NZ714044B2 - Cd19 specific chimeric antigen receptor and uses thereof - Google Patents
Cd19 specific chimeric antigen receptor and uses thereof Download PDFInfo
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- NZ714044B2 NZ714044B2 NZ714044A NZ71404414A NZ714044B2 NZ 714044 B2 NZ714044 B2 NZ 714044B2 NZ 714044 A NZ714044 A NZ 714044A NZ 71404414 A NZ71404414 A NZ 71404414A NZ 714044 B2 NZ714044 B2 NZ 714044B2
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Abstract
The present invention relates to chimeric antigen receptors (CAR). CARs are able to redirect immune cell specificity and reactivity toward a selected target exploiting the ligand-binding domain properties. In particular, the present invention relates to a Chimeric Antigen Receptor in which extracellular ligand binding is a scFV derived from a CD19 monoclonal antibody, preferably 4G7. The present invention also relates to polynucleotides, vectors encoding said CAR and isolated cells expressing said CAR at their surface. The present invention also relates to methods for engineering immune cells expressing 4G7-CAR at their surface which confers a prolonged "activated" state on the transduced cell. The present invention is particularly useful for the treatment of B-cells lymphomas and leukemia. ular ligand binding is a scFV derived from a CD19 monoclonal antibody, preferably 4G7. The present invention also relates to polynucleotides, vectors encoding said CAR and isolated cells expressing said CAR at their surface. The present invention also relates to methods for engineering immune cells expressing 4G7-CAR at their surface which confers a prolonged "activated" state on the transduced cell. The present invention is particularly useful for the treatment of B-cells lymphomas and leukemia.
Description
(12) Granted patent specificaon (19) NZ (11) 714044 (13) B2
(47) Publicaon date: 2021.12.24
(54) CD19 SPECIFIC CHIMERIC ANTIGEN RECEPTOR AND USES THEREOF
(51) Internaonal Patent Classificaon(s):
C12N 9/22 C12N 15/63 C07K 16/28 C07K 19/00 C07K 14/725
(22) Filing date: (73) Owner(s):
2014.05.12 CELLECTIS
(23) Complete specificaon filing date: (74) Contact:
2014.05.12 AJ PARK
(30) Internaonal Priority Data: (72) Inventor(s):
US 2013.05.13 GALETTO, Roman
US 13/892,805 2013.05.13 SMITH, Julianne
US 2013.05.13 SCHARENBERG, Andrew
US 61/888,259 2013.10.08 SCHIFFER-MANNIOUI, Cècile
(86) Internaonal Applicaon No.:
(87) Internaonal Publicaon number:
WO/2014/184143
(57) Abstract:
The present invenon relates to chimeric angen receptors (CAR). CARs are able to redirect
immune cell specificity and reacvity toward a selected target exploing the ligand-binding
domain properes. In parcular, the present invenon relates to a Chimeric Angen Receptor in
which extracellular ligand binding is a scFV derived from a CD19 monoclonal anbody, preferably
4G7. The present invenon also relates to polynucleodes, vectors encoding said CAR and
isolated cells expressing said CAR at their surface. The present invenon also relates to methods
for engineering immune cells expressing 4G7-CAR at their surface which confers a prolonged
"acvated" state on the transduced cell. The present invenon is parcularly useful for the
treatment of B-cells lymphomas and leukemia.
NZ 714044 B2
CD19 specific Chimeric Antigen Receptor and uses thereof
Field of the invention
The present invention generally relates to chimeric antigen receptors (CAR). CARs are able to
redirect immune cell specificity and reactivity toward a selected target exploiting the ligand-
binding domain properties. In particular, the present invention relates to a Chimeric Antigen
Receptor in which extracellular ligand binding is a scFV derived from a CD19 monoclonal
antibody, preferably 4G7. The present invention also relates to polynucleotides, vectors
encoding said CAR and isolated cells expressing said CAR at their surface. The present
invention also relates to methods for engineering immune cells expressing 4G7-CAR at their
surface which confers a prolonged “activated” state on the transduced cell. The CD19
specific chimeric antigen receptors of the present invention are particularly useful for the
treatment of B-cells lymphomas and leukemia.
Background of the invention
Adoptive immunotherapy, which involves the transfer of autologous antigen-specific T cells
generated ex vivo, is a promising strategy to treat viral infections and cancer. The T cells
used for adoptive immunotherapy can be generated either by expansion of antigen-specific
T cells or redirection of T cells through genetic engineering (Park, Rosenberg et al. 2011).
Transfer of viral antigen specific T cells is a well-established procedure used for the
treatment of transplant associated viral infections and rare viral-related malignancies.
Similarly, isolation and transfer of tumor specific T cells has been shown to be successful in
treating melanoma.
Novel specificities in T cells have been successfully generated through the genetic transfer of
transgenic T cell receptors or chimeric antigen receptors (CARs) (Jena, Dotti et al. 2010).
CARs are synthetic receptors consisting of a targeting moiety that is associated with one or
more signaling domains in a single fusion molecule. In general, the binding moiety of a CAR
consists of an antigen-binding domain of a single-chain antibody (scFv), comprising the light
and variable fragments of a monoclonal antibody joined by a flexible linker. Binding moieties
based on receptor or ligand domains have also been used successfully. The signaling
domains for first generation CARs are derived from the cytoplasmic region of the CD3zeta or
the Fc receptor gamma chains. First generation CARs have been shown to successfully
redirect T cell cytotoxicity, however, they failed to provide prolonged expansion and anti-
tumor activity in vivo. Signaling domains from co-stimulatory molecules including CD28, OX-
40 (CD134), and 4-1BB (CD137) have been added alone (second generation) or in
combination (third generation) to enhance survival and increase proliferation of CAR
modified T cells. CARs have successfully allowed T cells to be redirected against antigens
expressed at the surface of tumor cells from various malignancies including lymphomas and
solid tumors (Jena, Dotti et al. 2010).
CD19 is an attractive target for immunotherapy because the vast majority of B-acute
lymphoblastic leukemia (B-ALL) uniformly express CD19, whereas expression is absent on
non hematopoietic cells, as well as myeloid, erythroid, and T cells, and bone marrow stem
cells. Clinical trials targeting CD19 on B-cell malignancies are underway with encouraging
anti-tumor responses. Most infuse T cells genetically modified to express a chimeric antigen
receptor (CAR) with specificity derived from the scFv region of a CD19-specific mouse
monoclonal antibody FMC63 (Nicholson, Lenton et al. 1997; Cooper, Topp et al. 2003;
Cooper, Jena et al. 2012) (International application: WO2013/126712). However, there is still
a need to improve construction of CARs that show better compatibility with T-cell
proliferation, in order to allow the cells expressing such CARs to reach significant clinical
advantage. It is an object of the invention to go at least some way toward meeting this
need; and/or to at least provide the public with a useful choice.
Summary of the invention
In a first aspect, the invention relates to a CD19 specific chimeric antigen receptor
comprising at least one extracellular ligand binding domain, a transmembrane domain and
at least one intracellular signalling domain wherein said extracellular domain comprises a
single chain FV fragment derived from the monoclonal antibody 4G7, specific for CD19, said
single chain FV fragment comprising the variable fragment of the CD19 monoclonal antibody
4G7 immunoglobulin gamma 1 heavy chain of SEQ ID NO: 3 and the variable fragment of the
CD19 monoclonal antibody 4G7 immunoglobulin kappa light chain of SEQ ID NO: 4 or SEQ ID
NO: 5.
In a second aspect, the invention relates to a polynucleotide encoding said chimeric antigen
receptor according to the first aspect.
In a third aspect, the invention relates to an expression vector comprising a polynucleotide
according to the second aspect.
In a fourth aspect, the invention relates to a genetically engineered isolated immune cell
expressing at the cell surface membrane a CD19 specific chimeric antigen receptor
comprising at least one extracellular ligand binding domain and at least one intracellular
signalling domain wherein said extracellular domain comprises a single chain FV fragment
derived from a monoclonal antibody 4G7, specific for CD19, said single chain FV fragment
comprising the variable fragments of the CD19 monoclonal antibody 4G7 immunoglobulin
gamma 1 heavy chain of SEQ ID NO: 3 and the variable fragments of the CD19 monoclonal
antibody 4G7 immunoglobulin kappa light chain of SEQ ID NO: 4 or SEQ ID NO: 5.
In a fifth aspect, the invention relates to a genetically engineered isolated immune cell
expressing at the cell surface membrane a CD19 specific chimeric antigen receptor according
to the first aspect.
In a sixth aspect, the invention relates to use of the genetically engineered isolated immune
cell according to the fourth or fifth aspect in the manufacture of a medicament for the
treatment of a cancer in a patient in need thereof.
In a seventh aspect, the invention relates to an in vitro or ex vivo method of engineering an
immune cell comprising:
(a) Providing an immune cell,
(b) Expressing at the surface of said cell at least one CD19 specific chimeric antigen
receptor according to the first aspect.
Brief description
The inventors have generated a CD19 specific CAR (4G7-CAR) comprising a scFV derived from
the CD19 specific monoclonal antibody, 4G7, and have surprisingly found that introduction
of the resulting 4G7-CAR into primary T cells could confer a prolonged “activated” state on
the transduced cell independently of antigen binding. Following non-specific activation in
vitro (e.g. with anti CD3/CD28 coated beads and recombinant IL2), these cells displayed an
increased cell size (blast formation) as well as the expression of activation markers (CD25)
over an extended time period compared to cells transduced with a similar CAR comprising
the FMC63 scFV. This long-term activation permits extended proliferation and provides an
antigen-independent mechanism for expansion of 4G7-CAR cells in vitro.
