asp Val Val Ser Ser Lys Pro65 70 75 80Cys Lys Pro Cys Thr
Trp Cys Asn Leu Arg Ser Gly Ser Glu Arg Lys 85 90 95Gln Leu Cys Thr
Ala Thr Gln Asp Thr Val Cys Arg Cys Arg Ala Gly 100 105 110Thr Gln
Pro Leu Asp Ser Tyr Lys Pro Gly Val Asp Cys Ala Pro Cys 115 120
125Pro Pro Gly His Phe Ser Pro Gly Asp Asn Gln Ala Cys Lys Pro Trp
130 135 140Thr Asn Cys Thr Leu Ala Gly Lys His Thr Leu Gln Pro Ala
Ser Asn145 150 155 160Ser Ser Asp Ala Ile Cys Glu Asp Arg Asp Pro
Pro Ala Thr Gln Pro 165 170 175Gln Glu Thr Gln Gly Pro Pro Ala Arg
Pro Ile Thr Val Gln Pro Thr 180 185 190Glu Ala Trp Pro Arg Thr Ser
Gln Gly Pro Ser Thr Arg Pro Val Glu 195 200 205Val Pro Gly Gly Arg
Ala Val Ala Ala Ile Leu Gly Leu Gly Leu Val 210 215 220Leu Gly Leu
Leu Gly Pro Leu Ala Ile Leu Leu Ala Leu Tyr Leu Leu225 230 235
240Arg Arg Asp Gln Arg Leu Pro Pro Asp Ala His Lys Pro Pro Gly Gly
245 250 255Gly Ser Phe Arg Thr Pro Ile Gln Glu Glu Gln Ala Asp Ala
His Ser 260 265 270Thr Leu Ala Lys Ile 2758199PRTHomo sapiens 8Met
Lys Ser Gly Leu Trp Tyr Phe Phe Leu Phe Cys Leu Arg Ile Lys1 5 10
15Val Leu Thr Gly Glu Ile Asn Gly Ser Ala Asn Tyr Glu Met Phe Ile
20 25 30Phe His Asn Gly Gly Val Gln Ile Leu Cys Lys Tyr Pro Asp Ile
Val 35 40 45Gln Gln Phe Lys Met Gln Leu Leu Lys Gly Gly Gln Ile Leu
Cys Asp 50 55 60Leu Thr Lys Thr Lys Gly Ser Gly Asn Thr Val Ser Ile
Lys Ser Leu65 70 75 80Lys Phe Cys His Ser Gln Leu Ser Asn Asn Ser
Val Ser Phe Phe Leu 85 90 95Tyr Asn Leu Asp His Ser His Ala Asn Tyr
Tyr Phe Cys Asn Leu Ser 100 105 110Ile Phe Asp Pro Pro Pro Phe Lys
Val Thr Leu Thr Gly Gly Tyr Leu 115 120 125His Ile Tyr Glu Ser Gln
Leu Cys Cys Gln Leu Lys Phe Trp Leu Pro 130 135 140Ile Gly Cys Ala
Ala Phe Val Val Val Cys Ile Leu Gly Cys Ile Leu145 150 155 160Ile
Cys Trp Leu Thr Lys Lys Lys Tyr Ser Ser Ser Val His Asp Pro 165 170
175Asn Gly Glu Tyr Met Phe Met Arg Ala Val Asn Thr Ala Lys Lys Ser
180 185 190Arg Leu Thr Asp Val Thr Leu 1959220DNAHomo sapiens
9attggggagt cccagccttg gggattcccc aactccgcag tttcttttct ccctctccca
60acctatgtag ggtccttctt cctggatact cacgacgcgg acccagttct cactcccatt
120gggtgtcggg tttccagaga agccaatcag tgtcgtcgcg gtcgcggttc
taaagtccgc 180acgcacccac cgggactcag attctcccca gacgccgagg
22010197DNAHomo sapiens 10ggggaggcgc agcgttgggg attccccact
cccctgagtt tcacttcttc tcccaacttg 60tgtcgggtcc ttcttccagg atactcgtga
cgcgtcccca cttcccactc ccattgggta 120ttggatatct agagaagcca
atcagcgtcg ccgcggtccc agttctaaag tccccacgca 180cccacccgga ctcagag
19711216DNAHomo sapiens 11cactggggag gcgccgcgtt gaggattctc
cactcccctc agtttcactt cttctcccaa 60cctgcgtcgg gtccttcttc ctgaatactc
atgacgcgtc cccaattccc actcccattg 120ggtgtcgggt tctagagaag
ccaatcagcg tctccgcagt cccggtctaa agtccccagt 180cacccacccg
gactcagatt ctccccagac gccgag 21612205DNAHomo sapiens 12taagaactgc
tgattgctgg gaaactctgc agtttcccgt tcctctcgta acctggtcat 60gtgtccttct
tcctggatac tcatgacgca gactcagttc tcattcccaa tgggtgtcgg
120gtttctagag aagccaatca gcgtcgccac gactcccgac tataaagtcc
ccatccggac 180tcaagaagtt ctcaggactc agagg 20513252DNAHomo sapiens
13aggccccgag gcggtgtctg gggttggaag gctcagtatt gagaattccc catctcccca
60gagtttctct ttctctccca acccgtgtca ggtccttcat cctggatact cataacgcgg
120ccccatttct cactcccatt gggcgtcgcg tttctagaga agccaatcag
tgtcgccgca 180gttcccaggt tctaaagtcc cacgcacccc gcgggactca
tatttttccc agacgcggag 240gttggggtca tg 25214192DNAHomo sapiens
14aacatcacga gactctaaga aaaggaaact gaaaacggga aagtccctct ctctaacctg
60gcactgcgtc gctggcttgg agacaggtga cggtccctgc gggccttgtc ctgattggct
120gggcacgcgt ttaatataag tggaggcgtc gcgctggcgg gcattcctga
agctgacagc 180attcgggccg ag 1921533DNAArtificial SequenceSynthetic
PCR primer 15cttactagtt ggaagggcta attcactccc aac
331632DNAArtificial SequenceSynthetic PCR primer 16cattctagaa
ctgctagaga ttttccacac tg 321742DNAArtificial SequenceSynthetic PCR
primer 17taccccgggc catggcctcc aaaaaagcct cctcactact tc
421844DNAArtificial SequenceSynthetic PCR primer 18actcccgggt
aatttttttt atttatgcag aggccgaggc cgcc 441935DNAArtificial
SequenceSynthetic PCR primer 19tacacgcgtg gagttccgcg ttacataact
tacgg 352039DNAArtificial SequenceSynthetic PCR primer 20cgtggatccg
atcgcggtgt cttctatgga ggtcaaaac 392134DNAArtificial
SequenceSynthetic PCR primer 21gccggcgcgc cgagaaaccc tgcagggaat
tccc 342241DNAArtificial SequenceSynthetic PCR primer 22cgtggatccg
atcgctcggc ccgaatgctg tcagcttcag g 4123517DNAArtificial
SequenceSynthetic Promoter 23gagaaaccct gcagggaatt ccccagctgt
agttataaac agaagttctc cttctgctag 60gtagcattca aagatcttaa tcttctgggt
ttccgttttc tcgaatgaaa aatgcaggtc 120cgagcagtta actggcgggg
gcaccattag caagtcactt agcatctctg gggccagtct 180gcaaagcgag
ggggcagcct taatgtgcct ccagcctgaa gtcctagaat gagcgcccgg
240tgtcccaagc tggggcgcgc accccagatc ggagggcgcc gatgtacaga
cagcaaactc 300acccagtcta gtgcatgcct tcttaaacat cacgagactc