The present description thus includes a chimeric antigen receptor comprising at least one
extracellular ligand binding domain, a transmembrane domain and at least one signal
transducing domain, wherein said extracellular ligand binding domain comprises a scFV
derived from specific monoclonal antibody, 4G7. In particular, the CAR of the present
description once transduced into an immune cell contributes to antigen independent
activation and proliferation of the cell. The present description also relates to nucleic acid,
vectors encoding the CAR comprising a scFV derived from the CD19 specific monoclonal
antibody 4G7 and methods of engineering immune cells comprising introducing into said cell
the 4G7 CAR. The present description also relates to genetically modified immune cells
expressing at their surface the 4G7, particularly immune cells which proliferate
independently of antigen mechanism. The genetically modified immune cells of the present
description are particularly useful for therapeutic applications such as B-cell lymphoma or
leukemia treatments.
Brief description of the figures
Figure 1: Proliferation of TCR alpha inactivated T cells (KO) transduced with 4G7-CAR
lentiviral vector compared to non transduced KO T cells (NTD). Proliferation was followed
during 30 days after (IL2+CD28) or not (IL2) a step of reactivation with soluble anti-CD28.
Figure 2: CD25 activation marker expression analysis at the surface of inactivated TCR alpha
T cells transduced with 4G7-CAR lentiviral vector, gated on the basis of 4G7-CAR expression
(CAR+, CAR-) and compared to CD25 expression on TCR alpha positive non electroporated
(NEP) or TCR alpha disrupted but non tranduced (NTD) cells. CD25 expression was analyzed
after (IL2+CD28) or not (IL2) a step of reactivation with soluble anti-CD28.
Figure 3: CAR expression analysis at the surface of T cells transduced with a lentiviral vector
encoding either the 4G7-CAR or the FMC63-CAR. The analysis was done 3, 8 and 15 days post
transduction by flow cytometry. NT refers to no transduced T cells.
Figure 4: CD25 expression analysis at the surface of T cells transduced with a lentiviral vector
encoding either the 4G7-CAR or the FMC63-CAR. The analysis was done 3, 8 and 15 days post
transduction by flow cytometry. NT refers to no transduced T cells.
Figure 5: Size analysis of T cells transduced with a lentiviral vector encoding either the 4G7-CAR
or the FMC63-CAR. The analysis was done 3, 8 and 15 days post transduction by flow cytometry.
NT refers to no transduced T cells.
Figure 6: Proliferation of T cells transduced with 4G7-CAR compared to FMC63 lentiviral
vector. Proliferation was followed during 20 days after (CD28) or not (-) a step of reactivation
with soluble anti-CD28. NTD refers to no transduced T cells.
Detailed description of the invention
Unless specifically defined herein, all technical and scientific terms used have the same
meaning as commonly understood by a skilled artisan in the fields of gene therapy,
biochemistry, genetics, and molecular biology.
All methods and materials similar or equivalent to those described herein can be used in the
practice or testing of the present invention, with suitable methods and materials being
described herein. All publications, patent applications, patents, and other references
mentioned herein are incorporated by reference in their entirety. In case of conflict, the
present specification, including definitions, will prevail. Further, the materials, methods, and
examples are illustrative only and are not intended to be limiting, unless otherwise specified.
The practice of the present invention will employ, unless otherwise indicated, conventional
techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology,
recombinant DNA, and immunology, which are within the skill of the art. Such techniques
are explained fully in the literature. See, for example, Current Protocols in Molecular Biology
(Frederick M. AUSUBEL, 2000, Wiley and son Inc, Library of Congress, USA); Molecular
Cloning: A Laboratory Manual, Third Edition, (Sambrook et al, 2001, Cold Spring Harbor, New
York: Cold Spring Harbor Laboratory Press); Oligonucleotide Synthesis (M. J. Gait ed., 1984);
Mullis et al. U.S. Pat. No. 4,683,195; Nucleic Acid Hybridization (B. D. Harries & S. J. Higgins
eds. 1984); Transcription And Translation (B. D. Hames & S. J. Higgins eds. 1984); Culture Of
Animal Cells (R. I. Freshney, Alan R. Liss, Inc., 1987); Immobilized Cells And Enzymes (IRL
Press, 1986); B. Perbal, A Practical Guide To Molecular Cloning (1984); the series, Methods In
ENZYMOLOGY (J. Abelson and M. Simon, eds.-in-chief, Academic Press, Inc., New York),
specifically, Vols.154 and 155 (Wu et al. eds.) and Vol. 185, "Gene Expression Technology"
(D. Goeddel, ed.); Gene Transfer Vectors For Mammalian Cells (J. H. Miller and M. P. Calos
eds., 1987, Cold Spring Harbor Laboratory); Immunochemical Methods In Cell And Molecular
Biology (Mayer and Walker, eds., Academic Press, London, 1987); Handbook Of Experimental
Immunology, Volumes I-IV (D. M. Weir and C. C. Blackwell, eds., 1986); and Manipulating the
Mouse Embryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986).
CD19 specific Chimeric Antigen Receptor
The present description relates to a chimeric antigen receptor (CAR) comprising an
extracellular ligand-binding domain, a transmembrane domain and a signaling transducing
domain.
The term “extracellular ligand-binding domain” as used herein is defined as an oligo- or
polypeptide that is capable of binding a ligand. Preferably, the domain will be capable of
interacting with a cell surface molecule. For example, the extracellular ligand-binding
domain may be chosen to recognize a ligand that acts as a cell surface marker on target cells
associated with a particular disease state.
In a preferred embodiment, said extracellular ligand-binding domain comprises a single
chain antibody fragment (scFv) comprising the light (V ) and the heavy (V ) variable fragment
of a target antigen specific monoclonal antibody joined by a flexible linker. In a preferred
embodiment, said scFV is derived from the CD19 monoclonal antibody 4G7 (Peipp, Saul et al.
2004), preferably said scFV of the present description comprises a part of the CD19
monoclonal antibody 4G7 immunoglobulin gamma 1 heavy chain (GenBank: CAD88275.1;
SEQ ID NO: 1) and a part of the CD19 monoclonal antibody 4G7 immunoglobulin kappa light
chain (GenBank: CAD88204.1; SEQ ID NO: 2), preferably linked together by a flexible linker.
In a preferred embodiment, said scFV of the present description comprises the variable
fragments of the CD19 monoclonal antibody 4G7 immunoglobulin gamma 1 heavy chain
(SEQ ID NO: 3) and the variable fragments of the CD19 monoclonal antibody 4G7
immunoglobulin kappa light chain (SEQ ID NO: 4 or SEQ ID NO: 5) linked together by a
flexible linker. In particular embodiment said flexible linker has the amino acid sequence
(SEQ ID NO: 6).
In other words, said CAR comprises an extracellular ligand-biding domain which comprises a
single chain FV fragment derived from a CD19 specific monoclonal antibody 4G7. In a
particular embodiment, said scFV comprises a part of amino acid sequences selected from
the group consisting of: SEQ ID NO: 1 to 5. In a preferred embodiment said scFV comprises at
least 70%, preferably at least 80%, more preferably at least 90 %, 95 % 97 % or 99 %
sequence identity with amino acid sequence selected from the group consisting of SEQ ID
NO: 7 and SEQ ID NO: 8.
The signal transducing domain or intracellular signaling domain of the CAR according to the
present invention is responsible for intracellular signaling following the binding of
extracellular ligand binding domain to the target resulting in the activation of the immune
cell and immune response. In other words, the signal transducing domain is responsible for
the activation of at least one of the normal effector functions of the immune cell in which
the CAR is expressed. For example, the effector function of a T cell can be a cytolytic activity
or helper activity including the secretion of cytokines. Thus, the term “signal tansducing
domain” refers to the portion of a protein which transduces the effector signal function
signal and directs the cell to perform a specialized function.
Preferred examples of signal transducing domain for use in a CAR can be the cytoplasmic
sequences of the T cell receptor and co-receptors that act in concert to initiate signal
transduction following antigen receptor engagement, as well as any derivate or variant of
these sequences and any synthetic sequence that has the same functional capability. Signal
transduction domain comprises two distinct classes of cytoplasmic signaling sequence, those
that initiate antigen-dependent primary activation, and those that act in an antigen-
independent manner to provide a secondary or co-stimulatory signal. Primary cytoplasmic
signaling sequence can comprise signaling motifs which are known as immunoreceptor
tyrosine-based activation motifs of ITAMs. ITAMs are well defined signaling motifs found in
the intracytoplasmic tail of a variety of receptors that serve as binding sites for syk/zap70
class tyrosine kinases. Examples of ITAM used in the description can include as non limiting
examples those derived from TCRzeta, FcRgamma, FcRbeta, FcRepsilon, CD3gamma,
CD3delta, CD3epsilon, CD5, CD22, CD79a, CD79b and CD66d. In a preferred embodiment,
the signaling transducing domain of the CAR can comprise the CD3zeta signaling domain
which has amino acid sequence with at least 70%, preferably at least 80%, more preferably
at least 90 %, 95 % 97 % or 99 % sequence identity with amino acid sequence selected from
the group consisting of (SEQ ID NO: 10).
In particular embodiment the signal transduction domain of the CAR of the present
invention comprises a co-stimulatory signal molecule. A co-stimulatory molecule is a cell
surface molecule other than an antigen receptor or their ligands that is required for an
efficient immune response. “Co-stimulatory ligand” refers to a molecule on an antigen
presenting cell that specifically binds a cognate co-stimulatory molecule on a T-cell, thereby
providing a signal which, in addition to the primary signal provided by, for instance, binding
of a TCR/CD3 complex with an MHC molecule loaded with peptide, mediates a T cell
response, including, but not limited to, proliferation activation, differentiation and the like. A
co-stimulatory ligand can include but is not limited to CD7, B7-1 (CD80), B7-2 (CD86), PD-L1,
PD-L2, 4-1BBL, OX40L, inducible costimulatory igand (ICOS-L), intercellular adhesion
molecule (ICAM, CD30L, CD40, CD70, CD83, HLA-G, MICA, M1CB, HVEM, lymphotoxin beta
receptor, 3/TR6, ILT3, ILT4, an agonist or antibody that binds Toll ligand receptor and a
ligand that specifically binds with B7-H3. A co-stimulatory ligand also encompasses, inter
alia, an antibody that specifically binds with a co-stimulatory molecule present on a T cell,
such as but not limited to, CD27, CD28, 4-1BB, OX40, CD30, CD40, PD-1, ICOS, lymphocyte
function-associated antigen-1 (LFA-1), CD2, CD7, LTGHT, NKG2C, B7-H3, a ligand that
specifically binds with CD83.
A “co-stimulatory molecule” refers to the cognate binding partner on a T-cell that specifically
binds with a co-stimulatory ligand, thereby mediating a co-stimulatory response by the cell,
such as, but not limited to proliferation. Co-stimulatory molecules include, but are not
limited to an MHC class I molecule, BTLA and Toll ligand receptor. Examples of costimulatory
molecules include CD27, CD28, CD8, 4-1BB (CD137), OX40, CD30, CD40, PD-1, ICOS,
lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3 and a
ligand that specifically binds with CD83 and the like.
In a preferred embodiment, the signal transduction domain of the CAR of the present
invention comprises a part of co-stimulatory signal molecule selected from the group
consisting of fragment of 4-1BB (GenBank: AAA53133.) and CD28 (NP_006130.1). In
particular the signal transduction domain of the CAR of the present invention comprises
amino acid sequence which comprises at least 70%, preferably at least 80%, more preferably
at least 90 %, 95 % 97 % or 99 % sequence identity with amino acid sequence selected from
the group consisting of SEQ ID NO: 11 and SEQ ID NO: 12.
The CAR according to the present invention is expressed on the surface membrane of the
cell. Thus, the CAR can comprise a transmembrane domain. The distinguishing features of
appropriate transmembrane domains comprise the ability to be expressed at the surface of
a cell, preferably in the present invention an immune cell, in particular lymphocyte cells or
Natural killer (NK) cells, and to interact together for directing cellular response of immune
cell against a predefined target cell. The transmembrane domain can be derived either from
a natural or from a synthetic source. The transmembrane domain can be derived from any
membrane-bound or transmembrane protein. As non limiting examples, the transmembrane
polypeptide can be a subunit of the T cell receptor such as α, β, γ or δ, polypeptide
constituting CD3 complex, IL2 receptor p55 (α chain), p75 (β chain) or γ chain, subunit chain
of Fc receptors, in particular Fc γ receptor III or CD proteins. Alternatively the transmembrane
domain can be synthetic and can comprise predominantly hydrophobic residues such as
leucine and valine. In a preferred embodiment said transmembrane domain is derived from
the human CD8 alpha chain (e.g. NP_001139345.1). The transmembrane domain can further
comprise a stalk region between said extracellular ligand-binding domain and said
transmembrane domain. The term “stalk region” used herein generally means any oligo- or
polypeptide that functions to link the transmembrane domain to the extracellular ligand-
binding domain. In particular, stalk region are used to provide more flexibility and
accessibility for the extracellular ligand-binding domain. A stalk region may comprise up to
300 amino acids, preferably 10 to 100 amino acids and most preferably 25 to 50 amino acids.
Stalk region may be derived from all or part of naturally occurring molecules, such as from all
or part of the extracellular region of CD8, CD4 or CD28, or from all or part of an antibody
constant region. Alternatively the stalk region may be a synthetic sequence that corresponds
to a naturally occurring stalk sequence, or may be an entirely synthetic stalk sequence. In a
preferred embodiment said stalk region is a part of human CD8 alpha chain (e.g.
NP_001139345.1). In another particular embodiment, said transmembrane and hinge
domains comprise a part of human CD8 alpha chain, preferably which comprises at least
70%, preferably at least 80%, more preferably at least 90 %, 95 % 97 % or 99 % sequence
identity with amino acid sequence selected from the group consisting of SEQ ID NO: 13.
In a particular embodiment, said Chimeric Antigen Receptor of the present invention
comprises a scFV derived from the CD19 monoclonal antibody 4G7, a CD8 alpha human
hinge and transmembrane domain, the CD3 zeta signaling domain and 4-1BB signaling
domain. Preferably, the 4G7 CAR of the present invention comprises at least 70%, preferably
at least 80%, more preferably at least 90 %, 95 % 97 % or 99 % sequence identity with amino
acid sequence selected from the group consisting of SEQ ID NO: 14 and 15.
Downregulation or mutation of target antigens is commonly observed in cancer cells,
creating antigen-loss escape variants. Thus, to offset tumor escape and render immune cell
more specific to target, the CD19 specific CAR can comprise another extracellular ligand-
binding domains, to simultaneously bind different elements in target thereby augmenting
immune cell activation and function. In one embodiment, the extracellular ligand-binding
domains can be placed in tandem on the same transmembrane polypeptide, and optionally
can be separated by a linker. In another embodiment, said different extracellular ligand-
binding domains can be placed on different transmembrane polypeptides composing the
CAR. In another embodiment, the present description relates to a population of CARs
comprising each one different extracellular ligand binding domains. In a particular, the
present description relates to a method of engineering immune cells comprising providing
an immune cell and expressing at the surface of said cell a population of CAR each one
comprising different extracellular ligand binding domains. In another particular
embodiment, the present description relates to a method of engineering an immune cell
comprising providing an immune cell and introducing into said cell polynucleotides encoding
polypeptides composing a population of CAR each one comprising different extracellular
ligand binding domains. By population of CARs, it is meant at least two, three, four, five, six
or more CARs each one comprising different extracellular ligand binding domains. The
different extracellular ligand binding domains according to the present description can
preferably simultaneously bind different elements in target thereby augmenting immune cell
activation and function. The present description also relates to an isolated immune cell
which comprises a population of CARs each one comprising different extracellular ligand
binding domains.
Polynucleotides, vectors:
The present invention also relates to polynucleotides, vectors encoding the above described
CAR according to the invention. In a preferred embodiment, the present invention relates to
a polynucleotide comprising the nucleic acid sequence SEQ ID NO: 17. In a preferred
embodiment, the polynucleotide has at least 70%, preferably at least 80%, more preferably
at least 90 %, 95 % 97 % or 99 % sequence identity with nucleic acid sequence selected from
the group consisting of SEQ ID NO: 17.
The polynucleotide may consist in an expression cassette or expression vector (e.g. a plasmid
for introduction into a bacterial host cell, or a viral vector such as a baculovirus vector for
transfection of an insect host cell, or a plasmid or viral vector such as a lentivirus for
transfection of a mammalian host cell).
In a particular embodiment, the different nucleic acid sequences can be included in one
polynucleotide or vector which comprises a nucleic acid sequence encoding ribosomal skip
sequence such as a sequence encoding a 2A peptide. 2A peptides, which were identified in
the Aphthovirus subgroup of picornaviruses, causes a ribosomal "skip" from one codon to
the next without the formation of a peptide bond between the two amino acids encoded by
the codons (see (Donnelly and Elliott 2001; Atkins, Wills et al. 2007; Doronina, Wu et al.
2008)). By "codon" is meant three nucleotides on an mRNA (or on the sense strand of a DNA
molecule) that are translated by a ribosome into one amino acid residue. Thus, two
polypeptides can be synthesized from a single, contiguous open reading frame within an
mRNA when the polypeptides are separated by a 2A oligopeptide sequence that is in frame.
Such ribosomal skip mechanisms are well known in the art and are known to be used by
several vectors for the expression of several proteins encoded by a single messenger RNA.
To direct, transmembrane polypeptide into the secretory pathway of a host cell, a secretory
signal sequence (also known as a leader sequence, prepro sequence or pre sequence) is
provided in polynucleotide sequence or vector sequence. The secretory signal sequence is
operably linked to the transmembrane nucleic acid sequence, i.e., the two sequences are
joined in the correct reading frame and positioned to direct the newly synthesized
polypeptide into the secretory pathway of the host cell. Secretory signal sequences are
commonly positioned 5' to the nucleic acid sequence encoding the polypeptide of interest,
although certain secretory signal sequences may be positioned elsewhere in the nucleic acid
sequence of interest (see, e.g., Welch et al., U.S. Patent No. 5,037,743; Holland et al., U.S.