taagaaaagg aaactgaaaa 360cgggaaagtc cctctctcta acctggcact
gcgtcgctgg cttggagaca ggtgacggtc 420cctgcgggcc ttgtcctgat
tggctgggca cgcgtttaat ataagtggag gcgtcgcgct 480ggcgggcatt
cctgaagctg acagcattcg ggccgag 5172447DNAArtificial
SequenceSynthetic PCR primer 24atgcatgcgt cgacctcgag ttaatcctca
tcctgtctac ttgccac 472565DNAArtificial SequenceSynthetic PCR primer
25gcatgcatcg gccggggcgg cgactggtga gaggccacca tgggtgcgag agcgtcagta
60ttaag 652619DNAArtificial SequenceSynthetic PCR primer
26cccaagaacc caaggaaca 192725DNAArtificial SequenceSynthetic PCR
primer 27agacaagata gaggaagagc aaaac 252824DNAArtificial
SequenceSynthetic PCR primer 28cggtgaggat cttcatgagg tagt
242920DNAArtificial SequenceSynthetic PCR primer 29aacaccccag
ccatgtacgt 203022DNAArtificial SequenceSynthetic Probe 30ccagccaggt
ccagacgcag ga 2231160PRTArtificial SequenceSynthetic Hook peptide
31Met Asp Pro Ser Lys Asp Ser Lys Ala Gln Val Ser Ala Ala Glu Ala1
5 10 15Gly Ile Thr Gly Thr Trp Tyr Asn Gln Leu Gly Ser Thr Phe Ile
Val 20 25 30Thr Ala Gly Ala Asp Gly Ala Leu Thr Gly Thr Tyr Glu Ser
Ala Val 35 40 45Gly Asn Ala Glu Ser Arg Tyr Val Leu Thr Gly Arg Tyr
Asp Ser Ala 50 55 60Pro Ala Thr Asp Gly Ser Gly Thr Ala Leu Gly Trp
Thr Val Ala Trp65 70 75 80Lys Asn Asn Tyr Arg Asn Ala His Ser Ala
Thr Thr Trp Ser Gly Gln 85 90 95Tyr Val Gly Gly Ala Glu Ala Arg Ile
Asn Thr Gln Trp Leu Leu Thr 100 105 110Ser Gly Thr Thr Glu Ala Asn
Ala Trp Lys Ser Thr Leu Val Gly His 115 120 125Asp Thr Phe Thr Lys
Val Lys Pro Ser Ala Ala Ser Ile Asp Ala Ala 130 135 140Lys Lys Ala
Gly Val Asn Asn Gly Asn Pro Leu Asp Ala Val Gln Gln145 150 155
16032160PRTArtificial SequenceSynthetic Hook peptide 32Met Asp Pro
Ser Lys Asp Ser Lys Ala Gln Val Ser Ala Ala Glu Ala1 5 10 15Gly Ile
Thr Gly Thr Trp Tyr Asn Gln Leu Gly Ser Thr Phe Ile Val 20 25 30Thr
Ala Gly Ala Asp Gly Ala Leu Thr Gly Thr Tyr Glu Ser Ala Val 35 40
45Gly Asn Ala Glu Ser Arg Tyr Thr Leu Thr Gly Arg Tyr Asp Ser Ala
50 55 60Pro Ala Thr Asp Gly Ser Gly Thr Ala Leu Gly Trp Arg Val Ala
Trp65 70 75 80Lys Asn Asn Tyr Arg Asn Ala His Ser Ala Thr Thr Trp
Ser Gly Gln 85 90 95Tyr Val Gly Gly Ala Glu Ala Arg Ile Asn Thr Gln
Trp Thr Leu Thr 100 105 110Ser Gly Thr Thr Glu Ala Asn Ala Trp Lys
Ser Thr Leu Arg Gly His 115 120 125Asp Thr Phe Thr Lys Val Lys Pro
Ser Ala Ala Ser Ile Asp Ala Ala 130 135 140Lys Lys Ala Gly Val Asn
Asn Gly Asn Pro Leu Asp Ala Val Gln Gln145 150 155 16033260PRTHomo
sapiens 33Met Ala Arg Pro His Pro Trp Trp Leu Cys Val Leu Gly Thr
Leu Val1 5 10 15Gly Leu Ser Ala Thr Pro Ala Pro Lys Ser Cys Pro Glu
Arg His Tyr 20 25 30Trp Ala Gln Gly Lys Leu Cys Cys Gln Met Cys Glu
Pro Gly Thr Phe 35 40 45Leu Val Lys Asp Cys Asp Gln His Arg Lys Ala
Ala Gln Cys Asp Pro 50 55 60Cys Ile Pro Gly Val Ser Phe Ser Pro Asp
His His Thr Arg Pro His65 70 75 80Cys Glu Ser Cys Arg His Cys Asn
Ser Gly Leu Leu Val Arg Asn Cys 85 90 95Thr Ile Thr Ala Asn Ala Glu
Cys Ala Cys Arg Asn Gly Trp Gln Cys 100 105 110Arg Asp Lys Glu Cys
Thr Glu Cys Asp Pro Leu Pro Asn Pro Ser Leu 115 120 125Thr Ala Arg
Ser Ser Gln Ala Leu Ser Pro His Pro Gln Pro Thr His 130 135 140Leu
Pro Tyr Val Ser Glu Met Leu Glu Ala Arg Thr Ala Gly His Met145 150
155 160Gln Thr Leu Ala Asp Phe Arg Gln Leu Pro Ala Arg Thr Leu Ser
Thr 165 170 175His Trp Pro Pro Gln Arg Ser Leu Cys Ser Ser Asp Phe
Ile Arg Ile 180 185 190Leu Val Ile Phe Ser Gly Met Phe Leu Val Phe
Thr Leu Ala Gly Ala 195 200 205Leu Phe Leu His Gln Arg Arg Lys Tyr
Arg Ser Asn Lys Gly Glu Ser 210 215 220Pro Val Glu
Pro Ala Glu Pro Cys His Tyr Ser Cys Pro Arg Glu Glu225 230 235
240Glu Gly Ser Thr Ile Pro Ile Gln Glu Asp Tyr Arg Lys Pro Glu Pro
245 250 255Ala Cys Ser Pro 26034261PRTHomo sapiens 34Met Ile Glu
Thr Tyr Asn Gln Thr Ser Pro Arg Ser Ala Ala Thr Gly1 5 10 15Leu Pro
Ile Ser Met Lys Ile Phe Met Tyr Leu Leu Thr Val Phe Leu 20 25 30Ile
Thr Gln Met Ile Gly Ser Ala Leu Phe Ala Val Tyr Leu His Arg 35 40
45Arg Leu Asp Lys Ile Glu Asp Glu Arg Asn Leu His Glu Asp Phe Val
50 55 60Phe Met Lys Thr Ile Gln Arg Cys Asn Thr Gly Glu Arg Ser Leu
Ser65 70 75 80Leu Leu Asn Cys Glu Glu Ile Lys Ser Gln Phe Glu Gly
Phe Val Lys 85 90 95Asp Ile Met Leu Asn Lys Glu Glu Thr Lys Lys Glu
Asn Ser Phe Glu 100 105 110Met Gln Lys Gly Asp Gln Asn Pro Gln Ile
Ala Ala His Val Ile Ser 115 120 125Glu Ala Ser Ser Lys Thr Thr Ser
Val Leu Gln Trp Ala Glu Lys Gly 130 135 140Tyr Tyr Thr Met Ser Asn
Asn Leu Val Thr Leu Glu Asn Gly Lys Gln145 150 155 160Leu Thr Val
Lys Arg Gln Gly Leu Tyr Tyr Ile Tyr Ala Gln Val Thr 165 170 175Phe
Cys Ser Asn Arg Glu Ala Ser Ser Gln Ala Pro Phe Ile Ala Ser 180 185
190Leu Cys Leu Lys Ser Pro Gly Arg Phe Glu Arg Ile Leu Leu Arg Ala
195 200 205Ala Asn Thr His Ser Ser Ala Lys Pro Cys Gly Gln Gln Ser
Ile His 210 215 220Leu Gly Gly Val Phe Glu Leu Gln Pro Gly Ala Ser
Val Phe Val Asn225 230 235 240Val Thr Asp Pro Ser Gln Val Ser His
Gly Thr Gly Phe Thr Ser Phe 245 250 255Gly Leu Leu Lys Leu
260351224DNAArtificial SequenceSynthetic Hook sequence 35atgcaccgga
ggagatcacg ctcttgtagg gaggaccaga aacctgtcac cggtgaccct 60agcaaagact
caaaagctca ggtgtccgct gccgaggctg gcattactgg aacatggtac
120aatcagctcg ggagcacctt tattgtgact gctggagccg atggagccct
caccggaaca 180tacgaatctg ctgtgggaaa cgccgaatca cggtacgtcc
tcactggccg atacgatagt 240gcccctgcca ccgacggatc tgggactgcc
ctgggatgga ctgtcgcttg gaaaaacaac 300taccggaatg ctcattctgc
cacaacatgg agtggacagt acgtgggagg cgctgaggct 360agaatcaata
cacagtggct gctcacatct ggcacaaccg aggcaaatgc ttggaaatcc
420accctggtgg gacatgacac attcaccaaa gtgaaaccct ccgccgcttc
aatcgatgcc 480gccaaaaaag ccggagtcaa caacggcaat cctctggatg
ccgtccagca ggtcgactat 540ccgtacgacg taccagacta cgcagtcgga
ccgatggacg atcagaggga cctcattagc 600aacaacgaac agctgcctat
gctgggacgg cgacctggag cccctgaatc caaatgctct 660aggggagcac
tgtacactgg cttctccatt ctcgtgacac tgctgctggc cgggcaggct
720actactgctt acttcctgta ccagcagcag gggcggctgg acaaactcac
tgtgacatct 780cagaacctcc agctggaaaa tctgaggatg aaactgccca
aaccccctaa acccgtgtcc 840aaaatgagga tggccacacc tctgctcatg
caggcactgc caatgggagc cctgccccag 900gggcccatgc agaatgccac
caagtatggc aacatgacag aggaccatgt gatgcacctg 960ctccagaatg
ctgaccccct gaaggtgtac ccgccactga aggggagctt cccggagaac
1020ctgagacacc ttaagaacac catggagacc atagactgga aggtctttga
gagctggatg 1080caccattggc tcctgtttga aatgagcagg cactccttgg
agcaaaagcc cactgacgct 1140ccaccgaaag agtcactgga actggaggac
ccgtcttctg ggctgggtgt gaccaagcag 1200gatctgggcc cagtccccat gtga
122436477DNAArtificial SequenceSynthetic Hook sequence 36gaccctagca
aagactcaaa agctcaggtg tccgctgccg aggctggcat tactggaaca 60tggtacaatc
agctcgggag cacctttatt gtgactgctg gagccgatgg agccctcacc
120ggaacatacg aatctgctgt gggaaacgcc gaatcacggt acgtcctcac
tggccgatac 180gatagtgccc ctgccaccga cggatctggg actgccctgg
gatggactgt cgcttggaaa 240aacaactacc ggaatgctca ttctgccaca
acatggagtg gacagtacgt gggaggcgct 300gaggctagaa tcaatacaca
gtggctgctc acatctggca caaccgaggc aaatgcttgg 360aaatccaccc
tggtgggaca tgacacattc accaaagtga aaccctccgc cgcttcaatc
420gatgccgcca aaaaagccgg agtcaacaac ggcaatcctc tggatgccgt ccagcag
4773727DNAArtificial SequenceSynthetic HA TAG 37tatccgtacg
acgtaccaga ctacgca 27381216DNAHomo sapiens 38ggcctccgcg ccgggttttg
ggcctcccgc gggcgccccc ctcctcacgg cgagcgctgc 60cacgtcagac gaagggcgca
gcgagcgtcc tgatccttcc gcccggacgc tcaggacagc 120ggcccgctgc
tcataagact cggccttaga accccagtat cagcagaagg acattttagg
180acgggacttg ggtgactcta gggcactggt tttctttcca gagagcggaa
caggcgagga 240aaagtagtcc cttctcggcg attctgcgga gggatctccg
tggggcggtg aacgccgatg 300attatataag gacgcgccgg gtgtggcaca
gctagttccg tcgcagccgg gatttgggtc 360gcggttcttg tttgtggatc
gctgtgatcg tcacttggtg agtagcgggc tgctgggctg 420gccggggctt
tcgtggccgc cgggccgctc ggtgggacgg aagcgtgtgg agagaccgcc
480aagggctgta gtctgggtcc gcgagcaagg ttgccctgaa ctgggggttg
gggggagcgc 540agcaaaatgg cggctgttcc cgagtcttga atggaagacg
cttgtgaggc gggctgtgag 600gtcgttgaaa caaggtgggg ggcatggtgg
gcggcaagaa cccaaggtct tgaggccttc 660gctaatgcgg gaaagctctt
attcgggtga gatgggctgg ggcaccatct ggggaccctg 720acgtgaagtt
tgtcactgac tggagaactc ggtttgtcgt ctgttgcggg ggcggcagtt
780atggcggtgc cgttgggcag tgcacccgta cctttgggag cgcgcgccct
cgtcgtgtcg 840tgacgtcacc cgttctgttg gcttataatg cagggtgggg
ccacctgccg gtaggtgtgc 900ggtaggcttt tctccgtcgc aggacgcagg
gttcgggcct agggtaggct ctcctgaatc 960gacaggcgcc ggacctctgg
tgaggggagg gataagtgag gcgtcagttt ctttggtcgg 1020ttttatgtac
ctatcttctt aagtagctga agctccggtt ttgaactatg cgctcggggt
1080tggcgagtgt gttttgtgaa gttttttagg caccttttga aatgtaatca
tttgggtcaa 1140tatgtaattt tcagtgttag actagtaaat tgtccgctaa
attctggccg tttttggctt 1200ttttgttaga ccgatc 1216391518DNAArtificial
SequenceSynthetic CAR 39atggccctgc ctgtgacagc cctgctgctg cccctggctc
tcctgctgca tgccgccaga 60cccgctagcg acatccagat gacccagacc accagcagcc
tgagcgccag cctgggcgac 120agagtgacca tcagctgccg ggccagccag
gacatcagca agtacctgaa ctggtatcag 180cagaaacccg acggcaccgt
gaagctgctg atctaccaca ccagccggct ccacagcggc 240gtgcccagca
gattttctgg cagcggcagc ggcaccgact acagcctgac catctccaac
300ctggaacagg aagatatcgc tacctacttc tgtcagcaag gcaacaccct
gccctacacc 360ttcggcggag gcaccaagct ggaaatcacc ggcggaggcg
gaagtggagg tggaggatct 420ggcggcggag gctccgaagt gaagctgcag
gaaagcggcc ctggcctcgt ggcccctagc 480cagagcctgt ccgtgacctg
taccgtgtcc ggcgtgtccc tgcccgacta cggcgtgtcc 540tggatcagac
agcctcccag aaagggcctg gaatggctgg gcgtgatctg gggcagcgag
600acaacctact acaacagcgc cctgaagtcc cggctgacca tcatcaagga
caacagcaag 660agccaggtgt tcctgaagat gaacagcctg cagaccgacg
acaccgccat ctactactgc 720gccaagcact actactacgg cggcagctac
gccatggact actggggcca gggcaccagc 780gtgaccgtgt ccagccatat
ggccctgagc aacagcatca tgtacttcag ccacttcgtg 840cccgtgtttc