Patent No. 5,143,830). In a preferred embodiment the signal peptide comprises the amino
acid sequence SEQ ID NO: 18 and 19.
Those skilled in the art will recognize that, in view of the degeneracy of the genetic code,
considerable sequence variation is possible among these polynucleotide molecules.
Preferably, the nucleic acid sequences of the present invention are codon-optimized for
expression in mammalian cells, preferably for expression in human cells. Codon-optimization
refers to the exchange in a sequence of interest of codons that are generally rare in highly
expressed genes of a given species by codons that are generally frequent in highly expressed
genes of such species, such codons encoding the amino acids as the codons that are being
exchanged.
In a preferred embodiment, the polynucleotide according to the present invention
comprises the nucleic acid sequence of: SEQ ID NO: 17. The present description relates to
polynucleotides comprising a nucleic acid sequence that has at least 70%, preferably at least
80%, more preferably at least 90 %, 95 % 97 % or 99 % sequence identity with nucleic acid
sequence selected from the group consisting of SEQ ID NO: 17.
Methods of engineering an immune cell:
In encompassed particular embodiment, the description relates to a method of preparing
immune cells for immunotherapy comprising introducing into said immune cells the CAR
according to the present invention and expanding said cells. In particular embodiment, the
description relates to a method of engineering an immune cell comprising providing a cell
and expressing at the surface of said cell at least one CAR as described above. In particular
embodiment, the method comprises transforming the cell with at least one polynucleotide
encoding CAR as described above, and expressing said polynucleotides into said cell.
In a preferred embodiment, said polynucleotides are included in lentiviral vectors in view of
being stably expressed in the cells.
In another embodiment, said method further comprises a step of genetically modifying said
cell by inactivating at least one gene expressing one component of the TCR, a target for an
immunosuppressive agent, HLA gene and/or an immune checkpoint gene such as PDCD1 or
CTLA-4. In a preferred embodiment, said gene is selected from the group consisting of
TCRalpha, TCRbeta, CD52, GR, PD1 and CTLA-4. In a preferred embodiment said method
further comprises introducing into said T cells a rare-cutting endonuclease able to selectively
inactivate by DNA cleavage said genes. In a more preferred embodiment said rare-cutting
endonuclease is TALE-nuclease or Cas9 endonuclease.
Delivery methods
The different methods described above involve introducing CAR into a cell. As non-limiting
example, said CAR can be introduced as transgenes encoded by one plasmidic vector. Said
plasmid vector can also contain a selection marker which provides for identification and/or
selection of cells which received said vector.
Polypeptides may be synthesized in situ in the cell as a result of the introduction of
polynucleotides encoding said polypeptides into the cell. Alternatively, said polypeptides
could be produced outside the cell and then introduced thereto. Methods for introducing a
polynucleotide construct into cells are known in the art and including as non limiting
examples stable transformation methods wherein the polynucleotide construct is integrated
into the genome of the cell, transient transformation methods wherein the polynucleotide
construct is not integrated into the genome of the cell and virus mediated methods. Said
polynucleotides may be introduced into a cell by for example, recombinant viral vectors (e.g.
retroviruses, adenoviruses), liposome and the like. For example, transient transformation
methods include for example microinjection, electroporation or particle bombardment. Said
polynucleotides may be included in vectors, more particularly plasmids or virus, in view of
being expressed in cells.
Engineered immune cells
The present description also relates to isolated cells or cell lines susceptible to be obtained
by said method to engineer cells. In particular said isolated cell comprises at least one CAR as
described above. In another embodiment, said isolated cell comprises a population of CARs
each one comprising different extracellular ligand binding domains. In particular, said
isolated cell comprises exogenous polynucleotide sequence encoding CAR. Genetically
modified immune cells of the present description are activated and proliferate
independently of antigen binding mechanisms.
In the scope of the present description is also encompassed an isolated immune cell,
preferably a T-cell obtained according to any one of the methods previously described. Said
immune cell refers to a cell of hematopoietic origin functionally involved in the initiation
and/or execution of innate and/or adaptative immune response. Said immune cell according
to the present description can be derived from a stem cell. The stem cells can be adult stem
cells, non-human embryonic stem cells, more particularly non-human stem cells, cord blood
stem cells, progenitor cells, bone marrow stem cells, induced pluripotent stem cells,
totipotent stem cells or hematopoietic stem cells. Representative human cells are CD34+
cells. Said isolated cell can also be a dendritic cell, killer dendritic cell, a mast cell, a NK-cell, a
B-cell or a T-cell selected from the group consisting of inflammatory T-lymphocytes,
cytotoxic T-lymphocytes, regulatory T-lymphocytes or helper T-lymphocytes. In another
embodiment, said cell can be derived from the group consisting of CD4+ T-lymphocytes and
CD8+ T-lymphocytes. Prior to expansion and genetic modification of the cells of the
invention, a source of cells can be obtained from a subject through a variety of non-limiting
methods. Cells can be obtained from a number of non-limiting sources, including peripheral
blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymus tissue, tissue
from a site of infection, ascites, pleural effusion, spleen tissue, and tumors. In certain
embodiments of the present invention, any number of T cell lines available and known to
those skilled in the art, may be used. In another embodiment, said cell can be derived from a
healthy donor, from a patient diagnosed with cancer or from a patient diagnosed with an
infection. In another embodiment, said cell is part of a mixed population of cells which
present different phenotypic characteristics. In the scope of the present description is also
encompassed a cell line obtained from a transformed T- cell according to the method
previously described. Modified cells resistant to an immunosuppressive treatment and
susceptible to be obtained by the previous method are encompassed in the scope of the
present description.
In another embodiment, said isolated cell according to the present description comprises a
polynucleotide encoding CAR.
Activation and expansion of T cells
Whether prior to or after genetic modification of the T cells, even if the genetically modified
immune cells of the present description are activated and proliferate independently of
antigen binding mechanisms, the immune cells, particularly T-cells of the present description
can be further activated and expanded generally using methods as described, for example, in
U.S. Patents 6,352,694; 6,534,055; 6,905,680; 6,692,964; 5,858,358; 6,887,466; 6,905,681;
7,144,575; 7,067,318; 7,172,869; 7,232,566; 7,175,843; 5,883,223; 6,905,874; 6,797,514;
6,867,041; and U.S. Patent Application Publication No. 20060121005. T cells can be
expanded in vitro or in vivo.
Generally, the T cells of the description are expanded by contact with an agent that
stimulates a CD3 TCR complex and a co-stimulatory molecule on the surface of the T cells to
create an activation signal for the T-cell.
For example, chemicals such as calcium ionophore A23187, phorbol 12-myristate 13-acetate
(PMA), or mitogenic lectins like phytohemagglutinin (PHA) can be used to create an
activation signal for the T-cell.
As non limiting examples, T cell populations may be stimulated in vitro such as by contact
with an anti-CD3 antibody, or antigen-binding fragment thereof, or an anti-CD2 antibody
immobilized on a surface, or by contact with a protein kinase C activator (e.g., bryostatin) in
conjunction with a calcium ionophore. For co-stimulation of an accessory molecule on the
surface of the T cells, a ligand that binds the accessory molecule is used. For example, a
population of T cells can be contacted with an anti-CD3 antibody and an anti-CD28 antibody,
under conditions appropriate for stimulating proliferation of the T cells. Conditions
appropriate for T cell culture include an appropriate media (e.g., Minimal Essential Media or
RPMI Media 1640 or, X-vivo 5, (Lonza)) that may contain factors necessary for proliferation
and viability, including serum (e.g., fetal bovine or human serum), interleukin-2 (IL-2),
insulin, IFN-g , 1L-4, 1L-7, GM-CSF, -10, - 2, 1L-15, TGFp, and TNF- or any other additives for
the growth of cells known to the skilled artisan. Other additives for the growth of cells
include, but are not limited to, surfactant, plasmanate, and reducing agents such as N-acetyl-
cysteine and 2-mercaptoethanoi. Media can include RPMI 1640, A1M-V, DMEM, MEM, a-
MEM, F-12, X-Vivo 1, and X-Vivo 20, Optimizer, with added amino acids, sodium pyruvate,
and vitamins, either serum-free or supplemented with an appropriate amount of serum (or
plasma) or a defined set of hormones, and/or an amount of cytokine(s) sufficient for the
growth and expansion of T cells. Antibiotics, e.g., penicillin and streptomycin, are included
only in experimental cultures, not in cultures of cells that are to be infused into a subject.
The target cells are maintained under conditions necessary to support growth, for example,
an appropriate temperature (e.g., 37° C) and atmosphere (e.g., air plus 5% C02). T cells that
have been exposed to varied stimulation times may exhibit different characteristics
In another particular embodiment, said cells can be expanded by co-culturing with tissue or
cells. Said cells can also be expanded in vivo, for example in the subject’s blood after
administrating said cell into the subject.
Therapeutic applications
In another embodiment, isolated cell obtained by the different methods or cell line derived
from said isolated cell as previously described can be used as a medicament. In another
embodiment, said medicament can be used for treating cancer, particularly for the
treatment of B-cell lymphomas and leukemia in a patient in need thereof. In another
embodiment, said isolated cell according to the description or cell line derived from said
isolated cell can be used in the manufacture of a medicament for treatment of a cancer in a
patient in need thereof.