tgcccgccaa gcccaccacc acccctgccc ctagacctcc caccccagcc
900ccaacaatcg ccagccagcc tctgtccctg cggcccgaag cctgtagacc
tgctgccggc 960ggagccgtgc acaccagagg cctggatatc tacatctggg
cccctctggc cggcacctgt 1020ggcgtgctgc tgctgagcct ggtgatcaca
aagcggggca gaaagaagct gctgtacatc 1080ttcaagcagc cattcatgcg
gcccgtgcag accacccagg aagaggacgg ctgcagctgc 1140cggttccccg
aggaagagga aggcggctgc gaactgccca agctgtgcta cctgctggac
1200ggcatcctgt tcatctatgg cgtgatcctg accgccctgt tcctgagagt
gaagttcagc 1260agaagcgccg acgcccctgc ctaccagcag ggccagaacc
agctgtacaa cgagctgaac 1320ctgggcagac gggaagagta cgacgtgctg
gacaagcgga gaggccggga ccctgagatg 1380ggcggcaagc cccagcggcg
gaagaaccct caggaaggcc tgtataacga actgcagaaa 1440gacaagatgg
ccgaggccta cagcgagatc ggcatgaagg gcgagcggcg gagaggcaag
1500ggccacgatg gcctgtac 1518401641DNAArtificial SequenceSynthetic
CAR 40atggccctgc ctgtgacagc cctgctgctg cccctcgctc tgctgctgca
tgccgccaga 60cccgctagcg acatccagat gacccagacc accagcagcc tgagcgccag
cctgggcgac 120agagtgacca tcagctgccg ggccagccag gacatcagca
agtacctgaa ctggtatcag 180cagaaacccg acggcaccgt gaagctgctg
atctaccaca ccagccggct ccacagcggc 240gtgcccagca gattttctgg
cagcggcagc ggcaccgact acagcctgac catctccaac 300ctggaacagg
aagatatcgc tacctacttc tgtcagcaag gcaacaccct gccctacacc
360ttcggcggag gcaccaagct ggaaatcacc ggcggaggcg gaagtggagg
gggaggatct 420ggcggcggag gctccgaagt gaagctgcag gaaagcggcc
ctggcctggt ggcccctagc 480cagagcctgt ccgtgacctg taccgtgtcc
ggcgtgtccc tgcccgacta cggcgtgtcc 540tggatcagac agccccccag
aaagggcctg gaatggctgg gcgtgatctg gggcagcgag 600acaacctact
acaacagcgc cctgaagtcc cggctgacca tcatcaagga caacagcaag
660agccaggtgt tcctgaagat gaacagcctg cagaccgacg acaccgccat
ctactactgc 720gccaagcact actactacgg cggcagctac gccatggact
actggggcca gggcaccagc 780gtgaccgtgt ccagccatat ggccctgagc
aacagcatca tgtacttcag ccacttcgtg 840cccgtgtttc tgcccgccaa
gcccaccacc acccctgccc ctagacctcc caccccagcc 900ccaacaatcg
ccagccagcc tctgtccctg aggcccgaag cctgtagacc tgctgccggc
960ggagccgtgc acaccagagg cctggatatc tacatctggg cccctctggc
cggcacctgt 1020ggcgtgctgc tgctgagcct ggtgatcacc cggtccaagc
ggagcagact gctgcactcc 1080gactacatga acatgacccc cagacggcct
ggccccaccc ggaagcacta ccagccttac 1140gcccctcccc gggacttcgc
cgcctacaga agcaagcggg gcagaaagaa gctgctgtac 1200atcttcaagc
agcccttcat gcggcccgtg cagaccaccc aggaagagga cggctgcagc
1260tgccggttcc ccgaggaaga ggaaggcggc tgcgaactgc ccaagctgtg
ctacctgctg 1320gacggcatcc tgttcatcta tggcgtgatc ctgaccgccc
tgttcctgag agtgaagttc 1380agcagaagcg ccgacgcccc tgcctaccag
cagggccaga accagctgta caacgagctg 1440aacctgggca gacgggaaga
gtacgacgtg ctggacaagc gcagaggccg ggaccctgag 1500atgggcggca
agcctcagcg gcggaagaac cctcaggaag gcctgtataa cgaactgcag
1560aaagacaaga tggccgaggc ctacagcgag atcggcatga agggcgagcg
gcggagaggc 1620aagggccacg atggcctgta c 164141300DNAHomo sapiens
41gtctagaagt cagattgggg ttaaagagtc tgtccgtgat tgactaacag tcttaaatac
60ttgatttgtt gttgttgttg tcctgtttgt ttaagaactt tacttcttta tccaatgaac
120ggagtatctt gtgtcctgga ccctttgcaa gaacccttcc cctagcaaca
gatgcgtcat 180ctcaaaatat ttttctgatt ggccaaagag taattgattt
gcattttaat ggtcagactc 240tattacaccc cacattctct tttcttttat
tcttgtctgt tctgcctcac tcccgagctc 30042407PRTArtificial
SequenceSynthetic Hook peptide 42Met His Arg Arg Arg Ser Arg Ser
Cys Arg Glu Asp Gln Lys Pro Val1 5 10 15Thr Gly Asp Pro Ser Lys Asp
Ser Lys Ala Gln Val Ser Ala Ala Glu 20 25 30Ala Gly Ile Thr Gly Thr
Trp Tyr Asn Gln Leu Gly Ser Thr Phe Ile 35 40 45Val Thr Ala Gly Ala
Asp Gly Ala Leu Thr Gly Thr Tyr Glu Ser Ala 50 55 60Val Gly Asn Ala
Glu Ser Arg Tyr Val Leu Thr Gly Arg Tyr Asp Ser65 70 75 80Ala Pro
Ala Thr Asp Gly Ser Gly Thr Ala Leu Gly Trp Thr Val Ala 85 90 95Trp
Lys Asn Asn Tyr Arg Asn Ala His Ser Ala Thr Thr Trp Ser Gly 100 105
110Gln Tyr Val Gly Gly Ala Glu Ala Arg Ile Asn Thr Gln Trp Leu Leu
115 120 125Thr Ser Gly Thr Thr Glu Ala Asn Ala Trp Lys Ser Thr Leu
Val Gly 130 135 140His Asp Thr Phe Thr Lys Val Lys Pro Ser Ala Ala
Ser Ile Asp Ala145 150 155 160Ala Lys Lys Ala Gly Val Asn Asn Gly
Asn Pro Leu Asp Ala Val Gln 165 170 175Gln Val Asp Tyr Pro Tyr Asp
Val Pro Asp Tyr Ala Val Gly Pro Met 180 185 190Asp Asp Gln Arg Asp
Leu Ile Ser Asn Asn Glu Gln Leu Pro Met Leu 195 200 205Gly Arg Arg
Pro Gly Ala Pro Glu Ser Lys Cys Ser Arg Gly Ala Leu 210 215 220Tyr
Thr Gly Phe Ser Ile Leu Val Thr Leu Leu Leu Ala Gly Gln Ala225 230
235 240Thr Thr Ala Tyr Phe Leu Tyr Gln Gln Gln Gly Arg Leu Asp Lys
Leu 245 250 255Thr Val Thr Ser Gln Asn Leu Gln Leu Glu Asn Leu Arg
Met Lys Leu 260 265 270Pro Lys Pro Pro Lys Pro Val Ser Lys Met Arg
Met Ala Thr Pro Leu 275 280 285Leu Met Gln Ala Leu Pro Met Gly Ala
Leu Pro Gln Gly Pro Met Gln 290 295 300Asn Ala Thr Lys Tyr Gly Asn
Met Thr Glu Asp His Val Met His Leu305 310 315 320Leu Gln Asn Ala
Asp Pro Leu Lys Val Tyr Pro Pro Leu Lys Gly Ser 325 330 335Phe Pro