In another embodiment, the present description relies on methods for treating patients in
need thereof, said method comprising at least one of the following steps:
(a) providing an immune-cell obtainable by any one of the methods previously
described;
(b) Administrating said transformed immune cells to said patient,
On one embodiment, said T cells of the description can undergo robust in vivo T cell
expansion and can persist for an extended amount of time.
Said treatment can be ameliorating, curative or prophylactic. It may be either part of an
autologous immunotherapy or part of an allogenic immunotherapy treatment. By
autologous, it is meant that cells, cell line or population of cells used for treating patients are
originating from said patient or from a Human Leucocyte Antigen (HLA) compatible donor.
By allogeneic is meant that the cells or population of cells used for treating patients are not
originating from said patient but from a donor.
Cells that can be used with the disclosed methods are described in the previous section. Said
treatment can be used to treat patients diagnosed with cancer. Cancers that may be treated
may comprise nonsolid tumors (such as hematological tumors, including but not limited to
pre-B ALL (pedriatic indication), adult ALL, mantle cell lymphoma, diffuse large B-cell
lymphoma and the like. Types of cancers to be treated with the CARs of the invention
include, but are not limited to certain leukemia or lymphoid malignancies. Adult
tumors/cancers and pediatric tumors/cancers are also included.
It can be a treatment in combination with one or more therapies against cancer selected
from the group of antibodies therapy, chemotherapy, cytokines therapy, dendritic cell
therapy, gene therapy, hormone therapy, laser light therapy and radiation therapy.
According to a preferred embodiment of the description, said treatment can be
administrated into patients undergoing an immunosuppressive treatment. Indeed, the
present description preferably relies on cells or population of cells, which have been made
resistant to at least one immunosuppressive agent due to the inactivation of a gene
encoding a receptor for such immunosuppressive agent. In this embodiment, the
immunosuppressive treatment should help the selection and expansion of the T-cells
according to the invention within the patient.
The administration of the cells or population of cells according to the present invention may
be carried out in any convenient manner, including by aerosol inhalation, injection,
ingestion, transfusion, implantation or transplantation. The compositions described herein
may be administered to a patient subcutaneously, intradermaliy, intratumorally,
intranodally, intramedullary, intramuscularly, by intravenous or intralymphatic injection, or
intraperitoneally. In one embodiment, the cell compositions of the present invention are
preferably administered by intravenous injection.
The administration of the cells or population of cells can consist of the administration of 10 -
9 5 6
cells per kg body weight, preferably 10 to 10 cells/kg body weight including all integer
values of cell numbers within those ranges. The cells or population of cells can be
administrated in one or more doses. In another embodiment, said effective amount of cells
are administrated as a single dose. In another embodiment, said effective amount of cells
are administrated as more than one dose over a period time. Timing of administration is
within the judgment of managing physician and depends on the clinical condition of the
patient. The cells or population of cells may be obtained from any source, such as a blood
bank or a donor. While individual needs vary, determination of optimal ranges of effective
amounts of a given cell type for a particular disease or conditions within the skill of the art.
An effective amount means an amount which provides a therapeutic or prophylactic benefit.
The dosage administrated will be dependent upon the age, health and weight of the
recipient, kind of concurrent treatment, if any, frequency of treatment and the nature of the
effect desired.
In another embodiment, said effective amount of cells or composition comprising those cells
are administrated parenterally. Said administration can be an intravenous administration.
Said administration can be directly done by injection within a tumor.
In certain embodiments of the present description, cells are administered to a patient in
conjunction with (e.g., before, simultaneously or following) any number of relevant
treatment modalities, including but not limited to treatment with agents such as antiviral
therapy, cidofovir and interleukin-2, Cytarabine (also known as ARA-C) or nataliziimab
treatment for MS patients or efaliztimab treatment for psoriasis patients or other
treatments for PML patients. In further embodiments, the T cells of the invention may be
used in combination with chemotherapy, radiation, immunosuppressive agents, such as
cyclosporin, azathioprine, methotrexate, mycophenolate, and FK506, antibodies, or other
immunoablative agents such as CAM PATH, anti-CD3 antibodies or other antibody therapies,
cytoxin, fludaribine, cyclosporin, FK506, rapamycin, mycoplienolic acid, steroids, FR901228,
cytokines, and irradiation. These drugs inhibit either the calcium dependent phosphatase
calcineurin (cyclosporine and FK506) or inhibit the p70S6 kinase that is important for growth
factor induced signaling (rapamycin) (Henderson, Naya et al. 1991; Liu, Albers et al. 1992;
Bierer, Hollander et al. 1993). In a further embodiment, the cell compositions of the
present description are administered to a patient in conjunction with (e.g., before,
simultaneously or following) bone marrow transplantation, T cell ablative therapy using
either chemotherapy agents such as, fludarabine, external-beam radiation therapy (XRT),
cyclophosphamide, or antibodies such as OKT3 or CAMPATH, In another embodiment, the
cell compositions of the present description are administered following B-cell ablative
therapy such as agents that react with CD20, e.g., Rituxan. For example, in one embodiment,
subjects may undergo standard treatment with high dose chemotherapy followed by
peripheral blood stem cell transplantation. In certain embodiments, following the transplant,
subjects receive an infusion of the expanded immune cells of the present invention. In an
additional embodiment, expanded cells are administered before or following surgery.
Other definitions
- Unless otherwise specified, "a," "an," "the," and "at least one" are used interchangeably
and mean one or more than one.- Amino acid residues in a polypeptide sequence are
designated herein according to the one-letter code, in which, for example, Q means Gln or
Glutamine residue, R means Arg or Arginine residue and D means Asp or Aspartic acid
residue.
The term “comprising” as used in this specification and claims means “consisting at least in part of”.
When interpreting statements in this specification, and claims which include the term “comprising”,
it is to be understood that other features that are additional to the features prefaced by this term in
each statement or claim may also be present. Related terms such as “comprise” and “comprised”
are to be interpreted in similar manner.
- Amino acid substitution means the replacement of one amino acid residue with another,
for instance the replacement of an Arginine residue with a Glutamine residue in a peptide
sequence is an amino acid substitution.
- Nucleotides are designated as follows: one-letter code is used for designating the base of a
nucleoside: a is adenine, t is thymine, c is cytosine, and g is guanine. For the degenerated
nucleotides, r represents g or a (purine nucleotides), k represents g or t, s represents g or c,
w represents a or t, m represents a or c, y represents t or c (pyrimidine nucleotides), d
represents g, a or t, v represents g, a or c, b represents g, t or c, h represents a, t or c, and n
represents g, a, t or c.
- “As used herein, “nucleic acid” or “polynucleotides” refers to nucleotides and/or
polynucleotides, such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA),
oligonucleotides, fragments generated by the polymerase chain reaction (PCR), and
fragments generated by any of ligation, scission, endonuclease action, and exonuclease
action. Nucleic acid molecules can be composed of monomers that are naturally-occurring
nucleotides (such as DNA and RNA), or analogs of naturally-occurring nucleotides (e.g.,
enantiomeric forms of naturally-occurring nucleotides), or a combination of both. Modified
nucleotides can have alterations in sugar moieties and/or in pyrimidine or purine base
moieties. Sugar modifications include, for example, replacement of one or more hydroxyl
groups with halogens, alkyl groups, amines, and azido groups, or sugars can be
functionalized as ethers or esters. Moreover, the entire sugar moiety can be replaced with
sterically and electronically similar structures, such as aza-sugars and carbocyclic sugar
analogs. Examples of modifications in a base moiety include alkylated purines and
pyrimidines, acylated purines or pyrimidines, or other well-known heterocyclic substitutes.
Nucleic acid monomers can be linked by phosphodiester bonds or analogs of such linkages.
Nucleic acids can be either single stranded or double stranded.
- By chimeric antigen receptor (CAR) is intended molecules that combine a binding domain
against a component present on the target cell, for example an antibody-based specificity
for a desired antigen (e.g., tumor antigen) with a T cell receptor-activating intracellular
domain to generate a chimeric protein that exhibits a specific anti-target cellular immune
activity. Generally, CAR consists of an extracellular single chain antibody (scFvFc) fused to
the intracellular signaling domain of the T cell antigen receptor complex zeta chain (scFvFc:ζ)
and have the ability, when expressed in T cells, to redirect antigen recognition based on the
monoclonal antibody's specificity. One example of CAR used in the present description is a
CAR directing against CD19 antigen and can comprise as non limiting example the amino acid
sequence : SEQ ID NO: 14.
- The term “endonuclease” refers to any wild-type or variant enzyme capable of catalyzing
the hydrolysis (cleavage) of bonds between nucleic acids within a DNA or RNA molecule,
preferably a DNA molecule. Endonucleases do not cleave the DNA or RNA molecule
irrespective of its sequence, but recognize and cleave the DNA or RNA molecule at specific
polynucleotide sequences, further referred to as “target sequences” or “target sites”.