Glu Asn Leu Arg His Leu Lys Asn Thr Met Glu Thr Ile Asp 340 345
350Trp Lys Val Phe Glu Ser Trp Met His His Trp Leu Leu Phe Glu Met
355 360 365Ser Arg His Ser Leu Glu Gln Lys Pro Thr Asp Ala Pro Pro
Lys Glu 370 375 380Ser Leu Glu Leu Glu Asp Pro Ser Ser Gly Leu Gly
Val Thr Lys Gln385 390 395 400Asp Leu Gly Pro Val Pro Met
405431224DNAArtificial SequenceSynthetic Hook sequence 43atgcaccgga
ggagatcacg ctcttgtagg gaggaccaga aacctgtcac cggtgaccct 60agcaaagact
caaaagctca ggtgtccgct gccgaggctg gcattactgg aacatggtac
120aatcagctcg ggagcacctt tattgtgact gctggagccg atggagccct
caccggaaca 180tacgaatctg ctgtgggaaa cgccgaatca cggtacgtcc
tcactggccg atacgatagt 240gcccctgcca ccgacggatc tgggactgcc
ctgggatgga ctgtcgcttg gaaaaacaac 300taccggaatg ctcattctgc
cacaacatgg agtggacagt acgtgggagg cgctgaggct 360agaatcaata
cacagtggct gctcacatct ggcacaaccg aggcaaatgc ttggaaatcc
420accctggtgg gacatgacac attcaccaaa gtgaaaccct ccgccgcttc
aatcgatgcc 480gccaaaaaag ccggagtcaa caacggcaat cctctggatg
ccgtccagca ggtcgactat 540ccgtacgacg taccagacta cgcagtcgga
ccgatggacg atcagaggga cctcattagc 600aacaacgaac agctgcctat
gctgggacgg cgacctggag cccctgaatc caaatgctct 660aggggagcac
tgtacactgg cttctccatt ctcgtgacac tgctgctggc cgggcaggct
720actactgctt acttcctgta ccagcagcag gggcggctgg acaaactcac
tgtgacatct 780cagaacctcc agctggaaaa tctgaggatg aaactgccca
aaccccctaa acccgtgtcc 840aaaatgagga tggccacacc tctgctcatg
caggcactgc caatgggagc cctgccccag 900gggcccatgc agaatgccac
caagtatggc aacatgacag aggaccatgt gatgcacctg 960ctccagaatg
ctgaccccct gaaggtgtac ccgccactga aggggagctt cccggagaac
1020ctgagacacc ttaagaacac catggagacc atagactgga aggtctttga
gagctggatg 1080caccattggc tcctgtttga aatgagcagg cactccttgg
agcaaaagcc cactgacgct 1140ccaccgaaag agtcactgga actggaggac
ccgtcttctg ggctgggtgt gaccaagcag 1200gatctgggcc cagtccccat gtga
122444574DNAArtificial SequenceSynthetic IRES 44gcccctctcc
ctcccccccc cctaacgtta ctggccgaag ccgcttggaa taaggccggt 60gtgcgtttgt
ctatatgtta ttttccacca tattgccgtc ttttggcaat gtgagggccc
120ggaaacctgg ccctgtcttc ttgacgagca ttcctagggg tctttcccct
ctcgccaaag 180gaatgcaagg tctgttgaat gtcgtgaagg aagcagttcc
tctggaagct tcttgaagac 240aaacaacgtc tgtagcgacc ctttgcaggc
agcggaaccc cccacctggc gacaggtgcc 300tctgcggcca aaagccacgt
gtataagata cacctgcaaa ggcggcacaa ccccagtgcc 360acgttgtgag
ttggatagtt gtggaaagag tcaaatggct ctcctcaagc gtattcaaca
420aggggctgaa ggatgcccag aaggtacgcc attgtatggg atctgatctg
gggcctcggt 480gcacatgctt tacatgtgtt tagtcgaggt taaaaaacgt
ctaggccccc cgaaccacgg 540ggacgtggtt ttcctttgaa aaacacgatg ataa
574451638DNAArtificial SequenceSynthetic CAR 45atggccctgc
ctgtgacagc cctgctgctg cccctggctc tcctgctgca tgccgccaga 60cccgctagcg
acatccagat gacccagacc accagcagcc tgagcgccag cctgggcgac
120agagtgacca tcagctgccg ggccagccag gacatcagca agtacctgaa
ctggtatcag 180cagaaacccg acggcaccgt gaagctgctg atctaccaca
ccagccggct ccacagcggc 240gtgcccagca gattttctgg cagcggcagc
ggcaccgact acagcctgac catctccaac 300ctggaacagg aagatatcgc
tacctacttc tgtcagcaag gcaacaccct gccctacacc 360ttcggcggag
gcaccaagct ggaaatcacc ggcggaggcg gaagtggagg tggaggatct
420ggcggcggag gctccgaagt gaagctgcag gaaagcggcc ctggcctcgt
ggcccctagc 480cagagcctgt ccgtgacctg taccgtgtcc ggcgtgtccc
tgcccgacta cggcgtgtcc 540tggatcagac agcctcccag aaagggcctg
gaatggctgg gcgtgatctg gggcagcgag 600acaacctact acaacagcgc
cctgaagtcc cggctgacca tcatcaagga caacagcaag 660agccaggtgt
tcctgaagat gaacagcctg cagaccgacg acaccgccat ctactactgc
720gccaagcact actactacgg cggcagctac gccatggact actggggcca
gggcaccagc 780gtgaccgtgt ccagccatat ggccctgagc aacagcatca
tgtacttcag ccacttcgtg 840cccgtgtttc tgcccgccaa gcccaccacc
acccctgccc ctagacctcc caccccagcc 900ccaacaatcg ccagccagcc
tctgtccctg cggcccgaag cctgtagacc tgctgccggc 960ggagccgtgc
acaccagagg cctggatatc tacatctggg cccctctggc cggcacctgt
1020ggcgtgctgc tgctgagcct ggtgatcacc accggtatgg acgagaaaac
caccggctgg 1080cggggaggcc acgtggtgga aggactggcc ggcgagctgg
aacagctgcg ggccagactg 1140gaacaccacc cccagggcca gagagagccc
aagcggggca gaaagaagct gctgtacatc 1200ttcaagcagc ccttcatgcg
gcccgtgcag accacccagg aagaggacgg ctgcagctgc 1260cggttccccg
aggaagagga aggcggctgc gaactgccca agctgtgcta cctgctggac
1320ggcatcctgt tcatctacgg cgtgatcctg accgccctgt tcctgagagt
gaagttcagc 1380agaagcgccg acgcccctgc ctaccagcag ggccagaacc
agctgtacaa cgagctgaac 1440ctgggcagac gggaagagta cgacgtgctg
gacaagcgga gaggccggga ccctgagatg 1500ggcggcaagc cccagcggcg
gaagaacccc caggaaggcc tgtataacga actgcagaaa 1560gacaagatgg
ccgaggccta cagcgagatc ggcatgaagg gcgagcggcg gagaggcaag
1620ggccacgatg gcctgtac 163846546PRTArtificial SequenceSynthetic
CAR 46Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu
Leu1 5 10 15His Ala Ala Arg Pro Ala Ser Asp Ile Gln Met Thr Gln Thr
Thr Ser
20 25 30Ser Leu Ser Ala Ser Leu Gly Asp Arg Val Thr Ile Ser Cys Arg
Ala 35 40 45Ser Gln Asp Ile Ser Lys Tyr Leu Asn Trp Tyr Gln Gln Lys
Pro Asp 50 55 60Gly Thr Val Lys Leu Leu Ile Tyr His Thr Ser Arg Leu
His Ser Gly65 70 75 80Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly
Thr Asp Tyr Ser Leu 85 90 95Thr Ile Ser Asn Leu Glu Gln Glu Asp Ile
Ala Thr Tyr Phe Cys Gln 100 105 110Gln Gly Asn Thr Leu Pro Tyr Thr
Phe Gly Gly Gly Thr Lys Leu Glu 115 120 125Ile Thr Gly Gly Gly Gly
Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 130 135 140Ser Glu Val Lys
Leu Gln Glu Ser Gly Pro Gly Leu Val Ala Pro Ser145 150 155 160Gln
Ser Leu Ser Val Thr Cys Thr Val Ser Gly Val Ser Leu Pro Asp 165 170
175Tyr Gly Val Ser Trp Ile Arg Gln Pro Pro Arg Lys Gly Leu Glu Trp
180 185 190Leu Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser
Ala Leu 195 200 205Lys Ser Arg Leu Thr Ile Ile Lys Asp Asn Ser Lys
Ser Gln Val Phe 210 215 220Leu Lys Met Asn Ser Leu Gln Thr Asp Asp
Thr Ala Ile Tyr Tyr Cys225 230 235 240Ala Lys His Tyr Tyr Tyr Gly
Gly Ser Tyr Ala Met Asp Tyr Trp Gly 245 250 255Gln Gly Thr Ser Val
Thr Val Ser Ser His Met Ala Leu Ser Asn Ser 260 265 270Ile Met Tyr
Phe Ser His Phe Val Pro Val Phe Leu Pro Ala Lys Pro 275 280 285Thr
Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala 290 295
300Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala
Gly305 310 315 320Gly Ala Val His Thr Arg Gly Leu Asp Ile Tyr Ile
Trp Ala Pro Leu 325 330 335Ala Gly Thr Cys Gly Val Leu Leu Leu Ser
Leu Val Ile Thr Thr Gly 340 345 350Met Asp Glu Lys Thr Thr Gly Trp
Arg Gly Gly His Val Val Glu Gly 355 360 365Leu Ala Gly Glu Leu Glu
Gln Leu Arg Ala Arg Leu Glu His His Pro 370 375 380Gln Gly Gln Arg
Glu Pro Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile385 390 395 400Phe
Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp 405 410
415Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu
420 425 430Pro Lys Leu Cys Tyr Leu Leu Asp Gly Ile Leu Phe Ile Tyr
Gly Val 435 440 445Ile Leu Thr Ala Leu Phe Leu Arg Val Lys Phe Ser
Arg Ser Ala Asp 450 455 460Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln
Leu Tyr Asn Glu Leu Asn465 470 475 480Leu Gly Arg Arg Glu Glu Tyr
Asp Val Leu Asp Lys Arg Arg Gly Arg 485 490 495Asp Pro Glu Met Gly
Gly Lys Pro Gln Arg Arg Lys Asn Pro Gln Glu 500 505 510Gly Leu Tyr
Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser 515 520 525Glu
Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly 530 535
540Leu Tyr545471638DNAArtificial SequenceSynthetic CAR 47atggccctgc
ctgtgacagc cctgctgctg cccctggctc tcctgctgca tgccgccaga 60cccgctagcg
acatccagat gacccagacc accagcagcc tgagcgccag cctgggcgac
120agagtgacca tcagctgccg ggccagccag gacatcagca agtacctgaa
ctggtatcag 180cagaaacccg acggcaccgt gaagctgctg atctaccaca
ccagccggct ccacagcggc 240gtgcccagca gattttctgg cagcggcagc
ggcaccgact acagcctgac catctccaac 300ctggaacagg aagatatcgc
tacctacttc tgtcagcaag gcaacaccct gccctacacc 360ttcggcggag
gcaccaagct ggaaatcacc ggcggaggcg gaagtggagg tggaggatct
420ggcggcggag gctccgaagt gaagctgcag gaaagcggcc ctggcctcgt
ggcccctagc 480cagagcctgt ccgtgacctg taccgtgtcc ggcgtgtccc
tgcccgacta cggcgtgtcc 540tggatcagac agcctcccag aaagggcctg
gaatggctgg gcgtgatctg gggcagcgag 600acaacctact acaacagcgc
cctgaagtcc cggctgacca tcatcaagga caacagcaag 660agccaggtgt
tcctgaagat gaacagcctg cagaccgacg acaccgccat ctactactgc
720gccaagcact actactacgg cggcagctac gccatggact actggggcca
gggcaccagc 780gtgaccgtgt ccagccatat ggccctgagc aacagcatca
tgtacttcag ccacttcgtg 840cccgtgtttc tgcccgccaa gcccaccacc
acccctgccc ctagacctcc caccccagcc 900ccaacaatcg ccagccagcc
tctgtccctg cggcccgaag cctgtagacc tgctgccggc 960ggagccgtgc
acaccagagg cctggatatc tacatctggg cccctctggc cggcacctgt
1020ggcgtgctgc tgctgagcct ggtgatcaca aagcggggca gaaagaagct
gctgtacatc 1080ttcaagcagc ccttcatgcg gcccgtgcag accacccagg
aagaggacgg ctgcagctgc 1140cggttccccg aggaagagga aggcggctgc
gagctgaccg gtatggacga gaaaaccacc 1200ggctggcggg gaggccacgt
ggtggaagga ctggccggcg agctggaaca gctgcgggcc 1260agactggaac
accaccccca gggccagagg gaacccccca agctgtgcta cctgctggac
1320ggcatcctgt tcatctacgg cgtgatcctg accgccctgt tcctgagagt
gaagttcagc 1380agaagcgccg acgcccctgc ctaccagcag ggccagaacc
agctgtacaa cgagctgaac 1440ctgggcagac gggaagagta cgacgtgctg
gacaagcgga gaggccggga ccctgagatg 1500ggcggcaagc cccagcggcg
gaagaacccc caggaaggcc tgtataacga actgcagaaa 1560gacaagatgg
ccgaggccta cagcgagatc ggcatgaagg gcgagcggcg gagaggcaag
1620ggccacgatg gcctgtac 163848546PRTArtificial SequenceSynthetic
CAR 48Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu
Leu1 5 10 15His Ala Ala Arg Pro Ala Ser Asp Ile Gln Met Thr Gln Thr
Thr Ser 20 25 30Ser Leu Ser Ala Ser Leu Gly Asp Arg Val Thr Ile Ser
Cys Arg Ala 35 40 45Ser Gln Asp Ile Ser Lys Tyr Leu Asn Trp Tyr Gln
Gln Lys Pro Asp 50 55 60Gly Thr Val Lys Leu Leu Ile Tyr His Thr Ser
Arg Leu His Ser Gly65 70 75 80Val Pro Ser Arg Phe Ser Gly Ser Gly