Endonucleases can be classified as rare-cutting endonucleases when having typically a
polynucleotide recognition site greater than 12 base pairs (bp) in length, more preferably of
14-55 bp. Rare-cutting endonucleases significantly increase HR by inducing DNA double-
strand breaks (DSBs) at a defined locus (Perrin, Buckle et al. 1993; Rouet, Smih et al. 1994;
Choulika, Perrin et al. 1995; Pingoud and Silva 2007). Rare-cutting endonucleases can for
example be a homing endonuclease (Paques and Duchateau 2007), a chimeric Zinc-Finger
nuclease (ZFN) resulting from the fusion of engineered zinc-finger domains with the catalytic
domain of a restriction enzyme such as FokI (Porteus and Carroll 2005), a Cas9 endonuclease
from CRISPR system (Gasiunas, Barrangou et al. 2012; Jinek, Chylinski et al. 2012; Cong, Ran et al.
2013; Mali, Yang et al. 2013) or a chemical endonuclease (Eisenschmidt, Lanio et al. 2005;
Arimondo, Thomas et al. 2006). In chemical endonucleases, a chemical or peptidic cleaver is
conjugated either to a polymer of nucleic acids or to another DNA recognizing a specific
target sequence, thereby targeting the cleavage activity to a specific sequence. Chemical
endonucleases also encompass synthetic nucleases like conjugates of orthophenanthroline,
a DNA cleaving molecule, and triplex-forming oligonucleotides (TFOs), known to bind specific
DNA sequences (Kalish and Glazer 2005). Such chemical endonucleases are comprised in the
term “endonuclease” according to the present description.
- By a “TALE-nuclease” (TALEN) is intended a fusion protein consisting of a nucleic acid-
binding domain typically derived from a Transcription Activator Like Effector (TALE) and one
nuclease catalytic domain to cleave a nucleic acid target sequence. The catalytic domain is
preferably a nuclease domain and more preferably a domain having endonuclease activity,
like for instance I-TevI, ColE7, NucA and Fok-I. In a particular embodiment, the TALE domain
can be fused to a meganuclease like for instance I-CreI and I-OnuI or functional variant
thereof. In a more preferred embodiment, said nuclease is a monomeric TALE-Nuclease. A
monomeric TALE-Nuclease is a TALE-Nuclease that does not require dimerization for specific
recognition and cleavage, such as the fusions of engineered TAL repeats with the catalytic
domain of I-TevI described in WO2012138927. Transcription Activator like Effector (TALE)
are proteins from the bacterial species Xanthomonas comprise a plurality of repeated
sequences, each repeat comprising di-residues in position 12 and 13 (RVD) that are specific
to each nucleotide base of the nucleic acid targeted sequence. Binding domains with similar
modular base-per-base nucleic acid binding properties (MBBBD) can also be derived from
new modular proteins recently discovered by the applicant in a different bacterial species.
The new modular proteins have the advantage of displaying more sequence variability than
TAL repeats. Preferably, RVDs associated with recognition of the different nucleotides are
HD for recognizing C, NG for recognizing T, NI for recognizing A, NN for recognizing G or A,
NS for recognizing A, C, G or T, HG for recognizing T, IG for recognizing T, NK for recognizing
G, HA for recognizing C, ND for recognizing C, HI for recognizing C, HN for recognizing G, NA
for recognizing G, SN for recognizing G or A and YG for recognizing T, TL for recognizing A, VT
for recognizing A or G and SW for recognizing A. In another embodiment, critical amino acids
12 and 13 can be mutated towards other amino acid residues in order to modulate their
specificity towards nucleotides A, T, C and G and in particular to enhance this specificity.
TALE-nuclease have been already described and used to stimulate gene targeting and gene
modifications (Boch, Scholze et al. 2009; Moscou and Bogdanove 2009; Christian, Cermak et
al. 2010; Li, Huang et al. 2011). Engineered TAL-nucleases are commercially available under
the trade name TALEN (Cellectis, 8 rue de la Croix Jarry, 75013 Paris, France).
The rare-cutting endonuclease according to the present description can also be a Cas9
endonuclease. Recently, a new genome engineering tool has been developed based on the
RNA-guided Cas9 nuclease (Gasiunas, Barrangou et al. 2012; Jinek, Chylinski et al. 2012; Cong, Ran
et al. 2013; Mali, Yang et al. 2013) from the type II prokaryotic CRISPR (Clustered Regularly
Interspaced Short palindromic Repeats) adaptive immune system (see for review (Sorek,
Lawrence et al. 2013)). The CRISPR Associated (Cas) system was first discovered in bacteria
and functions as a defense against foreign DNA, either viral or plasmid. CRISPR-mediated
genome engineering first proceeds by the selection of target sequence often flanked by a
short sequence motif, referred as the proto-spacer adjacent motif (PAM). Following target
sequence selection, a specific crRNA, complementary to this target sequence is engineered.
Trans-activating crRNA (tracrRNA) required in the CRISPR type II systems paired to the crRNA
and bound to the provided Cas9 protein. Cas9 acts as a molecular anchor facilitating the
base pairing of tracRNA with cRNA (Deltcheva, Chylinski et al. 2011). In this ternary complex,
the dual tracrRNA:crRNA structure acts as guide RNA that directs the endonuclease Cas9 to
the cognate target sequence. Target recognition by the Cas9-tracrRNA:crRNA complex is
initiated by scanning the target sequence for homology between the target sequence and
the crRNA. In addition to the target sequence-crRNA complementarity, DNA targeting
requires the presence of a short motif adjacent to the protospacer (protospacer adjacent
motif - PAM). Following pairing between the dual-RNA and the target sequence, Cas9
subsequently introduces a blunt double strand break 3 bases upstream of the PAM motif
(Garneau, Dupuis et al. 2010).
Rare-cutting endonuclease can be a homing endonuclease, also known under the name of
meganuclease. Such homing endonucleases are well-known to the art (Stoddard 2005).
Homing endonucleases recognize a DNA target sequence and generate a single- or double-
strand break. Homing endonucleases are highly specific, recognizing DNA target sites ranging
from 12 to 45 base pairs (bp) in length, usually ranging from 14 to 40 bp in length. The
homing endonuclease according to the description may for example correspond to a
LAGLIDADG endonuclease, to a HNH endonuclease, or to a GIY-YIG endonuclease. Preferred
homing endonuclease according to the present description can be an I-CreI variant.
- By “ delivery vector” or “ delivery vectors” is intended any delivery vector which can be
used in the present description to put into cell contact ( i.e “contacting”) or deliver inside
cells or subcellular compartments (i.e “introducing”) agents/chemicals and molecules
(proteins or nucleic acids) needed in the present description. It includes, but is not limited to
liposomal delivery vectors, viral delivery vectors, drug delivery vectors, chemical carriers,
polymeric carriers, lipoplexes, polyplexes, dendrimers, microbubbles (ultrasound contrast
agents), nanoparticles, emulsions or other appropriate transfer vectors. These delivery
vectors allow delivery of molecules, chemicals, macromolecules (genes, proteins), or other
vectors such as plasmids, peptides developed by Diatos. In these cases, delivery vectors are
molecule carriers. By “delivery vector” or “delivery vectors” is also intended delivery
methods to perform transfection.
- The terms "vector" or “vectors” refer to a nucleic acid molecule capable of transporting
another nucleic acid to which it has been linked. A “vector” in the present invention
includes, but is not limited to, a viral vector, a plasmid, a RNA vector or a linear or circular
DNA or RNA molecule which may consists of a chromosomal, non chromosomal, semi-
synthetic or synthetic nucleic acids. Preferred vectors are those capable of autonomous
replication (episomal vector) and/or expression of nucleic acids to which they are linked
(expression vectors). Large numbers of suitable vectors are known to those of skill in the art
and commercially available.
Viral vectors include retrovirus, adenovirus, parvovirus (e. g. adenoassociated viruses),
coronavirus, negative strand RNA viruses such as orthomyxovirus (e. g., influenza virus),
rhabdovirus (e. g., rabies and vesicular stomatitis virus), paramyxovirus (e. g. measles and
Sendai), positive strand RNA viruses such as picornavirus and alphavirus, and double-
stranded DNA viruses including adenovirus, herpesvirus (e. g., Herpes Simplex virus types 1
and 2, Epstein-Barr virus, cytomegalovirus), and poxvirus (e. g., vaccinia, fowlpox and
canarypox). Other viruses include Norwalk virus, togavirus, flavivirus, reoviruses,
papovavirus, hepadnavirus, and hepatitis virus, for example. Examples of retroviruses
include: avian leukosis-sarcoma, mammalian C-type, B-type viruses, D type viruses, HTLV-
BLV group, lentivirus, spumavirus (Coffin, J. M., Retroviridae: The viruses and their
replication, In Fundamental Virology, Third Edition, B. N. Fields, et al., Eds., Lippincott-Raven
Publishers, Philadelphia, 1996).
- By “lentiviral vector” is meant HIV-Based lentiviral vectors that are very promising for gene
delivery because of their relatively large packaging capacity, reduced immunogenicity and
their ability to stably transduce with high efficiency a large range of different cell types.
Lentiviral vectors are usually generated following transient transfection of three (packaging,
envelope and transfer) or more plasmids into producer cells. Like HIV, lentiviral vectors enter
the target cell through the interaction of viral surface glycoproteins with receptors on the
cell surface. On entry, the viral RNA undergoes reverse transcription, which is mediated by
the viral reverse transcriptase complex. The product of reverse transcription is a double-
stranded linear viral DNA, which is the substrate for viral integration in the DNA of infected
cells. By “integrative lentiviral vectors (or LV)”, is meant such vectors as nonlimiting example,
that are able to integrate the genome of a target cell. At the opposite by “non-integrative
lentiviral vectors (or NILV)” is meant efficient gene delivery vectors that do not integrate the
genome of a target cell through the action of the virus integrase.
- Delivery vectors and vectors can be associated or combined with any cellular
permeabilization techniques such as sonoporation or electroporation or derivatives of these
techniques.
- By cell or cells is intended any eukaryotic living cells, primary cells and cell lines derived
from these organisms for in vitro cultures.
- By “primary cell” or “primary cells” are intended cells taken directly from living tissue (i.e.
biopsy material) and established for growth in vitro, that have undergone very few
population doublings and are therefore more representative of the main functional
components and characteristics of tissues from which they are derived from, in comparison
to continuous tumorigenic or artificially immortalized cell lines.
As non limiting examples cell lines can be selected from the group consisting of CHO-K1 cells;
HEK293 cells; Caco2 cells; U2-OS cells; NIH 3T3 cells; NSO cells; SP2 cells; CHO-S cells; DG44
cells; K-562 cells, U-937 cells; MRC5 cells; IMR90 cells; Jurkat cells; HepG2 cells; HeLa cells;
HT-1080 cells; HCT-116 cells; Hu-h7 cells; Huvec cells; Molt 4 cells.
All these cell lines can be modified by the method of the present description to provide cell
line models to produce, express, quantify, detect, study a gene or a protein of interest; these
models can also be used to screen biologically active molecules of interest in research and
production and various fields such as chemical, biofuels, therapeutics and agronomy as non-
limiting examples.
- by “mutation” is intended the substitution, deletion, insertion of up to one, two, three,
four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, twenty,
twenty five, thirty, fourty, fifty, or more nucleotides/amino acids in a polynucleotide (cDNA,
gene) or a polypeptide sequence. The mutation can affect the coding sequence of a gene or
its regulatory sequence. It may also affect the structure of the genomic sequence or the
structure/stability of the encoded mRNA.
- by “variant(s)”, it is intended a repeat variant, a variant, a DNA binding variant, a TALE-
nuclease variant, a polypeptide variant obtained by mutation or replacement of at least one
residue in the amino acid sequence of the parent molecule.
- by “functional variant” is intended a catalytically active mutant of a protein or a protein
domain; such mutant may have the same activity compared to its parent protein or protein
domain or additional properties, or higher or lower activity.
-"identity" refers to sequence identity between two nucleic acid molecules or polypeptides.
Identity can be determined by comparing a position in each sequence which may be aligned
for purposes of comparison. When a position in the compared sequence is occupied by the
same base, then the molecules are identical at that position. A degree of similarity or
identity between nucleic acid or amino acid sequences is a function of the number of
identical or matching nucleotides at positions shared by the nucleic acid sequences. Various
alignment algorithms and/or programs may be used to calculate the identity between two
sequences, including FASTA, or BLAST which are available as a part of the GCG sequence
analysis package (University of Wisconsin, Madison, Wis.), and can be used with, e.g., default
setting. For example, polypeptides having at least 70%, 85%, 90%, 95%, 98% or 99% identity
to specific polypeptides described herein and preferably exhibiting substantially the same
functions, as well as polynucleotide encoding such polypeptides, are contemplated.
- “similarity” describes the relationship between the amino acid sequences of two or more
polypeptides. BLASTP may also be used to identify an amino acid sequence having at least
70%, 75%, 80%, 85%, 87.5%, 90%, 92.5%, 95%, 97.5%, 98%, 99% sequence similarity to a
reference amino acid sequence using a similarity matrix such as BLOSUM45, BLOSUM62 or
BLOSUM80. Unless otherwise indicated a similarity score will be based on use of BLOSUM62.
When BLASTP is used, the percent similarity is based on the BLASTP positives score and the
percent sequence identity is based on the BLASTP identities score. BLASTP “Identities”
shows the number and fraction of total residues in the high scoring sequence pairs which are
identical; and BLASTP “Positives” shows the number and fraction of residues for which the
alignment scores have positive values and which are similar to each other. Amino acid
sequences having these degrees of identity or similarity or any intermediate degree of
identity of similarity to the amino acid sequences disclosed herein are contemplated and
encompassed by this disclosure. The polynucleotide sequences of similar polypeptides are
deduced using the genetic code and may be obtained by conventional means. A
polynucleotide encoding such a functional variant would be produced by reverse translating
its amino acid sequence using the genetic code.
- “signal-transducing domain” or “co-stimulatory ligand” refers to a molecule on an antigen
presenting cell that specifically binds a cognate co-stimulatory molecule on a T-cell, thereby
providing a signal which, in addition to the primary signal provided by, for instance, binding
of a TCR/CD3 complex with an MHC molecule loaded with peptide, mediates a T cell
response, including, but not limited to, proliferation activation, differentiation and the like. A
co-stimulatory ligand can include but is not limited to CD7, B7-1 (CD80), B7-2 (CD86), PD-L1,
PD-L2, 4-1BBL, OX40L, inducible costimulatory igand (ICOS-L), intercellular adhesion
molecule (ICAM, CD30L, CD40, CD70, CD83, HLA-G, MICA, M1CB, HVEM, lymphotoxin beta
receptor, 3/TR6, ILT3, ILT4, an agonist or antibody that binds Toll ligand receptor and a
ligand that specifically binds with B7-H3. A co-stimulatory ligand also encompasses, inter
alia, an antibody that specifically binds with a co-stimulatory molecule present on a T cell,
such as but not limited to, CD27, CD28, 4-IBB, OX40, CD30, CD40, PD-1, ICOS, lymphocyte
function-associated antigen-1 (LFA-1), CD2, CD7, LTGHT, NKG2C, B7-H3, a ligand that
specifically binds with CD83.
A “co-stimulatory molecule” refers to the cognate binding partner on a Tcell that specifically
binds with a co-stimulatory ligand, thereby mediating a co-stimulatory response by the cell,
such as, but not limited to proliferation. Co-stimulatory molecules include, but are not
limited to an MHC class I molecule, BTLA and Toll ligand receptor.
A “co-stimulatory signal” as used herein refers to a signal, which in combination with
primary signal, such as TCR/CD3 ligation, leads to T cell proliferation and/or upregulation or
downregulation of key molecules.
-The term “extracellular ligand-binding domain” as used herein is defined as an oligo- or
polypeptide that is capable of binding a ligand. Preferably, the domain will be capable of
interacting with a cell surface molecule. For example, the extracellular ligand-binding
domain may be chosen to recognize a ligand that acts as a cell surface marker on target cells
associated with a particular disease state. Thus examples of cell surface markers that may
act as ligands include those associated with viral, bacterial and parasitic infections,
autoimmune disease and cancer cells.
The term "subject" or “patient” as used herein includes all members of the animal kingdom
including non-human primates and humans.
The above written description of the invention provides a manner and process of making
and using it such that any person skilled in this art is enabled to make and use the same, this
enablement being provided in particular for the subject matter of the appended claims,
which make up a part of the original description.
Where a numerical limit or range is stated herein, the endpoints are included. Also, all values
and subranges within a numerical limit or range are specifically included as if explicitly
written out.
The above description is presented to enable a person skilled in the art to make and use the
invention, and is provided in the context of a particular application and its requirements.
Various modifications to the preferred embodiments will be readily apparent to those skilled
in the art, and the generic principles defined herein may be applied to other embodiments
and applications without departing from the spirit and scope of the invention. Thus, this
invention is not intended to be limited to the embodiments shown, but is to be accorded
the widest scope consistent with the principles and features disclosed herein.
Having generally described this invention, a further understanding can be obtained by
reference to certain specific examples, which are provided herein for purposes of illustration
only, and are not intended to be limiting unless otherwise specified.
Examples
Example 1: Proliferation of TCRalpha inactivated cells expressing a 4G7-CAR.
Heterodimeric TALE-nuclease targeting two 17-bp long sequences (called half targets)
separated by an 15-bp spacer within T-cell receptor alpha constant chain region (TRAC) gene
were designed and produced. Each half target is recognized by repeats of the half TALE-
nucleases listed in Table 1.
Target Target sequence Repeat sequence Half TALE-nuclease
TTGTCCCACAGATATCC Repeat TRAC_T01-L TRAC_T01-L TALEN
Agaaccctgaccctg (SEQ ID NO: 21) (SEQ ID NO: 23)
TRAC_T01
CCGTGTACCAGCTGAGA Repeat TRAC_T01-R TRAC_T01-R TALEN
(SEQ ID NO: 20) (SEQ ID NO: 22) (SEQ ID NO: 24)
Each TALE-nuclease construct was subcloned using restriction enzyme digestion in a
mammalian expression vector under the control of the T7 promoter. mRNA encoding TALE-
nuclease cleaving TRAC genomic sequence were synthesized from plasmid carrying the
coding sequence downstream from the T7 promoter.
Purified T cells preactivated during 72 hours with antiCD3/CD28 coated beads were
transfected with each of the 2 mRNAs encoding both half TRAC_T01 TALE-nucleases. 48
hours post-transfection, T cells were transduced with a lentiviral vector encoding 4G7-CAR
(SEQ ID NO: 14). 2 days post-transduction, CD3 cells were purified using anti-CD3
magnetic beads and 5 days post-transduction cells were reactivated with soluble anti-CD28
(5 µg/ml).