Ser Gly Thr Asp Tyr Ser Leu 85 90 95Thr Ile Ser Asn Leu Glu Gln Glu
Asp Ile Ala Thr Tyr Phe Cys Gln 100 105 110Gln Gly Asn Thr Leu Pro
Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu 115 120 125Ile Thr Gly Gly
Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 130 135 140Ser Glu
Val Lys Leu Gln Glu Ser Gly Pro Gly Leu Val Ala Pro Ser145 150 155
160Gln Ser Leu Ser Val Thr Cys Thr Val Ser Gly Val Ser Leu Pro Asp
165 170 175Tyr Gly Val Ser Trp Ile Arg Gln Pro Pro Arg Lys Gly Leu
Glu Trp 180 185 190Leu Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr
Asn Ser Ala Leu 195 200 205Lys Ser Arg Leu Thr Ile Ile Lys Asp Asn
Ser Lys Ser Gln Val Phe 210 215 220Leu Lys Met Asn Ser Leu Gln Thr
Asp Asp Thr Ala Ile Tyr Tyr Cys225 230 235 240Ala Lys His Tyr Tyr
Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly 245 250 255Gln Gly Thr
Ser Val Thr Val Ser Ser His Met Ala Leu Ser Asn Ser 260 265 270Ile
Met Tyr Phe Ser His Phe Val Pro Val Phe Leu Pro Ala Lys Pro 275 280
285Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala
290 295 300Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala
Ala Gly305 310 315 320Gly Ala Val His Thr Arg Gly Leu Asp Ile Tyr
Ile Trp Ala Pro Leu 325 330 335Ala Gly Thr Cys Gly Val Leu Leu Leu
Ser Leu Val Ile Thr Lys Arg 340 345 350Gly Arg Lys Lys Leu Leu Tyr
Ile Phe Lys Gln Pro Phe Met Arg Pro 355 360 365Val Gln Thr Thr Gln
Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu 370 375 380Glu Glu Glu
Gly Gly Cys Glu Leu Thr Gly Met Asp Glu Lys Thr Thr385 390 395
400Gly Trp Arg Gly Gly His Val Val Glu Gly Leu Ala Gly Glu Leu Glu
405 410 415Gln Leu Arg Ala Arg Leu Glu His His Pro Gln Gly Gln Arg
Glu Pro 420 425 430Pro Lys Leu Cys Tyr Leu Leu Asp Gly Ile Leu Phe
Ile Tyr Gly Val 435 440 445Ile Leu Thr Ala Leu Phe Leu Arg Val Lys
Phe Ser Arg Ser Ala Asp 450 455 460Ala Pro Ala Tyr Gln Gln Gly Gln
Asn Gln Leu Tyr Asn Glu Leu Asn465 470 475 480Leu Gly Arg Arg Glu
Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg 485 490 495Asp Pro Glu
Met Gly Gly Lys Pro Gln Arg Arg Lys Asn Pro Gln Glu 500 505 510Gly
Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser 515 520
525Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly
530 535 540Leu Tyr545491638DNAArtificial SequenceSynthetic CAR
49atggccctgc ctgtgacagc cctgctgctg cccctggctc tcctgctgca tgccgccaga
60cccgctagcg acatccagat gacccagacc accagcagcc tgagcgccag cctgggcgac
120agagtgacca tcagctgccg ggccagccag gacatcagca agtacctgaa
ctggtatcag 180cagaaacccg acggcaccgt gaagctgctg atctaccaca
ccagccggct ccacagcggc 240gtgcccagca gattttctgg cagcggcagc
ggcaccgact acagcctgac catctccaac 300ctggaacagg aagatatcgc
tacctacttc tgtcagcaag gcaacaccct gccctacacc 360ttcggcggag
gcaccaagct ggaaatcacc ggcggaggcg gaagtggagg tggaggatct
420ggcggcggag gctccgaagt gaagctgcag gaaagcggcc ctggcctcgt
ggcccctagc 480cagagcctgt ccgtgacctg taccgtgtcc ggcgtgtccc
tgcccgacta cggcgtgtcc 540tggatcagac agcctcccag aaagggcctg
gaatggctgg gcgtgatctg gggcagcgag 600acaacctact acaacagcgc
cctgaagtcc cggctgacca tcatcaagga caacagcaag 660agccaggtgt
tcctgaagat gaacagcctg cagaccgacg acaccgccat ctactactgc
720gccaagcact actactacgg cggcagctac gccatggact actggggcca
gggcaccagc 780gtgaccgtgt ccagccatat ggccctgagc aacagcatca
tgtacttcag ccacttcgtg 840cccgtgtttc tgcccgccaa gcccaccacc
acccctgccc ctagacctcc caccccagcc 900ccaacaatcg ccagccagcc
tctgtccctg cggcccgaag cctgtagacc tgctgccggc 960ggagccgtgc
acaccagagg cctggatatc tacatctggg cccctctggc cggcacctgt
1020ggcgtgctgc tgctgagcct ggtgatcaca aagcggggca gaaagaagct
gctgtacatc 1080ttcaagcagc ccttcatgcg gcccgtgcag accacccagg
aagaggacgg ctgcagctgc 1140cggttccccg aggaagagga aggcggctgc
gaactgccca agctgtgcta cctgctggac 1200ggcatcctgt tcatctacgg
cgtgatcctg accgccctgt tcctgagagt gaagttcagc 1260agaagcgccg
acgcccctgc ctaccagcag ggccagaacc agctgtacaa cgagctgaac
1320ctgggcagac gggaagagta cgacgtgctg gacaagcgga gaggccggga
ccctgagatg 1380ggcggcaagc cccagcggcg gaagaacccc caggaaggcc
tgtataacga actgcagaaa 1440gacaagatgg ccgaggccta cagcgagatc
ggcatgaagg gcgagcggcg gagaggcaag 1500ggccacgatg gcctgtacac
cggtatggac gagaaaacca ccggctggcg gggaggccac 1560gtggtggaag
gactggccgg cgagctggaa cagctgcggg ccagactgga acaccacccc
1620cagggccaga gggaaccc 163850546PRTArtificial SequenceSynthetic
CAR 50Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu
Leu1 5 10 15His Ala Ala Arg Pro Ala Ser Asp Ile Gln Met Thr Gln Thr
Thr Ser 20 25 30Ser Leu Ser Ala Ser Leu Gly Asp Arg Val Thr Ile Ser
Cys Arg Ala 35 40 45Ser Gln Asp Ile Ser Lys Tyr Leu Asn Trp Tyr Gln
Gln Lys Pro Asp 50 55 60Gly Thr Val Lys Leu Leu Ile Tyr His Thr Ser
Arg Leu His Ser Gly65 70 75 80Val Pro Ser Arg Phe Ser Gly Ser Gly
Ser Gly Thr Asp Tyr Ser Leu 85 90 95Thr Ile Ser Asn Leu Glu Gln Glu
Asp Ile Ala Thr Tyr Phe Cys Gln 100 105 110Gln Gly Asn Thr Leu Pro
Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu 115 120 125Ile Thr Gly Gly
Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 130 135 140Ser Glu
Val Lys Leu Gln Glu Ser Gly Pro Gly Leu Val Ala Pro Ser145 150 155
160Gln Ser Leu Ser Val Thr Cys Thr Val Ser Gly Val Ser Leu Pro Asp
165 170 175Tyr Gly Val Ser Trp Ile Arg Gln Pro Pro Arg Lys Gly Leu
Glu Trp 180 185 190Leu Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr
Asn Ser Ala Leu 195 200 205Lys Ser Arg Leu Thr Ile Ile Lys Asp Asn
Ser Lys Ser Gln Val Phe 210 215 220Leu Lys Met Asn Ser Leu Gln Thr
Asp Asp Thr Ala Ile Tyr Tyr Cys225 230 235 240Ala Lys His Tyr Tyr
Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly 245 250 255Gln Gly Thr
Ser Val Thr Val Ser Ser His Met Ala Leu Ser Asn Ser 260 265 270Ile
Met Tyr Phe Ser His Phe Val Pro Val Phe Leu Pro Ala Lys Pro 275 280
285Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala
290 295 300Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala
Ala Gly305 310 315 320Gly Ala Val His Thr Arg Gly Leu Asp Ile Tyr
Ile Trp Ala Pro Leu 325 330 335Ala Gly Thr Cys Gly Val Leu Leu Leu
Ser Leu Val Ile Thr Lys Arg 340 345 350Gly Arg Lys Lys Leu Leu Tyr
Ile Phe Lys Gln Pro Phe Met Arg Pro 355 360 365Val Gln Thr Thr Gln
Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu 370 375 380Glu Glu Glu
Gly Gly Cys Glu Leu Pro Lys Leu Cys Tyr Leu Leu Asp385 390 395
400Gly Ile Leu Phe Ile Tyr Gly Val Ile Leu Thr Ala Leu Phe Leu Arg
405 410 415Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln
Gly Gln 420 425 430Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg
Glu Glu Tyr Asp 435 440 445Val Leu Asp Lys Arg Arg Gly Arg Asp Pro
Glu Met Gly Gly Lys Pro 450 455 460Gln Arg Arg Lys Asn Pro Gln Glu
Gly Leu Tyr Asn Glu Leu Gln Lys465 470 475 480Asp Lys Met Ala Glu
Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg 485 490 495Arg Arg Gly
Lys Gly His Asp Gly Leu Tyr Thr Gly Met Asp Glu Lys 500 505 510Thr
Thr Gly Trp Arg Gly Gly His Val Val Glu Gly Leu Ala Gly Glu 515 520
525Leu Glu Gln Leu Arg Ala Arg Leu Glu His His Pro Gln Gly Gln Arg
530 535 540Glu Pro54551506PRTArtificial SequenceSynthetic CAR 51Met
Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu1 5 10
15His Ala Ala Arg Pro Ala Ser Asp Ile Gln Met Thr Gln Thr Thr Ser
20 25 30Ser Leu Ser Ala Ser Leu Gly Asp Arg Val Thr Ile Ser Cys Arg
Ala 35 40 45Ser Gln Asp Ile Ser Lys Tyr Leu Asn Trp Tyr Gln Gln Lys
Pro Asp 50 55 60Gly Thr Val Lys Leu Leu Ile Tyr His Thr Ser Arg Leu
His Ser Gly65 70 75 80Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly
Thr Asp Tyr Ser Leu 85 90 95Thr Ile Ser Asn Leu Glu Gln Glu Asp Ile
Ala Thr Tyr Phe Cys Gln 100 105 110Gln Gly Asn Thr Leu Pro Tyr Thr
Phe Gly Gly Gly Thr Lys Leu Glu 115 120 125Ile Thr Gly Gly Gly Gly
Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 130 135 140Ser Glu Val Lys
Leu Gln Glu Ser Gly Pro Gly Leu Val Ala Pro Ser145 150 155 160Gln
Ser Leu Ser Val Thr Cys Thr Val Ser Gly Val Ser Leu Pro Asp 165 170
175Tyr Gly Val Ser Trp Ile Arg Gln Pro Pro Arg Lys Gly Leu Glu Trp
180 185 190Leu Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser
Ala Leu 195 200 205Lys Ser Arg Leu Thr Ile Ile Lys Asp Asn Ser Lys
Ser Gln Val Phe 210 215 220Leu Lys Met Asn Ser Leu Gln Thr Asp Asp
Thr Ala Ile Tyr Tyr Cys225 230 235 240Ala Lys His Tyr Tyr Tyr Gly
Gly Ser Tyr Ala Met Asp Tyr Trp Gly 245 250 255Gln Gly Thr Ser Val
Thr Val Ser Ser His Met Ala Leu Ser Asn Ser 260 265 270Ile Met Tyr
Phe Ser His Phe Val Pro Val Phe Leu
Pro Ala Lys Pro 275 280 285Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr
Pro Ala Pro Thr Ile Ala 290 295 300Ser Gln Pro Leu Ser Leu Arg Pro
Glu Ala Cys Arg Pro Ala Ala Gly305 310 315 320Gly Ala Val His Thr
Arg Gly Leu Asp Ile Tyr Ile Trp Ala Pro Leu 325 330 335Ala Gly Thr
Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Lys Arg 340 345 350Gly
Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro 355 360
365Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu
370 375 380Glu Glu Glu Gly Gly Cys Glu Leu Pro Lys Leu Cys Tyr Leu
Leu Asp385 390 395 400Gly Ile Leu Phe Ile Tyr Gly Val Ile Leu Thr
Ala Leu Phe Leu Arg 405 410 415Val Lys Phe Ser Arg Ser Ala Asp Ala
Pro Ala Tyr Gln Gln Gly Gln 420 425 430Asn Gln Leu Tyr Asn Glu Leu
Asn Leu Gly Arg Arg Glu Glu Tyr Asp 435 440 445Val Leu Asp Lys Arg
Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro 450 455 460Gln Arg Arg
Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys465 470 475
480Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg
485 490 495Arg Arg Gly Lys Gly His Asp Gly Leu Tyr 500
5055227DNAArtificial SequenceSynthetic Probe 52aaccattagg
agtagcaccc accaagg 27
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References