Cell proliferation was followed for up to 30 days after reactivation by counting cell 2 times
per week. The Figure 1 shows the fold induction in cell number respect to the amount of
cells present at day 2 post reactivation for two different donors. Increased proliferation in
TCR alpha inactivated cells expressing the 4G7-CAR, especially when reactivated with anti-
CD28, was observed compared to non transduced cells.
To investigate whether the human T cells expressing the 4G7-CAR display activated state,
the expression of the activation marker CD25 was analyzed by FACS 7 days post
transduction. As indicated in Figure 2, purified cells transduced with the lentiviral vector
encoding 4G7-CAR expressed considerably more CD25 at their surface than the non
transduced cells. Increased CD25 expression is observed both in CD28 reactivation or no
reactivation conditions.
Example 2: Comparison of basal activation of primary human T cells expressing the 4G7-
CAR and the classical FMC63-CAR.
To determine whether 4G7 scFV confers a prolonged “activated” state on the transduced
cell, basal activation of T cell transduced with CAR harboring a 4G7 scFV (SEQ ID NO: 17
encoded SEQ ID NO: 15) or a classical FMC63 scFV (SEQ ID NO: 16) was compared.
Purified human T cells were transduced according to the following protocol: briefly, 1x10
CD3+ cells preactivated during 3 days with anti CD3/CD28 coated beads and recombinant IL2
were transduced with lentiviral vectors encoding the 4G7-CAR (SEQ ID NO: 15) and the FMC63-
CAR (SEQ ID NO: 16) at an MOI of 5 in 12-well non tissue culture plates coated with 30µg/ml
retronectin. 24 hours post transduction the medium was removed and replaced by fresh
medium. The cells were then maintained at a concentration of 1x10 cells/ml throughout the
culture period by cell enumeration every 2-3 days.
3, 8 and 15 days post transduction with the lentiviral vector encoding either the 4G7-CAR or the
FMC63-CAR, the percentage of CAR expressing cells was assessed by flow cytometry. It was
observed that the efficiency of transduction was relatively equivalent with the two lentiviral
vectors Figure 3.
It was then investigated whether the human T cells expressing the 4G7-CAR exhibited a more
activated state than the human T cells expressing the FMC63-CAR. For that purpose the
expression of the activation marker CD25 was compared at the surface of T cells transduced with
the 2 lentiviral vectors at different time points. As indicated in the Figure 4, 3 and 8 days post
transduction, the cells transduced with the lentiviral vector encoding the 4G7-CAR expressed
considerably more CD25 at their surface than the cells transduced with the lentiviral vector
encoding the FMC63-CAR.
The size of the 4G7-CAR or FMC63-CAR transduced cells was also assessed by flow cytometry at
different time points. It was observed that the cells expressing the 4G7-CAR were bigger than the
cells expressing the FMC63-CAR 3, 8 and 15 days post transduction Figure 5.
Following non-specific activation in vitro, 4G7-CAR transduced cells display an increased cell
size (blast formation) as well as the expression of activation markers (CD25) over an
extended time period. This long-term activation permits extended proliferation compared to
cells transduced with a similar CAR containing the FMC63 ScFv.
Example 3: Comparison of proliferation of primary human T cells expressing the 4G7-CAR
and the classical FMC63-CAR.
To determine whether 4G7 scFV confers a higher proliferation activity, proliferation of T cell
transduced with CAR harboring a 4G7 scFV (SEQ ID NO: 17 encoded SEQ ID NO: 15) or a
classical FMC63 scFV (SEQ ID NO: 16) was followed up to 20 days by counting cell two times
per week . Purified human T cells were transduced according to the following protocol:
briefly, 1x10 CD3+ cells preactivated during 3 days with anti CD3/CD28 coated beads and
recombinant IL2 were transduced with lentiviral vectors encoding the 4G7-CAR (SEQ ID NO:
15) and the FMC63-CAR (SEQ ID NO: 16). The cells were then maintained under classical
conditions and were reactivated at Day 12. Cells were seeded at the same density and were
counted two times per week during 20 days. As represented in figure 6, proliferation activity
of T-cells expressing the 4G7-CAR is twofold higher compared to those of cells expressing the
classical FMC63-CAR.
Certain statements that appear herein are broader than what appears in the statements of the
invention. These statements are provided in the interests of providing the reader with a better
understanding of the invention and its practice. The reader is directed to the accompanying claim set
which defines the scope of the invention.
In this specification where reference has been made to patent specifications, other external
documents, or other sources of information, this is generally for the purpose of providing a context
for discussing the features of the invention. Unless specifically stated otherwise, reference to such
external documents is not to be construed as an admission that such documents, or such sources of
information, in any jurisdiction, are prior art, or form part of the common general knowledge in the
art.
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Claims (17)
1. A CD19 specific chimeric antigen receptor comprising at least one extracellular ligand binding domain, a transmembrane domain and at least one intracellular signalling domain wherein said extracellular domain comprises a single chain FV fragment derived from the monoclonal antibody 4G7, specific for CD19, said single chain FV fragment comprising the variable fragment of the CD19 monoclonal antibody 4G7 immunoglobulin gamma 1 heavy chain of SEQ ID NO: 3 and the variable fragment of the CD19 monoclonal antibody 4G7 immunoglobulin kappa light chain of SEQ ID NO: 4 or SEQ ID NO: 5.
2. The CD19 specific chimeric antigen receptor of claim 1 wherein said single chain FV fragment comprises the amino acid sequence of SEQ ID NO: 7 or 8.
3. The CD19 specific chimeric antigen receptor of claim 1 or 2 wherein said intracellular signalling domain comprises a CD3 zeta signalling domain.
4. The CD 19 specific chimeric antigen receptor according to any one of claims 1 to 3 wherein said intracellular signalling domain comprises a 4-1BB domain.
5. The CD19 specific chimeric antigen receptor according to any one of claims 1 to 4 comprising a human CD8 alpha chain transmembrane and stalk domain.
6. The CD19 specific chimeric antigen receptor according to any one of claims 1 to 5 comprising an amino acid sequence having at least 75% sequence identity with the amino acid sequence SEQ ID NO: 14 or 15.
7. The CD19 specific chimeric antigen receptor according to any one of claims 1 to 5 comprising an amino acid sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 14 or 15.
8. The CD19 specific chimeric antigen receptor according to any one of claims 1 to 5 comprising an amino acid sequence having at least 85% sequence identity with the amino acid sequence of SEQ ID NO: 14 or 15.
9. The CD19 specific chimeric antigen receptor according to any one of claims 1 to 5 comprising an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 14 or 15.
10. The CD19 specific chimeric antigen receptor according to any one of claims 1 to 5 comprising an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 14 or 15.
11. The CD19 specific chimeric antigen receptor according to any one of claims 1 to 10 further comprising another extracellular ligand binding domain which is not specific for CD19.
12. A polynucleotide encoding said chimeric antigen receptor according to any one of claims 1 to 11.
13. A polynucleotide of claim 12 comprising a nucleic acid sequence having at least 75% sequence identity with the nucleic acid sequence SEQ ID NO: 17.
14. An expression vector comprising a polynucleotide of claim 12 or 13.
15. A genetically engineered isolated immune cell expressing at the cell surface membrane a CD19 specific chimeric antigen receptor comprising at least one extracellular ligand binding domain and at least one intracellular signalling domain wherein said extracellular domain comprises a single chain FV fragment derived from a monoclonal antibody 4G7, specific for CD19, said single chain FV fragment comprising the variable fragments of the CD19 monoclonal antibody 4G7 immunoglobulin gamma 1 heavy chain of SEQ ID NO: 3 and the variable fragments of the CD19 monoclonal antibody 4G7 immunoglobulin kappa light chain of SEQ ID NO: 4 or SEQ ID NO: 5.
16. A genetically engineered isolated immune cell expressing at the cell surface membrane a CD19 specific chimeric antigen receptor according to any one of claims 1 to 11.
17. The genetically engineered isolated immune cell of claim 15 or 16 further comprising another chimeric antigen receptor which is not specific for CD19.
Applications Claiming Priority (9)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
USPCT/US2013/040766 | 2013-05-13 | ||
USPCT/US2013/040755 | 2013-05-13 | ||
PCT/US2013/040766 WO2013176916A1 (en) | 2012-05-25 | 2013-05-13 | Use of pre t alpha or functional variant thereof for expanding tcr alpha deficient t cells |
PCT/US2013/040755 WO2013176915A1 (en) | 2012-05-25 | 2013-05-13 | Methods for engineering allogeneic and immunosuppressive resistant t cell for immunotherapy |
US13/892,805 | 2013-05-13 | ||
US13/892,805 US11603539B2 (en) | 2012-05-25 | 2013-05-13 | Methods for engineering allogeneic and immunosuppressive resistant T cell for immunotherapy |
US201361888259P | 2013-10-08 | 2013-10-08 | |
US61/888,259 | 2013-10-08 | ||
PCT/EP2014/059662 WO2014184143A1 (en) | 2013-05-13 | 2014-05-12 | Cd19 specific chimeric antigen receptor and uses thereof |
Publications (2)
Publication Number | Publication Date |
---|---|
NZ714044A NZ714044A (en) | 2021-08-27 |
NZ714044B2 true NZ714044B2 (en) | 2021-11-30 |
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